Note: Descriptions are shown in the official language in which they were submitted.
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SMALL MOLECULE INHIBITORS OF ONCOGENIC CHD1L
FOR THE TREATMENT OF CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application
63/250,803, filed September 30,
2021 and the benefit of U.S. application 17,953,221, filed September 26, 2022.
Each of the listed
applications is incorporated by reference herein in its entirety.
STATEMENT REGARDING U.S. GOVERNMENT SUPPORT
This invention was made with Government support under grant number
W81XWH1810142
awarded by the Department of Defense (DoD). The U.S. Government has certain
rights in this
invention.
BACKGROUND
The integrity of the genome is maintained by conformational changes to
chromatin structure that
regulate accessibility to DNA for gene expression and replication. Chromatin
structure is
maintained by post-translational modifications of histones and rearrangement
of nucleosomes.
[Lorch et al., 2010; Kumar et al., 2016; Swygert et al., 2014] ATP-dependent
chromatin remodelers
are enzymes that alter chromatin by changing histone composition, and by
evicting or translocating
nucleosomes along DNA. Their activity plays a critical role in cellular
function by regulating gene
expression and the accessibility of DNA for replication, transcription, and
DNA repair. [Fidel et al.,
2011; Brownlee et al., 2015] Dysregulation of chromatin remodeling is
associated with human
disease, particularly cancer. [Zhang et al., 2016; Valencia & Kadoch, 2019]
In the last decade, the chromatin remodeler known as CHD1L (chromodomain
helicase/ATPase
DNA binding protein 1-like), also known as ALC1 (amplified in liver cancer 1),
has emerged as an
oncogene implicated in the pathology of prominent human cancers.(Ma et al.,
2008; Cheng et a I.,
2013] CHD1L is also involved in multi-drug resistance, ranging from
upregulation of drug
resistance efflux pumps (e.g., ABCB1) [Li et al., 2019] to PARP1 mediated DNA
repair [Pines et
al., 2012; Tsuda et al., 2017], and anti-apoptotic activity. [Li et al., 2013;
Chen et al., 2009]
Moreover, amplification or overexpression of CHD1L are correlated with poor
prognoses for
patients, including low overall survival (OS) and metastatic disease. [He et
al., 2015; Hyeon et al.,
2013; Su et al., 2014; Mu et al., 2015; Su et al., 2015; Li et al., 2013; He
et al., 2012; Chen et al.,
2010] CHD1L overexpression has also been implicated in tumor progression and
as a predictor of
poor patient survival. [Ji et al., 2013] The multifunctional oncogenic
mechanisms of CHD1L make
it an attractive therapeutic target in cancer. While the cancer driving
mechanisms of CHD1L have
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been studied in liver [Li et al., 2019], breast [Wu et al., 2014], and lung
[Li et al., 2019] cancer, little
is known about the pathological mechanisms associated with CHD1L in colorectal
cancer (CRC).
CRC is the third most prevalent cancer diagnosed each year and CRC patients
have the second
highest mortality rate worldwide. [Jemal et al., 2011] Early detection of CRC
combined with surgery
and 5-fluorouracil (5FU) based combination chemotherapy has minimally improved
the overall
survival rate. [Jemal et al., 2011; Fakih, 2015] The current chemo and
targeted therapies are
largely ineffective against metastatic CRC (mCRC), evidenced by a low 11% 5-
year overall survival
rate. [Jemal et al., 2011; Fakih, 2015] There is an unmet need in the art to
identify and characterize
targets involved in the pathology of CRC tumor progression and metastasis.
A majority of CRC patients possess mutations in the Wnt signaling pathway,
leading to aberrant T-
Cell Factor/Lymphoid Enhancer Factor-transcription or TCF-complex. [Kinzler &
Voelstein, 1996;
Cancer Genome Atlas, 2012] Such mutations can lead to constitutive p-catenin
translocation and
transactivation of TCF-transcription. [Clevers, 2006; Korinek et al., 1997]
The TCF-complex is
orchestrated by TCF4 (a.k.a. TCFL2), which is activated through interactions
with an array of
coactivators such as p-catenin, PARP1, and CREB Binding protein (CBP).
[Shitashige et al., 2008]
Recently, TCF4 was shown to be a specific driver of both early metastasis from
adenomas (i.e.
polyps) and from late stage mCRC. [Hyeo et al., 2013; Su et al., 2014]
It has been reported that TCF transcription functions as a master regulator of
epithelial-
mesenchymal transition (EMT) [Sanchez-Tilla et al., 2011; Zhou et al., 2016;
Abraham et al.,
2019]. This process can transform relatively benign epithelial tumor cells
into mesenchymal cells
with increased cancer stem cell (CSC) sternness and other malignant properties
that drive mCRC.
[Chaffer et al., 2016] It has recently been reported that alterations in
certain CRC driver genes are
common in both primary and metastatic tumor pairs. [Hu et al., 2019] More
specifically, aberrant
TCF4 is reported to be a specific driver of mCRC. [Hu et al., 2019] and CRC
can metastasize in
early adenomas (i.e., polyps [see also Magri & Bardelli, 2019] which is likely
caused by TCF-driven
EMT. [Chaffer et al. 2016; Chaffer & Weinberg, 2011] These reports indicate
that TCF-transcription
is a driving force at all stages of CRC progression and metastasis.
EMT is a major driving force in numerous human diseases, especially solid
tumor progression,
drug and radiation therapy resistance, evasion of the immune response and
immunotherapy, and
promotion of metastasis. [Chaffer et al. 2016; Chaffer & Weinberg, 2011;
Scheel & Weinberg,
2012]
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Due to the significance of the Wnt signaling pathway and TCF-transcription in
cancer and other
diseases [Clevers, 2006], small molecule drugs that inhibit the Wnt signaling
pathway and TCF-
transcription have been examined. [Lee et al., 2011; Polakis, 2012]
Therapeutic strategies
considered include receptor targets (e.g., Frizzled); preventing Wnt ligand
secretion (e.g.,
porcupine); inhibiting p-catenin destruction complex (e.g., tankyrases); and
protein-protein
inhibition (PPI) with p-catenin and co-activators (e.g., CBP). While clinical
trials may be underway,
no drug has as yet been clinically approved that targets the Wnt/TCF pathway.
[Lu et al., 2016] In
contrast, the present invention describes a new therapeutic strategy,
particularly for identifying
small molecule drugs, for treatment of Wnt/TCF driven CRC in which CHD1L is
identified as a DNA
binding factor required for TCF-transcription regulating the malignant
phenotype in CRC.
For example, U.S. Patent 9,616,047 reports small molecule inhibitors of 6-
catenin or disruptors of
a 6-catenin/TCF-4 complex which are said to attenuate colon carcinogenesis.
Inhibitors of 13-
catenin reported therein include esculetin, as well as, compounds designated
HI-B1¨HI-B20, HI-
HI-B26, HI-B32 and HI-B34, the structures of each of which is provided in the
patent. The patent further describes, in a number of generic chemical formulae
therein,
compounds said to be useful as p-catenin inhibitors and for the treatment of
colon carcinogenesis.
This patent is incorporated by reference herein in its entirety for the
structures of specific
compounds, generic formulae and variable definitions of compounds said therein
to be useful in
the invention therein. The compounds identified herein are structurally
distinct from those
described herein.
CN109761909 published May 17, 2019 reports (as described in the Espacenet Eng.
Abstract
thereof) certain N-(4-(pyrimidine-4-amino)phenyl)sulfonamide compounds or
salts of a certain
formula inhibit Hsp9O-Cdc37 (heat shock protein Hsp90 and its auxiliary
chaperone Cdc37)
interactive client protein expression, and are reported useful for treating or
preventing various
diseases mediated by an Hsp90 signal channel. The formula given in the
published application is:
..H
N
(::) /9 1,
N
R f N
(I) R3
where variables are defined according to the Espacenet English machine
translation as follows:
R1 is mes-trimethylphenyl, 4-methylphenyl, 4-trifluoromethylphenyl, naphthyl,
2,3,4,5,-
tetramethylphenyl, 4-methoxyphenyl, 4-tert-butylphenyl, 2,4-dimethoxyphenyl,
2,5-
dimethoxyphenyl or 4-phenoxphenyl;
R2 is hydrogen, methyl acetate, acetate, aminoacetyl, 4-formic acid benzyl, 4-
isopropylbenzyl, 4-
chlorobenzyl or 4-nnethoxybenzyl; and
3
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R3 is chlorine, -0Ra or ¨NRbRc, where, Ra is a chain 01-3 alkyl, C5-6
cycloalkyl, C1-2 alkoxy,
mono- or di-C1-2 alkylannino, or 05-6 nitrogen-containing or oxygen-containing
heterocyclic group;
and Rb and Rc are C1-5 alkyl groups, respectively. More specifically, R3 is
chlorine, 2-
hydroxytetrahydropyrrolyl, ethanolamino, 2,3-dihydroxy-1-methylpropylamino,
2,3-
dihydroxypropylamino, piperazinyl, N-methylpiperazinyl, azepyl, piperidinyl, 2-
methylpropylamino,
propoxy, methylamino, ethylamino, cyclopropylamino, 1-ethylpropylamino,
tetrahydropyran-4-
ylmethoxy or 2-methoxyethoxy. The reference also refers to a compound of
formula 1-5:
NHNN
0% "0
NH
This published application is incorporated by reference herein in its entirety
for the structures of
specific compounds, generic formulae and variable definitions of compounds
said therein to be
useful in the invention therein. Structures disclosed in this published
application can be excluded
from any chemical formula of the present application.
The present invention examines the clinicopathological characteristics of
CHD1L in CRC, and the
results herein indicate that CHD1L is a druggable target involved in TCF-
transcription. A
mechanism for CHD1L-mediated TCF-transcription is also proposed herein. Small
molecule
inhibitors of CHD1L are identified herein which are able to prevent TCF
transcription, reverse EMT,
and other malignant properties in a variety of cell models including tumor
organoids and patient
derived tumor organoids (PDT0s). Certain CHD1L inhibitors identified herein
display drug-like
pharmacological properties, including in vivo pharmacokinetic (PK) and
pharmacodynamic (PD)
profiles, important for translational development towards the treatment of CRC
and other cancers.
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SUMMARY
This invention relates to the treatment of CHD1L-driven cancers, more
specifically TCF
transcription-driven cancers and yet more specifically EMT-driven cancers.
CHD1L is found to be
an essential component of the TCF transcription complex. Small molecule
inhibitors of CHDL1
which inhibit CHD1L ATPase and inhibit CHD1L-dependent TCF-transcription have
been identified.
CHD1L inhibitors are believed to prevent the TCF-complex from binding to Wnt
response elements
and promoter sites. More specifically, CHD1L inhibitors induce the reversion
of EMT. CHD1L
inhibitors are useful in the treatment of various cancers and particularly CRC
and m-CRC.
Particularly with respect to CRC, CHD1L inhibitors are shown in embodiments to
inhibit cancer
stern cell (CSC) sternness and invasive potential. In embodiments, CHD1L
inhibitors induce
cytotoxicity in CRC PDT0s. In specific embodiments, the CHD1L-driven cancer is
CRC, breast
cancer, including BRCA-mutated breast cancer and metastatic breast cancer,
ovarian cancer,
including BRCA-mutated ovarian cancer, pancreatic cancer, including BRCA-
mutated pancreatic
cancer, glioma, liver cancer, lung cancer, prostate cancer, or
gastrointestinal (GI) cancers. In
specific embodiments, the TCF transcription-driven cancer is CRC, including
mCRC. In specific
embodiments, the EMT-driven cancer is CRC, including mCRC. In specific
embodiments, the
cancer that is treated is breast cancer, including BRCA-mutated breast cancer
and metastatic
breast cancer. In specific embodiments, the cancer that is treated is ovarian
cancer. In specific
embodiments, the cancer that is treated is pancreatic cancer.
The invention provides a method for treatment of CHD1L-driven cancers, more
specifically TCF
transcription-driven cancers and yet more specifically EMT-driven cancers,
including GI cancer,
particularly CRC and mCRC, which comprises administration to a patient in need
thereof of an
amount of a CHD1L inhibitor which is effective for CHD1L inhibition, effective
inhibition of aberrant
TCF transcription or effective for induction of EMT reversion. In embodiments,
the CHD1L inhibitor
is a compound of any one of formulas l- XXIII or XXX-XVII. In embodiments, the
CHD1L inhibitor
is a compound of any one of formulas I, II, or XX-XXIII. In embodiments, the
CHD1L inhibitor is a
compound of either of formulas XLV or XLVI. In an embodiment, the CHD1L
inhibitor is any one of
compounds 1-177. In an embodiment, the CHD1L inhibitor is any one of compounds
8-177 or any
one of compounds 9-117. In an embodiment, the CHD1L inhibitor is any one of
compounds 28,
31, 52, 54, 57, 75, 118, 126, 131, 150, or 169. In more specific embodiments,
the compound is
selected from compounds 52, 118, 126, 131, 150, or 169. In embodiments, the
compound is
selected from compounds 28, 31, 54, 57, or 75. In embodiments, the compound is
one or more of
compounds 28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169. In embodiments,
the compound is
one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169. More
specifically, the
invention provides a method of inhibiting aberrant TCF-transcription,
particularly in CRC, by
administration of an effective amount of a CHD1L inhibitor. Yet more
specifically, the invention
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provides a method of inducing reversion of EMT, particularly in CRC or mCRC,
by administration of
an effective amount of a CHD1L inhibitor. The invention provides a method of
inhibiting Cancer
Stem Cell (CSC) stemness and/or invasive potential, particularly in CRC, by
administration of an
effective amount of a CHD1L inhibitor. The invention provides a method for
treatment of cancerous
tumors of CHD1L-driven cancers, or TCF transcription-driven cancers or EMT-
driven cancers,
particularly in CRC, by administration of an effective amount of a CHD1L
inhibitor. The invention
provides a method for treatment of cancerous solid tumors of CHD1L-driven
cancers, or TCF
transcription-driven cancers or EMT-driven cancers, particularly in CRC, by
administration of an
effective amount of a CHD1L inhibitor. The invention provides a method for
treatment of breast
cancer, including BRCA-mutated breast cancer, by administration of an
effective amount of a
CHD1L inhibitor. The invention provides a method for treatment of ovarian
cancer by
administration of an effective amount of a CHD1L inhibitor. The invention
provides a method for
treatment of pancreatic cancer by administration of an effective amount of a
CHD1L inhibitor.
In embodiments, CHD1L inhibitors are selective for inhibition of CHD1L. In
embodiments, CHD1L
inhibitors herein are not PARP inhibitors. In embodiments, CHD1L inhibitors
herein are not
inhibitors of topoisomerases. In particular, CHD1L inhibitors herein are not
inhibitors of DNA
topoisomerase. In particular, CHD1L inhibitors herein are not inhibitors of
topoisomerase type Ila.
In embodiments, CHD1L inhibitors herein are not inhibitors of p-catenin,
particularly inhibitors such
as described in U.S. Patent 9,616,047. In embodiments, CHD1L inhibitors herein
are not inhibitors
of Hsp9O-Cdc37 interactive client protein expression, particularly inhibitors
as described in
CN109761909.
In embodiments, invention also provides a method to prevent tumor growth,
invasion and/or
metastasis in CHD1L-driven, TCF-transcription, or EMT-driven cancers by
administering to a
patient in need thereof of an amount of a CHD1L inhibitor of this invention
which is effective for
CHD1L inhibition, inhibition of aberrant TCF transcription, or effective for
reversion of EMT. In
specific embodiments, tumors are solid tumors. In specific embodiments, tumors
are those
associated with GI cancer. In embodiments, tumors are those associated with
CRC. In
embodiments, tumors are those associated with mCRC. In embodiments, tumors are
those
associated with breast cancer. In embodiments, tumors are those associated
with BRAC-mutated
breast cancer. In embodiments, tumors are those associated with ovarian
cancer. In
embodiments, tumors are those associated with pancreatic cancer. In
embodiments, tumors are
those associated with lung cancer. In embodiments, tumors are those associated
with liver cancer.
In specific embodiments, the invention provides a method for treatment of CRC,
including mCRC,
which comprises administration to a patient in need thereof of an amount of a
CHD1L inhibitor
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which is effective for inhibition of CHD1L. In specific embodiments, the
invention provides a
method for treatment of CRC, including mCRC, which comprises administration to
a patient in
need thereof of an amount of a CHD1L inhibitor which is effective for
inhibition of aberrant TCF
transcription. In specific embodiments, the invention provides a method for
treatment of CRC,
including mCRC, which comprises administration to a patient in need thereof of
an amount of a
CHD1L inhibitor which is effective for induction of reversion of EMT.
In specific embodiments, the invention provides a method for inducing
apoptosis in cancer cells
which comprises contacting a cancer cell with an effective amount of a CHD1L
inhibitor. In an
embodiment, the CHD1L inhibitor is provided in an amount effective for
inhibition of aberrant TCF
transcription. In an embodiment, the CHD1L inhibitor is provided in an amount
effective for
induction of reversion of EMT. In an embodiment, the cancer cells are CRC
cancer cells. In an
embodiment, the cancer cells are mCRC cancer cells. In an embodiment, the
cancer cells are
breast cancer cells. In an embodiment, the cancer cells are breast cancer
cells carry a BRCA
mutation. In an embodiment, the cancer cells are ovarian cancer cells. In an
embodiment, the
cancer cells are pancreatic cancer cells. In an embodiment, the cancer cells
are lung cancer cells.
In an embodiment, the cancer cells are liver cancer cells. In an embodiment,
the method is applied
in vivo. In an embodiment, the method is applied in vivo in a patient. In an
embodiment, the
method is applied in vitro.
In embodiments of the methods herein comprising administration of the CHD1L
inhibitor, the
CHD1L inhibitor is administered by any known administration method and dosing
schedule to
achieve desired benefits. In an embodiment, administration is oral
administration. In an
embodiment, administration is by intravenous injection.
In embodiments, oral administration employs oral dosage forms comprising
pharmaceutically
acceptable polyethylene glycol (PEG). In such embodiments, the
pharmaceutically acceptable
PEG may be combined with a pharmaceutically acceptable organic solvent,
particularly a
pharmaceutically acceptable polar, aprotic solvent. In embodiments, the
organic solvent is
pharmaceutically acceptable DMSO. In embodiments, oral administration employs
oral dosage
forms comprising low molecular weight polyethylene glycol having molecular
weight less than 600
g/mole. In more specific embodiments, oral administration employs PEG 400. In
more specific
embodiments, oral administration employs PEG 200.
In embodiments, the invention in addition1i provides a method of treatment of
drug-resistant
cancer which comprises administering to a patient in need thereof of an amount
of a CHD1L
inhibitor, which is effective for CHD1L inhibition, inhibition of aberrant TCF
transcription or
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induction of reversion of EMT, in combination with a known treatment to which
the cancer has
become resistant. In specific embodiments, the treatment to which the cancer
has become
resistant is conventional chemotherapy and other targeted therapies. In
specific embodiments, the
invention provides a method of increasing the efficacy of a DNA-damaging drug
in cancer which
comprises combined treatment of the cancer with the DNA damaging drug and a
CHD1L inhibitor
where the CHD1L is administered in an amount effective for decreasing
resistance to the DNA-
damaging drug. In an embodiment, the DNA-damaging drug is a topoisomerase
inhibitor. In
particular, the DNA-damaging drug is a DNA topoisomerase inhibitor. In
particular, the DNA-
damaging drug is a topoisomerase type Ila inhibitor. In particular, the DNA-
damaging drug is
etoposide or teniposide. In particular, the DNA-damaging drug is SN38 or a
prodrug thereof. In an
embodiment, the DNA-damaging drug is a thymidylate synthase inhibitor. In an
embodiment, the
thymidylate synthase inhibitor is a folate analogue. In an embodiment, the
thymidylate synthase
inhibitor is a nucleotide analogue. In specific embodiments, the thymidylate
synthase inhibitor is
raltitrexed, pemetrexed, nolatrexed or ZD9331. In a particular embodiment, the
DNA-damaging
drug is 5-fluorouracil or capecitabine.
In an embodiment, the drug-resistant cancer is a CHD1L-driven cancer. In an
embodiment, the
drug-resistant cancer is a TCF transcription-driven cancer. In an embodiment,
the drug-resistant
cancer is an EMT-driven cancer. In an embodiment, the treatment is for drug-
resistant CRC. In an
embodiment, the treatment is for drug-resistant mCRC. In embodiments, the
treatment is for drug-
resistant breast cancer, drug-resistant ovarian cancer, drug-resistant
pancreatic cancer, drug-
resistant lung cancer or drug-resistant liver cancer. In embodiments, the DNA-
damaging drug and
the CHD1I inhibitor are administered by any known method on a dosing schedule
appropriate for
realizing the combined therapeutic benefit. In embodiments, the CHD1L
inhibitor is administered
orally and the DNA-damaging drug is administered by any known administration
method and
dosing schedule. In embodiments, the CHD1L inhibitor is administered prior to
administration of
the DNA-damaging drug. In embodiments, the CHD1L inhibitor is administered
prior to and
optionally after administration of the DNA-damaging drug. In embodiments, the
CHD1L inhibitor is
administered orally prior to and optionally after administration of the DNA-
damaging drug by
intravenous injection.
The invention provides methods for treatment of CHD1L-driven cancer, TCF-
transcription-driven
cancer, or EMT-driven cancer which comprises administration to a patient in
need thereof of an
amount of a CHD1L inhibitor which is effective for CHD1L inhibition or
inhibition of aberrant TCF
transcription or induction of reversion of EMT in combination with an
alternative method of
treatment for the cancer. In an embodiment, the cancer is GI cancer or more
specifically CRC
cancer and yet more specifically is mCRC. In an embodiment, the alternative
method for treatment
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is administration of one or more of 5-fluorouracil, 5-fluorouracil in
combination with folinic acid (also
known as leucovorin), a topoisomerase inhibitor, or a cytotoxic or
antineoplastic agent. In
embodiments, the CHD1L inhibitor is administered in combination with 5-
fluorouracil or in
combination with 5-fluorouracil and folinic acid. In embodiments, the CHD1L
inhibitor is
administered in combination with a topoisomerase inhibitor and in particular
with irinotecan (a
prodrug of SN38 also known as camptothecin) or any other known prodrug of
SN38. In
embodiments, the combined treatment using a CHD1L inhibitor and a
topoisomerase inhibitor
exhibits at least additive activity against the cancer. In embodiments, the
combined treatment of a
CHD1L inhibitor with a topoisomerase inhibitor exhibits synergistic activity
(greater than additive
activity) against the cancer.
In embodiments, the CHD1L inhibitor is administered in combination with a
cytotoxic or
antineoplastic agent, in particular a platinum-based antineoplastic agent and
more particularly
cisplatin, carboplatin or oxaliplatin. In embodiments, the combined treatment
using a CHD1L
inhibitor and a platinum-based antineoplastic agent exhibits at least additive
activity against the
cancer. In embodiments, the combined treatment of a CHD1L inhibitor with a
platinum-based
antineoplastic agent exhibits synergistic activity (greater than additive
activity) against the cancer.
In embodiments, the platinum-based antineoplastic agent and the CHD1I
inhibitor are administered
by any known method on a dosing schedule appropriate for realizing the
combined therapeutic
benefit. In embodiments, the CHD1L inhibitor is administered orally and the
platinum-based
neoplastic agent is administered by any known administration method and dosing
schedule. In
embodiments, the CHD1L inhibitor is administered prior to administration of
the platinum-based
neoplastic agent. In embodiments, the CHD1L inhibitor is administered prior to
and optionally after
administration of the platinum-based antineoplastic agent. In embodiments, the
CHD1L inhibitor is
administered orally prior to and optionally after administration of the
platinum-based neoplastic
agent by intravenous injection.
In embodiments, the CHD1L inhibitor is administered in combination with a
chemotherapy regimen
(administration of an alternative cancer cytotoxic agent or antineoplastic
agent or adnninintration of
an antineoplastic procedure) for treatment of cancer, including without
limitation GI cancer,
particularly CRC, and mCRC. In embodiments, the CHD1L inhibitor is
administered in combination
with the chemotherapy regimen designated FOLFOX. In embodiments, the CHD1L
inhibitor is
administered in combination with the chemotherapy regimen designated FOLFIRI.
In
embodiments, the chemotherapy regime and the CHD1I inhibitor are administered
by any known
method on a dosing schedule appropriate for realizing the combined therapeutic
benefit. In
embodiments, the CHD1L inhibitor is administered orally and the chemotherapy
regime is
administered by any known administration method and dosing schedule. In
embodiments, the
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CHD1L inhibitor is administered prior to administration of the chemotherapy
regime. In
embodiments, the CHD1L inhibitor is administered prior to and optionally after
administration of the
chemotherapy regime. In embodiments, the CHD1L inhibitor is administered
orally prior to and
optionally after administration of the PARP inhibitor by intravenous
injection.
The invention provides a method for treatment of cancers that are sensitive to
Poly(ADP)-ribose)
polymerase I (PARPI) in which a CHD1L inhibitor is used in combination with a
PARP inhibitor. In
embodiments, an amount of a CHD1L inhibitor effective for CHD1L inhibition,
inhibition of aberrant
TCF transcription or induction of reversion of EMT is used in combination with
an amount of a
PARP inhibitor effective for treating cancer to at least enhance the
effectiveness of the cancer
treatment. In embodiments, the combined treatment using a CHD1L inhibitor and
a PARP inhibitor
exhibits at least additive activity against the cancer. In embodiments, the
combined treatment of a
CHD1L inhibitor with a PARP inhibitor exhibits synergistic activity (greater
than additive activity)
against the cancer. In embodiments, the cancer is a cancer sensitive to
treatment by a PARP
inhibitor. In embodiments, the cancer is a cancer that is or has become
resistant to treatment by a
PARP inhibitor. In embodiments, the cancer is a cancer sensitive to treatment
by a PARP inhibitor
or which has become resistant to treatment by a PARP inhibitor and which is a
CHD1L-driven, a
TCF-driven or an EMT-driven cancer. In embodiments, the cancer is a homologous
recombination
deficient cancer (see, for example, Zhou et al. BioRxiv 2020). In embodiments,
the cancer treated
is a cancer sensitive to a PARP inhibitor and more particularly is breast or
ovarian cancer. In
specific embodiments, the cancer is a BRCA-deficient cancer (BRCA-mutated
cancer), for
example, BRCA-deficient breast cancer (BRCA-mutated breast cancer), BRCA-
deficient ovarian
cancer (BRCA-mutated ovarian cancer or BRCA-defcient pancreatic cancer (BRCA-
mutated
pancreatic cancer). In specific embodiments, the cancer is pancreatic cancer.
In specific
embodiments, the cancer is lung or liver cancer. In embodiments, the cancer is
prostate cancer. In
embodiments, the cancer treated is GI cancer, stomach cancer, CRC or mCRC. In
embodiments,
combined treatment of the CHD1L inhibitor with the PARP inhibitor reverses
resistance of the
cancer to treatment by the PARP inhibitor. In embodiments, the PARP inhibitor
is olaparib,
veliparib or talozoparib. In embodiments, the PARP inhibitor is rucaparib or
niraparib. The
invention also provides a method for treating a cancer which comprises
administration of an
amount of a PARP inhibitor effective for treatment of the cancer combined with
administration of an
amount of a CHD1L inhibitor effective for inhibiting CHD1L. In embodiments,
the PARP inhibitor
and the CHD1L inhibitor are administered by any known method on a dosing
schedule appropriate
for realizing the combined therapeutic benefit. In embodiments, the CHD1L
inhibitor is
administered orally and the PARP inhibitor is administered by intravenous
injection. In
embodiments, the CHD1L inhibitor and the PARP inhibitor are both administered
by intravenous
injection. In embodiments, the CHD1L inhibitor is administered prior to
administration of the PARP
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inhibitor. In embodiments, the CHD1L inhibitor is administered prior to and
optionally after
administration of the PARP inhibitor. In embodiments, the CHD1L inhibitor is
administered after
administration of the PARP inhibitor. In embodiments, the CHD1L inhibitor is
administered orally
prior to and optionally after administration of the PARP inhibitor by
intravenous injection.
In embodiments, CHD1L inhibitors are combined with one or more agent that
induces DNA
damage to treat neoplastic disease, including various cancers. In specific
embodiments, CHD1L
inhibitors exhibit more than additivity anticancer activity when combined with
one or more agent
that induces DNA damage. In specific embodiments, CHD1L inhibitors exhibit
synergistic
anticancer activity when combined with one or more agent that induces DNA
damage. This
combined axtivity of CHD1L inhibitors can be assessed in combination with
methylmethane
sulfonate (an alkylating agent), which is an exemplary agent that induces DNA
damage.
The invention also provides a method for identifying a CHD1L inhibitor, which
inhibits CHD1L-
dependent TCF transcription which comprises determining if a selected compound
inhibits a
CHD1L ATPase, as described in examples herein. In specific embodiments,
inhibition of cat-
CHD1L ATPase is determined. In embodiments, compounds exhibiting % inhibition
of 30% or
greater are selected as inhibiting a CHD1L ATPase. In embodiments, compounds
exhibiting A
inhibition of 80% or greater are selected as inhibiting a CHD1L ATPase. In
specific embodiments,
CHD1L inhibitors exhibit IC50 less than 10 pM in dose response assays against
CHD1L ATPase,
particularly cat-CHD1L ATPase. In specific embodiments, CHD1L inhibitors
exhibit IC50 less than
5 pM in dose response assays against CHD1L ATPase, particularly cat-CHD1L
ATPase. In
specific embodiments, CHD1L inhibitors exhibit IC50 less than 5 pM in dose
response assays
against CHD1L ATPase, particularly cat-CHD1L ATPase. In specific embodiments,
CHD1 L
inhibitors exhibit 1050 less than 5 pM. In specific embodiments, CHD1L
inhibitors exhibit IC50 less
than 3 pM. In specific embodiments, CHD1L inhibitors exhibit IC50 less than 1
pM.
In specific embodiments, CHD1L inhibitors are assessed for inhibition of TCF-
transcription in a 2D
cancer cell model, particularly using one or more CRC cell lines, such as
described in examples
herein. In specific embodiments, inhibition of TCF-transcription is determined
using a TOPflash
reporter construct and more specifically a TOPflash luciferase reporter
construct as described
herein. In specific embodiments, inhibition of TCF-transcription by CHD1L
inhibitors in the cell
model is dose-dependent. In specific embodiments, inhibition of TCF-
transcription by CHD1L
inhibitors in the cell model is dose-dependent in the range of 1 to 50 pM. In
specific embodiments,
a CHD1L inhibitor exhibits % TCF-transcription normalized to cell viability of
75% or less at 20 pM.
In specific embodiments, a CHD1L inhibitor exhibits % TCF-transcription
normalized to cell viability
of 50% or less at 40 pM. In specific embodiments, CHD1L inhibitors exhibit
dose dependent
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inhibition of TOE-transcription with IC50 less than 10 pM assayed with
TOPflash reporter in a
cancer cell line. In specific embodiments, CHD1L inhibitors exhibit dose
dependent inhibition of
TCF-transcription with 1050 less than 5 pM assayed with TOPflash reporter in a
cancer cell line. In
specific embodiments, CHD1L inhibitors exhibit dose dependent inhibition of
TOE-transcription with
1050 less than 3 pM assayed with TOPflash reporter in a cancer cell line. In
embodiments, the
cancer cell line is a CRC cancer cell, a breast cancer cell, a glioma cell, a
liver cancer cell, a lung
cancer cell or a GI cancer cell. In an embodiment, the cancer cell line is a
CRC cancer cell line. In
a specific embodiment, the CRC cancer cell line is SW620.
In specific embodiments, CHD1L inhibitors are assessed for their ability to
reverse or inhibit EMT.
In specific embodiments, CHD1L inhibitors are assessed for their ability to
reverse EMT in tumor
organoids. In embodiments, reversion or inhibition of EMT is assessed in tumor
organoids
expressing vimentin where dose-dependent decrease in vimentin expression
indicates reversion or
inhibition of EMT. In embodiments, reversion of EMT is assessed in tumor
organoids expressing
E-cadherin where dose-dependent increase in E-cadherin expression indicates
reversion or
inhibition of EMT. In embodiments, reversion of EMT is assessed in tumor
organoids expressing
E-cadherin, vimentin or both, where dose-dependent decrease in vimentin and
dose-dependent
increase in E-cadherin expression indicates reversion or inhibition of EMT. In
specific
embodiments, dose-dependent reversion or inhibition of EMT is measured over a
compound
concentration of 0.1 to 100 pM. In specific embodiments, dose-dependent
reversion of EMT is
measured over a compound concentration of 0.3 to 50 pM.
In specific embodiments, CHD1L inhibitors are assessed for their ability to
inhibit clonogenic colony
formation which is a well-established assay to measure cancer stem cell
stemness. In
embodiments, cells are pretreated with a selected concentration of CHD1L
inhibitors prior to
plating. In embodiments, cells are cultured at low density such that only CSC
will form colonies
over 10 days in culture. In embodiments, cells are pretreated for 12-36 h. In
embodiments, cells
are pretreated for 24 h. In embodiments, cells are pretreated with CHD1L
inhibitors at
concentration in the range of 0.5-50 pM with appropriate controls. In
embodiments, CHD1L
inhibitors exhibit 40% or more inhibition of clonogenic colony counts,
compared to no compound
control, for CHD1L concentration of 40 pM. In embodiments, CHD1L inhibitors
exhibit 40% or
more inhibition of clonogenic colony counts, compared to no compound control,
for CHD1L
concentration of 20 pM. In embodiments, CHD1L inhibitors exhibit 40% or more
inhibition of
clonogenic colony counts, compared to no compound control, for CHD1L
concentration of 2 pM. In
embodiments, inhibition of clonogenic colony formation lasts over 10 days in
culture.
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In specific embodiments, CHD1L inhibitors are further assessed for loss of
invasive potential
employing any known method and particularly employing a method as described in
the examples
herein.
In specific embodiments, CHD1L inhibitors are further assessed for antitumor
activity as measured
by induction of cytotoxicity in tumor organoids. In embodiments, cells are
treated for a selected
time (e.g., 24-96 h, preferably 72 h) with selected concentration of CHD1L
inhibitor (1-100 pM). In
embodiments, cytotoxicity is measured using any of a variety of cytotoxicity
reagents known in the
art, such as small molecules which, enter damaged cells and exhibit a
measurable change on
entry (e.g., fluorescence, such as, CellToxTm Green reagent (Promega, Madison,
WI) or
IncuCyteCytotox reagents (Sartorius, France). In embodiments, cytotoxicity is
measured by
measurement of LDH (lactate dehydrogenase) released from dead cells.
In embodiments, the CHD1L inhibitors useful in methods of treatment,
pharmaceutical
compositions and pharmaceutical combinations herein are those of formulas I-
XXIII, XXX-XLII and
XLV-XLVI or pharmaceutically acceptable salts or solvates thereof. In
embodiments, the invention
provides novel compounds of any formula herein and in particular of of
formulas I-XXIII, XXXV-XLII
or salts or solvates thereof. In embodiments, the CHD1L inhibitors are those
of formula I, II, )(X-
XXII. In embodiments, the CHD1L inhibitors are those of formula XX. In
embodiments, the
CHD1L inhibitors are those of formulas 1-IX, XI-XIX, XX, XXI, XXII, XXIII or
XXXV-XLII. In
embodiments, the CHD1L inhibitors are those of formula XLV or XLVI.
In specific embodiments, the methods, pharmaceutical compositions and
pharmaceutical
combinations of the invention employ CHD1L inhibitors that are selected from
one or more of
compounds 1-177 or pharmaceutically acceptable salts or solvates thereof. Two
or more CHD1L
inhibitors can be employed in combination in the methods herein. In specific
embodiments, the
CHD1L inhibitors employed in the invention are selected from one or more of
compounds 6-39 or
pharmaceutically acceptable salts thereof. In specific embodiments, the CHD1L
inhibitors
employed in the methods of the invention are selected from one or more of
compounds 40-51 or
pharmaceutically acceptable salts or solvates thereof. In specific
embodiments, the CHD1L
inhibitors employed in the methods of the invention are selected from one or
more of compounds
52-68 or pharmaceutically acceptable salts or solvates thereof. In specific
embodiments, the
CHD1L inhibitors employed in the methods of the invention are selected from
one or more of
compounds 70-73 or pharmaceutically acceptable salts or solvates thereof. In
specific
embodiments, the CHD1L inhibitors employed in the methods of the invention are
selected from
one or more of compounds 74-101 or pharmaceutically acceptable salts or
solvates thereof. In
specific embodiments, the CHD1L inhibitors employed in the methods of the
invention are selected
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from one or more of compounds 102-103 or pharmaceutically acceptable salts or
solvates thereof.
In specific embodiments, the CHD1L inhibitors employed in the methods of the
invention are
selected from one or more of compounds 104-116 or pharmaceutically acceptable
salts or solvates
thereof. In specific embodiments, the CHD1L inhibitors employed in the methods
of the invention
are selected from one or more of compounds 117-142 or pharmaceutically
acceptable salts or
solvates thereof. In specific embodiments, the CHD1L inhibitors employed in
the methods of the
invention are selected from one or more of compounds 143-177 or
pharmaceutically acceptable
salts or solvates thereof. In specific embodiments, the CHD1L inhibitors
employed in the methods
of the invention are selected from one or more of compounds 150-154 or
pharmaceutically
acceptable salts or solvates thereof. In specific embodiments, the CHD1L
inhibitors employed in
the methods of the invention are selected from one or more of compounds 155-
159 or
pharmaceutically acceptable salts or solvates thereof. In specific
embodiments, the CHD1L
inhibitors employed in the methods of the invention are selected from one or
more of compounds
28-39, 74-75, 52, 54, 62-66 or 74-75 or pharmaceutically acceptable salts or
solvates thereof. In
embodiments, the compound is selected from compounds 28, 31, 52, 54, 57, 75,
118, 126, 131,
150, or 169 or pharmaceutically acceptable salts or solvates thereof. In more
specific
embodiments, the compound is selected from compounds 52, 118, 126, 131, 150,
or 169 or
pharmaceutically acceptable salts or solvates thereof. In embodiments, the
compound is selected
from compounds 28, 31, 54, 57, or 75 or pharmaceutically acceptable salts or
solvates thereof. In
embodiments, the compound is one or more of compounds 28, 31, 52, 54, 57, 75,
118, 126, 131,
150, or 169 or pharmaceutically acceptable salts or solvates thereof. n
embodiments, the
compound is one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or
169 or
pharmaceutically acceptable salts or solvates thereof. In specific
embodiments, the CHD1L
inhibitors employed in the methods of the invention are selected from compound
52 or
pharmaceutically acceptable salts or solvates thereof. In specific
embodiments, the forgoing
specifically recited CHD1L inhibitors can be combined with one or more
alternative cancer cytoxic
or antineoplastic agents for treatment or pharmaceutical combination. More
specifically the
alternative cancer cytoxic or antineoplastic agents include, without
limitation, one or more PARP
inhibitor, one or more topoisomerase inhibitor, one or more thynnidylate
synthase inhibitor or one or
more platinum-based antineoplastic agent.
In specific embodiments, the CHD1L inhibitors employed in methods of this
invention are
compounds 6, 8, 52, 54, 56, 61, 62, 65 or 66 or pharmaceutically acceptable
salts or solvates
thereof. In specific embodiments, the CHD1L inhibitors employed in methods of
this invention are
compounds 6, 8 or pharmaceutically acceptable salts or solvates thereof. In
specific
embodiments, the CHD1L inhibitors employed in methods of this invention are
compounds 52, 54
or pharmaceutically acceptable salts or solvates thereof. In specific
embodiments, the CHD1L
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inhibitors employed in methods of this invention are compounds 22, 23, 26 or
27 or
pharmaceutically acceptable salts thereof.
In specific embodiments, the methods of the invention employ CHD1L inhibitors
of formula II and
include all embodiments described herein for formula II. The invention also
provides novel
compounds of formula II, salts thereof and pharmaceutical compositions
contains such compounds
and salts.
In specific embodiments, the methods of the invention employ CHD1L inhibitors
of formula XX and
include all embodiments described herein for formula XX. The invention also
provides novel
compounds of formula XX, salts thereof and pharmaceutical compositions
containing such
compounds and salts.
In specific embodiments, the methods of the invention employ CHD1L inhibitors
of formula XXI and
include all embodiments described herein for formula XXI. The invention also
provides novel
compounds of formula XXI, salts thereof and pharmaceutical compositions
containing such
compounds and salts.
In specific embodiments, the methods of the invention employ CHD1L inhibitors
of formula XXII
and include all embodiments described herein for formula XXII. The invention
also provides novel
compounds of formula XXII, salts thereof and pharmaceutical compositions
containing such
compounds and salts.
In specific embodiments, the methods of the invention employ CHD1L inhibitors
of formula XXIII
and include all embodiments described herein for formula XXIII. The invention
also provides novel
compounds of formula XXII, salts thereof and pharmaceutical compositions
containing such
compounds and salts.
In specific embodiments, the methods of the invention employ CHD1L inhibitors
of formula XLV
and include all embodiments described herein for formula XXIII. The invention
also provides novel
compounds of formula XLV salts thereof and pharmaceutical compositions
containing such
compounds and salts.
In specific embodiments, the methods of the invention employ CHD1L inhibitors
of formula XLVI
and include all embodiments described herein for formula XLVI. The invention
also provides novel
compounds of formula XXII, salts thereof and pharmaceutical compositions
containing such
compounds and salts.
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In embodiments, the invention is also directed to CHD1L inhibitors of this
invention and
pharmaceutically-acceptable compositions comprising any such inhibitors. In
embodiments,
pharmaceutically-acceptable compositions comprise one or more CHD1L inhibitors
and a
pharmaceutically-acceptable excipient.
In embodiments, the invention is directed to any compound or pharmaceutically
acceptable salt or
solvate thereof as described in chemical formulas herein which is novel. In
particular, the invention
is directed to CHD1L inhibitors and pharmaceutically acceptable salts thereof
as described in
formulas herein with the exception that the CHD1L inhibitor is other than
compounds 1-8 or salts or
solvates thereof. In particular, the invention is directed to CHD1L inhibitors
and pharmaceutically
acceptable salts thereof as described in formulas herein with the exception
that the CHD1L
inhibitor is other than compounds 1-9 or salts thereof. In embodiments, the
invention is directed to
any one of compounds 9-39, 40-68, 69-73, 74-101, 102-103, 104-116, 117-142, or
143-177 or
pharmaceutically acceptable salts or solvates thereof or pharmaceutically
acceptable compositions
that contains such compounds or salts or solvates. In embodiments, the
invention is directed to
any one of compounds 10-39, 40-73, 74-116, 117-142 or 43-177 or
pharmaceutically acceptable
salts or solvates thereof or pharmaceutically acceptable compositions that
contains such
compounds or salts or solvates. In embodiments, the invention is directed to
any one of
compounds 52-73 or pharmaceutically acceptable salts or solvates thereof or
pharmaceutically
acceptable compositions that contains such compounds or salts or solvates. In
embodiments, the
invention is directed to any one of compounds 28-39, 74,75, 52, 54, 62-66, or
74-75 or
pharmaceutically acceptable salts or solvates thereof or pharmaceutically
acceptable compositions
that contains such compounds or salts or solvates. In embodiments, the
invention is directed to
any one of compounds 10-39 or pharmaceutically acceptable salts or solvates
thereof or
pharmaceutically acceptable compositions that contains such compounds or salts
or solvates. In
embodiments, the invention is directed to any one of compounds 40-73 or
pharmaceutically
acceptable salts or solvates thereof or pharmaceutically acceptable
compositions that contains
such compounds or salts or solvates. In embodiments, the invention is directed
to any one of
compounds 74-116 or pharmaceutically acceptable salts or solvates thereof or
pharmaceutically
acceptable compositions that contains such compounds or salts or solvates. In
embodiments, the
invention is directed to any one of compounds 117-142 or pharmaceutically
acceptable salts or
solvates thereof or pharmaceutically acceptable compositions that contains
such compounds or
salts or solvates. In embodiments, the invention is directed to any one of
compounds 143-177 or
pharmaceutically acceptable salts or solvates thereof or pharmaceutically
acceptable compositions
that contains such compounds or salts or solvates. In embodiments, the
invention is directed to
one or more of compounds 10-177 of Scheme 1 or pharmaceutically acceptable
salts or solvates
thereof or pharmaceutically-acceptable compositions that contains such
compounds or salts or
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solvates. In embodiments, the invention is directed to one or more of
compounds 150-154 or
pharmaceutically acceptable salts or solvates thereof or pharmaceutically
acceptable compositions
that contains such compounds or salts or solvates. In embodiments, the
invention is directed to
one or more of compounds 155-159 or pharmaceutically acceptable salts or
solvates thereof or
pharmaceutically acceptable compositions that contains such compounds or salts
or solvates. In
embodiments, the compound is selected from compounds 28, 31, 52, 54, 57, 75,
118, 126, 131,
150, or 169 or pharmaceutically acceptable salts or solvates thereof or
pharmaceutically
acceptable compositions that contains such compounds or salts or solvates. In
more specific
embodiments, the compound is selected from compounds 52, 118, 126, 131, 150,
or 169 or
pharmaceutically acceptable salts or solvates thereof or pharmaceutically
acceptable compositions
that contains such compounds or salts or solvates. In embodiments, the
compound is selected
from compounds 28, 31, 54, 57, or 75 or pharmaceutically acceptable salts or
solvates thereof or
pharmaceutically acceptable compositions that contains such compounds or salts
or solvates. In
embodiments, the compound is one or more of compounds 28, 31, 52, 54, 57, 75,
118, 126, 131,
150, or 169 or pharmaceutically acceptable salts or solvates thereof or
pharmaceutically
acceptable compositions that contains such compounds or salts or solvates. In
embodiments, the
compound is one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or
169 or
pharmaceutically acceptable salts or solvates thereof or pharmaceutically
acceptable compositions
that contains such compounds or salts or solvates. In specific embodiments,
the CHD1L inhibitors
employed in the methods of the invention are selected from compound 52 or
pharmaceutically
acceptable salts or solvates thereof. In embodiments, a CHD1L inhibitor of the
invention has Clog
P of 5 or less. In embodiments, a CHD1L inhibitor of the invention has Clog P
of 3-4.
In specific embodiments the invention is directed to the following compounds
and to methods
herein employing these compounds for the treatment of cancer, particularly CRC
and mCRC: any
one of compounds 52-73; compound 52 or 53; compounds 54, 55 or 67; compounds
57, 58 or 59;
or pharmaceutically acceptable salts or solvates thereof; any one of compound
8, compound 52,
compound 53, compound 54, compound 55, compound 56, compound 57, compound 58,
compound 59, compound 61, compound 62, compound 65, compound 66, or compound
67 or
pharmaceutically acceptable salts or solvates thereof. In specific embodiments
the invention is
directed to the following compounds and to methods herein employing these
compounds for the
treatment of cancer, particularly CRC and mCRC: any one of compounds 28, 31,
52, 54, 57, 75,
118, 126, 131, 150, 0r169; any one of compounds 52, 118, 126, 131, 150, or
169;.any one of
compounds 28, 31, 54, 57, or 75; any one of compounds 28, 31, 52, 54, 57, 75,
118, 126, 131,
150, or 169; one or more of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131,
150, 01169.
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In embodiments, the invention provides a pharmaceutical combination of one or
more CHD1L
inhibitor and one or more alternative cancer cytotoxic or antineoplastic
agent. In embodiments, the
components of the pharmaceutical combination can be together or separate. In
embodiments, the
pharmaceutical combination is a pharmaceutical compositions containing one or
more CHDL1
inhibitor and one or more PARP inhibitor or one or more topoisomerase
inhibitor or one or more
thymidylate synthase inhibitor. In embodiments, the pharmaceutical combination
is a
pharmaceutical compositions containing one or more CHDL1 inhibitor and one or
more platinum-
based antineoplastic agent. In embodiments, the pharmaceutical combination is
two or more
separate pharmaceutical compositions each containing different components of
the pharmaceutical
combination. In embodiments, the pharmaceutical combination is two separate
pharmaceutical
compositions, one containing one or more CHD1L inhibitors and one containing
one or more PARP
inhibitors or one or more topoisomerase inhibitor or one or more thymidylate
synthase inhibitor. In
embodiments, the pharmaceutical combination is two separate pharmaceutical
compositions, one
containing one or more CHD1L inhibitors and one containing one or more
platinum-based
antineoplastic agent. In embodiments, the pharmaceutical combination is a
single pharmaceutical
composition, containing one or more CHD1L inhibitors and one containing one or
more PARP
inhibitor. In embodiments, the pharmaceutical combination is a single
pharmaceutical composition,
containing one or more CHD1L inhibitors and one containing one or more
topoisomerase inhibitor.
In embodiments, the pharmaceutical combination is a single pharmaceutical
composition,
containing one or more CHD1L inhibitors and one containing one or more
thymidylate synthase
inhibitor. More specifically, the invention relates to pharmaceutical
combinations as described
herein which comprise one or more CHD1L inhibitor of any one of formulas l-
XXIII, XXX-XLII anf
XLV-XLVI or pharmaceutically acceptable salts or solvates thereof. More
specifically, the invention
relates to pharmaceutical combinations as described herein which comprise one
or more CHD1L
inhibitor of any one of formulas I, II, XX-XXIII or pharmaceutically
acceptable salts or solvates
thereof. More specifically, the invention relates to pharmaceutical
combinations as described
herein which comprise one or more CHD1L inhibitor of any one of formulas XLV -
XLVI or
pharmaceutically acceptable salts or solvates thereof. More specifically, the
invention relates to
pharmaceutical combinations as described herein which comprise one or more of
CHDL1 inhibitors
of any of compounds 1-177 or any one of compounds 9-177 or pharmaceutically
acceptable salts
or solvates thereof. In specific embodiments, the CHD1L inhibitors of the
pharmaceutical
combination are compounds 52-73; compound 52 or 53; compounds 54, 55 or 67; or
compounds
57, 58 or 59; or pharmaceutically acceptable salts or solvates thereof; any
one of compound 8,
compound 52, compound 53, compound 54, compound 55, compound 56, compound 57,
compound 58, compound 59, compound 61, compound 62, compound 65, compound 66,
or
compound 67 or pharmaceutically acceptable salts or solvates thereof. In
embodiments, the
CHD1L inhibitors of the pharmaceutical combination are any one of compounds
28, 31, 52, 54, 57,
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75, 118, 126, 131, 150, or 169; any one of compounds 52, 118, 126, 131, 150,
or 169;.any one of
compounds 28, 31, 54, 57, or 75; any one of compounds 28, 31, 52, 54, 57, 75,
118, 126, 131,
150, or 169; one or more of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131,
150, or 169.
In embodiments, the invention also relates to the use of a CHD1L inhibitor in
the manufacture of a
medicament for the treatment of cancer, particularly for the treatment of
CHD1L-driven cancer,
TCF-driven cancer, or EMT-driven cancer, particularly GI cancer, and more
particularly CRC or
mCRC. In embodiments, the cancer to be treated is breast cancer, particularly
BRCA-mutated
breast cancer, ovarian cancer, particularly BRCA-mutated ovarian cancer,
pancreatic cancer,
particularly BRCA-mutated pancreatic cancer, lung cancer, prostate cancer or
liver cancer. More
specifically, the invention relates to the use of a CHD1L inhibitor of any one
of formulas l- XX, XXI,
XXII, XXIII, XXX-XLII and XLV-XLVI or pharmaceutically acceptable salts or
solvates thereof in the
manufacture of a medicament for the treatment of cancer, CHD1L-driven cancer,
TCF-driven
cancer, or EMT-driven cancer, particularly GI cancer, and more particularly
CRC or mCRC. In
embodiments, the CHD1L inhibitors are those of formulas 1-IX, XI-XIX, XX, XXI,
XXII, XXIII, XXXV-
XLII and XLV-XLVI. In embodiments, the CHD1L inhibitors are those of formula
I, formula II,
formula XX, formula XXI, formula XXII or formula XXIII. In embodiments, the
CHD1L inhibitors are
those of formula XLVor XLVI. In embodiments, the CHD1L inhibitor is one or
more of the
compounds 1-117 of Scheme 1. In embodiments, the CHD1L inhibitor is one or
more of the
compounds 118-177 of Scheme 1. In embodiments, the CHD1L inhibitors are
compounds 52-73;
compound 52 or 53; compounds 54, 55 or 67; or compounds 57, 58 or 59; or
pharmaceutically
acceptable salts or solvates thereof; any one of compound 8, compound 52,
compound 53,
compound 54, compound 55, compound 56, compound 57, compound 58, compound 59,
compound 61, compound 62, compound 65, compound 66, or compound 67 or
pharmaceutically
acceptable salts or solvates thereof. In embodiments, the CHD1L inhibitors of
the pharmaceutical
combination are any one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131,
150, or 169; any one
of compounds 52, 118, 126, 131, 150, or 169;.any one of compounds 28, 31, 54,
57, or 75; any
one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169; one or
more of compounds
28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169 or pharmaceutically
acceptable salts or solvates
thereof.
In embodiments, the invention also relates to the use of a CHD1L inhibitor in
combination with an
alternative cancer cytooxic or antineoplastic agent in ithe manufacture of a
medicament for the
combination treatment of cancer, particularly for the treatment of CHD1L-
driven cancer, TCF-
driven cancer, or EMT-driven cancer, particularly GI cancer, and more
particularly CRC or mCRC.
In embodiments, the cancer to be treated is breast cancer, particularly BRCA-
mutated breast
cancer or metastatic breast cancer, ovarian cancer, particularly BRCA-
mutatedovarian cancer,
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pancreatic cancer, particularly BRCA-mutated pancreatic cancer, lung cancer,
prostate cancer, or
liver cancer. More specifically, the invention relates to the use of a CHD1L
inhibitor of any one of
formulas 1- )0(111, )00(-XLII and XLV-XLVI or pharmaceutically acceptable
salts or solvates thereof
in the manufacture of a medicament for the combination treatment of cancer,
CHD1L-driven
cancer, TCF-driven cancer, or EMT-driven cancer, particularly GI cancer, and
more particularly
CRC or mCRC. In embodiments, the CHD1L inhibitors are those of formula!,
formula II, formula
XX, formula )0(1, formula XXII or formula XXIII. In embodiments, the CHD1L
inhibitors are those of
formula XLV-XLVI. In embodiments, the CHD1L inhibitors are those of formulas 1-
IX, XI-XIX, XX,
XXI, XXII, XXIII, XXII, XXIII, XXXV-XLII or XLV-XLVI. In embodiments, the
CHD1L inhibitor is one
or more of the compounds 1-117 or one or more of compounds 8-177, compounds 9-
177 or
compounds 118-177 of Scheme 1. In embodiments, the CHD1L inhibitors are
compounds 52-73;
compound 52 or 53; compounds 54, 55 or 67; or compounds 57, 58 or 59; or
pharmaceutically
acceptable salts or solvates thereof; any one of compound 8, compound 52,
compound 53,
compound 54, compound 55, compound 56, compound 57, compound 58, compound 59,
compound 61, compound 62, compound 65, compound 66, or compound 67 or
pharmaceutically
acceptable salts or solvates thereof. In embodiments, the CHD1L inhibitors of
the pharmaceutical
combination are any one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131,
150, or 169; any one
of compounds 52, 118, 126, 131, 150, or 169;.any one of compounds 28, 31, 54,
57, or 75; any
one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169; one or
more of compounds
28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169 or pharmaceutically
acceptable salts or solvates
thereof. In embodiments, the one or more CHD1L inhibitors are combined in the
medicament with
one or more PARP inhibitors, one or more topoisomerase inhibitors, one or more
thymidylate
synthase inhibitors or one or more platinum-based antineoplastic agents.
In embodiments, the invention further relates to a CHD1L inhibitor in
combination with one or more
alterntive cancer cytotoxic or antineoplastic agent for use in the treatment
of cancer, CHD1L-driven
cancer, TCF-driven cancer, or EMT-driven cancer, particularly GI cancer, and
more particularly
CRC or mCRC. In embodiments, the cancer to be treated is breast cancer,
ovarian cancer, and
pancreatic cancer, particularly BRCA-mutated breast cancer, BRCA-mutated
ovarian cancer,
BRCA-mutated pancreatic cancer, prostate cancer, stomach cancer, lung cancer,
or liver cancer.
More specifically, the invention relates to the use of a CHD1L inhibitor of
any one of formulas I-
XXIII, XXX-XLII and XLV-XLVI or pharmaceutically acceptable salts or solvates
thereof in the
manufacture of a medicament for the combination treatment of cancer, CHD1L-
driven cancer,
TCF-driven cancer, or EMT-driven cancer, particularly GI cancer, and more
particularly CRC or
mCRC. In embodiments, the CHD1L inhibitors are those of formula!, formula II,
formula )(X,
formula XXI, formula XXII or formula XXIII. In embodiments, the CHD1L
inhibitors are those of
formula XLV-XLVI. In embodiments, the CHD1L inhibitors are those of formulas 1-
IX, XI-XIX, XX,
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XXI, XXII, XXIII, XXII, )001, XXXV-XLII or XLV-XLVI. In embodiments, the CHD1L
inhibitor is one
or more of the compounds 1-117 or one or more of compounds 8-177, compounds 9-
177 or
compounds 118-177 of Scheme 1. In embodiments, the CHD1L inhibitors are
compounds 52-73;
compound 52 or 53; compounds 54, 55 or 67; or compounds 57, 58 or 59; or
pharmaceutically
acceptable salts or solvates thereof; any one of compound 8, compound 52,
compound 53,
compound 54, compound 55, compound 56, compound 57, compound 58, compound 59,
compound 61, compound 62, compound 65, compound 66, or compound 67 or
pharmaceutically
acceptable salts or solvates thereof. In embodiments, the CHD1L inhibitors of
the pharmaceutical
combination are any one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131,
150, or 169; any one
of compounds 52, 118, 126, 131, 150, or 169;.any one of compounds 28, 31, 54,
57, or 75; any
one of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169; one or
more of compounds
28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169 or pharmaceutically
acceptable salts or solvates
thereof. In embodiments, the alternitve cancer cytotoxic or antineoplastic
agent is one or more
PARP inhibitors, one or more topoisomerase inhibitors, one or more thymidylate
synthase
inhibitors or one or more platinum-based antineoplastic agents.
Other embodiments and aspects of the invention will be readily apparent to one
of ordinary skill in
the art on review of the drawings, detailed description and examples herein.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-B: Validation of CHD1L inhibitors identified from HTS. (FIG. 1A)
cat-CHD1L ATPase
050 dose responses with hits 1-7. Mean IC50 values are calculated from three
independent
experiments and representative graphs are shown. (FIG. 1B) SW620, HCT-16, and
DLD1CHD1L-
OE cells with TOPflash reporter were used to measure inhibition of TCF
transcription using 3
doses over 24h.
Figures 2A-2D: CHD1L inhibitors reverse EMT and the malignant phenotype in
CRC. Dose
responses for CHD1L inhibitors that modulate EMT measured by high-content
imaging of (FIG. 2A)
downregulation of VimPro-GFP reporter and (FIG. 2B) upregulation of EcadPro-
RFP reporter.
Mean E050 values SEM are calculated from three independent experiments (FIG.
2C) CSC
stemness measured by clonogenic colony formation after pretreatment with CHD1L
inhibitors in
DLD1CHD1L-OE and HCT-116 cells. (FIG. 2D) Inhibition of invasive potential of
HCT-116 cells
after treatment of CHD1L inhibitors. Welch's t-test statistical analysis was
used to determine
significance, where *= P 0.05, ** = P 0.01, ***= P 0.001, ****= P 0.0001.
Figures 3A-C: Compound 6 induces apoptosis in CRC cell lines and PDT0s. (FIG.
3A) Time
course evaluation of the induction E-cadherin expression using Ecad-ProRFP
reporter assay and
cytotoxicity using Cell-ToxTm Green cytotoxicity assay (Promega, Madison, WI).
(FIG. 3B) Annexin
V-FITC staining analysis of apoptosis after treatment of SN-38 and 6 for 12
hours. (FIG. 3C)
Cytotoxicity of 6 in PDTO CR0102 using CellTiter-Blue cell viability assay
(Promega, Madison,
WI). Mean E050 values s.d. are calculated from six independent experiments
and representative
graph is shown with inset of a representative PDTO. Welch's t-test statistical
analysis was used to
determine significance, where * = P 0.05, ** = P 0.01, *** = P 0.001, ****= P
0.0001.
Figure 4: Accumulation of Compound 6 in SW620 xenograft tumors. Compound 6 was
administered by i.p. injection to athymic nude mice QD for 5 days to measure
accumulation in
SW620 xenog raft tumors.
Figure 5: Proposed mechanism of action of CHD1L mediated TCF-transcription.
CHD1L is
activated through binding TCF-complex members PARP1 and TCF4 [Abbott et al.,
2020] (1) Once
activated, CHD1L is directed to hindered WREs localized on chromatin. (2)
Chromatin remodeling
and DNA translocation occurs exposing WRE sites. (3) TCF-complex binds to
exposed WREs
facilitated by CHD1L, promoting EMT genes and other genes associated with
mCRC. CHD1L
ATPase inhibitors effectively prevent step 1, leading to the reversion of EMT
and other malignant
properties of CRC.
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Figures 6A-E Evaluation of Compound 8. (FIG. 6A) Compound 8 displays potent
low pM dose-
dependent inhibition of TCF-transcription based on TOPFlash reported in SW260
cells cultures in
2D and over a 24 h time course. Compound 8 effectively reverses EMT in dual
reporter SW620
tumor organoids over 72 h evidenced by downregulation of vimentin (FIG. 6B)
and (FIG. 6C)
upregulation of E-cadherin promoter activity in a dose-dependent manner.
Compound 8
significantly inhibits (FIG. 6D) clonogenic colony formation over 10 days
after pre-treatment for 24
h and (FIG. 6E) HCT116 invasive potential over 48 h. The students t-test
indicates* P 0.05
Figures 7A-B: Viability of Colorectal Cancer Tumor Organoids after Treatment
with Exemplary
CHDIL Inhibitors. The figures illustrate representative graphs of % viability
as a function of log
concentration of the indicated compound. (FIG. 7A) Treatment with Compound
6.9; (FIG. 7B)
Treatment with Compound 6.11; Alternative compound numbers as used in Scheme 1
are given in
parenthesis. IC50, in some cases average IC50, are provided in each figure.
Viability data for a
number of exemplary compounds are provided in Table 3.
Figures 8A-B: Assessment of CHD1L-mediated DNA repair and "on target" effects
of CHD1L
inhibitor 6 alone and in combination with irinotecan (prodrug of SN38). CHD1L
is known to be
essential for PARP-1-mediated DNA repair, causing resistance to DNA damaging
chemotherapy
[Ahel et al., 2009; Tsuda et al., 2017]. DLD1 CRC cells that have low level
expression of CHD1L
(DLD1 Empty Vector, EV) compared to DLD1 cells that were engineered to
overexpress CHD1L
(DLD1 Overexpressing, OE) were used. FIG. 8A is a Western blot comparing
expression of
CHD1L in DLD1(EV) to DLD1(0E) in view of control expression of oc-tubulin in
these cells. FIG. 8B
presents a graph of y-H2AX intensity (relative to DMSO) for compound alone,
SN38 alone, and a
combination of the two in DLD1 empty vector cells and DLD1 overexpressing
cells. Compound 6.0
alone does not induce significant DNA damage, nor does it synergize with SN38
in DLD1 cells with
low expression of CHD1L. This graph demonstrates CHD1L inhibitor "on target"
effects that
synergize with SN38 inducing DNA damage in DLD1 cells overexpressing CHD1L.
Figures 9A- 9C: Synergy studies with exemplary CHD1L inhibitors and irinotecan
(Prodrug of
SN38). (FIG. 9A) Synergy studies with compounds 6 and 6.3 in SW620 Colorectal
Cancer (CRC)
Tumor Organoids. (FIG. 9B) Synergy studies with compound 6.9 in SW620
Colorectal Cancer
(CRC) Tumor Organoids. (FIG. 9C) Synergy studies with compound 6.11 in SW620
Colorectal
Cancer (CRC) Tumor Organoids. SN38 combinations of 6, and 6.3 are 50-fold, and
150-fold more
potent, respectively, than SN38 alone in killing colon SW620 tumor organoids.
SN38 combination
of 6.9 and 6.11 are both over 100-fold more potent than SN38 alone. Each of
compounds 6,6.3,
6.9 and 6.11 exhibit synergism with irinotecan (and SN38) for killing SW620
tumor organoids.
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Figure 10: In vivo synergy studies of compound 6 in combination with
irinotecan in mice. Figure
includes a graph of tumor volume (fold) SW620 tumor xenografts as a function
of days (up to 28
days) of treatment with compound 6 alone (2), irinotecan alone (3) or a
combination thereof (4),
compared to control (1). A data Table is also provided showing data
statistical significance. The
5 combination of irinotecan and compound 6 significantly inhibit colon
SW620 tumor xenografts to
almost no tumor volume within 28 days of treatment compared to the single
agent treatment
groups.
Figure 11: In vivo synergy of CHD1L inhibitor compound 6 and irinotecan
continues post
10 treatment. Figure 11 includes a graph of tumor volume (fold) SW620 tumor
xenografts as a
function of days (up to 41 days) of treatment with irinotecan alone (1) or a
combination of
compound 6 and irinotecan (2). A data Table is also provided showing data
statistical significance.
The combination of irinotecan and compound 6 significantly inhibits colon
SW620 tumors to almost
no tumor volume beyond the last treatment (day 28) compared to irinotecan
alone. Within 2-weeks
of the last treatment of irinotecan alone tumor volume rose to above the
volume of the last
treatment, signifying tumor recurrence. In contrast the combination maintained
a lower tumor
volume.
Figure 12: Compound 6 alone and in combination with irinotecan significantly
increases the
survival of CRC tumor-bearing mice compared to vehicle and irinotecan alone.
Figure 12 includes
a graph of survival (%) as a function of time up to 52 days after last
treatment on day 28 with
compound 6 alone (2), irinotecan alone (3) or a combination thereof (4),
compared to control (1). A
data Table is also provided showing data statistical significance. Survival
rate was significantly
higher with the combination treatment compared to single dosage compounds or
control.
Figure 13: In vivo synergy studies of compound 6.11 incombination with
irinotecan in mice. Figure
13 includes a graph of tumor volume (fold) SW620 tumor xenografts as a
function of days (up to 20
days) of treatment with compound 6.11 alone (2), irinotecan alone (3) or a
combination thereof (4),
compared to control (1). A data Table is also provided showing data
statistical significance. The
combination of irinotecan and compound 6.11 significantly inhibit colon SW620
tumor xenografts to
almost no tumor volume within 20 days of treatment compared to the irinotecan
alone.
Figure 14: In vivo synergy of CHD1L inhibitor compound 6.11 and irinotecan
continues post
treatment. Figure 14 includes a graph of tumor volume (fold) SW620 tumor
xenografts as a
function of days (up to 41 days) of treatment with irinotecan alone (1) or a
combination of
compound 6 and irinotecan (2). Treatment was stopped at day 33 (Tx released).
The
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combination of irinotecan and compound 6.11 significantly inhibits colorectal
SW620 tumors
beyond the last treatment (day 33) compared to irinotecan alone.
Figure 15:/n vivo synergy of CHD1L Inhibitor 6.11 and irinotecan significantly
increases survival
benefit. Compound 6.11 in combination with irinotecan significantly Increases
the survival of CRC
tumor-bearing mice compared to vehicle and irinotecan alone. Figure 15
includes a graph of
survival (%) as a function of time up to 50 days after last treatment on day
33 with compound 6
alone (2), irinotecan alone (3) or a combination thereof (4), compared to
control (1). A data Table
is also provided showing data statistical significance. Survival rate was
significantly higher with the
combination treatment compared to irinotecan alone or control.
Figures 16A and 16B. Enzymatic inhibition of CHD1L and SW620 tumor organoid
cytotoxicity.
(FIG. 16A) Quantification of the catalytic domain of CHD1L recombinant
protein. (FIG. 16B) Dose-
response of CHD1L inhibitor compounds measuring 5W620 tumor organoid
viability. Data is
normalized to DMSO control and is shown as mean SEM of triplicate
experiments.
Figures 17A-17C. CHD1L Inhibitors downregulate CHD1L mediated TCF-
transcription in M-
Phenotype cells. (FIG. 17A) TCF-transcriptional activity in isolated SW620 and
HCT116 EMT
phenotypes. P-values were calculated by one-way ANOVA where *P<0.05. Dose-
response
graphs of SW620 (FIG. 17B) and HCT116 (FIG. 17C) M-Phenotype monolayer cell
culture treated
with listed CHD1L inhibitors for 24h, measuring TCF-transcription via the
TOPflash luminescent
reporter assay. Data is normalized to cell viability and is shown as mean
SEM of duplicate
experiments.
Figures 18A-18D. CHD1L inhibitors are potent cytotoxic agents in CRC cell line
and patient tumor
organoids. (FIGs. 18A and 18B) Dose-response graphs of lead CHD1Li, measuring
cell viability
after 72 h of treatment of isolated M-phenotype SW620 and HCT116 tumor
organoids. (FIGs. 18C
and 18D) Dose-response graphs of lead CHD1Li, measuring cell viability after
72 h of treatment of
CRC042 and CRC102 patient-derived tumor organoids (PDTO). The data was
normalized to
DMSO (vehicle) and is shown as the mean SEM of triplicate experiments with
technical
replicates (n = 3) for each experiment.
Figures 19A-19E. CHD1L1 induce MET in M-phenotype 5W620 and HCT116 tumor
organoids.
(FIGs. 19A and 19C) Dose-response graphs of the downregulation of VimPro-GFP
promoter
activity measured by EGFP fluorescence of SW620 and HCT116 tumor organoids
treated with lead
CHD1L inhibitors. (FIGs. 19B and 19D) Fold change upregulation of EcadPro-RFP
promoter
activity measured through REP fluorescent signal in SW620 and HCT116 tumor
organoids after
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treatment with lead CHD1L inhibitors. (FIG. 19E) Representative maximum
projection confocal
images of HCT116 tumor organoids after treatment with compound 6.5 for both
VimPro-GFP and
EcadPro-RFP promoter activity. Data is shown in mean SEM of duplicate
experiments.
Figures 20A-20B. Cancer cell sternness is greatly reduced by CHD1L inhibitors.
(FIG. 20A)
Number of clonogenic colonies formed after continuous lead CHD1Li treatment in
SW620 cells.
(FIG. 20B) Number of clonogenic colonies formed after continuous treatment
with lead CHD1L
inhibitors in HCT116 cells. The data is represented as the mean SEM of
duplicate experiments
using triplicate technical replicates.
Figures 21A and 21B. Oral Bioavailable Efficacy of Compound 6.11 Against SW620
Quasi-
Mesaenchymal (GFP+) Tumor Xenographs. FIG. 21A is a graph of tumor volume as a
function of
days after initiation of treatment for control vehicle only (=, closed
circles), 6.11 75 mg/kg (squares)
and 6.11 125 mg/kg. Data point significance assessed using 2-Way ANOVA
(multiple
comparison), where *P<0,05, **P<0.01, ***P<0.001, ****P<0.0001, 4P<0.05,
4444P<0.0001. FIG.
21B is a graph of average mouse body weight as a function of days after
initiation of treatment.
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DETAILED DESCRIPTION
The invention relates generally to the characterization of a relatively new
oncogene, CHD1L, as a
tumorigenic factor associated with poor prognosis and survival in CRC. A new
biological function
for CHD1L as a DNA binding factor for the TCF transcription complex required
for promoting TCF-
driven EMT and other malignant properties has been demonstrated. Abbott et
al., 2020 and the
supplementary information for this article, which is available from the
journal web site
(mct.aacrjournals.org), provide description of a portion of the experiments
and data presented
herein and are each incorporated by reference herein in its entirety. Prigaro
et al., 2022 and the
supporting information for this article, which is available from the journal
web site
(pubs.acs.org/doi/10.1021/acs.jmedchem.1c01778) provide additional description
of a portion of
the experiments and data presented herein and are each incorporated by
reference herein in its
entirety.
CHD1L is amplified (Chr1q21) and overexpressed in many types of cancer (e.g.,
breast, bladder,
colorectal, esophageal, fibrosarcoma, liver, ovarian, and gastrix cancer). [Ma
et al., 2008; Cheng et
al., 2013] CHD1L overexpression has been characterized as a marker for poor
prognosis and
metastasis in numerous cancers. [Ma et al., 2008; Cheng et al., 2008; Hyeon et
al., 2013; Su et
al., 2014] While the collective literature demonstrating CHD1L as an oncogene
and driver of
malignant cancer is compelling, the rigor of the prior research and the
hypothesis that CHD1L is an
oncogene with potential as a molecular target in CRC is tested herein. In
silico analyses of
transcriptome data from a large cohort of 585 CRC patients obtained over 15
years was reported.
[Marisa et al., 2013] CHD1L expression was correlated with poor survival, with
low-CHD1L patients
living significantly longer than high-CHD1L patients. Using the same cohort,
Marisa et al., 2013
identified six distinct subtypes for improved clinical stratification of CRC
and CHD1L is universally
expressed in all six subtypes, indicating its potential as a therapeutic
target for CRC. CHD1L also
correlated with tumor node metastasis, with increased expression moving from
NO (no regional
spread) to N3 (distant regional spread). Transcriptonne data from a UCCC
patient cohort (n=25)
was analyzed and it was found that CHD1L expression significantly correlated
with stage IV and
mCRC. Literature reports and the work herein demonstrate that CHD1L is an
oncogene promoting
malignant CRC and its high expression correlates with poor prognosis and
survival of CRC
patients.
A new biological function for CHD1L as a DNA binding factor for the TCF-
transcription complex
required for promoting TCF-driven EMT and other malignant properties is
demonstrated herein.
Using HTS drug discovery the first known inhibitors of CHD1L have been
identified and
characterized which display good pharmacological efficacy in cell-based models
of CRC, including
PDT0s. CHD1L inhibitors effectively prevent CHD1L-mediated TCF-transcription,
leading to the
reversion of EMT and other malignant properties, including CSC stemness and
invasive potential.
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Notably, CHD1L inhibitor 6 displays the ability to induce cell death that is
consistent with the
reversion of EMT and induction of cleaved E-cadherin mediated extrinsic
apoptosis through death
receptors. Furthermore, compound 6 synergizes with SN38 (i.e., irinotecan)
displaying potent DNA
damage induction compared to SN38 alone, which is consistent with the
inhibition of
PARP1/CHD1L mediated DNA repair. CHD1L inhibitors having drug-like
physicochemical
properties and favorable in vivo PK/PD disposition with no acute liver
toxicity have been identified.
Based on the data presented herein, a mechanism of action for CHD1L-mediated
TCF-driven EMT
involved in CRC tumor progression and metastasis is presented (FIG. 5). In
this mechanism, TCF-
complex specifically recruits CHD1L to dynamically regulate metastatic gene
expression. Central to
this mechanism, CHD1L binds to nucleosome hindered WREs when directed by the
TCF-complex
via protein interactions with PARP1 and TCF4. Importantly, PARP1 is
characterized as the major
cellular activator of CHD1L through macro domain binding that releases auto
inhibition. [Lehmann
et al., 2017; Gottschalk et al., 2009] Moreover, PARP1 is a required component
of the TCF-
complex forming interactions with 13-catenin and TCF4. [Idogawa et al., 2005]
Therefore, the
mechanism indicates that CHD1L is recruited by the TCF-complex and activated
by PARP1 and
TCF4. Once activated, CHD1L exposes WREs by nucleosorne translocation,
facilitating TCF-
complex binding to WREs and transcription of malignant genes promoting EMT.
CHD1L inhibitors
have a unique mechanism of action by inhibiting CHD1L ATPase activity, which
prevents exposure
of WREs to the TCF-complex, inhibiting transcription of TCF-target genes
associated with EMT
and particularly with mCRC.
Small molecule inhibitors of CHD1L, as described herein, have been identified
in screens based on
inhibition of CHD1L ATPase activity. Certain inhibitors identified exhibit
drug-like physicochemical
properties and favorable in vivo PK/PD disposition with no acute liver
toxicity. Such inhibitors are
effective as a treatment for CRC and mCRC (metastatic CRC) among other CHD1L-
driven
cancers.
Well-known methods for assessment of drugability can be used to further assess
active
compounds of the invention for application to given therapeutic application.
The term "drugability"
relates to pharmaceutical properties of a prospective drug for administration,
distribution,
metabolism and excretion. Drugability is assessed in various ways in the art.
For example, the
"Lipinski Rule of 5" for determining drug-like characteristics in a molecule
related to in vivo
absorption and permeability can be applied [Lipinski et al., 2001; Ghose, et
al., 1999]
The invention provides methods for combination therapy in which administration
of CHD1L inhibitor
is combined with administration of one or more anticancer agent which is not a
CHD1L inhibitor. In
embodiments, the other anticancer agents is a topoisomerase inhibitor, a
platinum-based
antineoplastic agent, a PARP inhibitor or combinations of two or more of such
inhibitors and
agents. In embodiments, the combination therapy combines administration of a
CHD1L inhibitor
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with a topoisomerase inhibitor. In embodiments, the combination therapy
combines administration
of a CHD1L inhibitor with a platinum-based antineoplastic agent. In
embodiments, the
combination therapy combines administration of a CHD1L inhibitor with a PARP
inhibitor. In
embodiments, the combination therapy combines administration of a CHD1L
inhibitor with a
topoisomerase inhibitor and administration of a PARP inhibitor. In
embodiments, the combination
therapy combines administration of a CHD1L inhibitor with chemotherapy for the
specific cancer
being treated. In embodiments herein, the combination of a CHD1L inhibitor and
the other
antineoplastic agent exhibits synergistic activity in combination.
In embodiments herein, therapy employing CHD1L can be combined with radiation
therapy
suitable for a given cancer.
Various PARP inhibitors are known in the art. [See, for example Rouleau et
al., 2010; Yi et al.,
2019; Zhou et al., 2020; Wahlberg et al., 2012; D'Andrea, 2018] Each of these
references is
incorporated by reference herein it is entirety for descriptions of PARP
inhibitors, the mechanism of
PARP inhibitor action, cancers treated using PARP inhibitors, and resistance
to PARP inhibitors.
In a specific embodiment herein, PARP-resistance cancer is treated with a
combination of a
CHD1L inhibitor and the PARP inhibitor.
Various topoisomerase inhibitors are known in the art and have been employed
clinically. (See, for
example, Hevener, 2018; Bailly, 2012; Nitiss J, 2009) "Targeting DNA
topoisomerase ll in cancer
chemotherapy," Nature Rev. Cancer, 9:338-350). Each of these references is
incorporated by
reference herein in its entirety for descriptions of types of topoisomerase
inhibitors, specific
topoisomerase inhibitors, mechanisms of topoisomerase inhibition, cancers
treated using
topoisomerase inhibitors and combination therapies using topoisomerase
inhibitors. In
embodiments, topoisomerase inhibitors useful in methods and compositions
hereinare
topoisomerase I inhibitors. In embodiments, topoisomerase inhibitors useful in
methods and
compositions herein include cannptothecin and prodrugs thereof, irinotecan,
topotecan, belotecan,
indotecan, or indimitecan. In embodiments, topoisomerase inhibitors useful in
methods and
compositions herein include etoposide or teniposide. In embodiments,
topoisomerase inhibitors
useful in methods, pharmaceutical combinaions and combined cancer therapy
herein include
namitecan, silatecan, vosaroxin, aldoxorubicin, doxorubicin, becatecarin, or
edotecarin.
In embodiments, topoisomerase inhibitors useful in methods and compositions
hereinare
topoisomerase I inhibitors. Exemplary topoisomerase II-alpha inhibitors are,
for example, reported
in Published PCT application W02020/0205991, published October 8, 2020, and
its priority
document U.S. provisional application 62/827,818, filed April 1,2019. Each of
these references is
incorporated by reference herein in its entirety for descriptions of types of
topoisomerase inhibitors,
specific topoisomerase inhibitors, mechanisms of topoisomerase inhibition,
cancers treated using
topoisomerase inhibitors and combination therapies using topoisomerase
inhibitors.
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Various platinum-based antineoplastic agents (also called platins) are known
in the art and have
been employed clinically or are in clinical trials. [See, for example, Wheate
et al., 2010] This
reference is incorporated by reference herein in its entirety for descriptions
of types of platinum-
based antineoplastic agents, specific platinum-based antineoplastic agents,
mechanisms of action
of such agents, cancers treated using such agents and combination therapies
using platinum-
based antineoplastic agents. In embodiments, platinum-based antineoplastic
agents useful in
methods and compostions herein include cisplatin, carbon platin, oxaliplatin,
nedaplatin, lobaplatin,
or heptaplatin. In embodiments, platinum-based antineoplastic agents include
satraplatin, or
picoplatin. Platinum-based antineoplastic agents may be liposomally
encapsulated (e.g.,
LypoplatinTM) or bound in nanopolymers (e.g., ProLindacn").
Various thymidylate synthase inhibitors are known in the art and have been
employed clinically
particularly in the treatment of CRC [Papamichael, 2009; Lehman, 2002].
Thymidylate synthase
inhibitors useful in the methods and compositons herein include without
limitation folate analogues
and nucleotide analogues. In specific embodiments, the thymidylate synthase
inhibitor is
raltitrexed, pemetrexed, nolatrexed or ZD9331. In more specific embodiments,
the thymidylate
synthase inhibitor is 5-fluorouracil or capecitabine.
The invention provides CHD1L inhibitors of the following formulas:
Compounds useful in the methods, pharmaceutical compositions or pharmaceutical
combinations
of this invention include those of formula I:
yRP
r- X
(1--)x
RA A
(L2)y
RH
or salts, or solvates thereof,
where:
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the B ring is an optionally-substituted at least divalent heteroaryl ring or
ring system having one,
two or three 5- or 6-member rings, any two or three of which can be fused
rings, where the rings
are carbocyclic, heterocyclic, aryl or heteroaryl rings and at least one of
the rings is heteroaryl;
in the B ring, each X is independently selected from N or CH and at least one
X is N;
Rp is an optionally-substituted primary or secondary amine group [-N(R2)(R3)]
or is a -(M)x-P
group, where P is -N(R2)(R3) or an aryl or heteroaryl group, where x is 0 or 1
to indicate the
absence or present of M and M is an optionally substituted linker -(CH2)n- or -
N(R)(CH2)n-, where
each n is independently an integer from 1-6 (inclusive);
Y is a divalent atom or group selected from the group consisting of 0 , S ,
N(R1)-,
-CON(Ri)-, -N(Ri)C0-, ¨N(Ri)CON(Ri) ¨SO2N(Ri)-, or -N(Ri)S02-;
Li is an optional 1-4 carbon linker that is optionally substituted and is
saturated or contains
a double bond (which can be cis or trans), where x is 0 or 1 to indicate the
absence or
presence of Li;
the A ring is an optionally-substituted at least divalent carbocyclic or
heterocyclic ring or ring
system having one, two or three rings, two or three of which can be fused,
each ring having 3-10
carbon atoms and optionally 1-6 heteroatonns and wherein each ring is
optionally saturated,
unsaturated or aromatic;
Z is a divalent group containing at least one nitrogen substituted with a R
group,
where in embodiments, Z is a divalent group selected from -N(R')-, -CON(R')-, -
N(R)CO-, -
CSN(R')-, -N(R')CS-, -N(R)CON(R')-, -SO2N(R.)-, -N(R')S02-, -CH(CF3)N(R)-,
-N(R')CH(CF3)-, -N(R)C1-12CON(R')CH2-, -N(R)COCH2N(R)CH2-,
0
,
or the divalent Z group comprises a 5- or 6-member heterocyclic ring having at
least one nitrogen
ring member, for example,
µcr\ljr\I=N
N¨N
, , or
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N-0
=
L2 is an optional 1-4 carbon linker that is optionally substituted and is
saturated or contains
a double bond (which can be cis or trans), where z is 0 or 1 to indicate the
absence or
presence of L2;
R is selected from the group consisting of hydrogen, an aliphatic group, a
carbocyclyl group, an
aryl group, a heterocyclyl group and a heteroaryl group, each of which groups
is optionally
substituted;
each R' is independently selected from the group consisting of hydrogen, an
aliphatic group, a
carbocyclyl group, an aryl group, a heterocyclyl group and a heteroaryl group,
each of which
groups is optionally substituted;
R1 is selected from the group consisting of hydrogen, an aliphatic group, a
carbocyclyl group, an
aryl group, a heterocyclyl group and a heteroaryl group, each of which groups
is optionally
substituted;
R2 and R3 are independently selected from the group consisting of hydrogen, an
aliphatic group, a
carbocyclyl group, an aryl group, a heterocyclyl group and a heteroaryl group,
each of which
groups is optionally substituted or
R2 and R3 together with the N to which they are attached form an optionally
substituted 5- to 10-
member heterocyclic ring which is a saturated, partially unsaturated or
aromatic ring;
RA and RD represent hydrogens or 1-10 non-hydrogen substituents on the
indicated A and B ring or
ring systems, respectively, wherein RA and RD substituents are independently
selected from
hydrogen, halogen, hydroxyl, cyano, nitro, amino, mono- or disubstituted amino
(-NRcRD), alkyl,
alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, acyl,
haloalkyl, ¨COORc, ¨000R0,
¨CONRcRo, -000NRcRo, -NRcCORD, -SRc, -SORc, -SO2Rc,and ¨SO2NRcRo, where alkyl,
alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl, are
optionally substituted;
each Rc and RD is selected from hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkenyl, heterocyclyl,
aryl, or heteroaryl, each of which groups is optionally substituted with one
or more halogen, alkyl,
alkenyl, haloalkyl, alkoxy, aryl, heteroaryl, heterocyclyl, aryl-substituted
alkyl, or heterocyclyl-
substituted alkyl; and
RH is an optionally substituted aryl or heteroaryl group;
wherein optional substitution includes, substitution with one or more halogen,
nitro, cyano, amino,
mono- or di-C1-C3 alkyl substituted amino, C1-03 alkyl, C2-C4 alkenyl, C3-C6
cycloalkyl, C3-C6-
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cycloalkenyl, C1-C3 haloalkyl, 01-06 acyl. C1-C6 acyloxy, C1-C6
alkoxylcarbonyl. 06-012 aryl,
05-012 heteroaryl, 03-012 heterocyclyl. 01-03 alkoxy, 01-06 acyl, ¨COORE,
¨OCORE,
¨CONRERF, -OCONRERD, -NRECORF, -SRE, -SORE, -SO2RE, and ¨SO2NRERF, where
alkyl,
alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl, are
optionally substituted and
each RE and RF is selected from hydrogen, C1-C3 alkyl, C2-C4 alkenyl, C3-C6
cycloalkyl, C3-C6-
cycloalkenyl, 01-03 haloalkyl, 06-012 aryl, 05-012 heteroaryl, 03-012
heterocyclyl. 01-03
alkoxy, 01-06 acyl, each of which groups is optionally substituted with one or
more halogen, nitro,
cyano, amino, mono- or di-C1-03 alkyl substituted amino, 01-03 alkyl, 02-04
alkenyl, 03-06
cycloalkyl, 03-06-cycloalkenyl, 01-03 haloalkyl, 06-012 aryl, 05-012
heteroaryl, 03-012
heterocyclyl. 01-03 alkoxy, C1-06 acyloxy, 01-06 alkoxycarbonyl and 01-06
acyl.
In embodiments of formula I:
R is selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,
heterocyclyl, aryl, or
heteroaryl, each of which groups is optionally substituted;
each R' is independently selected from hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkenyl,
heterocyclyl, aryl, or heteroaryl, each of which groups is optionally
substituted;
R1-R3 are independently selected from hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkenyl,
heterocyclyl, aryl, or heteroaryl, each of which groups is optionally
substituted;
One or more of R1-R3 is cycloalkyl substituted alkyl, for example, a
cyclopropylmethyl, a
cyclopentylmethyl, or a cyclohexylmethyl;
R is hydrogen or a 01-03 alkyl;
each R' is independently hydrogen or C1-C3 alkyl;
R1 is hydrogen or C1-03 alkyl;
R2 and R3 are independently selected from hydrogen, or a C1-03 alkyl; or
R2 and R3 together with the N to which they are attached form a 5-7nnennber
heterocycliuc ring
which is saturated.
In embodiments of formula I, the A ring is divalent and is a single 6-member
aromatic ring which
can contain 1 or 2 heteroatoms, particularly 1 or 2 nitrogen. In an
embodiment, the divalent Aring
is 1,4-phenylene or 2, 5-pyridylene.
In an embodiment of formula I, the divalent B ring is substituted with at
least one electronegative
substituent. In an embodiment, the electronegative substituent is a halogen.
In an embodiment,
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the electronegative substituent is a haloalkyl group having 1-3 carbon atoms.
In an embodiment,
the electronegative substituent is fluorine. In an embodiment, the
electronegative substituent is
tifluoromethyl (CF3-). In furhter embodiments of the forgoing embodiments, x
is 1. In related
embodiments of formula I, the B ring is substituted with at least one halogen,
and x is 1 and Li is ¨
CH¨. In related embodiments of formula I, the B ring is substituted with at
least one fluorine, xis
1 and Li is ¨CH2¨.
In an embodiment of formula I, the divalent B ring is substituted with at
least one C1-C3 alkyl
group. In an embodiment of formula I, the B ring is substituted with at least
one methyl group.
In specific embodiments of formula I:
Ring A is an optionally substituted phenylene;
Ring A is an optionally substituted 1,4-disubstituted phenylene
Ring A is an optionally substituted naphthylene;
Ring A is an optionally substituted 2,6-disubstituted naphthylene;
Ring A is an optionally substituted pyridylene;
Ring A is an optionally substituted 2,5-pyridylene;
Ring B is an optionally substituted pyridylene,
Ring B is an optionally substituted pyrimidylene;
Ring B is an optionally substituted pyrazinylene;
Ring B is an optionally substituted triazinylene;
Ring B is an optionally substituted quinazolinylene;
Ring B is an optionally substituted pteridinylene;
Ring B is an optionally substituted quinolinylene;
Ring B is an optionally substituted isoquinolinyenel;
Ring B is an optionally substituted naphthyridinylene;
Ring B is an optionally substituted pyridopyrimidylene;
Ring B is an optionally substituted pyrimidopyridylene;
Ring B is an optionally substituted pryanopyridylene;
Ring B is an optionally substituted pyranopyrimidylene;
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Ring B is an optionally substituted purinylene;
Ring B is an optionally substituted 6,8-disubstituted purinylene;
Ring A is an optionally substituted phenylene and Ring B is an optionally
substituted
pyrimidinylene; or
Ring A is an optionally substituted phenylene and Ring B is an optionally
substituted pteridinylene.
In embodiments, RA represents H at all available ring positions.
In embodiments, RA represents one C1-03 alkyl substituebt at an available ring
position.
In embodiments, RA represents one methyl substituebt at an available ring
position.
In embodiments, RA represents one halogen substituted at an available ring
position.
In embodiments, RA represents one fluorine substituted at an available ring
position.
In embodiments, RA represents one C1-C3 haloalkyl substituted at an available
ring position;
In embodiments, RA represents one trifluoromethyl group at an available ring
position.
In embodiments, RB represents H at all available ring positions.
Preferred A and B ring substitution includes one or more C1-C3 alkyl, 03-07
cycloalkyl, C4-C10
cycloalkyl substituted alkyl, 02-04 alkenyl, 01-03 alkoxy, 01-03 acyl, a 01-04
alkoxycarbonyl, a
C1-C4 acyloxy, carboxyl, halogen, hydroxyl, C1-C3 haloalkyl, mono- or
disubstituted phenyl or
mono- or disubstituted benzyl. More specific A and B ring substitution
includes methyl, ethyl,
isopropyl, cyclopropyl, cyclopropylmethyl, methont, ethoxy, phenyl, benzyl,
halophenyl,
halobenzyl, Cl, Br, F, CF3-, HO-, CF30-, CH3C0- , HOOC-, CH3OCO-and CHCO-.
In an embodiment, the divalent A ring is other than a phenyl ring or a benzyl
ring. In an
embodiment, the A ring is other than a phenyl ring. In an embodiment, the A
ring is other than an
unsubstituted phenyl ring or an unsubstituted benzyl ring. In an embodiment,
the A ring is other
than an unsubstituted phenyl ring.
In embodiments, the divalent B ring has one of the structures illustrated in
Scheme 4, RBI-RB17.
In embodiments, the divalent B ring has structrure RB2-RB5, wherein RB
represents optional
substitution as described for formula I. The ring is bonded to Rp or Y at
positions indicated. More
specifically, RB represents optional substitution at ring carobns with one or
more of C1-C3 alkyl,
halogen or 01-03 haloalkyl and more specifically 01-03 fluoroalkyl and more
specifically with one
or more methyl, trifluoromethyl or fluorine. In embodiments, the divalent B
ring has structrure RB6,
which is bonded to Rp or Y are the positions indicated and wherein RB
represents optional
substitution as described for formula I. More specifically, RB represents
optional substitution at ring
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carobns with one or more of C1-C3 alkyl, halogen or C1-C3 haloalkyl and more
specifically C1-C3
fluoroalkyl and more specifically with one or more methyl, trifluoromethyl or
fluorine. In
embodiments, the B ring is as illustrated in RB7-RB17 which is bonded to RP
and bonded to Y at
the position indicated and wherein RB represents optional substitution as
described for formula I.
More specifically, RB represents optional substitution at ring carobns with
one or more of C1-C3
alkyl, halogen or 01-03 haloalkyl and more specifically 01-03 fluoroalkyl and
more specifically
with one or more methyl, trifluoromethyl or fluorine. In specific embodiments,
the B ring is as
shown in RB14-17.
In embodiments, the divalent B ring has structure as shown in Scheme 4,
formula RBI, where X1
and X2 are selected from CH and N and at least one of X.1 and X2 is N and X3-
X6 are selected from
CH, CH2, 0, S, N and NH where the illustrated B ring is saturated, unsaturated
or aromatic,
dependent upon choice of 1-X6 and RB represents optional substitution as
defined for formula I. In
embodiments, RB represents hydrogens and the B ring is unsubstituted. In
embodiments, RB
represents one or more halogen, C1-C3 alkyl, C1-03 acyl, C1-C3 alkoxy. In
embodiments, RB
represents one or more F, Cl or Br, methyl, ethyl, acetyl or methoxy or
combinations thereof. In
embodiments, of formula I the B ring is selected from any of RB2-RB5, as shown
in Scheme 4.
In embodiments of formula I:
xis 1 and Li is ¨(CH2)n¨, where n is 1,2 0r3;
x is 1 and Li is ¨(CH2)n¨, where n is 1 or 2;
x is 0 and L1 is absent;
y is 1 and L2 is ¨(CH2)n¨, where n is 1,2 0r3;
y is 1 and L2 is ¨(CH2)n¨, where n is 1 or 2;
y is 1, and L2 is -CH=CH-;
y is 1, and L2 is trans -CH=CH-;
both of x and y are 0;
x is 1 and y is 0 and Li is ¨(CH2)n¨, where n is 1 or 2;
y is 1 and x is 0 and L2 is ¨(CH2)n¨, where n is 1 or 2; or
both of x and y are 1 and both of L2 and Li are ¨(CH2)n¨, where n is 1 or 2.
In embodiments of formula I:
Y is ¨ , S , N(Ri)¨, ¨CON(Ri)¨, ¨N(R1)C0¨ or ¨N(Ri)CON(Ri)¨;
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Y is ¨ , ---------- S , NH , CONH¨, or ¨NHCO¨ or ¨N(Ri)CON(Ri)¨;
Y is ¨N(Ri)¨, ¨CON(Ri)¨,¨N(Ri)C0¨ or ¨N(Ri)CON(Ri)¨;
Y is ¨N(Ri)¨, ¨CON(Ri)¨, or¨N(Ri)CO¨
Y is ¨N(Ri)CON(Ri)¨;
Y is ¨N(H)¨, ¨CON(H)¨,¨N(H)C0¨ or ¨N(H)CON(H)¨;
Y is ¨N(H)¨, ¨CON(H)¨, or ¨N(H)CO¨
Y is ¨N(H)CON(H)¨;
R1 is hydrogen, a C1-C3 alkyl or a C1-C3 haloalkyl, particularly C1-C3
fluoroalkyl;
R1 is hydrogen, a methyl group or CF3-;
Ri is hydrogen;
Y is ¨N(Ri)¨, ¨CON(Ri)¨, or¨N(Ri)CO¨ and Ri is hydrogen, methyl or CF3-;
Y is ¨N(Ri)¨ and R1 is hydrogen, C1-C3 alkyl or C1-C3 haloalkyl, particularly
C1-C3 fluoroalkyl; or
Y is ¨N(Ri)¨ and Ri is hydrogen, methyl or CF3-.
In embodiments of formula I, both x and y are 0 and Y is ¨N(Ri)¨.
In embodiments of formula I, both x and y are 0 and Y is ¨NH¨.
In embodiments of formula I, both x and y are 0 and Y is ¨CONH-.
In embodiments of formula 1, both x and y are 0 and Y is ¨NHCO-.
In embodiments of formala 1, both x and y are 0 and Y is ¨NHCONH-.
In embodiments of formula I:
Z is ¨N(R')¨, ¨CON(R')¨, or ¨N(R)C0¨;
Z is ¨CH(CF3)N(R)¨;
Z is ¨SO2N(R)¨;
Z is ¨N(R')CON(R')¨;
Z is ¨N(R)CH200N(R)CH2¨;
Z is
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0
xr(N¨NIN v\IJN=N
Z is R' or R' =
N¨N N-0
Z is or \----(N)-----1=
R' is hydrogen, a C1-C6 alkyl or a C1-C3 haloalkyl, particularly a C1-C3
fluoroalkyl;
R' is hydrogen or a 01-03 alkyl;
R' is hydrogen, methyl or CF3-;
R' is hydrogen or methyl;
R' is hydrogen;
Z is -N(R')-, -CON(R')-, or -N(R)CO- and R' is hydrogen or methyl;
Z is -N(R')-, -CON(R')-, -N(R')C0- or -N(R')CON(R') and R' is hydrogen;
Z is -CON(R')- or -N(R)CO- and R' is hydrogen or methyl;
Z is -N(R')CON(R')- and both R' are hydrogen;
µ.(N¨N)N µcr\iir\IN
Z is R' or R' and R' is hydrogen; or
N¨N N-0
Z is 0 or N and R' is hydrogen.
In embodiments of formula I,
xis 0;
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x is 1 and L2 is -(CH2),-, where n is 1-3;
y is 0, x is 1 and L2 is -(CH2)n-, where n is 1-3;
x is 0 and Z is -N(R')-, -CON(R')-,-N(R')C0- or -N(R)CON(R)-;
x is 0, and Z is -N(R')-, -CON(R')-,-N(R')C0- or -N(R')CON(R')- and R' is
hydrogen or methyl;
x is 0, and Z is -N(H)-, -CON(H)-,-N(H)C0- or -N(H)CON(H)-;
x is 0 and Z is-CON(R)-;
x is 0, and Z is -CON(R')- or -N(R')C0- and R' is hydrogen or methyl;
x is 0, and Z is -CONH- or -NHCO-;
x is 0 and Z is -CONH-, -NHCO- or -NHCONH-.
x is 1, L2 is -(CH2)n-, where n is 1-3, and Z is -N(Ri)-, -CON(R')-, -N(R)CO-
or
-N(R')CON(R')- ;
x is 1, L2 is -(CH2)n-, where n is 1-3, and Z is -NH-, -CONH-, -NHCO- or
-NHCONH-;
x is 1, L2 is -CH2- and Z is -NH-, -CONH-, -NHCO- or -NHCONH-;
x is 1, L2 is -CH2-CH2-, and Z is -NH-, -CONH-, -NHCO- or -NHCONH-;
x is 1, L2 is -CH2-CH2-CH2-, and Z is -NH-, -CONH-, -NHCO- or -NHCONH-;
x is 1, L2 is -(CH2)n-, where n is 1-3, and Z is -CON(R')-;
x is 1, L2 is -CH2- and Z is -CON(R')-;
x is 1, L2 is -CH2-CH2- and Z is -CON(R')-;
x is 1, L2 is -CH2- and Z is -CONH-;
x is 1, L2 is -CH2-CH2- and Z is -CONH-;
x is 1, L2 is -CH2-CH2-CH2- and Z is -CONH-;
x is 0 or 1, L2, if present, is -CH2- or -CH2-CH2- and Z is -CON(R)-;
x is 0 or 1, L2, if present, is -CH2- or -CH2-CH2-, and Z is -CONH-;
x is 0 or 1, L2, if present, is -CH2- or -CH2-CH2- and Z is -N(R)C0-;
x is 0 or 1, L2, if present, is -CH2- or -CH2-CH2-, and Z is -NHCO-;
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x is 0 or 1, L2 if present, is ¨CH2¨ or ¨CH2-CH2¨, and Z is ¨NHCONH-;
y is 0, x is 0 or 1, L2, if present, is ¨CH2¨ or ¨CH2-CH2¨, and Z is ¨CONH¨;
y is 0, Y is ¨N(Ri)¨, x is 0 or 1, L2, if present, is ¨CH2¨ or ¨0H2-CH2¨, and
Z is ¨CONH¨;
y is 0, Y is ¨NH¨, x is 0 or 1, L2, if present, is ¨CH2¨ or ¨CH2-CH2¨, Z is
¨CONH-;
y is 0, Y is ¨NH¨ , x is 0 or 1, L2, if present, is ¨CH2¨ or ¨CH2-0H2¨, Z is
¨CONH-, -NHCO-, or ¨
NHCONH-;
In embodiments of formula I,
Rp contains at least one nitrogen; or
when Rp is ¨(M)x-P, and x = 0, then P is ¨N(R2)(R3) or P is a heterocyclic or
heteroaryl group
having at least one ring N; or
when Rp is ¨(M)x-P, x = 1, and M = -(CH2)n-, then P is ¨N(R2)(R3) or P is a
heterocyclic or
heteroaryl group having at least one ring N.
In embodiments of formula I, Rp is:
¨N(R2)(R3);
¨(M)-N(R2)(R3), where M is an optionally substituted linker ¨(CH2)n¨ or
¨N(R)(CH2)n¨, where each
n is independently an integer from 1-6 (inclusive) and R is hydrogen or an
optionally substituted
alkyl group having 1-3 carbon atoms;
¨(M)-N(R2)(R3), M is an optionally substituted linker ¨(CH2),¨, where each n
is independently an
integer from 1-6 (inclusive) and R is hydrogen or an optionally substituted
alkyl group having 1-3
carbon atoms;
¨(M)-N(R2)(R3), M is an optionally substituted linker ¨(CH2),¨, where each n
is independently an
integer from 1-6 (inclusive) and R is hydrogen or an optionally substituted
alkyl group having 1-3
carbon atoms, where optional substitution is substitution with one or more
halogen or one or more
01-03 alkyl groups;
¨(M)-N(R2)(R3), M is optionally substituted ¨N(R)(CH2)n¨, where each n is
independently an
integer from 1-6 (inclusive and R is hydrogen) and R is hydrogen or an
optionally substituted alkyl
group having 1-3 carbon atoms;
¨(M)-N(R2)(R3), M is optionally substituted ¨N(R)(CH2)n¨, where each n is
independently an
integer from 1-6 (inclusive) and R is H or an optionally substituted alkyl
group having 1-3 carbon
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atoms, where optional substitution is substitution with one or more halogen or
one or more C1-C3
alkyl groups;
¨(M)-N(R2)(R3), where M is an optionally substituted linker ¨(CH2)n¨ and n is
1, 2 or 3;
¨(M)-N(R2)(R3), where M is an optionally substituted linker ¨N(R)(CH2)¨ and n
is 1, 2 or 3;
¨(M)-N(R2)(R3), where M is ¨(CH2)n¨ and n is 1, 2 or 3;
¨(M)-N(R2)(R3), where M is ¨N(R)(CH2)n¨ and n is 1, 2 or 3;
¨(M))(-P group, where P is a aryl or heteroaryl group, where x is 0 or 1 to
indicate the absence or
presence of M and M is an optionally substituted linker ¨(CH2)n¨ or
¨N(R)(CH2)n¨, where each n is
independently an integer from 1-6 (inclusive) and R is H or an optionally
substituted alkyl group
having 1-3 carbon atoms;
¨(M)-P group, where P is a aryl or heteroaryl group, and M is an optionally
substituted linker
¨(CH2)n¨ or ¨N(R)(CH2)n¨, where each n is independently an integer from 1-6
(inclusive) and R is
H or an optionally substituted alkyl group having 1-3 carbon atoms;
¨(M)-P group, where P is a aryl or heteroaryl group, and M is an optionally
substituted linker
¨N(R)(CH2)n¨, where each n is independently an integer from 1-6 (inclusive)
and R is H or an
optionally substituted alkyl group having 1-3 carbon atoms;
¨(M)-P group, where P is a aryl or heteroaryl group, and M is an optionally
substituted linker
¨(CH2)n¨, where each n is independently an integer from 1-3 (inclusive) and R
is H or an optionally
substituted alkyl group having 1-3 carbon atoms;
P is an optionally substituted phenyl or naphthyl;
P is an optionally substituted phenyl or naphthyl and optional substitution
is with one or more
halogen, C1-C3 alkyl, C1-C3 alkoxy or C1-C3 haloalkyl;
P is an optionally substituted heteroaryl group having a 5- or 6-member
ring or two fused 5- or 6-
member rings;
P is an optionally substituted heteroaryl group having a 5- or 6-member ring
or two fused 5- or 6-
member rings and having 1 to 3 nitrogen ring members;
R2 in Rp is hydrogen (i.e., -N(R2)(R3) is a primary amine group);
both R2 and R3 in Rp are groups other than hydrogen (i.e., -N(R2)R3) is a
secondary amine group);
R2 is hydrogen and R3 is an optionally substituted 3-8-member cycloalkyl
group;
R2 is hydrogen and R3 is a C1-C3 alkyl group substituted with a 3-8-member
cycloalkyl group;
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R2 is hydrogen and R3 is an optionally substituted aryl group having 6-12
carbon atoms;
R2 is hydrogen and R3 is an optionally substituted heteroaryl group having 6-
12 carbon atoms and
1-3 heteroatoms (N, 0, or S);
R2 is hydrogen and R3 is an optionally substituted heteroaryl group having 6-
12 carbon atoms and
1-3 ring nitrogens;
R2 and R3 together with the N to which they are attached form an optionally
substituted 5- to 10-
member heterocyclic ring which is a saturated, partially unsaturated or
aromatic ring;
R2 and R3 together with the N to which they are attached form a 5- to 10-
member heterocyclic
sultam ring;
Rp is ¨(CH2)n-N(R2)(R3), where n is 1 or 2 and R2 and R3 together with the N
to which they are
attached form an optionally substituted 5-to 10-member heterocyclic ring which
is a saturated,
partially unsaturated or aromatic ring;
Rp is ¨N(R)(CH2)n-N(R2)(R3), where n is 1 or 2, R is hydrogen or methyl and R2
and R3 together
with the N to which they are attached form an optionally substituted 5- to 10-
member heterocyclic
ring which is a saturated, partially unsaturated or aromatic ring;
Rp is ¨M-N(R2)(R3) and R2 and R3 together with the N to which they are
attached form an optionally
substituted 5-to 10-member heterocyclic ring which is a saturated, partially
unsaturated or
aromatic ring;
R2 and R3 together with the N to which they are attached form an optionally
substituted 5- to 10-
member heterocyclic ring which contains no double bonds;
Rp is -N(R2)(R3) and R2 and R3 together with the N to which they are attached
form an optionally
substituted 5-to 10-member heterocyclic ring which contains no double bonds;
R2 and R3 together with the N to which they are attached form an optionally
substituted 5- to 10-
member heterocyclic ring which contains one, two or three double bonds;
Rp is -N(R2)(R3) and R2 and R3 together with the N to which they are attached
form an optionally
substituted 5-to 10-member heterocyclic ring which contains one, two or three
double bonds;
R2 and R3 together with the N to which they are attached form an optionally
substituted 5- to 10-
member heteroaryl ring; or
Rp is -N(R2)(R3) and R2 and R3 together with the N to which they are attached
form an optionally
substituted 5-to 10-member heteroaryl ring.
In specific embodiments of formula I, Rp or ¨N(R2)(R3) is:
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any one of RN1-RN39 of Scheme 2;
RN1; RN3; RN2 or RN4; RN5 or RN6; RN7 or RN8; RN9; RN10; RN11; RN12; RN13;
RN14; RN15; RN16;
RN17 or RN18; RN19 or RN20; RN21; RN22; RN23 or RN24; RN25; RN26-RN29; RN27-
RN32; RN30;
RN31; RN33-RN36; RN37; RN38; RN39; or
RN1, RN2, RN3, RN4, RN11, RN13, or RN14; or
RN1-RN31 which is unsubstituted.
In embodiments of formula!, RH is:
optionally substituted phenyl; other than optionally substituted pheny;
unsubstituted phenyl; other
than unsubsttuted pheny; optionally substituted naphthyl; unsubstituted
naphthyl; optionally
substituted naphthy-2-y1; optionally substituted naphthy-1-y1; naphthy-2-y1;
naphthy-1-y1; optionally
substituted thiophenyl; halogen substituted thiophenyl; bromine substituted
thiophenyl; optionally
substituted thiophen-2-y1; halogen substituted thiophen-2-y1; bromine
substituted thiophen-2-y1; 4-
halothiophen-2-y1; 4-bromothiophen-2-y1; optionally substituted furyl;
optionally substituted fur-2-y';
optionally substituted indolyl; unsubstituted indolyl; indo1-3-y1; indo1-2-y1;
indo1-1-y1; optionally
substituted pyridinopyrrolyl; optionally substituted pyridinopyrrol-2-y1;
optionally substituted
pyridinopyrrolyl; optionally substituted pyridinopyrrol-3-y1; optionally
substituted quinolinyl;
optionally substituted quinolin-4-y1; optionally substituted isoquiolinyl;
optionally substituted
isoquinolin-4-yl, optionally substituted benzoimidazolyl; optionally
substituted benzoimidazol-1-y1;
optionally substituted 1H-pyrrolo[2,3-b]pyridin-3-yl: optionally substituted
pyridine-2-y'; optionally
substituted pyridine-3-y'; optionally substituted pyridine-4-y'; 1H-imidazol-1-
yl, 1H-imidazol-2-y1; or
1H-imidazole-5-yl.
In specific embodiments, optional substitution of RH is substitution with one
or more halogen, C1-
C3 alkyl, C1-C3 alkoxyl, C1-03 haloalkyl, C1-C3 fluoroalkyl, C4-C7
cycloalkylalkyl, OH, amino, 01-
06 acyl, ¨COORE, ¨OCORE, ¨CONRERF, -OCONRERD, -NRECORF, -SRE, -SORE, -
SO2RE,and
¨SO2NRERF, where RE and Re are as defined above and in particular are
hydrogen, 01-03 alkyl,
phenyl or benzyl. More specifically, optional substitution of RH is
substitution with one or more
halogen (particularly Br or Cl), 01-03 alkyl, C1-03 alkoxyl, Cl-C3 fluoroalkyl
(particularly CF3-).
In embodiments, RH has formula:
pp 1
, II _________ R'
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where:
Xii is CH, CRT or N; RT is optional RH ring substitution as described above
and R and R' are
independently hydrogen, C1-C6 alkyl group, C4-C10 cycloalkylalkyl group, aryl
group, heterocyclyl
group, or heteroaryl group each of which groups are optionally substituted. In
specific
embodiments, RT is hydrogen or substitution with one or more of halogen, OH,
Cl-C3 alkyl, C1-C3
alkoxy, or 01-03 alkyl substituted with a 03-06 cycloalkyl; R' is hydrogen, 01-
03 alkyl, 01-03
alkoxy, or 01-03 alkyl substituted with a C3-C6 cycloalkyl; and R is hydrogen,
C1-C3 alkyl, C1-C3
alkoxy, or C1-C3 alkyl substituted with a C3-C6 cycloalkyl.
In embodiments, RH has formula:
7-
RTjj /1 __
R'
X11 10
where:
Xii is CH, CRT or N; Xio is CH, CRT or N; RT is RH ring optional substitution
as described above
and R and R' are independently hydrogen, C1-C6 alkyl group, C4-C10
cycloalkylalkyl group, aryl
group, heterocyclyl group, or heteroaryl group each of which groups are
optionally substituted. In
specific embodiments, RT is hydrogen or substitution with one or more of
halogen, OH, 01-03
alkyl, 01-03 alkoxy, or 01-03 alkyl substituted with a 03-C6 cycloalkyl; R' is
hydrogen, 01-03
alkyl, 01-03 alkoxy, or 01-03 alkyl substituted with a C3-C6 cycloalkyl; and R
is hydrogen, C1-C3
alkyl, C1-03 alkoxy, or C1-03 alkyl substituted with a 03-C6 cycloalkyl.
In embodiments, RH in formula I or R12 in formula XX is selected from any one
of formulas R12-1
to R12-84. In embodiments, RH is selected from the following formulas in
Scheme 3:
R12-79 or R12-80; or
R12-81-R12-84; or
R12-70, R12-71, or R12-75-R12-78; or
R12-3, R12-4, R12-5, R12-7, R12-8, R12-10, R12-23, R12-25, R12-27, R12-29, or
R12-31; or
R12-12, R12-13, R2-145, R12-15, R12-16, R12-17, R12-18, R12-19, R12-20, R12-
21, R12-21 or
R12-22, where p is 0; or
R12-33, R12-34, R12-35, R12-36, R12-37, R12-38, R12-39 R12-40, R12-41, R12-42,
where p is 0;
or
R12-70 or R12-71, where p is 0; or
R12-75, R12-76, R12-77 or R12-78, where p is 0.
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In embodiments, RH is selected from 5-membered heterocyclic groups of general
formula:
r= T
YY1
where:
T, U, V, and W are selected from 0, S, C(R")(R"), C(R")¨/, C(R"), C¨I, N(R),
or N¨/;
where the group contains one or two double bonds dependent upon choice of T,
U, V, and W;
where the RH group is bonded to the ¨(L2)y-Z¨moiety in the compound of formula
I through
C(R")¨/, or N¨I; and
where R" indicates optional substitution on N or e_
More specifically, RH is selected from 5-membered heterocyclic groups of
formula:
i==-41
W"%a
V
YY2
where:
T is C(R"), C¨/, or N; or
U is 0, S, C(R")(R"), C(R")¨/, N(R), or N¨I;
V is CR", C¨I, or N and
W is CR", C¨/, N, where the RH group is bonded to the ¨(L2)y-Z¨moiety in the
compound of formula
I through C-I, C(R")¨/, or N¨I,
where the RH group is bonded to the ¨(L2)y-Z¨moiety in the compound of formula
I through C¨/,
C(R")¨/, or N¨/; and
where R" indicates optional substitution on N or C. The symbol "4" indicates
a monovalent bond through which the heterocyclic group is bonded in the
compounds herein e.g.,
C¨/ indicates a monovalent bond from a ring carbon through which the
heterocyclic group is
bonded into compounds herein.
In embodiments, RH is a fused ring heterocyclic group of formula:
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U' T'
= =
=,
.õv T'
v2-J,
W' vv
or
YY3 YY4
where:
U, V and W are selected from 0, S, N, C(R")(R"), C(R")¨/, C(R"), C¨/, N(R), or
N¨/;
T', U', V and W are selected from C(R"), C¨/, N(R), or N¨/;
where the RH group is bonded to the ¨(L2)y-Z¨moiety in the compound of formula
I through C-/,
0(R)¨I, or N¨/in the indicated ring;
where the group contains bonds dependent upon choice of, U, V, and W; and
where R" indicates optional substitution on N or C.
More specifically, RH is a fused heterocyclic group of formula:
õ
W' vv
or VV \AP
YY5 YY6
where:
U, and V are selected from N, C(R"), or C¨/,I;
W is selected from 0, S, C(R")(R"), C(R")¨/, N(R), or N¨/;
T', U', V and W are selected from C(R"), C¨I, N(R), or N¨/;
where the RH group is bonded to the ¨(L2)y-Z¨moiety in the compound of formula
I through C-/,
C(R")¨/, or N¨/in the indicated ring; and
where R" indicates optional substitution on N or C.
Each R", independently, is selected from hydrogen, halogen, nitro, cyano,
amino, mono- or di-C1-
C3 alkyl substituted amino, 01-03 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, 03-
06-cycloalkenyl, C1-
03 haloalkyl, 06-012 aryl, 05-012 heteroaryl, 03-012 heterocyclyl. 01-03
alkoxy, 01-06 acyl,
¨COORE, ¨OCORE, ¨CONRERF, -OCONRERD, -NRECORF, -SRE, -SORE, -SO2RE, and
¨SO2NRERF,
where alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy,
and acyl, are optionally
substituted;
where each RE and RF is selected from hydrogen, C1-C3 alkyl, C2-C4 alkenyl, C3-
C6 cycloalkyl,
03-06-cycloalkenyl, C1-03 haloalkyl, 06-012 aryl, 05-012 heteroaryl, 03-012
heterocyclyl. C1-
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03 alkoxy, C1-C6 acyl, each of which groups is optionally substituted with one
or more halogen,
nitro, cyano, amino, mono- or di-C1-03 alkyl substituted amino, 01-03 alkyl,
02-04 alkenyl, C3-C6
cycloalkyl, C3-C6-cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12
heteroaryl, C3-C12
heterocyclyl. C1-C3 alkoxy, and C1-C6 acyl.
In more specific embodiments, RH is selected from any one of:
--, NN R' --, X. R'
In,õT,,<./
r--. KT ----N
\R RH1 \R RH2
-1-
N, N RT., N R' ---__ N
R'
--µ /
µ R-r
R RH3 R RH4
7.- -1-
-_, ---._ RT----"Cz R'
/
D,,,
rx-F RH5 R RH6
N, N R' N
/N--; ---R'
RT-- \ / 0 / N
R' RH7 ' `B RH8
-7
N...._R' ---__
ff
c,(\_.1)___A
/
RT N N RH9 RT -----C- /
RH1 0
RT¨'
r---:.--...,,.........T.H., R
T 11.,,õ,,,,,,,,,<,
R ____________________________ RT
T-- ,..-
RH11 or RT RH12,
where:
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RT is RH ring optional substitution as described above and R and R' are
independently hydrogen,
01-06 alkyl group, C4-C10 cycloalkylalkyl group, aryl group, heterocyclyl
group, or heteroaryl
group each of which groups are optionally substituted. In specific
embodiments, RT is hydrogen or
substitution with one or more of halogen, OH, C1-C3 alkyl, Cl-C3 alkoxy, or C1-
C3 alkyl
substituted with a C3-C6 cycloalkyl; R' is hydrogen, C1-03 alkyl, 01-03
alkoxy, or C1-C3 alkyl
substituted with a 03-06 cycloalkyl; and R is hydrogen, 01-03 alkyl, 01-03
alkoxy, or C1-C3 alkyl
substituted with a 03-06 cycloalkyl. In specific embodiments, R and R' are
independently
hydrogen, C1-C3 alkyl or C4-C7 cycloalkylalkyl. In specific embodiments, RT
represents
hydrogens or substitution with one halogen, particularly Br.
In embodiments, RH is a 6-member optionally substituted heterocyclic or
heteroaryl group having
1-3 nitrogen in the ring, 1 or 2 oxygens, sulfurs or both in the ring, or 1 or
2 nitrogens and one
oxygen or sulfur in the ring, where optional substitution is defined as in
formula I. The heterocyclic
group can be unsaturated, partially unsaturated or a heteroaryl group.
In embodiments, RH is an optionally substituted fused heterocyclic or
heteroaryl group having two
fused 6-member rings having 1-5 nitrogens in the fused rings, 1-3 oxygens,
sulfurs or both in the
fused rings or 1-4 nitrogens and 1 or 2 oxygens, sulfurs or both in the fused
rings, where optional
substitution is defined as in formula I. In more specific embodiments, the
fused rings have 1, 2, 3
or 4 nitrogens in the fused rings. In more specific embodiments, the fused
rings have 1 or 2
oxygens or sulfurs in the fused rings. In more specific embodiments the fused
rings have 1 or 2
nitrogens and one oxygen or sulfur in the fused rings. The fused ring
heterocyclic group can be
unsaturated, partially unsaturated or a heteroaryl group.
In specific embodiments, the RH group is selected from phenyl, oxazinyl,
pyridinyl, pyrimidinyl,
thionyl, pyranyl, thiazinyl, 4H-pyranyl, naphthyl, quinolinyl, isoquinolinyl,
quinoxalinyl, quinazolinyl,
pteridinyl, purinyl and chromanyl, where the RH group is attached to the -
(L2)y-Z-moiety in the
compound of formula I at any available ring position. In specific embodiments,
the RH group is
attached to the -(L2)y-Z-moiety in the compound of formula I at a carbon in
the ring.
In embodiments of formula I, Rp is selected from the group of moieties RN1,
RN2, RN3, RN9, RN10,
RN11, RN13, RN14, RN36, RN37, RN38 or RN39 and RH is selected from the group
of moieties R12-
3, R12-5, R12-44. R13-45, R12-48, R12-58, R12-70, R12-72, R12-73, R12-75, R12-
79, R12-80,
R12-82, R12-83, or R12-84. In embodiments of formula! I, Rp is selected from
the group of
moieties RN1, RN2, RN3, RN9, RN10, RN11, RN13, RN14, RN36, RN37, RN38 or RN39,
RH is selected
from the group of moieties R12-3, R12-5, R12-44. R13-45, R12-48, R12-58, R12-
70, R12-72, R12-
73, R12-75, R12-79, R12-80, R12-82, R12-83, or R12-84, and the A ring is
unsubstituted 1,4-
phenylene or 2,5-pyridylene. In embodiments of formula! I, Rp is selected from
the group of
moieties RN1, RN2, RN3, RN9, RN10, RN11, RN13, RN14, RN36, RN37, RN38 or RN39,
RH is selected
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from the group of moieties R12-3, R12-5, R12-44. R13-45, R12-48, R12-58, R12-
70, R12-72, R12-
73, R12-75, R12-79, R12-80, R12-82, R12-83, or R12-84, the A ring is
unsubstituted 1,4-
phenylene or 2,5-pyridylene, Y is -NH-, -CONH, -NH-00- or -NH-CO-NH- and x is
0 or 1 and 1_1, if
present, is -(CH2)-. In embodiments of formulall, Rp is selected from the
group of moieties RN1,
RN2, RN3, RN9, RN10, RN11, RN13, RN14, RN36, RN37, RN38 or RN39, RH is
selected from the
group of moieties R12-3, R12-5, R12-44. R13-45, R12-48, R12-58, R12-70, R12-
72, R12-73, R12-
75, R12-79, R12-80, R12-82, R12-83, or R12-84, the A ring is unsubstituted 1,4-
phenylene or 2,5-
pyridylene, Y is -NH-, -CONH, -NH-00- or -NH-CO-NH-, x is 0 or 1,1_1, if
present, is -(CH2)-, Z is
-CONH, -NH-00- or -NH-CO-NH-, y is 0 or 1 and L2, if present is -(CH2)-. In
more specific
embodiments of the forgoing embodiments, the A ring is unsubstituted 1,4-
phenylene. In more
specific embodiments of the forgoing embodiments, Y is -NH-. In more specific
embodiments of
the forgoing embodiments, Z is -CONH-. In more specific embodiments of the
forgoing
embodiments, y is 1. In more specific embodiments of the forgoing embodiments,
x is 1.
In specific embodiments of formula!, -Z-(L2)y-RH is a group other than -NH-S02-
Rw, where Rw is
R1 is mes-trimethylphenyl, 4-methylphenyl, 4-trifluoromethylphenyl, naphthyl,
2,3,4,5,-
tetrannethylphenyl, 4-nnethoxyphenyl, 4-tert-butylphenyl, 2,4-dimethoxyphenyl,
2,5-
dimethoxyphenyl or 4-phenoxypheny. In specific embodiments of formula!, -Z-
(L2)y- is a moiety
other than -NRx-S02-, where Rx is H, hydrogen, methyl acetate, acetate,
aminoacetyl, 4-formic
acid benzyl, 4-isopropylbenzyl, 4-chlorobenzyl or 4-methoxybenzyl. In
embodiments of formula!, -
Z- is other than -NRx-S02-, where Rx is H, hydrogen, methyl acetate, acetate,
aminoacetyl, 4-
formic acid benzyl, 4-isopropylbenzyl, 4-chlorobenzyl or 4-methoxybenzyl,
In embodiments of formula!, RH is other than a phenyl group or an optionally
substituted phenyl
group. IN embodiments of formula!, RH is a heterocyclic group that is
substituted with a single
halogen, particularly a Br.
In embodiments of formula!, Rp or -N(R2)(R3) are optionally substituted amine
groups illustrated in
Scheme 2, RN1-RN39. Exemplary optional substitution of groups is illustrated
in Scheme 2. The
illustrated R substituent groups can be positioned on any available ring
position. In the moieties of
Scheme 2, preferred alkyl are C1-C3 alkyl, acyl includes formyl, preferred
acyl are C1-C6 acyl and
more preferably acetyl, acyloxy are preferably C1-C4 acyloxy, alkoxycarbonyl
are preferably C2-05
alkoxycarbonylõ hydroxyalkyl are C1-C6 hydroxyalkyl and preferably are -CH2-
CH2-0H, for amine
groups, preferred alkyl are C1-C3 alkyl, preferred alkyl for -S02alkyl are C1-
C3 alkyl and more
preferred is methyl.
In specific embodiments of formula!, -N(R2)(R3) is RN1. In specific
embodiments, -N(R2)(R3) is
RN3. In specific embodiments, -N(R2)(R3) is RN2 or RN4. In specific
embodiments, -N(R2)(R3) is
RN5 or RN6. In specific embodiments, -N(R2)(R3) is RN7 or RN8. In specific
embodiments,
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-N(R2)(R3) is RN9. In specific embodiments, -N(R2)(R3) is RN10. In specific
embodiments,
-N(R2)(R3) is R1 1. In specific embodiments, -N(R2)(R3) is RN12. In specific
embodiments,
-N(R2)(R3) is RN13. In specific embodiments, -N(R2)(R3) is RN14. In specific
embodiments,
-N(R2)(R3) is RN15. In specific embodiments, -N(R2)(R3) is RN16. In specific
embodiments,
-N(R2)(R3) is RN17 or RN18. In specific embodiments, -N(R2)(R3) is RN19 or
RN20. In specific
embodiments, -N(R2)(R3) is RN21. In specific embodiments, -N(R2)(R3) is RN22.
In specific
embodiments, -N(R2)(R3) is RN23 or RN24. In specific embodiments, -N(R2)(R3)
is RN25. In an
embodiment, N(R2)(R3) is RN1, RN2, RN3, RN4, RN11, RN13, or RN14. In an
embodiment,
-N(R2)(R3) is RN26-RN29. In an embodiment, -N(R2)(R3) is RN30. In an
embodiment, -N(R2)(R3) is
RN31.
In embodiments of formula I, RH is a moiety illustrated in Scheme 3 R12-1 to
R12-78. In
embodiments of formula I, RH is a moiety illustrated in Scheme 3 R12-1 to R12-
69. In
embodiments of formula I, RH is a moiety illustrated in Scheme 3 R12-1 to R12-
71. In embodiments
of formula I, RH is a moiety illustrated in Scheme 3 R12-72 to R12-78. In an
embodiment, RH is
R12-35-R12-42. In embodiments, RH is any of R12-43-R12-69. In embodiments, RH
is any of
R12-43-R12-45. In embodiments, RH is any of R12-46-R12-48. In embodiments, RH
is any of R12-
49-R12-51. In embodiments, RH is any of R12-52-R12-54. In embodiments, RH is
any of R12-55-
R12-58. In embodiments, RH is any of R12-59-R12-62 In embodiments, RH is any
of R12-63-
R12-66. In embodiments, RH is any of R12-67-R12-69. In embodiments, RH is R12-
72 or R12-73.
In embodiments, RH is R12-74. In embodiments, RH is t12-75 or R12-76. In
embodiments, RH is
R12-77. In embodiments, RH is R12-78. In moieties of Scheme 3, preferred alkyl
groups are C-C6
alkyl groups or more preferred C1-C3 alkyl groups, preferred halogen are F, Cl
and Br, acyl
includes formyl and preferred acyl are -CO-C1-C6 alky and more preferred is
acetyl, phenyl is
optionally substituted with one or more halogen, alkyl or acyl. More preferred
alkyl are methyl,
ethyl. Methyl cyclopropyl and cyclopropyl. More preferred halogen are Cl and
Br.
In specific embodiments, compounds useful in the methods herein include those
of formula II:
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LX
R5
(I-2)y
RH
or salts, or solvates thereof,
where both X, Rp, Y, x, L1, RA, Z, y, L2 and RH are as defined in formula I,
R4 and R5 are
independently selected from hydrogen, halogen, alkyl group, alkenyl group,
cycloalkyl group,
cycloalkenyl group, or heterocyclyl group, each of which groups is optionally
substituted or
R4 and R5 together form an optionally substituted 5- or 6-member heterocyclic
ring which can
contain one or two double bonds or be aromatic; and
the dotted line is a single or double bond dependent upon choice of R4 and Rs.
In embodiments, x is 1, and y is 1. In embodiments, both X are nitrogens. In
embodiments, Rp is
-N(R2)(R3). In embodiments, Li and L2 are -(CH2)n-, where n are independently
is 1, 2 or 3. In
embodiments, RH is a heterocyclic or heteroaryl group. In embodiments, Y is -
N(Ri)-, -CON(Ri)-,
or -N(Ri)C0-. In embodiments, Z is -CON(R')- or -N(R)C0-. In embodiments, R.
is hydrogen,
a 01-03 alkyl or a Ci-03 haloalky. In embodiments, R is hydrogen, methyl or
trifluoromethyl. In
embodiments, RA is hydrogen, halogen C1-C3 alkyl, C1-C3 alkoxyl, C1-C3 acyl,
or C1-C3
haloalkyl. In embodiments, R' is hydrogen, methyl, methoxy or trifluoromethyl.
In embodiments,
R4 and R5 are selected from hydrogen, halogen, 01-03 alkyl, C1-C3 alkoxyl, or
C1-C3 haloalkyl. In
embodiments, R4 and R5 together form a 5- or 6-member carbocyclic or
heterocyclic ring which is
saturated, partially unsaturated or is heteroaromatic. In embodiments, RH is
any one of RH1-
RH12.
In embodiments of formula II, Y is NH. In embodiments of formula II, Y is NH,
and xis 0. In
embodiments of formula II, Y is NH, xis 0 and R5 is other than an
electronegative group. In
embodiments of formula II, Y is NH, xis 0 and R5 is hydrogen. In embodiments
of formula II, Y is
NH, xis 0, R5 is hydrogen and R4 is a C1-C3 alkyl. In embodiments of formula
II, Y is NH, x is 0, R5
is hydrogen and R4 is methyl.
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In embodiments of formula II, Y is NH, x is 1 and Li is -(CH2)n-, where n is 1
or 2. In embodiments
of formula II, Y is NH, x is 1 and Li is -(CH2)n-, where n is 1 or 2, and R5
is an electronegative
group. In embodiments of formula II, Y is NH, x is 1 and Li is -(CH2)n-, where
n is 1 or 2, and R5
is a halogen. In embodiments of formula II, Y is NH, x is 1 and Li is -(CH2)-,
and R5 is a halogen.
In embodiments of formula II, Y is NH, x is 1 and Li is -(CH2)n-, where n is 1
or 2, and R5 is a
fluorine. In embodiments of formula II, Y is NH, xis 1 and Li is -(CH2)-, and
R5 iS a fluorine. In
embodiments of formula II, Y is NH, xis 1 and Li is -(CH2)-, R5 is a halogen
and R4 is 01-03 alkyl.
In embodiments of formula II, Y is NH, xis 1 and Li is -(CH2)-, R5 is a
halogen and R4 is methyl. In
embodiments of formula II, Y is NH, xis 1 and Li is -(CH2)-, R5 is a fluorine
and R4 is 01-03 alkyl.
In embodiments of formula II, Y is NH, xis 1 and Li is -(CH2)-, R5 is a
fluorine and R4 is methyl.
In specific embodiments, compounds useful in the methods herein include those
of formula III:
p
X
R5
(I-2)y
RH
or salts, or solvates thereof,
where variables are as defined in formula I and ll and the dotted line
represent a single or double
bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, Rp is -N(R2)(R3). In embodiments, L2 is -(CH2)n-, where n is 1, 2
or 3. In
embodiments, RH is a heterocyclyl or heteroaryl group. In embodiments, Y is -
N(Ri)-, -CON(Ri)-,
or -N(Ri)C0-. In embodiments, Z is -CON(R')- or -N(R)C0-. In embodiments, R.
is hydrogen,
a C1-C3 alkyl or a C1-C3 haloalky. In embodiments, R is hydrogen, methyl or
trifluoromethyl. In
embodiments, RA is hydrogen, halogen C1-C3 alkyl, C1-C3 alkoxyl, C1-C3 acyl,
or C1-C3
haloalkyl. In embodiments, R' is hydrogen, methyl, methoxy or trifluoromethyl.
In embodiments,
R4 and R5 are selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxyl, or
C1-C3 haloalkyl. In
embodiments, R4 and R5 together form a 5- or 6-member carbocyclic or
heterocyclic ring which is
saturated, partially unsaturated or is heteroaromatic. In embodiments, RH is
any one of RH1-RH12.
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In specific embodiments, compounds useful in the methods herein include those
of formula IV:
R5
RA< N
R1
(L2)y
RH
or salts or solvates thereof;
where variables are as defined in formula I and ll and the dotted line
represents a single or double
bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, Rp is ¨N(R2)(R3). In embodiments, L2 is ¨(CH2)n¨, where n is 1, 2
or 3. In
embodiments, RH is a heterocyclyl or heteroaryl group. In embodiments Ri is
hydrogen In
embodiments, R1 is hydrogen, methyl or trifluoromethyl. In embodiments, Z is
¨CON(R')¨ or
N(R)C0¨. In embodiments, R is hydrogen, a C1-C3 alkyl or a Ci-C3 haloalky. In
embodiments,
R' is hydrogen, methyl or trifluoromethyl. In embodiments, RA is hydrogen,
halogen C1-C3 alkyl,
C1-C3 alkoxyl, C1-C3 acyl, or C1-C3 haloalkyl. In embodiments, R' is hydrogen,
methyl, methoxy
or trifluoromethyl. In embodiments, R4 and R5 are selected from hydrogen,
halogen, C1-C3 alkyl,
01-03 alkoxyl, or C1-03 haloalkyl. In embodiments, R4 and R5 together form a 5-
or 6-member
carbocyclic or heterocyclic ring which is saturated, partially unsaturated or
is heteroaromatic. In
embodiments, RH is any one of RH1-RH12.
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In specific embodiments, compounds useful in the methods herein include those
of formula V:
X
R5
RA< N
R R1
R3
0=C
(L2)y
RH
or salts or solvates thereof;
where variables are as defined in formula I and ll and the dotted line
represents a single or double
bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, Rp is ¨N(R2)(R3). In embodiments, L2 is ¨(CH2)n¨, where n is 1, 2
or 3. In
embodiments, RH is a heterocyclyl or heteroaryl group. In embodiments Ri is
hydrogen In
embodiments, R1 is hydrogen, methyl or trifluoromethyl. In embodiments, Rs is
hydrogen, C1-C3
alkyl, optionally substituted C1-C3 alkyl, or aryl. In embodiments, RA is
hydrogen, halogen C1-C3
alkyl, C1-C3 alkoxyl, C1-C3 acyl, or C1-C3 haloalkyl. In embodiments, R is
hydrogen, methyl,
methoxy or trifluoromethyl. In embodiments, R4 and R5 are selected from
hydrogen, halogen, C1-
C3 alkyl, C1-C3 alkoxyl, or C1-C3 haloalkyl. In embodiments, R4 and R5
together form a 5- or 6-
member carbocyclic or heterocyclic ring which is saturated, partially
unsaturated or is
heteroaromatic. In embodiments, RH is any one of RH1-RH12.
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In specific embodiments, compounds useful in the methods herein include those
of formula VI:
R2
X
R5
Rs
R1
\N
0=C
(L2)y
RH
or salts or solvates thereof;
where variables are as defined in formula I and ll and the dotted line
represents a single or double
bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, x is 1 and ¨N-(CH2)n¨, where n is 1, 2 or 3. In embodiments, y is
0. In
embodiments, L2 is ¨(CH2)n¨, where n is 1, 2 or 3. In embodiments, RH is a
heterocyclyl or
heteroaryl group. In embodiments R1 is hydrogen In embodiments, R1 is
hydrogen, methyl or
trifluoromethyl. In embodiments, Rs is hydrogen, C1-C3 alkyl, optionally
substituted C1-C3 alkyl,
or aryl. In embodiments, RA is hydrogen, halogen C1-C3 alkyl, C1-C3 alkoxyl,
C1-C3 acyl, or C1-
C3 haloalkyl. In embodiments, R is hydrogen, methyl, methoxy or
trifluoromethyl. In
embodiments, R4 and R5 are selected from hydrogen, halogen, 01-03 alkyl, 01-03
alkoxyl, or 01-
03 haloalkyl. In embodiments, R4 and R5 together form a 5- or 6-member
carbocyclic or
heterocyclic ring which is saturated, partially unsaturated or is
heteroaromatic. In embodiments,
RH is any one of RH1-RH12.
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In specific embodiments, compounds useful in the methods herein include those
of formula VII:
R2
/N¨R3
X
R5
R1
Rs,
0=C
(L2)y
RH
or salts or solvates thereof;
where variables are as defined in formula I and ll and the dotted line
represents a single or a
double bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, x is 1 and ¨N-(CH2)n¨, where n is 1, 2 or 3. In embodiments, y is
0. In
embodiments, L2 is ¨(CH2)n¨, where n is 1, 2 or 3. In embodiments, RH is a
heterocyclyl or
heteroaryl group. In embodiments R1 is hydrogen In embodiments, R1 is
hydrogen, methyl or
trifluoromethyl. In embodiments, Rs is hydrogen, C1-C3 alkyl, optionally
substituted C1-C3 alkyl,
or aryl. In embodiments, RA is hydrogen, halogen C1-C3 alkyl, C1-C3 alkoxyl,
C1-C3 acyl, or C1-
03 haloalkyl. In embodiments, R is hydrogen, methyl, methoxy or
trifluoromethyl. In
embodiments, R4 and R5 are selected from hydrogen, halogen, 01-03 alkyl, C1-C3
alkoxyl, or C1-
C3 haloalkyl. In embodiments, R4 and R5 together form a 5- or 6-member
carbocyclic or
heterocyclic ring which is saturated, partially unsaturated or is
heteroaronnatic. In embodiments, RH
is any one of RH1-RH12.
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In specific embodiments, compounds useful in the methods herein include those
of formula VIII:
R2
R5
R6
R7 R1
Rs \
R8
0=C
R9
(L2)y
I Rm
or salts or solvates thereof;
where variables are as defined in formula I and II, the dotted line represents
a single or a double
bond, R6-R9 are independently selected from hydrogen and RA groups defined in
formula I. Rm
represents optional substitution on the fused ring and Rm takes the values of
RA in formula I.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, x is 1 and ¨N-(CH2)n¨, where n is 1, 2 or 3. In embodiments, x is
0. In
embodiments, L2 is ¨(CH2)n¨, where n is 1, 2 or 3. In embodiments R1 is
hydrogen In
embodiments, R1 is hydrogen, methyl or trifluoronnethyl. In embodiments, R7-R9
are independently
selected from hydrogen, C1-C3 alkyl, optionally substituted 01-03 alkyl, or
aryl. In embodiments,
R7-R9 are independently selected from hydrogen, halogen 01-03 alkyl, 01-03
alkoxyl, C1-C3 acyl,
or 01-03 haloalkyl. In embodiments, R7-R9 are all hydrogens. In embodiments,
R4 and R5 are
selected from hydrogen, halogen, C1-C3 alkyl, 01-03 alkoxyl, or 01-03
haloalkyl. In
embodiments, R4 and R5 together form a 5- or 6-member carbocyclic or
heterocyclic ring which is
saturated, partially unsaturated or is heteroaromatic. In embodiments, Rm is
one or more hydrogen,
halogen, C1-03 alkyl group, 04-07 cycloalkylalkyl group or C1-03 haloalkyl
group. In
embodiments, Rm is one or more hydrogen, halogen, particularly Br, methyl or
trifluoromethyl. In
embodiments, Rm is hydrogen.
In specific embodiments, compounds useful in the methods herein include those
of formula IX:
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R2
(M)--R3
X
Ry
R6
R7 R1
RS\
R8
0=C R9
(L2)y
Rm
or salts or solvates thereof; where variables are as defined in formula I, the
dotted line represents a
single or a double bond. R6-R9 are independently selected from hydrogen and RA
groups defined
in formula I and Rm represents optional substitution as defined in formula I.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, x is 1 and M is ¨N-(CH2)n¨, where n is 1, 2 or 3. In embodiments,
x is 1 and M is -
(CH2)n¨, where n is 1, 2 or 3. In embodiments, x is 0. In embodiments, L2 is
¨(CH2)n¨, where n is
1, 2 or 3. In embodiments R1 is hydrogen In embodiments, R1 is hydrogen,
methyl or
trifluoromethyl. In embodiments, R7-R9 are independently selected from
hydrogen, C1-C3 alkyl,
optionally substituted C1-C3 alkyl, or aryl. In embodiments, R7-1R9 are
independently selected from
hydrogen, halogen C1-C3 alkyl, C1-C3 alkoxyl, C1-C3 acyl, or C1-C3 haloalkyl.
In embodiments,
R7-R9 are all hydrogens. In embodiments, R4 and R5 are selected from hydrogen,
halogen, C1-C3
alkyl, C1-C3 alkoxyl, or C1-C3 haloalkyl. In embodiments, R4 and R5 together
form a 5- or 6-
member carbocyclic or heterocyclic ring which is saturated, partially
unsaturated or is
heteroaromatic. In embodiments, Rm is hydrogen, halogen, C1-C3 alkyl group or
C1-C3 haloalkyl
group. In embodiments, Rm is hydrogen, halogen, particularly Br, methyl or
trifluoromethyl.
In other embodiments, the invention provides a compound of formula XI:
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R2
R4 X
R3
X
R5
(I-1)x -N
RA
A
or salts, or solvates thereof,
where:
each X is independently selected from N or CH and at least one X is N;
the A ring is a carbocyclic or heterocyclic ring having 3-10 carbon atoms and
optionally 1-6
heteroatoms and which optionally is saturated, unsaturated or aromatic;
L1 is an optional 1-3 carbon linker that is optionally substituted, where xis
0 or 1 to indicate the
absence of presence of Li;
R1 is selected from the group consisting of hydrogen, alkyl group. alkenyl
group, cycloalkyl group,
cycloalkenyl group, heterocyclyl group, or aryl group, each of which groups is
optionally
substituted;
R2 and R3 are independently selected from hydrogen, alkyl, alkenyl,
cycloalkyl, cycloalkenyl, aryl or
heterocyclyl, each of which groups is optionally substituted or
R2 and R3 together form an optionally substituted 5- to 8-member heterocyclic
ring which is a
saturated, partially unsaturated or aromatic ring;
R4 and R5 are independently selected from hydrogen, halogen, alkyl, alkenyl,
cycloalkyl,
cycloalkenyl, aryl or heterocyclyl, each of which groups is optionally
substituted or
R4 and R5 together form an optionally substituted 5- or 6-member ring which
optionally contains
one or two double bonds or is aromatic and optionally contains 1-3
heteroatoms;
where the dotted line is a single or double bond dependent upon selection of
R4 and R5;
and
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RA represents hydrogens or 1-10 substituents on the indicated ring, wherein RA
substituents are
independently selected from hydrogen, halogen, hydroxyl, cyano, nitro, amino,
mono- or
disubstituted amino, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl,
heterocyclyl, -0R15, -COR15, -
000R15, -000R15, -CO-NR16R17, -OCON R16R17, -NR16-CO-R15, -SR15, -SOR15, -
S02R15, -SO2-
NRi6R17, R10, ¨NH-00-(L2)y-R12, or¨NH-CO-(L2)-R12, where L2 is an optional 1-6
carbon atom
linker group which linker is optionally substituted and wherein one or two
carbons of the liker are
optionally replaced with 0 or S, where y is 0 or 1 to show the absence or
presence of L2;
R10 is selected from alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl,
or aryl, each of which
groups is optionally substituted with one or more halogen, alkyl, alkenyl,
haloalkyl, alkoxy, aryl,
heteroaryl, heterocyclyl, aryl-substituted alkyl, or heterocyclyl-substituted
alkyl;
R12 is selected from cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or
aryl, each of which groups
is optionally substituted, or Ri2 is a C1-C3 alky substituted with cycloalkyl,
cycloalkenyl,
heterocyclyl, heteroaryl, or aryl each of which is optionally substituted and
where optional
substitution is one or more halogen, alkyl, alkenyl, haloalkyl, alkoxy, aryl,
heteroaryl, or
heterocyclyl;
each R15 is independently selected from hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkenyl, aryl or
heterocyclyl, arylalkyl (an alkyl group substituted with an aryl) and
heterocyclylalkyl, cycloalkylalkyl,
cycloalkenylalkyl, each of which groups is optionally substituted; and
each R16 and R17 is independently selected from hydrogen, alkyl, alkenyl,
cycloalkyl, cycloalkenyl,
aryl or heterocyclyl, arylalkyl (an alkyl group substituted with an aryl) and
heterocyclylalkyl,
cycloalkylalkyl, cycloalkenylalkyl, each of which groups is optionally
substituted;
wherein optional substitution includes substitution with one or more halogen,
nitro, cyano, amino,
mono- or di-C1-03 alkyl substituted amino, C1-03 alkyl, 02-04 alkenyl, 03-06
cycloalkyl, 03-C6-
cycloalkenyl, C6-C12 aryl, and C6-C12 heterocyclyl.
In embodiments of formula XI, R1 is H. In embodiments of formula XI, R1 is H,
and xis 0. In
embodiments of formula XI, R1 is H, xis 0 and R5 is other than an
electronegative group. In
embodiments of formula XI, R1 is H, x is 0 and R5 is hydrogen. In embodiments
of formula XI, R1 is
H, xis 0, R5 is hydrogen and R4 is a C1-C3 alkyl. In embodiments of formula
XI, R1 is H, xis 0, R5
is hydrogen and R4 is methyl.
In embodiments of formula XI, R1 is H, x is 1 and L1 is ¨(CH2)n-, where n is 1
or 2. In embodiments
of formula XI, R1 is H, x is 1 and L1 is ¨(CH2)n-, where n is 1 or 2, and R5
is an electronegative
group. In embodiments of formula XI, R1 is H, x is 1 and Li is ¨(CH2)n-, where
n is 1 or 2, and R5
is a halogen. In embodiments of formula XI, R1 is H, x is 1 and L1 is ¨(CH2)-,
and R5 is a
halogen. In embodiments of formula XI, R1 is H, x is 1 and L1 is ¨(CH2)n-,
where n is 1 or 2, and
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R5 is a fluorine. In embodiments of formula XI, Ri is H, x is 1 and Li is
¨(CH2)-, and R5 is a
fluorine. In embodiments of formula XI, Ri is H, x is 1 and Li is ¨(CH2)-, R5
is a halogen and R4 is
C1-C3 alkyl. In embodiments of formula XI, Ri is H, xis 1 and Li is ¨(CH2)-,
R5 is a halogen and
R4 is methyl. In embodiments of formula XI, Ri is H, xis 1 and Li is ¨(CH2)-,
R5 is a fluorine and R4
is 01-03 alkyl. In embodiments of formula XI, Ri is H, xis 1 and Li is ¨(CH2)-
, R5 is a fluorine and
R4 is methyl.
In an embodiment, the compound has formula XII:
R2
R4
R3
X
R5
R6
R7 R1
(1_2)Y R8
Rio
R9
or a salt or solvate thereof where variables are as defined for formula Xl.
In an embodiment, the compound has formula XIII:
R2
X
R3
RB-1
YY X
R1
RA A
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or a salt, or a solvate thereof, wherein variables are as defined in formula
XI and where;
each Y is independently selected from N or CH;
RB represents hydrogens or 1-10 substituents on the indicated ring, wherein RA
substituents are
independently selected from hydrogen, halogen, hydroxyl, cyano, nitro, amino,
mono- or
disubstituted amino, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl,
heterocyclyl, -0R15, -00R15, -
C00R15, -000R15, -CO-NR16R17, -000N R16R17, -NR16-CO-R15, -SR15, -50R15, -
S02R15, -SO2-
NR16R17, or -(L2)y-R1o, where L2 is an optional 1-6 carbon atom linker group
which linker is
optionally substituted, and where y is 0 or 1 to show the absence or presence
of L2.
In embodiments of formula XIII, Ri is H. In embodiments of formula XIII, Ri is
H, and xis 0. In
embodiments of formula XIII, Ri is H, x is 0 and the B ring is substituted
with other than an
electronegative group. In embodiments of formula XIII, R1 is H, x is 0 and the
B ring is substituted
with one or more hydrogens or C1-C3 alkyl groups. In embodiments of formula
XIII, Ri is H, x is 0,
the B ring is substituted with one or more hydrogens or methyl groups.
In embodiments of formula XIII, Ri is H, xis 1 and Li is ¨(CH2)n-, where n is
1 or 2. In
embodiments of formula XIII, Ri is H, x is 1 and Li is ¨(CH2)n-, where n is 1
or 2, and the B ring is
substituted with an electronegative group. In embodiments of formula XIII, Ri
is H, x is 1 and Li
is ¨(CH2)n-, where n is 1 01 2, and the B ring is substituted with a halogen.
In embodiments of
formula XIII, R1 is H, x is 1 and L1 is ¨(CH2)-, and the B ring is substituted
with a halogen. In
embodiments of formula XIII, Ri is H, x is 1 and Li is ¨(CH2)n-, where n is 1
0r2, and the B ring is
substituted with a fluorine. In embodiments of formula XIII, Ri is H, x is 1
and L1 is ¨(CH2)-, and
the B ring is substituted with a fluorine. In embodiments of formula XIII, R1
is H, x is 1 and L1 is ¨
(CH2)-, the B ring is substituted a halogen and a C1-C3 alkyl. In embodiments
of formula XIII, Ri is
H, x is 1 and Li is ¨(CH2)-7 and the B ring is substituted a halogen. In
embodiments of formula X1117
Ri is H, x is 1 and Li is ¨(CH2)-, the B ring is substituted with a fluorine
and R4 is C1-C3 alkyl. In
embodiments of formula XIII, Ri is H7 x is 1 and Li is ¨(CH2)-, the B ring is
substituted a halogen
and a methyl.
In embodiments, the compound has formula XIV or XV:
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R2
R2
X eN R B NN R3
RB-
N
=Nõ..
R1 R1
RA A RA
A
xiv XV
or a salt or solvate thereof,
where variables are as defined in formula XI, XII or XIII.
In embodiments of these formulas, xis 0 and R1 is hydrogen. In embodiments of
these formulas, x
is 1, Li is ¨(CH2)- and R1 is hydrogen.
In embodiments, the compound has formula XVI or XVII:
R2
R2
. .3
Ri2
Ri2
R6 R6
N N
R7 R1 R7 R8
(L2)Y R8
e7(L2)Y
R10 .10
R9 R9
XVI XVI I
or a salt or solvate thereof,
where variables are as defined in formula XI or XV, and
R11 and R12 are independently selected from hydrogen, halogen, alkyl group,
alkenyl group,
cycloalkyl group, cycloalkenyl group, or heterocyclyl group, each of which
groups is optionally
substituted.
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In embodiments, the compound has formula XVIII:
R2
R4
R3
R5
R6
R7
R1
0-2)Y R8
R10
R9
or salts (or solvates) thereof,
wherein:
Ri is selected from the group consisting of hydrogen, alkyl group. alkenyl
group, cycloalkyl group,
cycloalkenyl group, heterocyclyl group, or aryl group, each of which groups is
optionally substituted
(need to define substitution);
R2 and R3 together form an optionally substituted 5- or 6-member heterocyclic
ring which can
contain one or two double bonds or be aromatic;
R4 and R5 are independently selected from hydrogen, halogen, alkyl group,
alkenyl group,
cycloalkyl group, cycloalkenyl group, or heterocyclyl group, each of which
groups is optionally
substituted or
R4 and R5 together form an optionally substituted 5- or 6-member heterocyclic
ring which can
contain one or two double bonds or be aromatic;
the dotted line is a single or double bond dependent upon choice of R4 and R5,
R6-R5 are independently selected from hydrogen, halogen, alkyl group, alkenyl
group, cycloalkyl
group, cycloalkenyl group, or heterocyclyl group, each of which groups is
optionally substituted;
L is an optional 1-6 atom linker group, where x is 1 or 0 to show the presence
or absence of the L
group; and
R10 is selected from alkyl group. alkenyl group, cycloalkyl group,
cycloalkenyl group, heterocyclyl
group, or aryl group, each of which groups is optionally substituted.
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For example, L is a 2-6 atom linker group; (e.g., --CH2-0-, -CH2-CH2-0-, -0-
CH2-, -0-CH2-CH2-, -
CO-NH-, --NH-CO-, -CH2-CO-NH-, -CH2-CH2-CO-NH-)
In an embodiment, the compound is of formula XIX:
R2
R4
_3
N
R5
R6
R7 el R1
0
R8
R10¨(CH2)Y Rg
or salts (or solvates) thereof,
where:
R1-R0 are as defined above; the dotted line represents a single or double bond
dependent on
choice of R4 and R5;
y is 0 or an integer ranging from 1-3 inclusive; and
R10 is selected from alkyl group. alkenyl group, cycloalkyl group,
cycloalkenyl group, heterocyclyl
group, or aryl group, each of which groups is optionally substituted.
In embodiments, the CHD1L inhibitor is a compound of formula XX, )0(1, >0(11
or XXIII:
R4 RN
R5
R6
R7
Ri
R10 R8
Rg
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XX
R4
R6,.......... ^--....,...
N
R6
I
0
N
I
Ri FAN
R10 R8
R9
XXI
R4
R6-....õ..r......õ.õ-RN
1 N
N.\%.
R6
R7 0
Ri
Rio R8
R9
XXI I
R4
1=Z6 RN
1
N
R6 N \%
R7 aft, [1-18k-----N
Ri
R10 II R
R9
)0(111
and salts or solvates thereof, where Ri-R9 represent hydrogen or optional
substituents, Rio is a
moiety believed to be associated with potency; and RN is a moiety believed to
be associated with
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physicochemical properties such as solubility. L1 is as defined for formula 1
above and x is 0 or 1
to show the absence of presence of the L1 group. In embodiments, R5 is a
substituent other than
hydrogen which is believed to be associated with metabolic stability. In
specific embodiments, R5
is a halogen, particularly F or Cl, a C1-C3 alkyl group, particularly a methyl
group. In specific
embodiments, when x is 1, R5 is an electronegative substituent, particularly a
halogen, and more
preferably F or Cl. In specific embodiments, R5 is a halogen, particularly F
or Cl, and R4 is a 01-
03 alkyl group, particularly a methyl group. In specific embodiments, when x
is 1, R5 is an
electronegative substituent, particularly a halogen, and more preferably F or
Cl. In embodiments,
R4 is a substituent other than hydrogen and in particular is a C1-C3 alkyl
group, and more
particularly is a methyl group. In a specific embodiment, R5 is F and R4 is
methyl. In specific
embodiments L1 is -(CH2)n-, where n is 1 or 2 and more specifically where n is
1. In
embodiments, R6-R9 are selected from hydrogen, C1-C3-alkyl, halogen, hydroxyl,
01-03 alkoxy,
formyl, or 01-03 acyl. In embodiments, one or two of R6-R9 are moieties other
than hydrogen. In
an embodiment, one of R6-R9 is a halogen, particularly fluorine. In specific
embodiments, all of R6-
R9 are hydrogen. In embodiments, RN is an amino moiety -N(R2)(R3). In specific
embodiments, RN
is an optionally substituted heterocyclic group having a 5- to 7- member ring
optionally containing a
second heteroatoms (N, S 01 0). In embodiments, RN is optionally substituted
pyrrolidin-1-yl,
piperidin-1-yl, azepan-1-yl, piperazin-1-yl, or morpholino. In RN is
substituted with one substituent
selected from 01-03 alkyl, formyl, 01-03 acyl (particularly acetyl), hydroxyl,
halogen (particularly F
or Cl), hydroxyl, 01-03 alkyl (particularly -CH2-CH2-0H). In embodiments, RN
is unsubstituted
pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, piperazin-1-yl, or nnorpholino.
In embodiments, R10 is -NRy-00-(L2)y-R12 or -CO-NRy--(L2)y-R12, where y is 0
or 1 to indicate
the absence of presence of L2 which is an optional 1-6 carbon atom linker
group which linker is
optionally substituted and wherein one or two, carbons of the linker are
optionally replaced with 0,
NH, NRy or S, where Ry is hydrogen or a 1-3 carbon alkyl, and R12 is an aryl
group, cycloalkyl
group, heterocyclic group, or heteroaryl group, each of which is optionally
substituted. IN
embodiments, y is 1. L2 is -(CH2)p-, where p is 0-3. In embodiments, R12 is
thiophen-2-yl,
thiophen-3-yl, furany-2-yl, furan-3-yl, pyrrol-2-yl, pyrrol-3-yl,
indo1-2-yl, indo1-3-yl, benzofuran-2-yl, benzofuran-3-yl, benzo[b]thiophen-2-
yl, benzo[b]thiophen-3-
yl, isobenzofuran-1-yl, isoindo1-1-yl, or benzo[c]thiophen-1-yl. In
embodiments, R1 is hydrogen or
methyl. In embodiments, Ri2 is thiophen-2-yl, furany-2-yl, pyrrol-2-yl, oxazol-
4-yl, indo1-2-yl,
benzofuran-2-yl, or benzo[b]thiophen-2-yl. In embodiments, Ri2 is thiophen-2-
y1 or indo1-2-yl. In
embodiments, Ri is hydrogen or methyl.
In more general embodiments of formula XX -XXIII:
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R1 is selected from the group consisting of hydrogen, alkyl group, alkenyl
group, cycloalkyl group,
cycloalkenyl group, heterocyclyl group, or aryl group, each of which groups is
optionally
substituted;
RN is ¨NR2R3, R2 and R3 are independently selected from hydrogen, alkyl,
alkenyl, cycloalkyl,
cycloalkenyl, aryl or heterocyclyl, each of which groups is optionally
substituted or R2 and R3
together form an optionally substituted 5- to 8- member heterocyclic ring
which is a saturated,
partially unsaturated or aromatic ring;
R4 ¨Rs are independently selected from hydrogen, halogen, hydroxyl, cyano,
nitro, amino, mono-
or dialkyl substituted amino, optionally substituted alkyl, optionally
substituted alkenyl, optionally
substituted cycloalkyl, optionally substituted cycloalkenyl, optionally
substituted aryl, optionally
substituted heterocyclyl, -0R15, -00R15, -COOR15, -000R15, -CO-NR15R16, -
0C0NR15R16, -NR15-
CO-R16, -SR15, -SOR15, -S02R15, and -S02-NR151R16;
R113 is ¨NRy-00-(L2)y-R12, -CO-NRy-(L2)y-R12, where L2 is an optional 1-6
carbon atom linker group
which linker is optionally substituted and wherein one or two, carbons of the
liker are optionally
replaced with 0 or S, where y is 0 or 1 to show the absence or presence of L2,
R12 is selected from cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or
aryl, each of which groups
is optionally substituted, or R12 is a C1-C3 alky substituted with cycloalkyl,
cycloalkenyl,
heterocyclyl, heteroaryl, or aryl each of which is optionally substituted and
where optional
substitution is one or more halogen, alkyl, alkenyl, haloalkyl, alkoxy, aryl,
heteroaryl, or
heterocyclyl;
each R15 and R16 is independently selected from hydrogen, alkyl, alkenyl,
cycloalkyl, cycloalkenyl,
aryl or heterocyclyl, arylalkyl and heterocyclylalkyl, cycloalkylalkyl,
cycloalkenylalkyl, each of which
groups is optionally substituted; and
wherein optional substitution includes, substitution with one or more halogen,
nitro, cyano, amino,
mono- or di-C1-C3 alkyl substituted amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6
cycloalkyl, C3-C6-
cycloalkenyl, 06-C12 aryl, 06-012 heterocyclyl, -0R17, -00R17, -000R17, -
000R17, -CO-NR17R15,
-0C0NR17R10, -NR17-CO-R10, -SR17, -S0R17, -S02R17, and -S02-NR17R10, where R17
and R18 are
independently hydrogen or aC1-C6 alkyl.
In embodiments of formula XX and XXI, RN is an optionally substituted cyclic
amine group selected
from any of RN1-RN39 (Scheme 2). Exemplary optional substitution of groups is
illustrated in
Scheme 2. The illustrated R substituent groups can be positioned on any
available ring position.
In the moieties of Scheme 2, preferred alkyl are C1-C3 alkyl, acyl includes
fornnyl, preferred acyl
are C1-C6 acyl and more preferably acetyl, hydroxyalkyl are C1-C6 hydroxyalkyl
and preferably
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are -CH2-CH2-0H, for amine groups, preferred alkyl are C1-C3 alkyl, preferred
alkyl for -S02alkyl
are 01-03 alkyl and more preferred is methyl.
In specific embodiments of formula X,X, or )(XI, RN IS RN1. In specific
embodiments, RN is RN3. In
specific embodiments, RN IS RN2 or RN4. In specific embodiments, RN IS RN5 or
RN6. In specific
embodiments, RN is RN7 or RN8. In specific embodiments, RN is RN9. In specific
embodiments, RN
is RN10. In specific embodiments, RN is RN11. In specific embodiments, RN is
RN12. In specific
embodiments, RN IS RN13. In specific embodiments, RN IS RN14. In specific
embodiments, RN is
RN15. In specific embodiments, RN is RN16. In specific embodiments, RN IS RN17
or RN18. In
specific embodiments, RN IS RN19 or RN20. In specific embodiments, RN IS RN21.
In specific
embodiments, RN is RN22. In specific embodiments, RN is RN23 or RN24. In
specific embodiments,
RN is RN25. In an embodiment, RN is RN1, RN2, RN3, RN4, RN1 1, RN13, or RN14.
In an
embodiment, RN is RN26-RN29. In an embodiment, RN IS RN30. In an embodiment,
RN IS RN31.
In embodiments of formula XX-XXIII, R12 is an optionally-substituted thienyl,
thienylmethyl, fury!,
furylmethyl, indolyl or methylindolyl. In embodiments, R12 is a moiety
illustrated in Scheme 3 R12-
1 to R12-69, R12-1-R12-71 or R12-72-R12-78. In moieties of Scheme 3, preferred
alkyl groups
are 0-06 alkyl groups or more preferred 01-03 alkyl groups, preferred halogen
are F, Cl and Br,
acyl includes formyl and preferred acyl are -00-01-06 alky and more preferred
is acetyl, phenyl is
optionally substituted with one or more halogen, alkyl or acyl. In
embodiments, R12 is a methyl,
ethyl group or propyl substituted with a moiety as illustrated in Scheme 3 R12-
1 to R12-22. In an
embodiment, R12 is R12-1. In an embodiment, R12 is R12-2. In an embodiment,
R12 is R12-3. In an
embodiment, R12 IS R12-4. In an embodiment, R12 is R12-5. In an embodiment,
R12 is R12-6. In an
embodiment, R12 is R12-7. In an embodiment, R12 is R12-8. In an embodiment,
R12 is R12-9. In an
embodiment, R12 IS R12-10. In an embodiment, Ri2 is R12-11. In an embodiment,
Ri2 is R12-12.
In an embodiment, R12 is R12-13. In an embodiment, R12 is R12-14. In an
embodiment, R12 is
R12-15. In an embodiment, R12 is R12-16. In an embodiment, R12 is R12-17. In
an embodiment,
Ri2 is R12-18 In an embodiment, Ri2 is R12-19. In an embodiment, Ri2 is R12-
20. In an
embodiment, R12 is R12-21. In an embodiment, Ri2 is R12-22. In an embodiment,
Ri2 is one of
R12-23-R12-26. In an embodiment, Ri2 is one of R12-27-R12-30. In an
embodiment, Ri2 is one of
R12-31-R12-34. In an embodiment, R12 is one of R12-35-R12-42. In embodiments,
R12 is any
one of R12-43-R12-69. In embodiments, Ri2 is a methyl, ethyl group or propyl
group substituted
with a moiety R12-43-R12-69, as illustrated in Scheme 3. In embodiments, R12
is any one of R12-
43-R12-45. In embodiments, R12 is a methyl, ethyl group or propyl group
substituted with a moiety
R12-43-R12-45 as illustrated in Scheme 3. In embodiments, R12 is any one of
R12-46-R12-48. In
embodiments, Ri2 is a methyl, ethyl group or propyl group substituted with a
moiety R12-46-R12-
48 as illustrated in Scheme 3. In embodiments, R12 is any one of 12-49-R12-51.
In
embodiments, Ri2 is a methyl, ethyl group or propyl group substituted with a
moiety R12-49-R12-
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51 as illustrated in Scheme 3. In embodiments, R12 is any one of R12-52-R12-
54. In
embodiments, R12 is a methyl, ethyl group or propyl group substituted with a
moiety R12-52-R12-
54 as illustrated in Scheme 3. In embodiments, R12 is any one of R12-55-R12-
58. In
embodiments, R12 is a methyl, ethyl group or propyl group substituted with a
moiety R12-55-R12-
58 as illustrated in Scheme 3. In embodiments, R12 is any one of R12-59-R12-62
In
embodiments, R12 is a methyl, ethyl group or propyl group substituted with a
moiety R12-59-R12-
62 as illustrated in Scheme 3. In embodiments, R12 is any one of R12-63-R12-
66. In
embodiments, R12 is a methyl, ethyl group or propyl group substituted with a
moiety R12-63-R12-
66 as illustrated in Scheme 3. In embodiments, R12 is any one of R12-67-R12-
69. In
embodiments, R12 is a methyl, ethyl or propyl group substituted with a moiety
R12-67-R12-69 as
illustrated in Scheme 3. In embodiments, R12 is a moiety R12-70 or R12-71 as
illustrated in
Scheme 3. In embodiments, R12 is a moiety R12-72 or R12-73 as illustrated in
Scheme 3. In
embodiments, R12 is a moiety R12-74 as illustrated in Scheme 3. In
embodiments, R12 is a moiety
R12-75 or R12-76 as illustrated in Scheme 3. In embodiments, R12 is a moiety
R12-75 or R12-76,
where p is 1 or 2 as illustrated in Scheme 3. In embodiments, R12 is a moiety
R12-77 as illustrated
in Scheme 3. In embodiments, R12 is a moiety R12-77., where p is 1 or 2 as
illustrated in Scheme
3. In embodiments, R12 is a moiety R12-78 as illustrated in Scheme 3. In
embodiments, R12 is a
moiety R12-78., where p is 1 or 2 as illustrated in Scheme 3.
In embodiments herein of formula XX-XXIII, RN is an optionally substituted
cyclic amine group
selected from any of RN1-RN25 or RN26-RN39 (Scheme 2) and R12 is a thienyl,
thienylmethyl, fury!,
furylmethyl, indolyl or methylindoyl. In embodiments of formula XX-XXIII, RN
is RN1, RN2, RN3,
RN4, RN11, RN13, RN14 or RN25 and R12 is a thienyl, thienylmethyl, fury!,
furylmethyl, indolyl or
methylindoyl. In embodiments, RN is RN37 and R12 is a thienyl, thienylmethyl,
fury!, furylmethyl,
indolyl or methylindoyl. In embodiments, RN is RN38 or RN39 and R12 is a
thienyl, thienylmethyl,
fury!, furylmethyl, indolyl or methylindoyl. In embodiments, RN is RN26 and
R12 is a thienyl,
thienylmethyl, fury!, furylmethyl, indolyl or methylindoyl. In embodiments, RN
is RN27-RN32 and R12
is a thienyl, thienylmethyl, fury!, furylmethyl, indolyl or methylindoyl. In
embodiments, RN is RN33-
RN35 and R12 is a thienyl, thienylmethyl, fury!, furylmethyl, indolyl or
methylindoyl. In embodiments,
RN is RN36 and R12 is a thienyl, thienylmethyl, fury!, furylmethyl, indolyl or
methylindoyl.
In embodiments herein of formula XX-XXIII, RN is an optionally substituted
cyclic amine group of
formula RN37 (Scheme 2) and R12 is a thienyl, thienylmethyl, fury!,
furylmethyl, indolyl or
methylindoyl. In embodiments of formula XX-XXIII, RN is RN38 and R12 is a
thienyl, thienylmethyl,
fury!, furylmethyl, indolyl or methylindoyl. In embodiments of formula XX-
XXIII, RN is RN39 and R12
is a thienyl, thienylmethyl, fury!, furylmethyl, indolyl or methylindoyl.
In embodiments of formula XX-XXIII, Rio is ¨NHCOR12. In embodiments of formula
XX-XXIII, Rio
is ¨CONHR12. In embodiments herein of formula XX-XXIII, R10 is ¨CO-NH-R12 and
RN is any one
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of RN1-RN25 and R12 is any one of R12-1-R12-22. In embodiments herein of
formula XX-XXIII, Rio
is ¨CO-NH-R12 and RN is any one of RN1-RN25 and R12 is any one of R12-1-R12-
69.
In further embodiments of the forgoing embodiments of formula XXI or XXIII, x
is 1. In further
embodiments of the forgoing embodiments of formula XXI or XXIII, xis 1 and Li
is ¨(CH2)n-. In
further embodiments of the forgoing embodiments of formula XXI or XXIII, x is
1 and Li is ¨(CH2)n-
, where n is 1 0r2. In further embodiments of the forgoing embodiments of
formula )0(1 or XXIII, x
is 1 and Li is ¨(CH2)n-, where n is 1. In further embodiments of the forgoing
embodiments of
formula )0(1 or XXIII, x is 1 and Li is ¨(CH2)-, and R5 is an electronegative
group. In further
embodiments of the forgoing embodiments of formula XXI or )0(111, xis 1 and Li
is ¨(CH2)-, and R5
is a halogen. In further embodiments of the forgoing embodiments of formula
)0(1 or XXIII, x is 1
and Li is ¨(CH2)-, and R5 is a fluorine. In further embodiments of the
forgoing embodiments of
formula XXI or XXIII, xis 1 and Li is ¨(CH2)-, R5 is a fluorine and R4 is a 01-
03 alkyl group. In
further embodiments of the forgoing embodiments of formula XXI or XXIII, xis 1
and Li is ¨(CH2)-,
R5 is a fluorine and R4 is a methyl group.
In embodiments, the compound is of formula XXX:
R2
R11
R3
R12
R6
R7 R1
0
R8
R10¨(0H2)y R9
or salts (or solvates) thereof,
wherein:
Ri is selected from the group consisting of hydrogen, alkyl group. alkenyl
group, cycloalkyl group,
cycloalkenyl group, heterocyclyl group, or aryl group, each of which groups is
optionally substituted
(need to define substitution);
R2 and R3 together form an optionally substituted 5- or 6-member heterocyclic
ring which can
contain one or two double bonds or be aromatic;
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R6-R9 are independently selected from hydrogen, halogen, alkyl group, alkenyl
group, cycloalkyl
group, cycloalkenyl group, or heterocyclyl group, each of which groups is
optionally substituted;
Y is 0 or an integer ranging from 1-3 inclusive;
Rio is selected from alkyl group. alkenyl group, cycloalkyl group,
cycloalkenyl group, heterocyclyl
group, or aryl group, each of which groups is optionally substituted; and
R11 and R12 are independently selected from hydrogen, halogen, alkyl group,
alkenyl group,
cycloalkyl group, cycloalkenyl group, or heterocyclyl group, each of which
groups is optionally
substituted. In embodiments, R10 is any one of RH1-RH12.
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In specific embodiments, compounds useful in the methods herein include those
of formula XXXI:
R2
R5
R6
R7 R1
Rs \
R8
0=C
R9
(L2)y
Rm
or salts or solvates thereof; where variables are as defined in formula I, R6-
R9 are independently
selected from hydrogen and RA groups defined in formula I, Rm represents
optional substitution on
the fused ring and Rm takes the values of RA in formula I and Wi is N or CH.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, x is 1 and M is ¨N-(CH2)n¨, where n is 1, 2 or 3. In embodiments,
x is 1 and M is -
(CH2)n¨, where n is 1, 2 or 3. In embodiments, x is 0. In embodiments, L2 is
¨(CH2)n¨, where n is
1, 2 or 3. In embodiments R1 is hydrogen In embodiments, Ri is hydrogen,
methyl or
trifluoromethyl. In embodiments, R7-R9 are independently selected from
hydrogen, C1-C3 alkyl,
optionally substituted C1-C3 alkyl, or aryl. In embodiments, R7-R9 are
independently selected from
hydrogen, halogen Cl-C3 alkyl, C1-C3 alkoxyl, C1-C3 acyl, or C1-C3 haloalkyl.
In embodiments,
R7-R9 are all hydrogens. In embodiments, R4 and R5 are selected from hydrogen,
halogen, C1-C3
alkyl, C1-C3 alkoxyl, or C1-C3 haloalkyl. In embodiments, R4 and R5 together
form a 5- or 6-
member carbocyclic or heterocyclic ring which is saturated, partially
unsaturated or is
heteroaromatic. In embodiments, Rm is one or more hydrogen, halogen, 01-03
alkyl group or 01-
03 haloalkyl group. In embodiments, Rm is one or more hydrogen, halogen,
particularly Br, methyl
or trifluorornethyl. In embodiments, Rm is hydrogen.
In embodiments, compounds useful in the methods, pharmaceutical compositions
and
pharmaceutical combinations of this invention include compounds of formula
XXXII:
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R2
(M)--R3
RBi
R6
R7 R1
RS\
R8
0=C R9
\(L2)y
RH
or salts or solvates thereof,
where variables are as defined in formula I, RB represents optional
substitution as defined in
formula I and R6-R9 are hydrogen or take values of RA from formula I.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are
nitrogens. In
embodiments, x is 1 and M is ¨N-(CH2)n¨, where n is 1, 2 or 3. In embodiments,
x is 1 and M is -
(CH2)n¨, where n is 1, 2 or 3. In embodiments, x is 0. In embodiments, L2 is
¨(CH2)n¨, where n is
1, 2 or 3. In embodiments R1 is hydrogen In embodiments, R1 is hydrogen,
methyl or
trifluoromethyl. In embodiments, R6-R9 are independently selected from
hydrogen, C1-C3 alkyl,
optionally substituted C1-03 alkyl, or aryl. In embodiments, R6-R9 are
independently selected from
hydrogen, halogen C1-C3 alkyl, C1-C3 alkoxyl, C1-C3 acyl, or C1-C3 haloalkyl.
In embodiments,
R7-R9 are all hydrogens. In embodiments, R4 and R5 are selected from hydrogen,
halogen, C1-C3
alkyl, C1-C3 alkoxyl, or C1-C3 haloalkyl. In embodiments, RB is one or more
hydrogen, halogen,
C1-C3 alkyl group or C1-C3 haloalkyl group. In embodiments, RB is one or more
hydrogen,
halogen, particularly Br, methyl or trifluoromethyl. In embodiments, RB is
hydrogen. In
embodiments, RH is a heterocyclyl or heteroaryl group. In embodiments, RH is
optionally
substituted naphthyl, thiophene, indoyl, or pyridinopyrroyl.
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Compounds of formulas XXXV-XLII are useful in the methods, pharmaceutical
compositions and
pharmaceutical combinations herein:
R2
R5
R6
R7
R8
/N
0=C R9
s 2(L
X5 XXXV,
R2
R7 R1
Rs \
Rs
O¨C
R9
s (L2/y
X5 )(XXV I ,
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R2
N¨R3
R6
Nõ
R7 Ri
Rs \
0=C
R9
S
(L2)y
SI
X5 XXXV I I ,
R2
R5
Rs Ri
0=C
l (L2)y
SY
X5 )(XXV I I I ,
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X
R5
RS
0=C
(L2)y
X5 )(XXIX,
X
R5
7.0S _____________________________________ 11_2)y
X5 XL.
R5
RA(1-1)x
s Z
zL) 1-2)y
X5 XLI, or
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:yRp
RB
r.--X
(1-1)x
RA A
,¨S
71) _____________________________________ (L2)y
X5 XLII,
where variables are as defined in formulas I-XIX above and X5 is a halogen,
including F, Cl and Br
and in a specific embodiment is Br. In specific embodiments of formulas XXXV-
XLII, y is O. In
specific embodiments of formulas XXXV-XLII, y is 1 and L2 is ¨(CH2)n- and n is
1, 2 or 3. In
specific embodiments of formulas XXXV-XLII, the A ring is a phenyl ring where
RA is hydrogen. In
specific embodiments Rp is a group selected from any one of RN-1 to RN-31. In
specific
embodiments, the B ring of formula XLII is that of formula RBI as shown in
Scheme 4. In more
specific embodiments, the B ring of formula XLII is that of RB2-RB5 of Scheme
4.
The invention provides salts, particularly pharmaceutically acceptable salts
of each of the
compounds of any of formulas 1-IX, XI-XIX, )00W00(II, )00(V-XLII and formula
XX below. The
invention provides solvates and salts thereof, particularly pharmaceutically
acceptable solvates
and salts of each of the compounds of any of formulas I-XIX, )00(-)0(XII,
)00(V, )00(V-XLII and
formula XX and XXI below. A preferred solvate is a hydrate. The invention
provides
pharmaceutical compositions comprising any compound of any one of the formulas
herein.
In embodiments, compounds of formula XLV are useful in the methods,
pharmaceutical
compositions and pharmaceutical combinations herein:
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ncH2,a
C b
(C H2)
0
11110
(CH2)d H
RH
XLV
or salts or solvates thereof,
wherein:
X1 and X2 are independently CH or N;
Re is hydrogen, 01-03 alkyl or 01-03 fluoroalkyl;
a, b, c or d are zero or integers, where a is 1 0r2, b is 0 or 1, c is 0 or 1,
and d is 0 or 1; and
RH is selected from any one of the moieties of Scheme 3, R12-1 to R12-84.
In embodiments of formula XLV:
X1 is N and X2 is CH or X1 is CH and X2 is N;
X1 is N and X2 is CH;
X1 is CH and X2 is N;
a is 1 and X1 is N and X2 is CH or X1 is CH and X2 is N;
a is 1 and X1 is N and X2 is CH;
a is 1 and X1 is CH and X2 is N;
a is 2 and X1 is N and X2 is CH or X1 is CH and X2 is N;
a is 2 and X1 is N and X2 is CH; or
a is 2 and X1 is CH and X2 is N.
In embodiments of formula XLV and each of the forgoing embodiments of X1, X2,
and a therein:
b is 0 and c is 0, b is 0 and cis 1, b is 1 and c is 0, orb is 1 and c is 1;
b is 0 and c is 0;
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b is 0 and c is 1;
b is 1 and c is 0; or
b is 1 and c is 1.
In embodiments of formula XLV and each of the forgoing embodiments of X1, X2,
a, band c
therein:
d is 0 or
d is 1.
In embodiments of formula XLV and each of the forgoing embodiments of X1, X2,
a, b, c and d
therein:
RH is one of moieties R12-1 to R12-84 of Scheme 3; or
RH is one of moieties R12-5; R12-44; R12-45; R12-58; R12-62; R12-75, R12-79;
or R12-80; or
RH is.
vvvvvvv=
1>1R I ___________________________________ "R21
I
15 R20 = jj-=
JNOVVVV,
R21 R20
I ¨I ¨R21 R¨
; or 20
where:
20 Rzo and R21 are independently, a hydrogen, a C1-C3 alkyl, a C1-C3
fluoroalkyl or a halogen on the
indicated carbon or represents substitution on the indicated ring with one or
more of the listed
atoms or groups.
In embodiments of the foregoing embodiments of formula XLV, R20 and R21 are
both hydrogens or
represent hydrogens at all available ring positions.
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In embodiments of the foregoing embodiments of formula XLV, R20 and R21 are
independently a
hydrogen, methyl, trifluormethyl or halogen on the indicated carbon or
represents substitution on
the indicated ring with one or more of the listed atoms or groups.
In embodiments of the foregoing embodiments of formula XLV, R20 and R21 are
independently a
methyl, trifluormethyl or halogen on the indicated carbon above or represents
substitution on the
indicated ring with one or more of the listed atoms or groups.
In embodiments of the foregoing embodiments of formula XLV, R21 is hydrogen or
represents
hydrogen at all available positions on the indicated ring and R20 is a methyl,
trifluormethyl or
halogen on the indicated carbon above or represents substitution on the
indicated ring with one or
more of the listed atoms or groups.
In embodiments of the foregoing embodiments of formula XLV, R20 is hydrogen or
represents
hydrogen at all available positions on the indicated ring and R21 is a methyl,
trifluormethyl or
halogen on the indicated carbon above or represents substitution on the
indicated ring with one or
more of the listed atoms or groups.
In embodiments of the foregoing embodiments of formula XLV, the halogen of R20
or R21 is
independently fluorine, chlorine or bromine.
In embodiments of the foregoing embodiments of formula XLV, RH is R12-79; R12-
80; R12-44,
wherein R' represents hydrogens at all ring positions; R12-45, wherein R'
represents hydrogens at
all ring positions; R12-58, wherein R' represents hydrogens at all available
ring positions; R12-62,
wherein R' represents hydrogens at all available ring positions; 2-
haloquinolin-4-y1; 2-
chloroquinolin-4-y1; R12-75, where both Rs are hydrogen and X is a halogen,
R12-5, wherein R is
hydrogen and R' represents hydrogens on all ring positions; R12-5, where R is
hydrogen and R'
represents a halogen at the 6-ring position; 6-chloroquinolin-4-yl, 2-C1-
C3alky1-1H-indo1-3-y1 or 2-
methy1-1H-indo1-3-yl..
In embodiments of the foregoing embodiments of formula XLV, RH is R12-80; R12-
44, wherein R'
represents hydrogens at all ring positions; R12-58, wherein R' represents
hydrogens at all
available ring positions; 2-haloquinolin-4-y1; 2-chloroquinolin-4-y1; R12-5,
wherein R is hydrogen
and R' represents hydrogens on all ring positions; R12-5, where R is hydrogen
and R' represents a
halogen at the 6-ring position; 6-chloroquinolin-4-yl, 2-C1-C3 alky1-1H-indo1-
3-yl0r 2-methyl-IN-
indo1-3-yl.
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In embodiments of the foregoing embodiments of formula XLV, RH is naphth-1-yl.
In embodiments of the foregoing embodiments of formula XLV, RH is 4-
bromothiophen-2-yl.
In embodiments of the foregoing embodiments of formula XLV, RH is thiophen-2-
yl.
In embodiments of the foregoing embodiments of formula XLV, RH is 1 H-indo1-3-
yl.
In embodiments of the forgoing embodiments of formula XLV, RH is 6-chloro-1H-
indo1-3-yl.
In embodiments of the forgoing embodiments of formula XLV, RH is 2-methyl-1H-
indo1-3-yl.
In embodiments of the foregoing embodiments of formula XLV, RH is quinolin-4-
yl.
In embodiments of the foregoing embodiments of formula XLV, RH is 2-
chloroquinolin-4-yl.
In embodiments of formula XLV, the compound is selected from compounds 28, 31,
52, 54, 57, 75,
118, 126, 131, 150, or 169. In more specific embodiments, the compound is
selected from
compounds 52, 118, 126, 131, 150, or 169. In embodiments of formula XLV, the
compound is
selected from compounds 28, 31, 54, 57, or 75. In embodiments of formula XLV,
the compound is
one or more of compounds 28, 31, 52, 54, 57, 75, 118, 126, 131, 150, or 169.
In embodiments of
formula XLV, the compound is one of compounds 28, 31, 52, 54, 57, 75, 118,
126, 131, 150, or
169.
In embodiments, the compounds of formula XLVI are useful in the method,
pharmaceutical
compositions and pharmaceutical combinations as described herein:
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XN
RBXRP
(CH2)
0
(CH2)d H
RH
or salts or solvates thereof,
wherein variables are as defined for formula XLV and RH and Rp are as defined
in formula and
various embodiments thereof listed above. In embodiments, Rp is any of the
moieties RN1-RN39.
In embodiment of formula XLVI, Rp is any of RN1; RN3; RN2 or RN4; RN5 or RN6;
RN7 or RN8; RN9;
RN10; RN11; RN12; RN13; RN14; RN15; RN16; RN17 or RN18; RN19 or RN20; RN21;
RN22; RN23 or
RN24; RN25; RN26-RN29; RN27-RN32; RN30; RN31; RN33-RN36; RN37; RN38; RN39; or
RN1, RN2, RN3, RN4, RN11, RN13, or RN14; or
RN1-RN31 which is unsubstituted or
RN32-RN-39; or
RN37; or
RN38 or RN39.
An aliphatic compound is an organic compound containing carbon and hydrogen
joined together in
straight chains, branched chains, or non-aromatic rings and which may contain
single, double, or
triple bonds. Aliphatic compounds are distinguished from aromatic compounds.
The term aliphatic
group herein refers to a monovalent group containing carbon and hydrogen that
is not aromatic.
Aliphatic groups include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
and cycloalkynyl, as well as
aliphatic groups substituted with other aliphatic groups, e.g., alkenyl groups
substituted with alkyl
groups, alkyl groups substituted with cycloalkyl groups.
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The terms alkyl or alkyl group refer to a monoradical of a straight-chain or
branched saturated
hydrocarbon. Alkyl groups include straight-chain and branched alkyl groups.
Unless otherwise
indicated alkyl groups have 1-8 carbon atoms (C1-08 alkyl groups) and
preferred are those that
contain 1-6 carbon atoms (C1-C6 alkyl groups) and more preferred are those
that contain 1-3
carbon atoms (C1-C3 alkyl groups). Alkyl groups are optionally substituted
with one or more non-
hydrogen substituents as described herein. Exemplary alkyl groups include
methyl, ethyl, n-propyl,
iso-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, various branched-pentyl, n-
hexyl, various branched
hexyl, all of which are optionally substituted, where substitution is defined
elsewhere herein.
Substituted alkyl groups include fully halogenated or semihalogenated alkyl
groups, such as alkyl
groups having one or more hydrogens replaced with one or more fluorine atoms,
chlorine atoms,
bromine atoms and/or iodine atoms. Substituted alkyl groups include fully
fluorinated or
semifluorinated alkyl.
Cycloalkyl groups are alkyl groups having at least one 3- or higher member
carbon ring. Cycloalkyl
groups include those having 3-12-member carbon rings. Cycloalkyl groups
include those having 3-
20 carbon atoms and those having 3-12 carbon atoms. More specifically,
cycloalkyl groups can
have at least one 3-10-member carbon ring. Cycloalkyl groups can have a single
carbon ring
having 3-10 carbons in the ring. Cycloalkyl groups are optionally substituted.
Cycloalkyl groups
can be bicyclic having 6-12 carbons. Exemplary cycloalkyl groups include among
others,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
cyclononyl and cyclodecyl
groups. Bicyclic alkyl groups include fused bicycici grouos and bridged
bicyclic groups. Exemplary
bicycloalkyl groups include, among others, bicyclo[2.2.2]octyl, bicyclo[4.4.0]
decyl (decalinyl), and
bicyclo[2.2.2]heptyl (norbornyl).
Cycloalkylalkyl groups are alkyl groups as described herein which are
substituted with a cycloalkyl
group as dcribed herein. More specifically, the alkyl group is a methyl or an
ethyl group and the
cycloalkyl group is a cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl
group. Cycloalkyl groups
are optionally substituted. In specific embodiments, optional substitution
iincludes substitution with
one or more halogens, alkyl groups having 1-3 carbon atoms, alkoxy groups
having 1-3 carbo
atoms, hydroxyl and nitro groups
The term alkylene refers to a divalent radical of a straight-chain or branched
saturated
hydrocarbon. Alkylene groups can have 1-12 carbon atoms unless otherwise
indicated. Alkylene
groups include those having 2-12, 2-8, 2-6 or 2-4 carbon atoms. Linker groups
(L1) herein include
alkylene groups, particularly straight chain, unsubstituted alkylene groups, -
(CH2)n-, where n is 1-
12, n is 1-10, n is 1-9, n is 1-8, n is 1-7, n is 1-6, n is 1-5, n is 1-4, n
is 1-3, n is 2-10, n 1s2-9, n is
2-8, n is 2-7, n is 2-6, n is 2-5 or n is 2-4.
An alkoxy group is an alkyl group, as broadly discussed above, linked to
oxygen (Ralky1-0-). An
alkoxy grou is monovalent.
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An alkenylene group is a divalent radical of a straight-chain or branched
alkylene group which has
one or more carbon-carbon double bonds. In specific embodiments, the same
carbon atom is not
part of two double bonds. In an alkenylene group one or more CH2-CH2 moieties
of the alkylene
group are replaced with a carbon-carbon double bond. In specific embodiments,
an alkenylene
group contains 2-12 carbon atoms or more preferably 3-12 carbon atoms. In
specific
embodiments, an alkenylene group contains one or two double bonds. In specific
embodiments,
the alkenylene group contains one or two trans-double bonds. In specific
embodiments, the
alkenylene group contains one or two cis-double bonds. Exemplary alkenylene
groups include:
-(CH2)n-CH=CH-(CH2)n-, where n is 1-4 and more preferably is 2; and
-(CH2)n-CH=CH-CH=CH-(CH2)n-, where n is 1-4 and more preferably is 1 or 2.
An alkoxyalkyl group is an alkyl group in which one or more of the non-
adjacent internal ¨CH2-
groups are replaced with ¨0-, such a group may also be termed an ether group.
The alkoxyalkyl
group is monovalent. These groups may be straight-chain or branched, but
straight-chain groups
are preferred. Alkoxyalkyl groups include those having 2-12 carbon atoms and
1, 2, 3 or 4 oxygen
atoms. More specifically, alkoxyalkyl groups include those having 3 or 4
carbons and 1 oxygen, or
those having 4, 5 or 6 carbons and 2 oxygens. Each oxygen in the group is
bonded to a carbon in
the group. The group is bonded into a molecule via a bond to a carbon in the
group.
An alkoxyalkylene group is a divalent alkoxyalkyl group. This group can be
described as an
alkylene group in which one or more of the internal ¨CH2- groups are replaced
with an oxygen.
These groups may be straight-chain or branched, but straight-chain groups are
preferred.
Alkoxyalkylene groups include those having 2-12 carbon atoms and 1, 2, 3 or 4
oxygen atoms.
More specifically, alkoxyalkylene groups include those having 3 or 4 carbons
and 1 oxygen, or
those having 4, 5 or 6 carbons and 2 oxygens. Each oxygen in the group is
bonded to a carbon in
the group. The group is bonded into a molecule via bonds to a carbon in the
group. Linker groups
(L1) herein include alkoxyalkylene groups, particularly straight chain,
unsubstituted alkoxyalkylene
groups. Specific alkoxyalkylene groups include, among others, -CH2-0-CH2-, -
CH2.CH2-0-CH2-
CH2-, -CH2-CH2-CH2-0-CH2-CH2-CH2-,-CH2-CH2-0-CH2-, -CH2-0-CH2-CH2-, -CH2-CH2-0-
CH2-
CH2-0-CH2-, -CH2-CH2-0-CH2-CH2-0-CH2-CH2-, -CH2-CH2-CH2-0-CH2-CH2-CH2-0-CH2-,
and -
CH2-CH2-CH2-0-CH2-CH2-CH2-0-CH2-CH2-.
The term acyl group refers to the group ¨CO-R where R is hydrogen, an alkyl or
aryl group as
described herein.
Aryl groups include monovalent groups having one or more 5- or 6-member
aromatic rings. Aryl
groups can contain one, two or three, 6-member aromatic rings. Aryl groups can
contain two or
more fused aromatic rings. Aryl groups can contain two or three fused aromatic
rings. Aryl groups
are optionally substituted with one or more non-hydrogen substituents.
Substituted aryl groups
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include among others those which are substituted with alkyl or alkenyl groups,
which groups in turn
can be optionally substituted. Specific aryl groups include phenyl groups,
biphenyl groups, and
naphthyl groups, all of which are optionally substituted as described herein.
Substituted aryl
groups include fully halogenated or semihalogenated aryl groups, such as aryl
groups having one
or more hydrogens replaced with one or more fluorine atoms, chlorine atoms,
bromine atoms
and/or iodine atoms. Substituted aryl groups include fully fluorinated or
semifluorinated aryl
groups, such as aryl groups having one or more hydrogens replaced with one or
more fluorine
atoms.
Alkyl groups include arylalkyl groups in which an alkyl group is substituted
with an aryl group.
Arylalkyl groups include benzyl and phenethyl groups among others. Arylalkyl
groups are
optionally substituted as described herein. Substituted arylalkyl groups
include those in which the
aryl group is substituted with 1-5 non-hydrogen substituents and particularly
those substituted with
1, 2 or 3 non-hydrogen substituents Useful substituents include among others,
methyl, methoxy,
hydroxy, halogen, and nitro. Particularly useful substituents are one or more
halogens. Specific
substituents include F. Cl, and nitro.
An acyl group is an R-00- groups where R is alkyl, cycloalkyl or aryl as
defined herein each of
which is optionally substituted.
An acyl oxy group is an R-000- group where R is alkyl, cycloalkyl or aryl as
defined herein each of
which is optionally substituted.
An alkoxycarbonyl group is an RO-00- group where R is an alkyl or cycloalkyl
as defined herein
each of which is optionally substituted.
A carboxyl group is a ¨COOH group which may be in the ionized form ¨COO-.
A heterocyclic group is a monovalent group having one or more saturated or
unsaturated carbon
rings and which contains one or more heteroatoms (e.g., N, 0 or S) per ring.
In specific
embodiments, a heterocyclic group contains one to six heteroatoms (e.g., N, 0
or S). In specific
embodments, a heterocyclic groups contains one to three heteroatoms. These
groups optionally
contain one, two or three double bonds. To satisfy valence requirements, a
ring atom may be
bonded to one or more hydrogens or be substituted as described herein. One or
more carbons in
the heterocyclic ring can be ¨CO- groups. The heteroatoms in the ring may be
substituted with one
or more substituents dependent upon valency or sbstituted with one or more
oxygen atoms.
Heterocyclic ring members can include, for example, ¨N=, -NH-, -NR- , -SO-, or
-SO2-.
Heterocyclic groups include those having 3-12 carbon atoms, and 1-6,
heteroatoms, wherein 1 or 2
carbon atoms are replaced with a ¨CO- group. Heterocyclic groups include those
having 3-12 or 3-
10 ring atoms of which up to three can be heteroatoms other than carbon.
Heterocyclic groups can
contain one or more rings each of which is saturated or unsaturated.
Heterocyclic groups include
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bicyclic and tricyclic groups. Preferred heterocyclic groups have 5- or 6-
member rings.
Heterocyclic groups are optionally substituted as described herein.
Specifically, heterocyclic
groups can be substituted with one or more alkyl groups. Heterocyclic groups
include those having
5- and 6- member rings with one or two nitrogens and one or two double bonds.
Heterocyclic
groups include those having 5- and 6-member rings with an oxygen or a sulfur
and one or two
double bonds. Heterocyclic group include those having 5- or 6-member rings and
two different
heteroatoms, e.g., N and 0, 0 and S or N and S. Specific heterocyclic groups
include among
others among others, pyrrolidinyl, piperidyl, piperazinyl, pyrrolyl,
pyrrolinyl, fury!, thienyl,
morpholinyl, oxazolyl, oxazolinyl, oxazolidinyl, indolyl, triazoly, triazinyl
groups, sultam groups (e.g.,
1,1-dioxidoisothiazolidin-2-yl, 1,1-dioxidothiazinan-2-y1)
Heterocycylalky groups are alkyl groups substituted with one or more
heterocycyl groups wherein
the alkyl groups optionally carry additional substituents and the heterocycyl
groups are optionally
substituted. Specific groups are heterocycyl-substituted methyl or ethyl
groups.
Heteroaryl groups are monovalent groups having one or more aromatic rings in
which at least one
ring contains a heteroatom (a non-carbon ring atom). Heteroaryl groups include
those having one
or two heteroaromatic rings carrying 1, 2 or 3 heteroatoms and optionally have
one 6-member
aromatic ring. Heteroaryl groups can contain 5-20, 5-12 or 5-10 ring atoms.
Heteroaryl groups
include those having one aromatic ring contains a heteroatom and one aromatic
ring containing
carbon ring atoms. Heteroaryl groups include those having one or more 5- or 6-
member aromatic
heteroaromatic rings and one or more 6-member carbon aromatic rings.
Heteroaromatic rings can
include one or more N, 0, or S atoms in the ring. Heteroaromatic rings can
include those with one,
two or three N, those with one or two 0, and those with one or two S, or
combinations of one or
two or three N, 0 or S. Specific heteroaryl groups include fury!, pyridinyl,
pyrazinyl, pyrimidinyl,
quinolinyl, purinyl, indolyl groups. In a specific embodiment, the heteroaryl
group is an indolyl
group and more specifically is an indo1-3-y1 group:
Heteroatoms include 0, N, S, P or B. More specifically heteroatoms are N, 0 or
S. In specific
embodiments, one or more heteroatoms are substituted for carbons in aromatic
or carbocyclic
rings. To satisfy valence any heteroatoms in such aromatic or carbocyclic
rings may be bonded to
H or a substituent group, e.g., an alkyl group or other substituent.
Heteroarylalkyl groups are alkyl groups substituted with one or more
heteroaryl groups wherein the
alkyl groups optionally carry additional substituents and the aryl groups are
optionally substituted.
Specific alkyl groups are methyl and ethyl groups.
The term amino group refers to the species ¨N(H)2. The term alkylamino refers
to the species -
NHR" where R" is an alkyl group, particularly an alkyl group having 1-3 carbon
atoms. The term
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dialkylamino refers to the species ¨N(R")2 where each R" is independently an
alkyl group,
particularly an alkyl group having 1-3 carbon atoms.
Groups herein are optionally substituted. Most generally any alky, cycloalkyl,
aryl, heteroaryl and
heterocyclic groups can be substituted with one or more halogen, hydroxyl
group, nitro group,
cyano group, isocyano group, oxo group, thioxo group, azide group, cyanate
group, isocyanate
group, acyl group, haloakyl group, alkyl group, alkenyl group or alkynyl group
(particularly those
having 1-4 carbons), a phenyl or benzyl group (including those that are
halogen or alkyl
substituted), alkoxy, alkylthio, or nnercapto (HS-). In specific embodiments,
optional substitution is
substitution with 1-12 non-hydrogen substituents. In specific embodiments,
optional substitution is
substitution with 1-6 non-hydrogen substituents. In specific embodiments,
optional substitution is
substitution with 1-3 non-hydrogen substituents. In specific embodiments,
optional substituents
contain 6 or fewer carbon atoms. In specific embodiments, optional
substitution is substitution by
one or more halogen, hydroxy group, cyano group, oxo group, thioxo group,
unsubstituted C1-C6
alkyl group or unsubstituted aryl group. The term oxo group and thioxo group
refer to substitution
of a carbon atom with a =0 or a =S to form respectively ¨CO-- (carbonyl) or
¨CS- (thiocarbonyl)
groups.
Specific substituted alkyl groups include haloalkyl groups, particularly
trihalomethyl groups and
specifically trifluoromethyl groups. Specific substituted aryl groups include
mono-, di-, tri, tetra-
and pentahalo-substituted phenyl groups; mono-, di, tri-, tetra-, penta-, hexa-
, and hepta-halo-
substituted naphthalene groups; 3- or 4-halo-substituted phenyl groups, 3- or
4-alkyl-substituted
phenyl groups, 3- or 4-alkoxy-substituted phenyl groups, 3- or 4-RCO-
substituted phenyl, 5- or 6-
halo-substituted naphthalene groups. More specifically, substituted aryl
groups include
acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups,
particularly 3-
fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups, particularly 3-
chlorophenyl and 4-
chlorophenyl groups; methylphenyl groups, particularly 4-methylphenyl groups,
and methoxyphenyl
groups, particularly 4-methoxyphenyl groups.
The term aromatic as applied to cyclic groups refers to ring structures which
contain double bonds
that are conjugated around the entire ring structure, possibly through one or
more heteroatoms
such as an oxygen atom, sulfur atom or a nitrogen atom. Aryl groups, and
heteroaryl groups are
examples of aromatic groups. The conjugated system of an aromatic group
contains a
characteristic number of electrons, for example, 6 or 10 electrons that occupy
the electronic
orbitals making up the conjugated system, which are typically un-hybridized p-
orbitals.
The term carbocyclic refers to a monovalent group having a carbon ring or ring
system which
comprises 3 to 12 carbon atoms and may be monocyclic, bicyclic or tricyclic.
The ring does not
contain any heteroatoms. The ring may be unsaturated, partially unsaturated or
saturated.
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Compounds and substituent groups of formulas herein are optionally
substituted. A substituent
refers to a single atom (for example, a halogen atom) or a group of two or
more atoms that are
covalently bonded to each other, which are covalently bonded to an atom or
atoms in a molecule to
satisfy the valency requirements of the atom or atoms of the molecule,
typically in place of a
hydrogen atom. Examples of substituents include among others alkyl groups,
hydroxyl groups,
alkoxy groups, acyloxy groups, mercapto groups, and aryl groups. Substituent
groups may
themselves be substituted.
Substituted or substitution refer to replacement of a hydrogen atom of a
molecule or of an chemical
group or moiety with one or more additional substituents such as, but not
limited to, halogen, alkyl,
alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy,
aryloxy, aryl, arylalkyl,
heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino,
pyrrolidin-1-yl, piperazin-1-yl,
nitro, sulfato, or other R-groups.
Carbocyclic or heterocyclic rings are optionally substituted as described
generally for other groups,
such as alkyl and aryl groups herein. Substitution if present is typically on
ring C, ring N or both.
In addition, carbocyclic and heterocyclic ring can optionally contain a -CO-, -
00-0-, -CS- or ¨CS-
0- moiety in the ring.
As to any of the chemical groups herein that are substituted, i.e., contain
one or more non-
hydrogen substituents, it is understood, that such groups do not contain any
substitution or
substitution patterns which are sterically impractical and/or synthetically
non-feasible. In addition,
the compounds of this invention include all stereochemical isomers arising
from the substitution of
these compounds.
Protected derivatives of the disclosed compounds also are contemplated. A
variety of suitable
protecting groups for use with the disclosed compounds are disclosed in Greene
and Wuts,
Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York,
1999. In general,
protecting groups are removed under conditions which will not affect the
remaining portion of the
molecule. These methods are well known in the art and include acid hydrolysis,
hydrogenolysis,
and the like. One preferred method involves the removal of an ester, such as
cleavage of a
phosphonate ester using Lewis acidic conditions, such as in TMS-Br mediated
ester cleavage to
yield the free phosphonate. A second preferred method involves removal of a
protecting group,
such as removal of a benzyl group by hydrogenolysis utilizing palladium on
carbon in a suitable
solvent system such as an alcohol, acetic acid, and the like or mixtures
thereof. A t-butoxy-based
group, including t-butoxy carbonyl protecting groups can be removed utilizing
an inorganic or
organic acid, such as HCI or trifluoroacetic acid, in a suitable solvent
system, such as water,
dioxane and/or methylene chloride. Another exemplary protecting group,
suitable for protecting
amino and hydroxy functions amino is trityl. Other conventional protecting
groups are known, and
suitable protecting groups can be selected by those of skill in the art in
consultation with Greene
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and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons,
New York, 1999.
When an amine is deprotected, the resulting salt can readily be neutralized to
yield the free amine.
Similarly, when an acid moiety, such as a phosphonic acid moiety is unveiled,
the compound may
be isolated as the acid compound or as a salt thereof. Protected derivatives
of compounds herein
can, for example, be employed in the synthesis of structurally related
compounds herein.
The present invention provides novel therapeutic strategies for targeting TCF-
driven EMT, a
process that promotes tumor cell heterogeneity, MDR, and metastasis. The
inventors' structure-
based drug design has produced novel potent CHD1L inhibitors which in an
embodiment target
TCF-driven EMT. Reversion of EMT by CHD1L inhibitors may be an effective
treatment when
used in combination with cytotoxic chemotherapy and targeted antitumor drugs
as well as radiation
therapy. These EMT-targeting agents may also sensitize both primary tumors and
metastatic
lesions to clinically relevant therapies, and potentially inhibit tumor cell
metastasis.
Thus, one aspect of this invention are CHD1L inhibitors which can be used to
treat or prevent
metastasis of a wide variety of advanced solid tumors and blood cancers.
Pharmaceutically
acceptable salts, prodrugs, stereoisomers, and metabolites of all the CHD1L
inhibitor compounds
of this invention also are contemplated.
The invention expressly includes pharmaceutically usable solvates of compounds
according to
formulas herein. Specifically, useful solvates are hydrates. The compounds of
formula I or salts
thereof can be solvated (e.g., hydrated). The salvation can occur in the
course of the
manufacturing process or can take place (e.g., as a consequence of hygroscopic
properties of an
initially anhydrous compound of formulas herein (hydration)).
Compounds of the invention can have prodrug forms. Prodrugs of the compounds
of the invention
are useful in the methods of this invention. Any compound that will be
converted in vivo to provide
a biologically, pharmaceutically or therapeutically active form of a compound
of the invention is a
prodrug. Various examples and forms of prodrugs are well known in the art. A
prodrug is an
active or inactive compound that is modified chemically through in vivo
physiological action, such
as hydrolysis, metabolism and the like, into an active compound following
administration of the
prodrug to a subject. The term prodrug as used throughout this text means the
pharmacologically
acceptable derivatives such as esters, amides and phosphates, such that the
resulting in vivo
biotransformation product of the derivative is the active drug as defined in
the compounds
described herein. Prodrugs preferably have excellent aqueous solubility,
increased bioavailability,
and are readily metabolized into the active TOP2A inhibitors in vivo. Prodrugs
of compounds
described herein may be prepared by modifying functional groups present in the
compound in such
a way that the modifications are cleaved, either by routine manipulation or in
vivo, to the parent
compound. The suitability and techniques involved in making and using prodrugs
are well known
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by those skilled in the art. Examples of prodrugs are found, inter alia, in
Design of Prodrugs,
edited by H. Bundgaard, (Elsevier, 1985), Methods in Enzymology, Vol. 42, at
pp. 309-396, edited
by K. Widder, et. al. (Academic Press, 1985); A Textbook of Drug Design and
Development, edited
by Krosgaard-Larsen and H. Bundgaard, Chapter 5, "Design and Application of
Prodrugs," by H.
Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug Delivery
Reviews, Vol. 8, p. 1-
38 (1992); H. Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol. 77,
p. 285 (1988); and
Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University
Press, New York,
pages 388-392).
Administration of and administering a compound or composition should be
understood to mean
providing a compound or salt thereof, a prodrug of a compound, or a
pharmaceutical composition
comprising a compound. The compound or composition can be administered by
another person to
the patient (e.g., intravenously) or it can be self-administered by the
subject (e.g., tablets or
capsules). The term "patient" refers to mammals (for example, humans and
veterinary animals
such as dogs, cats, pigs, horses, sheep, and cattle). Administration of CHD1L
inhibitors herein in
combination with other agents, such as alternative anti-cancer, antineoplastic
or cancer cytotoxic
agents is contemplated. Such combined administration includes administration
of two or more
active ingredients at the same time or at times separated by minutes, hours or
days as is found to
be effective and consistent with the administration of any known alternative
treatments with which
the CHD1L inhibitor is to be combined. Combined administration further
includes administration by
the same method and/or location of the patient's body or by different methods
at different locations,
again as is consistent with and consistent with the administration of known
alternative treatments
with which the CHD1L inhibitor is to be combined.
In embodiments, the CHD1L inhibitors are administered together with an
alternative cancer
cytotoxic or cancer cytotoxic or antineoplastic agent or antineoplastic
procedure (e.g., radiation
treatment) in one or more acceptable pharmaceutical dosage forms or are
administered separately
within a selected time period to provide synergistic effect.
In embodiments, the CHD1L inhibitor(s) is (are) administered by the same route
as the alternative
cancer cytotoxic or cancer cytotoxic or antineoplastic agent. In embodiments,
the CHD1L
inhibitor(s) is administered by a route different from the alternative cancer
cytotoxic or
antineoplastic agent. In embodiments, the CHD1L inhibitor(s) are administered
orally or by
injection. In embodiments, the alternative cancer cytotoxic or antineoplastic
agent are
administered orally or by injection. In embodiments, the CHD1L inhibitor(s)
are administered
locally to tumors or systemically or a combination of both forms of
administration. In embodiments,
the alternative neoplastic agent is administered locally to tumors or
systemically or a combination
of both forms of administration.
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In embodiments, components of the pharmaceutical combination (one or more
CHD1L with one
alternative neoplastic agent) are administered to a subject in need thereof in
a joint therapeutic
amount to provide synergistic therapeutic effect. In embodiments, components
of the
pharmaceutical combination are administered by any appropriate mode of
administration to a
subject in need thereof in a joint therapeutic amount to provide synergistic
therapeutic effect. In
embodiments, components of the pharmaceutical combination are administered by
local or
systemic administration or by a combination of local and systemic
administration to a subject in
need thereof in a joint therapeutic amount to provide synergistic therapeutic
effect.
If a patient is to receive or is receiving multiple pharmaceutically active
compounds, the
compounds can be administered simultaneously or sequentially. For example, in
the case of
tablets, the active compounds may be found in one tablet or in separate
tablets, which are
administered at once or sequentially in any order. In addition, it should be
recognized that the
compositions may be in different dosage forms. For example, one or more
compounds may be
delivered via a tablet, while another is administered via injection or orally
as a syrup. All
combinations, delivery methods and administration sequences are contemplated.
In embodiments, the combination therapy herein comprises administration of one
or more CHD1L
inhibitor and administration of one or more alternative cancer cytotoxic or
antineoplastic agent to a
patient in need of treatment. Administration includes any form or forms of
administration which
achieves synergistic therapeutic action of the CHD1L inhibitor(s) and the
alternative cancer
cytotoxic or antineoplastic agent. Administration includes simultaneous,
concurrent, sequential,
successive, alternate or separate administration of inhibitor(s) CHD1L with
the alternative cancer
cytotoxic or antineoplastic agent. In embodiments, oral administration of
CHD1L inhibitor(s) may
be combined with administration of the alternative cancer cytotoxic or
antineoplastic agent orally or
by injection. The order (sequence) and relative timing of administration of
CHD1L inhibitor(s) and
administration of the alternative cancer cytotoxic or antineoplastic agent is
adjusted to achieve
synergistic therapeutic action. In embodiments, administration of CHD1L
inhibitor(s) is at the same
time (i.e., within up to 2 hours of each other) as administration of
alternative cancer cytotoxic or
antineoplastic agent. In embodiments, administration of CHD1L inhibitor(s) is
separate from
administration of the alternative cancer cytotoxic or antineoplastic agent
within a selected time
period of more than 2 hours of each other. In embodiments, administration of
CHD1L inhibitor(s) is
separate from administration of the alternative cancer cytotoxic or
antineoplastic agent, but within a
selected time period of 24 hours to 1 week.
In embodiments, the invention provides a pharmaceutical combination of one or
more CHD1L
inhibitor and one or more alternative cancer cytotoxic or antineoplastic
agent. In embodiments, the
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components of the pharmaceutical combination can be together or separate. In
embodiments, the
pharmaceutical combination is a pharmaceutical compositions containing one or
more CHDL1
inhibitor and one or more topoisomerase inhibitor, PARP inhibitor, or
thymidylate synthase inhibitor.
In embodiments, the pharmaceutical combination is two or more separate
pharmaceutical
compositions each containing different components of the pharmaceutical
combination. In
embodiments, the pharmaceutical combination is two separate pharmaceutical
compositions, one
containing one or more CHD1L inhibitors and one containing one or more
topoisomerase inhibitor,
one or more PARP inhibitor and/or one or more thymidylate synthase inhibitor.
In embodiments, the
pharmaceutical combination is a single pharmaceutical composition, containing
one or more CHD1L
inhibitors and one containing one or more inhibitor of PARP. In embodiments,
the pharmaceutical
combination is a single pharmaceutical composition, containing one or more
CHD1L inhibitors and
one containing one or more inhibitor of topoisomerase. In embodiments, the
pharmaceutical
combination is a single pharmaceutical composition, containing one or more
CHD1L inhibitors and
one containing one or more inhibitor of thymidylate synthase.
In embodiments, the components of the pharmaceutical combination are
administered together in a
single dosage form appropriate for the selected mode of administration, e.g_,
oral or by injection. In
embodiments, where the pharmaceutical combination is a single dosage form, the
relative amount
of the one or more CHD1L inhibitor and one or more alternative cancer
cytotoxic or antineoplastic
agent in the dosage form is fixed. In embodiments, the pharmaceutical
combination is administered
as two separate pharmaceutical compositions or dosage forms, one containing
one or more CHD1L
inhibitors and one containing one or more alternative cancer cytotoxic or
antineoplastic agent Such
separate administration may be in the same or different dosage form for
appropriate for the selected
mode of administration.
In embodiments, the components of the pharmaceutical combination are
administered in one or
more dosage form and may be administered at the same time or at different
times. In embodiments,
the components of the pharmaceutical combination can be administered
simultaneously,
concurrently or sequentially with or without specific time limits where such
administration provides
therapeutically effective combined amounts of the one or more CHD1L inhibitor
and the one or more
alternative cancer cytotoxic or antineoplastic agent. In embodiments, the
combined therapeutically
effective amount of the one or more CHD1L inhibitor and the one or more
alternative cancer cytotoxic
or antineoplastic agent exhibits greater than an additive therapeutic effect.
In embodiments, the
combined therapeutically effective amount of the one or more CHD1L inhibitor
and the one or more
alternative cancer cytotoxic or antineoplastic agent exhibits a synergistic
therapeutic effect.
In embodiments, the one or more CHD1L inhibitor and the one or more
alternative cancer cytotoxic
or antineoplastic agent are formulated separately and sold separately, but
administered to a subject
in need thereof as a pharmaceutical combination. In embodiments, the one or
more CHD1L inhibitor
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and the one or more alternative cancer cytotoxic or antineoplastic agent are
administered for
treatment of the same disorder or disease state. In specific embodiments, the
disorder or disease
state is a proliferative disorder and more specifically is cancer. In
embodiments, the components of
the pharmaceutical combination may be sold together or separately in the same
or different dosage
forms, in combination with instructions for simultaneous, concurrent or
sequential administration of
the components of the pharmaceutical combination.
Any forms of administration that achieve the desired combined therapeutic
effect can be employed.
For example, the combined administration can be local to the site of one or
more tumors or can be
systemically administered to the subject.
In embodiments, one or more components of the
pharmaceutical combination can be administered locally to one or more tumor
site and one or more
other components of the pharmaceutical combination can be administered
systemically to the
subject. Local or systemic administration can be by any appropriate mode of
administration. Local
administration can, for example, be by injection, infusion or by topical
application. Systemic
administration can, for example, be oral, topical or by injection.
One or more CHD1L inhibitors as described herein can be administered in
combination with
chemotherapy, radiotherapy, immunotherapy, surgery or any combination of such
therapies. The
combination therapy(ies) descibed herein can be administered in combination
with chemotherapy,
radiotherapy, immunotherapy, surgery or any combination of such therapies.
Pharmaceutical compositions herein comprise a named active ingredient or
combination of named
active ingredients in an amount effective for achieving the desired biological
activity for a given
form of administration to a given patient and optionally contain a
pharmaceutically acceptable
excipient or carrier. Pharmaceutical compositions can include an amount (for
example, a unit
dosage) of one or more of the disclosed compounds together with one or more
non-toxic
pharmaceutically acceptable additives, including carriers, diluents, and/or
adjuvants, and optionally
other biologically active ingredients. Such pharmaceutical compositions can be
prepared by
standard pharmaceutical formulation techniques such as those disclosed in
Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (19th Edition).
In embodiments, pharmaceutical compositions herein comprise one or more
compounds of any of
formulas I-XIX, )00(-XXXII, )0(XV, )00(V-XLII, XLV, XLVI and formula )(X and
XXI or
pharmaceutically acceptable salts, or solvates thereof and a pharmaceutically
acceptable
excipient. The term "excipient" means any pharmaceutically acceptable
additive, carrier, diluent,
adjuvant, or other ingredient, other than the active pharmaceutical ingredient
(API) or another
clearly designated active pharmaceutical ingredient, which is typically
included for formulation
and/or administration to a patient.
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Pharmaceutically acceptable carriers are those carriers that are compatible
with the other
ingredients in the formulation and are biologically acceptable. Carriers can
be solid or liquid.
In some embodiments, carriers are solids, for example, in which oral dosage
forms are pills. In
some embodiments, carriers are liquids, for example, in which oral dosage
forms are solutions or
suspensions. Carriers can include one or more substances that can also act as
solubilizers,
suspending agents, fillers, glidants, compression aids, binders, tablet-
disintegrating agents, or
encapsulating materials. Liquid carriers can be used in preparing solutions,
suspensions,
emulsions, syrups and elixirs. The active ingredient can be dissolved or
suspended in a
pharmaceutically acceptable liquid carrier such as water (of appropriate
purity, e.g., pyrogen-free,
sterile, etc.), an organic solvent, a mixture of both, or a pharmaceutically
acceptable oil or fat. The
liquid carrier can contain other suitable pharmaceutical additives such as,
for example, solubilizers,
emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending
agents, thickening
agents, colors, viscosity regulators, stabilizers or osmo-regulators.
Compositions for oral
administration can be in either liquid or solid form.
Suitable examples of liquid carriers for oral and parenteral administration
include water of
appropriate purity, aqueous solutions (particularly containing additives,
e.g., cellulose derivatives,
sodium carboxymethyl cellulose solution), alcohols (including monohydric
alcohols and polyhydric
alcohols e.g., glycols) and their derivatives, and oils. In specific examples,
liquid carriers for oral
administration include solutions of active ingredients (i.e., CHD1L inhibitors
preferably dissolved or
suspended in a liquid carrier. For parenteral administration, the carrier can
also be an oily ester
such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used
in sterile liquid form
compositions for parenteral administration. The liquid carrier for pressurized
compositions can be
halogenated hydrocarbon or other pharmaceutically acceptable propellant.
Liquid pharmaceutical
compositions that are sterile solutions or suspensions can be administered by,
for example,
intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions
can also be administered
intravenously. Compositions for oral administration can be in either liquid or
solid form. The carrier
can also be in the form of creams and ointments, pastes, and gels. The creams
and ointments can
be viscous liquid or semisolid emulsions of either the oil-in-water or water-
in-oil type.
In embodiments, administration of CHD1L inhibitors employs dosage forms
comprising
pharmaceutically acceptable polyethylene glycol (PEG). In such embodiments,
the
pharmaceutically acceptable PEG may be combined with a pharmaceutically
acceptable organic
solvent, particularly a pharmaceutically acceptable polar, aprotic solvent. In
embodiments, the
organic solvent is pharmaceutically acceptable DMSO. In embodiments, oral
administration
employs oral dosage forms comprising low molecular weight polyethylene glycol
having molecular
weight of 600 g/mole or less. In more specific embodiments, oral
administration employs PEG
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400. In more specific embodiments, oral administration employs PEG 200. In
embodiments, PEG
is described by its average Mn (number average) molecular weight. PEG having
Mn of 600 or less
are suitable for use in formulations of CHD1L inhibitors herein. More
specifically, PEG having Mn
of 400 or 200 are suitable for formulations herein. In embodiments,
administration employs oral
formulations comprising PEG, preferably low molecular weight PEG and more
specifically PEG
having Mn of 600 or less. In embodiments, oral formulations comprise a
therapeutically effective
amount of a CHD1L inhibitor in combination with PEG, particularly where the
CHD1L inhibitor
suspended or dissolved in the PEG. In embodiments, a combination of PEG and an
appropriate
pharmaceutically acceptable polar aprotic solvent. In embodiments, the polar
aprotic solvent is
pharmaceutically acceptable DMSO. In specific embodiment, the oral formulation
comprises PEG
and DMSO. In embodiments, the solvent combination of PEG and DMSO is miscible.
In
embodiments, the combination of PEG and DMSO dissolves the therapeutically
effective amount
of the CHD1L inhibitor. In embodiments, the volume ratio of PEG to DMSO in
oral formulations
ranges from 100 to 4. More specifically, the volume ratio of PEG to DMSO
ranges from 20 to 4, or
9 to 4 or 12 to 6 or 10 to 8. In specific embodiments, a solvent mixture of
90% by volume PEG,
particularly low molecular weight PEG, and 10% by volume DMSO is employed in
oral
formulations.
A "therapeutically effective amount" of the disclosed compounds is a dosage of
the compound that
is sufficient to achieve a desired therapeutic effect, such as an anti-tumor
or anti-metastatic effect.
It will be understood that the therapeutically effective amount of a given
compound depends upon
the compound, the route of administration and the dosage form as well as the
patient to be treated
(age, weight, etc.). In some examples, a therapeutically effective amount is
an amount sufficient
to achieve tissue concentrations at the site of action that are similar to
those that are shown to
modulate TCF-transcription and/or epithelial-mesenchymal transition (EMT) in
tissue culture, in
vitro, or in vivo. For example, a therapeutically effective amount of a
compound may be such that
the subject receives a dosage of about 0.1 pg/kg body weight/day to about 1000
mg/kg body
weight/day, for example, a dosage of about 1 pg/kg body weight/day to about
1000 pg/kg body
weight/day, such as a dosage of about 5 pg/kg body weight/day to about 500
pg/kg body
weight/day. In cases in which treatment using a CHD1L inhibitor of the
invention is combined with
treatment using another active ingredient or with another form of cancer
treatment or therapy, the
therapeutically effect amount of the CHD1L inhibitor may depend upon the
active ingredient,
treatment or therapy with which it is combined.
The term modulate refers to the ability of a disclosed compound to alter the
amount, degree, or
rate of a biological function, the progression of a disease, or amelioration
of a condition. For
example, modulating can refer to the ability of a compound to elicit an
increase or decrease in
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angiogenesis, to inhibit TCF-transcription and/or EMT, or to inhibit tumor
metastasis or
turnorigenesis.
Treatment refers to a therapeutic intervention that ameliorates a sign or
symptom of a disease or
pathological condition after it has begun to develop. As used herein, the
ternn ameliorating, with
reference to a disease or pathological condition, refers to any observable
beneficial effect of the
treatment. The beneficial effect can be evidenced, for example, by a delayed
onset of clinical
symptoms of the disease in a susceptible subject, a reduction in severity of
some or all clinical
symptoms of the disease, a slower progression of the disease, an improvement
in the overall
health or well-being of the subject, or by other parameters well known in the
art that are specific to
the particular disease. The phrase treating a disease is inclusive of
inhibiting the full development
of a disease or condition, for example, in a subject who is at risk for a
disease, or who has a
disease, such as cancer or a disease associated with a compromised immune
system. Preventing
a disease or condition refers to prophylactically administering a composition
to a subject who does
not exhibit signs of a disease or exhibits only early signs of the disease,
for the purpose of
decreasing the risk of developing a pathology or condition, or diminishing the
severity of a
pathology or condition.
In general the CHD1I inhibitors herein can be used to treat cancer alone or in
combination
therapies as described herien. Cancers which may generally be treated with
compounds of the
present invention include, without limitation, carcinomas such as cancer of
the bladder, breast,
colon, rectum, kidney, liver, lung (small cell lung cancer, and non-small-cell
lung cancer),
esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate,
and skin (including
squamous cell carcinoma); hematopoietic tumors of lymphoid lineage (including
leukemia, acute
lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic
leukemia, B-cell
lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy
cell lymphoma
and Burkett's lymphoma); hennatopoietic tumors of myeloid lineage (including
acute and chronic
myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia);
tumors of
mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma, and other
sarcomas, e.g.,
soft tissue and bone); tumors of the central and peripheral nervous system
(including astrocytoma,
neuroblastoma, glioma and schwannomas); and other tumors (including melanoma,
seminoma,
teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma,
thyroid follicular
cancer and Kaposi's sarcoma). Other cancers that can be treated with the
compound of the
present invention include endometrial cancer, head and neck cancer,
glioblastoma, malignant
ascites, and hennatopoietic cancers.
All references throughout this application, for example patent documents
including issued or
granted patents or equivalents; patent application publications; and non-
patent literature
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documents or other source material; are hereby incorporated by reference
herein in their entireties,
as though individually incorporated by reference. All patents and publications
mentioned in the
specification are indicative of the levels of skill of those skilled in the
art to which the invention
pertains. References cited herein are incorporated by reference herein in
their entirety to indicate
the state of the art, in some cases as of their filing date, and it is
intended that this information can
be employed herein, if needed, to exclude (e.g., to disclaim) specific
embodiments that are in the
prior art. For example, when a compound is claimed, it should be understood
that compounds
known in the prior art, including certain compounds disclosed in the
references disclosed herein
(particularly in referenced patent documents), are not intended to be included
in the claim.
Esquer et al., 2021 and any supplementary information for that journal article
are each
incorporated by reference herein in its entirety for descriptions of
biological and chemical methods
useful in making and assessing the activities and properties of the CHD1L
inhibitors herein.
Abbott et al., 2020 and the supplementary information for that journal article
are each incorporated
by reference herein in its entirety for descriptions of biological and
chemical methods useful in
making and assessing the activities and properties of the CHD1L inhibitors
herein.
Esquer et al., 2020 and any supplementary information for that journal article
are each
incorporated by reference herein in its entirety for descriptions of
biological and chemical methods
useful in making and assessing the activities and properties of the CHD1L
inhibitors herein.
Yang et al., 2020 and any supplementary information for that journal article
are each incorporated
by reference herein in its entirety for descriptions of biological and
chemical methods useful in
making and assessing the activities and properties of the CHD1L inhibitors
herein.
Abraham et al., 2019 and any supplementary information for that journal
article are each
incorporated by reference herein in its entirety for descriptions of
biological and chemical methods
useful in making and assessing the activities and properties of the CHD1L
inhibitors herein.
Zhou et al., 2020 and any supplementary information for that journal article
are each incorporated
by reference herein in its entirety for descriptions of biological and
chemical methods useful in
making and assessing the activities and properties of the CHD1L inhibitors
herein.
PCT/US2021/023981, filed March 24, 2021, U.S. provisional applications
62/994,259, filed March
24, 2020 and 63/139,394, filed January 20, 2021, are each incorporated by
reference herein in its
entirety.
Prigaro et al. 2022 and any supplementary information for that journal article
are each incorporated
by reference herein in its entirety for descriptions of biological and
chemical methods useful in
making compounds herein and assessing the activities and properties of the
CHD1L inhibitors
herein.
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When a group of substituents is disclosed herein, it is understood that all
individual members of the
group and all subgroups, including any isomers and enantionners of the group
members, and
classes of compounds that can be formed using the substituents are disclosed
separately. When a
compound is claimed, it should be understood that compounds known in the art
including the
compounds disclosed in the references disclosed herein are not intended to be
included. When a
Markush group or other grouping is used herein, all individual members of the
group and all
combinations and subcombinations possible of the group are intended to be
individually included in
the invention.
When a compound is described herein such that a particular isomer, enantiomer
or diastereomer
of the compound is not specified, for example, in a formula or in a chemical
name, that description
is intended to include each isomers and enantiomer (e.g., cis/trans isomers,
R/S enantiomers) of
the compound described individual or in any combination. Additionally, unless
otherwise specified,
all isotopic variants of compounds disclosed herein are intended to be
encompassed by the
invention. For example, it will be understood that any one or more hydrogens
in a molecule
disclosed can be replaced with deuterium or tritium. Isotopic variants of a
molecule are generally
useful as standards in assays for the molecule and in chemical and biological
research related to
the molecule or its use. Isotopic variants, including those carrying
radioisotopes, may also be
useful in diagnostic assays and in therapeutics. Methods for making such
isotopic variants are
known in the art.
Molecules disclosed herein may contain one or more ionizable groups [groups
from which a proton
can be removed (e.g., -COOH) or added (e.g., amines) or which can be
quaternized (e.g.,
amines)]. All possible ionic forms of such molecules and salts thereof are
intended to be included
individually in the invention herein. With regard to salts of the compounds
herein, one of ordinary
skill in the art can select from among a wide variety of available counterions
those that are
appropriate for preparation of salts of this invention for a given
application. In specific applications,
the selection of a given anion or cation for preparation of a salt may result
in increased or
decreased solubility of that salt.
CHD1L inhibitors of this invention are commercially available or can be
prepared without undue
experimentation by the methods disclosed herein or by routine adaptation of
such methods using
starting materials and reagents which are commercially available or which can
be made by known
methods. It will be appreciated that it may be necessary, dependent upon the
compound to be
synthesized, to protect potentially reactive groups in starting materials from
undesired conjugation.
Useful protective groups, for various reactive groups are known in the art,
for example as
described in Wutts & Greene, 2007.
Compounds herein can be in the form of salts, for example ammonium salts, with
a selected anion
or quaternized ammonium salts. The salts can be formed as is known in the art
by addition of an
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acid to the free base. Salts can be formed with inorganic acids such as
hydrochloric acid,
hydrobronnic acid, sulfuric acid, nitric acid, phosphoric acid and the like,
or organic acids such as
acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic
acid, malonic acid,
succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic
acid, N-acetylcystein
and the like.
In specific embodiments, compounds of the invention can contain one or more
negatively charged
groups (free acids) which may be in the form of salts. Exemplary salts of free
acids are formed
with inorganic base include, but are not limited to, alkali metal salts (e.g.,
Lit, Nat, Kt), alkaline
earth metal salts (e.g., Ca2+, Mg2+), non-toxic heavy metal salts and ammonium
(NH4) and
substituted ammonium (N(R)4+ salts, where R is hydrogen, alkyl, or substituted
alkyl, i.e.,
including, methyl, ethyl, or hydroxyethyl, specifically, trimethyl ammonium,
triethyl ammonium, and
triethanol ammonium salts), salts of cationic forms of lysine, arginine, N-
ethylpiperidine, piperidine,
and the like. Compounds of the invention can also be present in the form of
zwitterions.
Compound herein can be in the form of pharmaceutically acceptable salts, which
refers to those
salts which retain the biological effectiveness and properties of the free
bases or free acids, and
which are not biologically or otherwise undesirable.
The scope of the invention as described and claimed encompasses the racemic
forms of the
compounds as well as the individual enantiomers and non-racemic mixtures
thereof. The
compounds of the invention may contain one or more asymmetric carbon atoms, so
that the
compounds can exist in different stereoisomeric forms. The compounds can be,
for example,
racemates or optically active forms. The optically active forms can be
obtained by resolution of the
racemates or by asymmetric synthesis. In a preferred embodiment of the
invention, enantiomers of
the invention exhibit specific rotation that is + (positive). Preferably, the
(+) enantiomers are
substantially free of the corresponding (-) enantiomer. Thus, an enantiomer
substantially free of the
corresponding enantiomer refers to a compound which is isolated or separated
via separation
techniques or prepared free of the corresponding enantiomer. "Substantially
free," means that the
compound is made up of a significantly greater proportion of one enantiomer.
In preferred
embodiments the compound is made up of at least about 90% by weight of a
preferred enantiomer.
In other embodiments of the invention, the compound is made up of at least
about 99% by weight
of a preferred enantiomer. Preferred enantiomers may be isolated from racemic
mixtures by any
method known to those skilled in the art, including high performance liquid
chromatography
(HPLC) and the formation and crystallization of chiral salts or prepared by
methods described
herein. [See, for example, Jacques et al., 1981; Wilen et al., 1977; Elie!,
1962; Wilen, 1972.]
Compounds of the invention, and salts thereof, may exist in their tautomeric
form, in which
hydrogen atoms are transposed to other parts of the molecules and the chemical
bonds between
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the atoms of the molecules are consequently rearranged. It should be
understood that all
tautonneric forms, that may exist, are included within the invention.
Every formulation, compound or combination of components described or
exemplified herein can
be used to practice the invention, unless otherwise stated. Specific names of
compounds are
intended to be exemplary, as it is known that one of ordinary skill in the art
can name the same
compounds differently. When a compound is described herein such that a
particular isomer or
enantiomer of the compound is not specified, for example, in a formula or in a
chemical name, that
description is intended to include each isomers and enantionner of the
compound described
individual or in any combination.
It will be appreciated by one of ordinary skill in the art that chemical
compounds can be named
using various conventions and that even within a given convention chemical
names for a given
compound may vary, such that the same compound can be properly named in
different ways.
Where herein, there is an inconsistency between a compound name and a compound
structure, if
specifically provided, the compound structure is given precedence.
One of ordinary skill in the art will appreciate that methods, alternative
therapies, starting materials,
and synthetic methods other than those specifically exemplified can be
employed in the practice of
the invention without resort to undue experimentation. All art-known
functional equivalents, of any
such methods, device elements, starting materials, and synthetic methods are
intended to be
included in this invention. Whenever a range is given in the specification,
for example, a
temperature range, a time range, or a composition range, all intermediate
ranges and subranges,
as well as all individual values included in the ranges given are intended to
be included in the
invention.
The singular terms "a," "an," and "the" include plural referents unless
context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and" unless the
context clearly indicates
otherwise. The term "comprises" means "includes." Also, "comprising A or B"
means including A or
B, or A and B, unless the context clearly indicates otherwise. It is to be
further understood that all
molecular weight or molecular mass values given for compounds are approximate
and are
provided for description. Although methods and materials similar or equivalent
to those described
herein can be used in the practice or testing of this invention, suitable
methods and materials are
described below. In addition, the materials, methods, and examples are
illustrative only and not
intended to be limiting.
As used herein, "comprising" is synonymous with "including," "containing," or
"characterized by,"
and is inclusive or open-ended and does not exclude additional, unrecited
elements or method
steps. As used herein, "consisting of" excludes any element, step, or
ingredient not specified in
the claim element. As used herein, "consisting essentially of" does not
exclude materials or steps
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that do not materially affect the basic and novel characteristics of the
claim. More specifically, the
term 'consisting essentially of' is open to the listed component(s), excluding
(1) active ingredients
that do not function for the intended therapeutic application, and (2) other
components that
negatively affect the activity or combined activity of the listed components,
but not excluding
pharmaceutically acceptable excipients which do not negatively affect the
activity or combined
activity of the listed component(s). Any recitation herein of the term
"comprising", particularly in a
description of components of a composition or in a description of elements of
a device, is
understood to encompass those compositions and methods consisting essentially
of and
consisting of the recited components or elements.
The invention illustratively described herein suitably may be practiced in the
absence of any
element or elements, limitation or limitations which is not specifically
disclosed herein.
Without wishing to be bound by any particular theory, there can be discussion
herein of beliefs or
understandings of underlying principles relating to the invention. It is
recognized that regardless of
the ultimate correctness of any mechanistic explanation or hypothesis, an
embodiment of the
invention can nonetheless be operative and useful.
The terms and expressions that have been employed are used as terms of
description and not of
limitation, and there is no intention in the use of such terms and expressions
of excluding any
equivalents of the features shown and described or portions thereof, but it is
recognized that
various modifications are possible within the scope of the invention claimed.
Thus, it should be
understood that although the present invention has been specifically disclosed
by preferred
embodiments and optional features, modification and variation of the concepts
herein disclosed
may be resorted to by those skilled in the art, and that such modifications
and variations are
considered to be within the scope of this invention.
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THE EXAMPLES
Example 1: Clinicopathological characterization of CHD1L in patients with CRC
CHD1L expression is correlated with poor prognosis in several cancers, but
only limited
information about the pathology of CHD1L in CRC is known. This example
describes the
pathogenic characterization and mechanisms of pathology for CHD1L in CRC
patients. The
clinicopathological characteristics of 585 patients with CRC were analyzed
from the Cartes
d'Identite des Tunneurs (CIT) program with respect to CHD1L expression (GEO:
GSE39582).
[Marisa et al., 2013] These characteristics are summarized in Abbott et al.,
2020, supplementary
information.
Additional data for this example are found in Abbott et al., 2020 and its
supplementary information.
Follow up information was available for all patients in the CIT cohort over a
period of 15 years. For
the entire patient cohort, high CHD1L expression is associated with lower OS
(P= 0.0167) and
median survival (MS) of 8.8 years for high CHD1L patients. Median survival was
not reached in
the low CHD1L cohort as 72% (115/159) of patients were censored and 26%
(42/159) were
deceased. Patient data were evaluated using the TNM staging system. As Stage I
and IV patients
have a high likelihood of survival or death, respectively, survival of Stage
II and III CRC patients
was evaluated. High CHD1L expression was associated with a lower OS (P=
0.0191) and MS of
11 years for Stage II and III CRC, again median survival was not reach in the
low CHD1L cohort
Survival was also analyzed with respect to CHD1L expression for each stage of
CRC.
Stage II patients showed a significant difference in survival (P = 00319) with
a M.S. of 11
years, no significant difference was observed for Stage I, Ill or IV patients.
Analysis of
CHD1L expression indicated a significant difference in expression cancer
stage. Patients with
Stage I and ll colorectal cancer versus patients with Stage III and IV were
evaluated and showed a
significant increase in CHD1L expression in the Stage III and IV versus early
stage cohort (P =
0.0051). Analysis of CHD1L expression with respect to lymph node metastasis
suggests that
CHD1L is overexpressed in patients with increased regional lymph node
metastasis (Ni P=
0.0128, N2 P = 0.05 compared to NO). Although the trend of CHD1L expression
was the same for
the N3 cohort, no significance was determined due to the limited number of
patient samples
available. No significant difference in CHD1L expression with respect to tumor
size, metastasis or
location was found.
Evaluation of CHD1L in CRC molecular subtypes.
The association of CHD1L expression with six molecular subtypes of CRC [Marisa
et al., 2013]: Cl
(immune system down, n = 116), C2 (deficient mismatch repair, n = 104), C3
(KRAS mutant, n =
75), 04 (CSC, n = 59), 05 (activated WNT pathway, n = 152), and 06
(chromosomal instability
normal, n = 60) was investigated. There is a significant difference of CHD1L
expression among the
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six molecular subtypes (P < 0.001). CHD1L expression was high in C5, C4, and
03, and low in 02
and 06. The 02 subtype is associated with a decrease in the WNT signaling
pathway and deficient
for mismatch repair. The C4 and C6 subtypes are associated with poorer relapse-
free survival
compared to other subtypes. The C4 subtype is associated with increased CSC
stemness and the
05 subtype is associated with activated WNT signaling and deregulated EMT
pathways. The lower
CHD1L expression in the 02 (deficient mismatch repair) subtype is consistent
with its known
function in DNA damage response. [Ahel et al., 2009] Additionally, CHD1L
expression was lower
in patients with deficient mismatch repair than in patients without (P <
0.001). CHD1L expression
was also higher in patients with KRAS mutations (P = 0.049). The expression of
CHD1L in the C3,
04, and 05 molecular subtypes prompted a further investigation of the function
of CHD1L
expression in EMT, CSC stem ness, and the WNT/TCF pathway.
CHD1L expression correlates with Wnt/TCF associated genes
Utilizing a smaller cohort of CRC patients (n = 26) from the UCCC GI tumor
tissue bank, a similar
trend was observed as with the larger CIT cohort CHD1L expression
significantly correlated with
late stage and metastatic CRC compared to early stage and primary CRC (Abbott
et al., 2020,
Supplementary information). The expression is quantified as FPKM (fragments
per kilobase exon
per million fragments mapped). Metastatic tumor samples had significantly more
CHD1L than
primary tumors. Additionally, CHD1L levels were higher in Stage IV compared to
Stage II/III patient
cohorts. When analyzing CHD1L expression with genes involved in KEGG WNT
pathway, using
Spearman's correlation a significant positive correlation with 65 of 125 genes
was observed.
Among these were well-established genes involved in TCF-mediated transcription
such as
topoisomerase Ila (TOP2A) (r= 0.65, P= 0.004 [Zhou et al., 2016; Abraham et
al, 2019], and
TCF4 (r = 0.61, P= 0.0012) (Abbott et al., Supplementary Figure 2). Genes had
P< 0.05
correlation value. Transcript expression was Log2 normalized and quantified by
FPKM (fragments
per kilobase exon per million fragments mapped).
A significant positive correlation was observed between known CSC markers 0D44
(r = 0.43, P =
0.038), LGR5 (r = 0.55, P = 0.0075) and CHD1L. When comparing the CIT cohort
to the UCCC
cohort a significant correlation was observed for TOP2A (r = 404 0.1275, P =
0.0020) and TCF4 (r
= 0.1050, P = 0.011). Consistent with this result, it has been shown that
TOP2A is a required
component of the TCF-complex, promoting EMT in CRC. [Zhou et al., 2016;
Abraham et al., 2019]
Hence, CHD1L appears to be involved in TCF-transcription and EMT in CRC
patients.
Example 2: CHD1L mediates TCF-transcription in CRC
Based on the correlation of CHD1L with TCF-complex members, CHD1L may have a
mechanistic
role in TCF-transcription. To assess this role, SW620 and DLD1 cell lines,
which have high and low
endogenous CHD1L expression, respectively, were utilized. Additional data for
this example are
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found in Abbott et al, 2020, and its Supplemetary Information. Small hairpin
RNA (shRNA) was
used to knockdown CHD1L in SW620 cells (SW620CHD1L-KD). CHD1L was
overexpressed in DLD1
cells (DLD1CHD1L-OE). Using the TOPflash luciferase reporter [Morin et al.,
1997; Zhou et al., 2016]
transfected into SW620CHD1L-KD or DLD1CHD1L-0E, it was determined that
overexpression of CHD1L
produced a significant increase in TCF-transcription (P<0.0001) (Abbott et
al., 2020). Conversely,
sw620CHD1L-KD cells displayed a significant decrease in TCF-transcription (P=
0.0006). These
results indicate that CHD1L is a potential factor directly involved in TCF-
transcription. Each of
Morin et al., 1997 and Zhou et al., 2016 is incorporated by reference herein
in its entirety for
descriptions of the TOPflash reporter and assays employing it.
CHD1L directly interacts with the TCF-transcription complex
Activation of TCF-transcription is a dynamic process that involves the
shedding of co-repressor
proteins, binding of co-activator proteins, and remodeling of the chromatin
landscape. [Lorch et al.
2010, Shitashige et al., 2008] Co-immunoprecipitation (Co-IF) studies with
TCF4 were performed,
demonstrating that CHD1L directly binds to the TCF-complex [Abbott et al,
2020].
CHD1L has been well characterized as a binding partner with PARP1 in DNA
damage response.
[Pines, 2012; Ahel et al., 2009] PARP1 is also a component of the TCF-complex
binding to TCF4
and 13-catenin. [Idogawa et al. 2005] The results herein demonstrate that
CHD1L binds to the TCF-
complex, which is likely through interactions between TCF4 and PARP1.
To further characterize CHD1L as a component of the TCF-complex, chromatin
immunoprecipitation (ChIP) of CHD1L to TCF-complex WNT response elements
(WREs) was
performed in SW620 cells. [Abbott et al., 2020] CHD1L was enriched at c-Myc,
vimentin, slug,
LEF1, and N-cadherin WREs, further supporting that CHD1L is functioning
directly with the TCF-
complex. Taken together, the data implicate CHD1L as a critical component of
the TCF-
transcription.
CHD1L mediated TCF-transcription promotes EMT and CSC stemness in CRC
Previously, TCF-transcription was characterized as a master regulator of EMT
in CRC. [Zhou et
al., 2016] In addition, CHD1L localizes at WREs of EMT effector genes. [Abbott
et al, 2020]
Therefore, biomarker expression in SVV620CHD1L-KD and DLD1CHD1L-OE cells was
measured to
determine whether knockdown or overexpression of CHD1L modulates EMT.
Knockdown of
CHD1L induced reversion of EMT, decreasing vimentin and slug while increasing
E-cadherin
expression. [Abbott et al, 2020] Conversely, EMT was induced in DLD1CHD1L-OE
cells, evidenced by
a decrease in E-cadherin and an increase in vimentin and slug expression.
[Abbott et al, 2020]
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These results indicate that CHD1L is an EMT effector gene involved in
promoting the
nnesenchynnal phenotype in CRC. A hallmark of EMT is an increase in CSC
stennness.
Clonogenic colony formation assays [Abbott et al, 2020] were performed to
characterize the impact
of CHD1L expression on sternness. [Franken et al., 2006] CSC sternness
increased in DLD1 CHOI L-
QE (P = 0.0001) and decreased in sw6200HD1L-KD (P = 0.002) cells measured by
colony formation.
Example 3: Identification of Small Molecule Inhibitors of CHD1L
As established in Examples 1 and 2, herein, CHD1L is a driver of TCF-mediated
EMT. Based on
this, an assay to identify small molecule inhibitors of CHD1L is described
herein. The drug
discovery goal was to target CHD1L DNA translocation or interactions with DNA,
which are
dependent on CHD1L's catalytic domain ATPase activity. [Ryan & Owen-Hughes,
2011; Flaus et
al., 2011]
CHD1L belongs to the SNF2 (sucrose non-fermenter 2) ATPase superfamily of
chromatin
remodelers that contains a two-lobe ATPase domain. [Abbott et al, 2020 and its
Supplemental
Information] CHD1L also has a macro domain that is unique relative to other
chromatin
remodelers, which promotes an auto-inhibited state through interactions
between the macro and
the ATPase domains. [Lehmann et al., 2017; Gottschalk et al., 2009] However,
the macro domain
binds to PARP1, the major activator of CHD1L, alleviating auto-inhibition.
[Lehmann et al., 2017;
Gottschalk et al., 2009]
Using the methodology of Lehmann et al., 2017, full-length CHD1L (fl-CHD1L)
and the catalytic
ATPase domain (cat-CHD1L) were purified. [Abbott et al, 2020 and its
Supplementary Information]
Protein constructs were used for recombinant expression and purification of
CHD1L for in vitro
HTS, as illustrated in Abbott et al., 2020. An SDS page gel showed purified
cat-CHD1L (68 kD)
and fl-CHD1L (101 kD). Enzyme kinetics of cat-CHD11 versus fl-CHD1L were
compared. The cat-
CHD1L provides for a more robust ATPase assay compared to fl-CHD1L, which is
consistent with
the report from Lehman et al., 2017. Therefore, to identify direct inhibitors
of CHD1L ATPase, an
exemplary High-through-put screening (HTS) assay in the context of TCF-
transcription is described
which includes: cat-CHD1L, c-Myc DNA, ATP, and phosphate-binding protein that
fluoresces upon
binding inorganic phosphate (Pi).
This assay was validated and pilot screening was preformed against clinically
relevant kinase
inhibitors. [Abbott et al, 2020 and its Supplemental Information]. The pilot
screen found no hits,
demonstrating that CHD1L is not a likely target for kinase inhibitors. Once
validated, a primary
HTS was preformed using 20,000 compounds from the Life Chemicals Diversity
Set, which were
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screened at 20 pM in 1% DMSO with 10 mM EDTA as a positive control [Abbott et
al, 2020 and its
Supplementary Information] The screen provided robust statistics with an
average Z'-factor value
of 0.57 0.06 over 64 plates. The average compound activity was 92.3% 17.8.
As a result, the
hit limit was set to be 3 standard deviations from the mean at 39% ATPase
activity. This stringent
hit limit identified 64 hits, of which 53 hits were confirmed against
recombinant CHD1L ATPase
activity.
Example 4: Exemplary Inhibitors
A subset of seven confirmed hits (compounds 1-7, see Scheme 1) were purchased,
representing a
range of pharrinacophores with greater than 50% inhibition against cat-CHD1L
ATPase.
Compounds 1-7 were subjected to dose response studies against cat-CHD1L
ATPase, which
validated these hits as potent CHD1L inhibitors with activity between 900 nM
to 5 pM (Figure 1A).
Structures of additional exemplary compounds 8-73 are provided in Scheme 1,
where SEM
represents the protecting group trimethylsilylethoxy methyl. Structures of
additional exemplary
compounds 74- 116 are provided in Scheme 1. Note that in a number of cases in
Scheme 1, an
additional compound number is given in parenthesis which may be employed in
Tables and
Figures herein or in Abbott et al., 2020 and its Supplementary Information or
Prigaro et al. .
Compounds 1-7 were tested in HCT116, SW620, and DLD1CHD1L-OE cells for their
ability to inhibit
TCF-transcription using the TOPflash reporter system (Figure 1B). Compounds 1-
3 were shown to
have no significant activity in cells. Compound 4 was shown to have modest
activity in cells with no
dose dependent inhibition of TCF-activity. However, compounds 5-7 demonstrate
superior dose
dependent activity against TCF transcription in all three CRC cell lines.
Notably, decreased
inhibition of TCF-transcription was observed for 5-7 at the low 2 pM dose in
DLD1CHD1L-OE cells,
which is evidence of cellular CHD1L target engagement.
CHD1L inhibitors reverse EMT and malignant properties in CRC.
After validating hits 5-7 against CHD1L mediated TCF transcription, the
ability of these compounds
to reverse EMT and other malignant properties in CRC were evaluated. E-
cadherin and vimentin are putative biomarkers for the epithelial and
mesenchymal phenotypes,
respectively. [McDonald et al., 2015] Loss of E-cadherin and gain of vimentin
are also clinical
biomarkers of poor prognosis. [Yun et al., 2014; Richardson et al., 2012;
Dhanasekaran et al.,
2001; Kashiwagi et al., 2010; Toiyama et al., 2013] Accordingly, lentiviral
promoter driven
reporters for E-cadherin (pCDH1-EcadPro-RFP) and vimentin (pCDH1-VimPro-GFP)
were
developed, which faithfully report E-cadherin and vimentin protein expression,
respectively. [Zhou
et al., 2016; Abraham et al., 2019] SW620 cells transduced with either EcadPro-
RFP or VinnPro-
GFP were cultured as tumor organoids for 72 h, reaching a diameter of 600 pm.
Tumor organoids
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were treated with compounds 5-7 for an additional 72 h to determine the
effective concentration 50
percent (EC50) for modulating promoter activity. Changes in promoter
expression was quantified
using a 3D confocal image 507 based high-content analysis algorithm (Figure 2A-
26). [Zhou et al.,
2016; Abraham et al., 2019]
Compounds 5-7 effectively downregulated vimentin promoter activity with E050
values of 15.6 1.7
pM (5), 4.7 510 1.5 pM (6), and 12.8 1.3 pM (7). Conversely, E-cadherin
promoter activity was
upregulated with EC50 values of 11.9 0.3 pM (5), 11.4 0.3 pM (6), and 28
0.003 pM (7).
Representative images exhibiting reversion of EMT by compound 6 in SW620 tumor
organoids
measured by EMT reporter assays are shown in Abbott et al., 2020. These
results indicate that
small molecule inhibitors of CHD1L reverse TCF-driven EMT in CRC.
To confirm that CHD1L inhibitors reverse EMT, protein expression of two
additional putative
biomarkers of EMT, slug (mesenchymal) and zona occludens-1 (ZO-1, epithelial)
were evaluated.
Changes in slug and ZO-1 are considered major criteria for EMT. [Zeisberg &
Neilson, 2009]
SW620 tumor organoids treated with CHD1L inhibitors downregulate slug and
upregulate ZO-1,
further indicating a reversion of EMT. Western blot analysis showing protein
expression changes
of additional EMT biomarkers slug and ZO1 is shown in Abbott et al., 2020.
A hallmark of EMT is an increase in CSC sternness and cell invasion.
Therefore, the ability of
compounds 5-7 to inhibit migration and invasion in HCT-116 and DLD1CHD1L-OE
cells was tested. All
three compounds demonstrated a significant inhibition of CSC sternness (Figure
2C). However,
compounds 5 and 6.0 display more potent dose dependent inhibition. Note that
DLD1CHD1L-OE cells
form two times more colonies than HCT-116 cells, which have moderate CHD1L
expression. This
observation is consistent with CHD1L's oncogenic and tumorigenic properties.
Next, using HCT-
116 cells with uniform scratch wounds imbedded in 50% Matrigel0 matrix
(Corning Life Sciences,
Corning, NY) cells were treated with CHD1L inhibitors at concentrations
indicated and invasion
was monitored over 72 h. Compounds 5-7 exhibited a dose dependent inhibition
of invasion
(Figure 2D), with compound 6.0 displaying the most potent activity.
Example 5: Inhibition of CHD1L Efficacy of DNA Damaging Drugs
CHD1L is known to function in PARP1 mediated DNA damage response repair, which
is a
mechanism of with increased drug resistance to DNA damaging chemotherapy [Li
et al., 2019;
Ahel et al., 2009; Gottschalk et al., 2009]. For example, drug resistance to
cisplatin in lung cancer
was observed in cells overexpressing CHD1L. The efficacy of cisplatin was
restored after CHD1L
knockdown. [Li Y., et al., 2019] In addition, knockdown of CHD1L alone does
not increase DNA
damage. [Ahel D, et al., 2009] In order to determine if CHD1L inhibitors could
increase the efficacy
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of DNA damaging drugs against low CHD1L expressing DLD1 cells transduced with
empty vector
(DLD1CHD1L-EV) and overexpressing DLD1CHD1L-OE in CRC cells, compound 6 was
evaluated
alone as a single agent and in combination with SN-38 (active pharmacophore of
prodrug
irinotecan), oxaliplatin, and etoposide. To assess DNA damage, the
phosphorylation of H2AX (7-
H2AX) by immunofluorescence, a biomarker for DNA damaging chemotherapy, [Ahel
D, et al.,
2009], was measured as shown in Abbott et al., 2020 and its Supplementary
Information.
Compound 6 alone showed no significant DNA damage when treating cells at 10 pM
and
measuring y-H2AX activity, which is consistent with previously reported CHD1L
knockdown
studies. [Ahel D, et al., 2009]. However, combination treatments in DLD1CHD1L-
OE cells with
compound 6 synergized with etoposide (10 pM) and SN-38 (1 pM), significantly
increasing DNA
damage compared to etoposide and SN-38 alone. In DLD1CHD1L-EV cells only the
combination
of etoposide and compound 6 displayed significant synergy. Under the
experimental conditions
used we observed no synergy was observed with oxaliplatin. Nevertheless, SN-38
(i.e. irinotecan)
combination therapy, known as FOLFIRI, is a standard of care in the treatment
of CRC. Therefore,
the enhanced DNA damage that occurs with compounds 6 in combination with SN-38
supports the
hypothesis that CHD1L inhibitors can increase the efficacy of CRC standard of
care DNA
damaging chemotherapies.
Example 6: CHD1L inhibitors reverse EMT prior to the induction of cell death.
CHD1L has been reported to confer anti-apoptotic activity by inhibiting
activation of caspase-
dependent apoptosis. [Li et al., 2013; Sun et al., 2016] Additionally,
reversal or inhibition of EMT is
known to restore apoptotic activity of cancer cells. [Lu et al., 2014] To
determine if CHD1L
inhibitors reverse EMT prior to induction of cell death, E-cadherin expression
by EcadPro-RFP
reporter activity was monitored and cytotoxicity was measured using the
CellToxTm Green assay.
Cells were treated with CHD1L inhibitors for 72 h and imaged every 2 h. A
significant increase in
E-cadherin expression prior to induction of cytotoxicity for compound 6
relative to DMSO (Figure
3A).
To determine if CHD1L inhibitors are able to induce apoptosis in CRC, western
blots from SW620
tumor organoids were performed and it was observed that E-cadherin is cleaved
after treatment
with 5 and 6 [Abbott et al., 2020]. Cleavage of E-cadherin is a marker of
apoptosis [Steinhusen et
al., 2001]
The more potent CHD1L inhibitor 6, exhibited increases in cleaved PARP1,
cleaved caspase 8,
and cleaved caspase 3 relative to DMSO control [Abbott et al., 2020] These
results indicate that
compound 6 induces extrinsic apoptosis that is consistent with E-cadherin
mediated apoptosis
through death receptors. [Lu et al., 2014]
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To further characterize the apoptotic activity of CHD1L inhibitors, annexin-V
staining in SW620
cells over 12 h was examined. Compound 6.0 induced significant apoptosis
compared to DMSO
alone and had similar activity to the positive control SN-38, the active
metabolite of irinotecan
(Figure 3B)
CHD1L inhibitors are effective against patient-derived tumor organoids
(PDTOs). The use of
PDTOs in preclinical drug development has been established as a predictive in
vitro cell model for
clinical efficacy. [Drost J & Clevers H, 2018] After establishing the ability
of compound 6 to reverse
EMT and induce apoptosis using cell line based models, the efficacy of
compound 6 was evaluated
in PDTOs produced from patient sample CRC102 obtained from the University of
Colorado Cancer
Center (UCCC) gastrointestinal (GI) tissue bank (Figure 3C). Consistent with
the results in CRC
cell lines, compound 6.0 showed potent cytotoxicity in PDTOs with an EC50 of
11.6 2 pM
Example 7: In vitro and in vivo PK, PD, and liver toxicity of Exemplary
Inhibitor Compound
6.
To assess the drug-like potential and properties of compound 6.0 in silico, in
vitro, and in vivo PK
studies were conducted assessing CLogP, aqueous solubility, stability in mouse
liver microsomes,
and PK in CD-1 mice.
Table 1 provides a summary of in vivo and in vitro pharmacokinetic parameters
of compound 6.
The consensus LogP (CLogP) values were obtained using the SwissADME web tools.
[Daina et
al., 2017] Compound 6 was administered by i.p. injection to athymic nude mice
QD for 5 days to
measure accumulation in SW620 xenograft tumors (FIG. 4) and to assess
histopathology of liver
toxicity. Representative H&E-stained photomicrograph sections (5x
magnification) of liver in both
vehicle and compound 6 treated animals are shown in Abbott et al., 2020. The
images
demonstrate normal hepatic cord and lobule architecture, with no evidence of
hepatocyte
degeneration, necrosis, hyperplasia, or parenchymal inflammation. Compound 6.0
has an excellent
balance of lipophilicity (CLogP = 3.2) and aqueous solubility that is
relatively stable to liver
metabolizing enzymes, and an excellent PK disposition when administered to CD-
1 mice.
Compound 6.0 reaches a high plasma drug concentration Cmõ (-30,000 ng/mL) and
AUC
(-80,000 ng/mL/h) with a relatively long half-life (T1i2k) of 3 h after
intraperitoneal (i.p.)
administration.
In an initial study, compound 6, exhibited a half-life in liver microsomes of
less than 20 minutes. In
subsequent analogous in vitro liver microsome half-life experiments conducted
with a different liver
microsome preparation (data not shown), compound 6 exhibited a longer half-
life of 67 minutes
and compound 6.3 exhibited an improved (over 6) in vitro half-life of 98
minutes and compound
6.11 exhibited a further improved (over 6) in vitro half-life of 130 minutes.
The initial half-life studies
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with compound 6 were conducted with a different liver microsome preparation
and not comparable
to later in vitro microsonne half-live experiments. The results of the second
series of in vitro and in
vivo half-life measurements is provided in Table 2 which includes data for
several additional
compounds as indicated.
A second acute in vivo experiment was conducted using a maximum tolerated dose
of 6.0 (50
mg/kg) administered to athymic nude mice by i.p. QD over five days. The goals
of this experiment
were to (1) determine if compound 6.0 causes acute toxicity to livers, (2)
accumulates in VimPro-
GFP SW620 xenograft tumors, and (3) to determine PD effects. Compound 6.0
accumulates in
SW620 tumors at a concentration of 10,533 5,579 ng/mL (n=4). As expected,
when comparing
the ratio of compound 6.0 accumulation in tissue/plasma, 2.7 times more
accumulation in liver
compared to tumor was observed (FIG. 4). Howver, there was no apparent liver
toxicity resulting
from compound 6.0 at the dose and schedule administered (Table 3). Overall,
there were no
significant histological differences between the livers of vehicle or compound
6.0 treated mice. The
primary histological changes observed were minimal fibrosis and inflammation
of the hepatic
capsule in both vehicle and compound 6.0 treated animals. This suggests a very
low grade, sub-
clinical peritonitis, and is consistent with being secondary to i.p. drug
administration.
In accordance with accumulation of compound 6.0 in tumors, PD effects on tumor
tissue were
measured by Western blot analysis, indicating a significant downregulation of
mesenchymal
markers vimentin, vimentin reporter (VimPro-GFP), and slug [Abbott et al.,
2020]. Although not
statistically significant, upregulation of the epithelial marker ZO-1 and
induction of cleaved caspase
3 (the putative biomarker of apoptosis) were also observed. Taken together,
these observations of
PD effects by compound 6.0 indicate the reversion of EMT and apoptosis in vivo
that were
consistent with in vitro cell-based antitumor activity of compound 6. Compound
6.0 displays good
PK drug-like properties and the ability to alter EMT and induce apoptosis in
vivo with no observed
liver toxicity.
In contrast, compound 6.11 exhibits significantly longer half-life (Tv22)
compared to that of
compound 6 of much greater than 6 h after intraperitoneal (i.p.)
administration.
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Table 1: PK Parameters Compound 6
PK Parameters In Vitro In vivo
Comp. ClogP PBS Microsomal Crviax V2 AUCo_t CL T1/22.
Solubility Half-life (ng/mL) (L/kg) (ng/nriL (L/h/kg) (le)
(mg/mL) (min) x h)
6 3.2 0.70 17.0 29,900 2.7
80,333 0.62 2.97
Table 2: CHD1L Inhibitor Pharmacokinetics
Pharmacokinetics (PK)
CHD1L
Inhibitor In Vitro Half- In vivo Half-
Life (Min) Life (hr)
6 67 3
6.1 34.9
6.2 26.1
6.3 98
6.4 31.4
6.9 295
6.10 16.7
6.11 130 8
6.12 9.3
6.13 43.1
6.14 70.4
6.15 22.7
Table 3: Histological evaluation raw scores of livers from athymic nude mice
treated with
vehicle or compound 6.0 (50 mg/kg) QD for 5 days.
Animal Groups Organ Assessmentl
Inflammation score2
Vehicle - 1 Liver
Vehicle - 2 Liver A 1
Vehicle - 3 Liver
Vehicle - 4 Liver j A 1
Compound 6.0 - 1 Liver
Compound 6.0 - 2 Liver A 1
Compound 6.0 - 3 Liver
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Compound 6.0 - 3 Liver A 1
lAssessment: N = normal background lesion for mouse strain; A = abnormal.
2Inf1ammat10n score
(performed if abnormal tissue assessment): 0 = none, 1 = minimal, 2 = mild, 3
= moderate, 4 = severe
Example 8: Biological Evaluation of Compound 8
Compound 8 was evaluated in a number of biological assays described above.
Results are
presented in Figures 7A-E Compound 8 displays more potent dose dependent
inhibition of
CHD1L-mediated TCF-transcription (Fig. 7A) compared to compound 6. Likewise,
compound 8
reverses EMT, evidenced by the downregulation of vimentin and upregulation of
E-cadherin
promoter activity (Figs. 7B and 7C, respectively). Compound 8 significantly
inhibits clonogenic
colony formation over 10 days (Fig. 70). Compound 8 significantly inhibits
HCT116 invasive
potential over 48 h (FIG. 7E).
Example 9: Methods Applied in Examples herein
Additional Materials and Methods
Antibodies. Monoclonal mouse anti-TCF4 antibody was purchased from EMD
Millipore (Billerica,
MA, USA) (catalog# 05-511), a 1:1000 dilution was used for Western blot and 2
pg antibody per 300
pg of protein was used for IP. Monoclonal rabbit anti-CHD1L antibody was
purchased from Abcam
(Cambridge, MA, USA) (catalog #ab197019), a 1:5000 dilution was used for
Western blot, and 1.5
pg antibody per 300 pg of protein was used for IP. Monoclonal rabbit anti-
Vimentin (catalog# 5741),
anti-Slug (catalog #9585), anti-E-cadherin (catalog #3195), anti-ZO-1 (catalog
#8193), anti-Histone
H3 (catalog #4620) were purchased from Cell Signaling (Danvers, MA, USA) and
mouse anti-a-
tubulin (catalog# 3873) were purchased from Cell Signaling and a 1:1000
dilution was used for
Western blot. Monoclonal rabbit anti-13-catenin (catalog #9582) were purchased
from Cell Signaling,
a 1:1000 dilution was used for Western blot. Monoclonal rabbit anti-phospho-6-
catenin was
purchased from Cell Signaling (catalog# 5651). Monoclonal rabbit anti-TCF4
(catalog #2569) and
anti-Histone H3 (catalog #4620) were purchased from Cell Signaling and 2 pg
antibody per 1 mg of
protein was used for ChIP. Anti-rabbit IgG HRP-linked secondary antibody
(catalog #7074) was
purchased from Cell Signaling and a 1:3000 dilution was used for Western blot.
Anti-goat and anti-
mouse IgG HRP-linked secondary antibodies (catalog #805-035-180 and #115-035-
003) were from
Jackson ImmunoResearch (West Grove, PA), a 1:10,000 dilution was used for
Western blot.
Clinicopathological Characterization of CHD1L
Transcriptome expression data of 585 CRC patients from the CIT cohort (GEO:
GSE39582) were
used for in silico validation (G5E39582). [Marisa et al., 2013] Gene
expression analyses were
performed by the Affymetrix GeneChipTM Human Genome U133 Plus 2.0 Array
(Thermo Fisher
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Scientific, Waltham, MA). Robust Multi-Array Analysis (RMA) was used for data
preprocessing and
ConnBat (empirical Bayes regression) for batch correction. Signal intensity
was 1092 normalized.
The CHD1L cutoff for CRC risk stratification based on disease specific
survival was determined by
the receiver operating characteristic (ROC) curve. Cutoff for CHD1L expression
was set to 6.45.
Differences in OS were estimated by the Kaplan-Meier method and compared using
the log-rank
test. The Fisher's exact test was used for the comparison of categorical
variables. The Mann-
Whitney U test was used for 2 groups of continuous variables. In case of more
than two groups,
data was analyzed by the Kruskal-Wallis test. For all 2-sided P-values, the
unadjusted significance
level of 0.05 was applied.
The CHD1L cutoff and clinicopathologic characteristics were evaluated by
multiple cox regression
analysis. Only variables that were significant in univariate analyses were
integrated in the cox
regression model using the Wald forward algorithm for significance
determination. All variables
including more than 2 groups were categorized and the stepwise entry criterion
for covariates was
P<0.05 and the removal criterion was P>0.1. Statistical analysis was performed
using IBM SPSS
Statistics (IBM, Armonk, NY), Prism8 (GraphPad Software, San Diego, CA), JMPO
(SAS Institute,
Cary, NC), and RStudioTmIDE (RStudio Inc, Boston, MA).
UCCC Patient sample RNA-seq analysis
RNA-seq data from CRC patient tumor xenograft explants were obtained from the
UCCC
(University of Colorado Cancer Center) GI tumor tissue bank, and analyzed as
previously
described. [Scott et al, 2017] Briefly, gene expression was Log2 normalized
and measured by
FPKM (Fragments Per Kilobase of transcript per Million mapped reads). The Wnt
signaling
pathway defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used
as the
gene set in this study. Samples with expression of CHD1L <1 FPKM were
considered low
expression and were removed from this study. Genes with significant Spearman's
correlations
(P<0.05) were displayed as heatmap using matrix2png (gene-wise Z-normalized)
[See: Abbott et
al, 2020 and its Supplementary Information]
CHD1L overexpression and shRNA knockdown
Full length CHD1L was synthesized in a pGEX-6P-1 plasmid (GenScript,
Piscataway, NJ). The
CHD1L sequence flanked by EcoR/ and Not/ was digested out and ligated to a
lentiviral backbone
to create pCDH1-CMV-CHD1L-EF1-puro plasmid for overexpression of CHD1L in
human CRC
cells. Mission shRNA (Sigma-Aldrich Co. LLC, St. Louis, MO) (scrambled) and
TR0N0000013469 and TRCN0000013470 (sh69 and sh70) specific for CHD1L were
purchased
from Sigma-Aldrich (St. Louis, MO). Virus was produced in HEK293T cells using
TransITO-293
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reagent (Mirus, Madison, WI), and plasmids pHRdelta8.9 and pVSV-G. CRC cells
were transduced
with overexpression or shRNA knockdown virus and selected with 2 pg/ml
puronnycin for 7 days.
Western Blots
CRC cell lines and homogenized tumor tissue samples from mice were resuspended
in RIPA lysis
buffer (20 mM Tris-HCI (pH 7.5), 150 mM NaCI, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-
40, 1%
sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1
mM Na3 VO4,
0.1 mM PMSF. Protein concentration was determined using the Pierce TM BCA
protein assay kit
(ThermoFisher, Waltham, MA). Forty micrograms of sample were run on 10% Bis-
Tris gels.
Following electrophoresis, the proteins were transferred to a nitrocellulose
membrane. The
membranes were blocked at room temperature with 5% non-fat milk in TBS/Tween
20 (TBST
contains 20 mM Tris, 150 mM NaCI, and 0.1% Tween 20 (Croda International PLC,
Snaith, UK)
for 1 hour at room temperature. Membranes were washed three times with TBST.
Blots were
incubated with the appropriate primary antibody in 5% nonfat milk in TBST
overnight at 4 'C.
Membranes were washed three times with TBST and then incubated with
appropriate secondary
antibody for one hour. Membranes were washed again with TBST three times.
Blots were exposed
using SuperSignalTmWest Pico PLUS Chemiluminescent Substrate (ThermoFisher,
Waltham, MA)
and imaged using a ChemiDoc imaging system (Bio-Rad, Hercules, CA). [See:
Abbott et al., 2020
and its Supplementary Information for Western Blots]
TOPflash TCF-transcriptional reporter assay
TOPflash assay (Millipore, Billerica, MA) was used to evaluate TCF
transcriptional activity in CRC
cells. A total of 20,000 cells per well were plated into 96-well white plates
and transfected with
TransITO-LT1 transfection reagent (Mirus, Madison, WI). Cells were incubated
with transfection
mix for 24 h. Next, cells were washed with phosphate-buffered solution (PBS)
and a 1:1 ratio of
PBS: ONE-Glo TM luciferase reagent Promega (Madison, WI) was added and the
luminescence
was detected within 10 min. A duplicate experiment was conducted to measure
cell viability using
CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI), which
was used to
normalize TOPflash luminescence to obtain the fold change in TCF activity.
Experiments were
replicated 2x (n = 3 for each experiment).
Co-ImmunoPrecipitation (Co-IP)
Nuclear cell lysates were generated 138 from untreated SW620 cells. For the
input control, 100 pL
of 1 mg/mL nuclear extract was saved and used as the input. ImmunoPreciptation
(IP) was
conducted with Dynabeads TM Protein A IP Kit (ThermoScientific, Waltham, MA).
Briefly, 300 pg of
lysate incubated with 2 pg of the anti-TCF4 and anti-CHD1L IP antibody, anti-
rabbit IgG and anti-
mouse IgG were used as nonspecific binding controls and were rotated at 4 C
for 2 h. After
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preincubation, 50 pL of beads were transferred to the preincubated
antibody/lysate mixture
followed by overnight incubation at 4 C. The flow through was collected and
the beads were
washed 3x with PBST. Proteins were eluted with 20 pL of 50 mM glycine (pH =
2.8) at 70 C for 10
min.
Chromatin Immunoprecipitation (ChIP)
Using detailed methods previously described [Zhou et al., 2016], cells were
cross-linked with
1.42% formaldehyde for 15 min and quenching with 125 mM glycine for 5 min.
Cells were lysed
with Szak's RIPA (Radioimmunoprecipitation assay buffer) buffer and sonicated.
The IP steps were
conducted at 4 C as follows: 50 pL of protein A/G agarose beads were
prewashed with cold
Szak's RIPA buffer and incubated with 1 mg of lysate for 2 h. 0.3 mg/mL of
salmon sperm DNA
was added and incubated for 2 h. Lysate (100 pL) was set aside as the input
control. Anti-CHD1L
(2 pg) was added to the remainder and incubated overnight. Beads were washed
and the
supernatant was aspirated to 100 pL followed by the addition of 200 pL of 1.5x-
Talianidis elution
buffer (70 mM Tris-CI pH 8.0, 1 mM EDTA pH 8.0, 1.5% w/v SDS). To elute
immunocomplexes
and reverse crosslink, 12 pL of 5M NaCI was added and the mixture was
incubated at 65 C for 16
h. The supernatant was mixed with 20 pg of proteinase K and incubated for 30
min at 37 C. DNA
was extracted with phenol/chloroform and precipitated with ethanol. The IP
product was amplified
with PowerUpTM SYBRTM Green Master Mix (Applied Biosystems, Austin, TX) using
known
published primers. [Zhou et al., 2016]
Clonoaenic Assay
Colony formation was assessed after CHD1L knockdown in SW620 cells or
overexpression in
DLD1 cells as previously described. [Zhou et al., 2016; Abraham et al., 2019]
Cells were plated at
1,000 cells/well in six-well plates and medium was changed 2x per week over a
10-day time
course. Colony formation analysis was also performed as previously described.
[Zhou et al., 2016;
Abraham et al., 2019]
To assess CHD1L inhibitors for their ability to suppress CSC stemness, HCT-116
or CHD1L
overexpressing DLD1 cell lines were pre-treated in nnonolayer cultures for 24
h with vehicle control
(0.5% DMSO) or CHD1L inhibitors at the concentrations indicated in FIG. 2C.
Pretreated viable
cells were plated at 1,000 cells/well in 6-well plates or 200 cells/well in a
24-well plates. Colonies
were analyzed using the IncuCyte S3 2018A (Sartorius, France) software (with
the following
parameters modified from default: (1) for HCT116 cells segmentation adjustment
= 0.6; Min area
(pm2) = 3x104; Max area (pm2) = 1.6x106; Max eccentricity = 0.9; (2) for
DLD1CHD1L OF cells
segmentation adjustment = 1; Min area (pm2) = 1x104; Max area was not
constrained; Max
eccentricity = 0.95. Experiments were replicated 2x (n = 2 for each
experiment).
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Tumor organoid Culture
Cell lines were cultured [Zhou et al., 2016; Abraham et al., 2019] as tumor
organoids using phenol
red free RPM 1-1640 containing 5% FBS and by seeding 5,000 cells/well into un-
coated 96-well U-
bottom Ultra Low Attachment Microplates (Perkin-Elmer, Hopkinton, MA) followed
by centrifugation
for 15 min at 1,000 rpm to promote cells aggregation. A final concentration of
2% Matrigel matrix
(Corning Incorporated, Corning, New York) was added and tumor organoids were
allowed to self-
assemble over 72 h under incubation (5% CO2, 37 C, humidity) before
treatment, and maintained
under standard cell culture conditions during treatment time courses.
VimPro-GFP and EcadPro-RFP reporter 3D high-content imaging assays
Stable VimPro-GFP or EcadPro-RFP 5W620 reporter cells were generated using
pCDH imPro-
GFP-EF1-puro virus or pCDH-EcadPro-mCherry-EF1-puro virus as previously
reported. [Zhou et
al., 2016; Abraham et al., 2019] The stable fluorescently labeled reporter
cells were used to
generate tumor organoids as described herein. Tumor organoids were treated
with CHD1L
inhibitors at 10 pM for an additional 72 h. Following treatment, tumor
organoids were stained with
16 pM of Hoechst 33342 for 1 h (nuclei stain). Images were taken with a 5x air
objective. Z-stacks
were set at 26.5 pm apart for a total of 15 optical slices. Imaging and high-
content analysis were
performed using an Opera PhenixTM and Harmony software (PerkinElmer,
Hopkinton, MA).
Nuclei were identified within each layer and cells were found with either GFP
or mCherry channel.
The fluorescence intensities of each channel were calculated and thresholds
were set based on
the background intensities. Percentages of GFP or nnCherry RFP positive cells
were calculated
and normalized to the DMSO treated group.
Tumor organoid cytotoxicity.
SW620 tumor organoids were cultured as described herein. CellToxTm Green
cytotoxicity assay
solution was prepared per manufacturer's protocol (Promega, Madison, WI).
Briefly, tumor
organoids were treated for 72 h with CellToxTm Green reagent (0.5X) and
various doses of CHD1L
inhibitors over a range of 0-to-100 pM. Organoids were imaged using the Opera
PhenixTM 207
screening system (PerkinElmer Cellular Technologies, Hamburg, Germany) with
excitation at 488
nm and emission at 500-550 nm. Mean intensity of the whole well was utilized
for calculating
cytotoxicity with Lysis Buffer (Promega, Madison, WI) as the 100% cytotoxicity
control and 0.5%
DMSO as the 0% cytotoxicity control. Intensity values were normalized to these
controls using
Prism8 (GraphPad, San Diego, CA).
Invasion assays.
HCT116 cells were plated at 60,000 cells/well into an IncuCyte ImageLock 96-
well plate
(Sartorius, France) and allowed to attach overnight. A wound was created in
all wells using the
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IncuCytee WoundMaker then washed 2x with PBS. The plate was brought to 4 C
using a Corning
XT Cool Core to avoid polymerization of the Matrigel matrix (Corning Life
Sciences, Corning, NY)
during the preparation of the invasion conditions. Wells were coated with 50
pL of 50% Matrigel
matrix in RPMI-1640 media. Plates were centrifuged at 150 rpm at 4 C for 3
min, using a swing
bucket rotor to ensure even matrix coating with no air bubbles. Afterwards,
plates were placed on a
Corning XT CoolSink module prewarmed inside a cell culture incubator (5% CO2,
37 C, humidity)
for 10 min to evenly polymerize the matrix, followed by the addition of CHD1L
inhibitors dissolved
in 50 pL of RPM1-1640 media containing 5% FBS. Finally, the plate was placed
in an IncuCyte0
S3 live cell imager (Sartorius, France) for 48 h. The wound was imaged every
hour using the
phase contrast channel and 10x objective in wide mode.
Cloning and purification of recombinant human CHD1L
Cat-CHD1L (residues 16-61) and fl-CHD1L (residues 16-879) constructs were a
generous gift from
Helena Berglund at the Karolinska Institute, Department of Medical
Biochemistry and Biophysics.
Proteins were expressed in Rosetta TM 2 (DE3) pLysS cells (Novagen available
from Sigma-Aldrich,
St. Louis, MO) in Terrific Broth (ThermoFischer, Waltham, MA). Cultures were
induced with 0.2
mM IPTG at 0D600 = 2.0 at 18 C for 16 h. Cells were harvested and resuspended
in buffer-A,
containing 20 mM HEPES, pH 7.5, 500 mM NaCI, 50 mM KCI, 20 mM imidazole, 10 mM
MgCl2, 1
mM TCEP (tris(2-carboxyethyl)phosphine), 10% glycerol and 500 pM PMSF. Cells
were lysed by
sonication and cellular debris was removed by centrifugation. The supernatant
was loaded onto a
Ni-NTA resin column (Qiagen, Hilden, Germany). Protein bound to the column was
washed with lx
with buffer-A, lx with buffer-A containing 10 mM ATP, and washed an additional
time with buffer-A.
Proteins were eluted using buffer-B (buffer-A with 500 mM imidazole) with a
gradient from 20 to
500 mM imidazole. Following affinity purification, cat-CHD1L was dialyzed
overnight into 50 mM
Tris, pH 7.5, 200 mM NaCI, and 1 mM DTT. Similarly, fl-CHD1L was dialyzed
overnight into 20 mM
MES, pH 6.0, 300 mM NaCI, 10% glycerol, and 1 mM DTT. Protein was then
purified by ion-
exchange chromatography. cat-CHD1L was bound to a Q-sepharose column (GE
Healthcare,
Chicago, IL) and fl-CHD1L was bound to a 5-sepharose column (GE Healthcare,
Chicago, IL), and
proteins were eluted using a NaCI gradient of 0.2 ¨ 1M for cat-CHD1L and 0.3 -
1M for fl-CHD1L.
Pure fractions were pooled, concentrated, and further purified by size-
exclusion chromatography
using a SuperdexTM 200 column (GE Healthcare, Chicago, IL) with 20 mM HEPES,
pH 7.5, 100
mM NaCI, 1 mM TCEP, and 10% Glycerol. Protein purifications were conducted
using an ACTA
Start FPLC (GE Healthcare, Chicago, IL).
CHD1L ATPase assay
All reactions were carried out using low volume non-binding surface 384-well
plates (Corning Inc.,
Corning NY). cat-CHD1L or fl-CHD1L (100 nM) and 200 nM c-Myc DNA or
mononucelosome
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(Active Motif, Carlsbad, CA) were added to a buffer containing 50 mM Tris pH
7.5, 50 mM NaCI, 1
mM DTT, 5% glycerol, and the reaction was initiated by the addition of 10 pM
ATP (New England
Biolabs, Ipswich, MA) to a total volume of 10 pL and incubated at 37 C for 1
h. ATPase activity
was assayed by adding 500 nM of Phosphate Sensor (Life Technologies, Carlsbad,
CA),
containing labeled phosphate-binding protein, specifically labeled with the
fluorophore MDCC, and
measuring excitation (430 nm) and emission (450 nm) immediately on an EnVision
plate reader
(PerkinElmer, Hopkinton, MA). An inorganic phosphate standard curve was used
to convert the
fluorescence to [Pi], and enzyme kinetics were determined using Prism8
(GraphPad Software, San
Diego, CA).
HTS drug discovery for inhibitors of CHD1L
Assay composition was the same as described above using cat-CHD1L, except that
the reaction
mixture volume was modified to accommodate addition of drug or DMSO. Using a
Janus liquid
handler (PerkinElmer, Hopkinton, MA), a selected amount of compounds dissolved
in 100% DMSO
were mixed with 50 mM Tris pH 7.5, 50 mM NaCI, 1 mM DTT, 5% glycerol buffer to
200 pM in 10%
DMSO. Next, 1 pL of each compound was added to the enzyme mixture to give a
final
concentration of 20 pM. The negative control used was 1% DMSO and 10 mM EDTA
was used as
a positive control. Reactions were initiated with the addition of 10 pM ATP
and incubated at 37 C
for 1 h. ATPase activity was measured by fluorescence by adding 500 nM
Phosphate Sensor. cat-
CHD1L was screened against a 20,000-compound diversity set from Life Chemicals
(Woodbridge,
CT) and a Kinase Inhibitor library from Selleck Chemicals (Houston, TX). Both
libraries were
prescreened before purchase to remove Pan-assay interference compounds (PAINS)
which tend
to react nonspecifically with many biological targets rather than selectively
with a desired target.
[Baell & Nissink, 2018; Baell & Holloway, 2010]
Patient derived tumor organoid (PDTO) culture and viability assay
CRC patient tumor tissues were obtained from the UCCC GI tissue bank and
expanded following
established protocols. [Morin et al., 1997]. Briefly, cells were seeded at
5,000 cells per well in 96-
well plates and cultured by established methods [Franken et al., 2006]
allowing PDTO formation
over 72 h. PDTOs were treated with DMSO (0.5%) or compound 6.0 with various
concentrations
for an additional 72 h to obtain a dose response. PDTO cell viability was
measured using CellTiter-
Blue reagent (Promega, Madison, WI). Media (80 pL) was aspirated from wells
and 80 pL of the
reagent was added and incubated for 4 h and cell viability was measured by
fluorescence intensity
using excitation 560 excitation and 590 emission.
Evaluation of apoptosis
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SW620 cells were plated at 30,000 cells/well in 96-well plates. Cells were
treated with DMSO
(negative control), SN-38 (apoptosis positive control), and compound 6.0 at
concentrations
indicated for 12 h. Cells were then rinsed 2x with cold PBS, lx with cold
Annexin-V staining buffer
(10 mM HEPES, pH 7.4, 140 mM NaCI, 2.5 mM CaCl2), and then incubated with
Annexin-V FITC
at 1:100 for 30 min in the dark. Cells were then rinsed 2x with Annexin-V
staining buffer and FITC
intensity was measured using an EnVisione plate reader (PerkinElmer,
Hopkinton, MA).
Evaluation of DNA damage by y-H2AX
DLD1CH01L-0E cells were seeded into a 96-well PerkinElmer Cell Carrier plate
and allowed to
adhere overnight. Cells were then treated with the appropriate compound at 10
pM (0.5% DMSO)
or with CHD1L inhibitor in combination SN-38 (1 pM), oxaliplatin (10 pM), and
etoposide (10 pM).
Cells were treated for 6 h. Media was aspirated and cells were washed with
cold PBS. Cells were
then fixed with 3% paraformaldehyde for 15 min at room temperature, fixed
cells were washed with
PBS three times. Cells were blocked for 1 hour at room temperature in 5% BSA,
0.3% Triton X-100
in PBS. Cells were then immunostained with phospho-(S139)-g-H2AX rabbit mAb
using a 1:800
dilution in 1% BSA, 0.3% Triton X-100 in PBS at 4 C overnight. Primary
antibody was aspirated
and cells were washed with PBS. Cells were incubated for 2 h at room
temperature with goat anti-
rabbit Alexa Fluor PlusTM 647 fluorescent secondary antibody at a
concentration of 5 pg/mL in 1%
BSA, 0.3% Triton X-100 in PBS. Cells were then washed with PBS, Hoechst 33342
stain was
diluted to a concentration of 1:1000 in PBS, and added to cells for 15 min at
room temperature.
Cells were then imaged using a 20X water objective on the Opera PhenixTM HCS
imaging system
(PerkinElmer). Synergy was determined using the coefficient of drug
interaction (CD!) equation,
CD! = (A+B)/(AB). Synergy was defined in these experiments with a CD! <0.8.
Additivity was 0.8-
1.2 and antagonism was defined by a CD! > 1.2.
Aqueous solubility and CLoqP
Using a recently reported detailed method [Abraham et al., 2019], aqueous
solubility was
measured for compound 6. The PBS UV absorption spectra were compared to a
fully saturated
solution in 1-propanol and the solubility of compound 6.0 in 10% DMSO in PBS
(pH 7.4) was
determined using linear regression analysis. The measurement of solubility in
PBS was conducted
in duplicate experiments. The consensus LogP (CLogP) values were obtained
using the
SwissADME web tools. [Daina et al., 2017]
Microsome stability studies
The microsomal stability of compound 6.0 was determined using female CD-1
mouse microsomes
(M1500) purchased from Sekisui XenoTech (Kansas City, KS), following the
recently reported
method. [Abraham et al., 2019] Samples were centrifuged at 20,000g for 10 min
and the
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supernatant was transferred to an autosampler vial for LCMS analysis. The
following mass
transition (m/z, annu) was monitored for compound 6 (molecular weight =
393.5).
In vivo pharmacology
All animal studies were conducted in accordance with the animal protocol
procedures approved by
the Institutional Animal Care and Use Committee (IACUC) at the University of
Colorado Anschutz
Medical Campus (Aurora, CO) and Colorado State University (Fort Collins, CO).
Pharmacokinetics
Nine-week old female CD-1 mice, purchased from Charles River (Wilmington, MA),
were used for
PK studies using recently reported methods [Abraham et al., 2019] Briefly, the
PK studies were
designed to cover a range of 0.25-to-24 h with 3 mice/time point for a total
of 21 mice/compound 6.
Each mouse was dosed with a single i.p. injection of compound 6.0 at 50 mg/kg
prepared in 100%
DMSO. Whole blood was harvested at specific time points and the separated
plasma frozen at -80
C for storage or used for LC-MS/MS analysis.
Pharmacodynamics and liver toxicity
Two million VimPro-GFP 5W620 cells suspended in 100 pL of a 1:1 mixture of
Matrigel matrix
(Corning Life Sciences, Corning, NY) and RPM! 1640 were injected into the
flanks of 9-week old
female athymic nude mice (Nude-Foxnl nu (069)) (Envigo, Huntingdon,
Cambridgeshire, UK).
Growth was monitored with caliper measurements 3x per week. At four weeks,
mice were
randomized into 2 groups and treated with 50 mg/kg of compound 6.0 in 200 pL
of vehicle (10%
DMSO, 40% PEG 400, 50% PBS pH=7.4) or with vehicle control. Treatments were
administered
i.p. QD over five days. Mice were sacrificed 2 h after the final dose on day
five of the treatment.
Tumors and livers were collected for analysis of compound 6.0 accumulation
measured by LCMS,
Western blot analysis measuring effects on EMT and apoptosis, and liver
toxicity.
Statistical Analysis
Data were subjected to unpaired two-tailed Student's t-test with Welch's
correction statistical
analysis or as otherwise stated using Prism8 (GraphPad, LaJolla, CA). All
experiments were
replicated 3x (n=3) or as described in the methods.
Example 10: Additional Experimental Methods for Assessment of Compound
Activities
Microsome stability. CD-1 mouse microsomes were commercially purchased and the
reactions
were performed as previously desribed. Briefly, a master mix was prepared as
follows: Microsomes
(0.5 mg/mL), 10 pM CHD1Li solubilized in DMSO (0.1%), 5 mM UDPGA, 25 pg
alamethicin, and 1
mM MgCl2 in 100 mM phosphate buffer (pH 7.4). The master mix was pre-incubated
at 37 C for 5
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min, then 1 mM NADPH was added to start the microsomal activity reaction and
maintained at
37 C throughout the time course. Reactions were stopped at 0, 5, 15, 30, 45,
and 60 min by
adding 200 pL acetonitrile and analyzed by mass spectrometry. The appropriate
microsome
controls were also performed in the same reaction conditions.
y-H2AX DNA damage combination studies with irinotecan (SN38). CHD1L inhibitor
6 alone and in
combination with SN38 was assessed for DNA damage as previously reported
[Abbott et al., 2020;
Abraham et al., 2019]. Using DLD1 colorectal cancer cells that have low CHD1L
endogenous
expression and DLD1 cells engineered to overexpress CHD1L, DNA damage studies
were
conducted measuring the immunofluorescence of y-H2AX, a well-established
biomarker of DNA
damage [Ji et al., 2017; Ivashkevich et al., 2012]. Cells were seeded into a
96-well plate as
monolayers and treated with compound 6.0 at 101.1M (0.5% DMSO) or SN-38 (1
PM), or the
combination of 6.0 and SN38 over 6 hours. Media was aspirated and cells were
washed with cold
PBS. Cells were then fixed with 3% paraformaldehyde for 15 min at room
temperature and washed
with PBS three times. Cells were blocked for 1 hour at room temperature in 5%
BSA, 0.3% Triton
X-100 in PBS. Cells were then immunostained with phospho-(5139)-y-H2AX rabbit
mAb using a
1:800 dilution in 1% BSA, 0.3% Triton X-100 in PBS at 4 C overnight. Primary
antibody was
aspirated, and cells were washed with PBS. Cells were incubated for 2 hours at
room temperature
with goat anti-rabbit Alexa Fluor PlusTM 647 fluorescent secondary antibody at
a concentration of 5
1.tg/mL in 1% BSA, 0.3% Triton X-100 in PBS. Cells were then washed with PBS;
Hoechst 33342
stain was diluted to a concentration of 1:1000 in PBS and added to cells for
15 min at room
temperature. Cells were then imaged using a 20X water objective on the
PerkinElmer Phenix HCS
imaging system. We observed synergy between compound 6.0 and SN38 in inducing
damage in
DLD1 cells that overexpress CHD1L, determined using the coefficient of drug
interaction (CD!)
equation. CD! = (A+B)/(AB), synergy was determined with a CD! <0.8, additivity
was 0.8-1.2, and
antagonism was defined by a CD! > 1.2. Welch's t-test statistical analysis was
used to determine
significance, where **= P 0.01.
Cell based cytotoxicity dose response and combination studies. CHD1L
inhibitors and SN38 (the
active pharmacophore of irinotecan) were assessed for antitumor activity
against colorectal cancer
cell lines alone or in combination. Cell lines were cultured as monolayers or
3D tumor organoids
using RPMI-1640 containing 5% fetal bovine serum as previously reported
[Abbott et al. , 2020].
For 3D 5W620 tumor organoid cytotoxicity studies, 2,000 cells in 100 pL were
plated into each well
of the 96-well U-bottom ultra-low attachment microplates (Corning Inc.,
Corning, NY, USA). Plates
were centrifuged at 1,000 rpm for 15 minutes to promote cell aggregation. A
final 2% of Matrigel
concentration was reached by coating the centrifuged cells with 25 pL of 10%
Matrigel per well.
Plates were then incubated for 3 days before treatment. 3D organoids were
treated with 25 pL of
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various concentrations of drugs. 3 days after treatment, organoids with 40 pL
of medium were
manually transferred to 96-well white solid bottom plates. An equal amount of
Celltiter-glo 3D
(Promega) was added, and the plates were kept on a plate shaker for 45 minutes
at 400 rpm
before luminescence was read with Envision plate reader (PerkinElmer). For
combination studies,
synergy scores were determined using Combenefit analysis [De Veroli et al.,
2016].
In vivo studies. CHD1L inhibitors compound 6.0 and 6.11 were assessed
pharmacokinetically to
determine the plasma half-life in nine-week-old female CD-1 mice as previously
reported [Abbott et
al., 2020]. Compound 6 was further assessed for antitumor activity alone and
in combination with
irinotecan against SW620 tumor xenografts in athymic nude mice. Xenografts
were generated
using the methodology as previously reported [Zhou et al., 2016]. Briefly,
compound 6 was
administered at 5 mg/kg by intraperitoneal injection (i.p.) 2x/day 7 days/week
for a total of 5 weeks.
Irinotecan was administered i.p. at 60 mg/kg 1x/week for 3 weeks, starting
after the first week of
compound 6 treatment. Body weight and tumor volumes were monitored 2x/week.
Mice were
sacrificed and tissues collected when single tumors reached 2000 mm3 or the
total tumor volume
reached 3000mm [Ji et al., 2017]. Compound 6.11 was analogously assessed for
antitumor
activity alone and in combination with irinotecan against SW620 tumor
xenografts. It was recently
reported [Esquer et al., 2021 and its Supplementary Information] that the CRC
M-phenotype is
significantly more tumourogenic than other CRC EMT-phenotypes and that the M-
phenotype also
has significantly higher TCF-transcription. The xenografts used in this study
were generated using
isolated dual-reporter nnesenchynnal cells (M-phenotype) as described in
Esquer et al, 2021. The
half-life of compound 6.11 is 8 hours in CD-1 mice, which is 2.7-fold more
stable compared to
compound 6 (half-life = 3 hours). Thus, the number of treatments was reduced
from 2x/day to
1x/day. In addition, irinotecan was administered i.p. at 50 mg/kg.
FIGs. 7A and 7B illustrate representative single agent cytotoxicity dose
response studies in
SW620 colorectal cancer (CRC) tumor organoids and provide IC50 for exemplary
compounds as
indicated. Tables 4A and 4B below provides a summary of cytotoxicity data for
exemplary
compounds. Table 4A provides cytotoxicity data for representative single
compounds in several
different CRC tumor organaoids.
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Table 4A: Tumor Organoid Cytotoxicity
CHD1L Tumor Organoid Cytotoxicity IC50 (pM)
Inhibitor 5W620 HCT116 CRC042 CRC102
(PM) # (PM) (PM) (PM)
6 4.6 4.93 18.61 22.6
6.1 >40 >30 - -
6.2 >40 - - -
6.3 3.8 3.72
6.4 28.4 >30 - -
6.5 1.2 2.2 - -
6.6 >40 >30 - -
6.7 12,7 17.6 - -
6.8 3.6 6.85 - -
6.9 8.1 19.6 - -
6.10 22.6 >30
6.11 2.6 3.48 4.5 8.38
6.12 >20 >30 - -
6.13 >20 >30 - -
6.14 10.4 15.3
6.15 16.4 19.7 - -
6.16 1.4 2.43
6.17 5.5 3.86 - -
6.18 1.4 2.95 - -
6.19 7.7 >30
6.20 2.1 - - -
6.21 1.6
6.22 12.6 - - -
6.23 5.7 - - -
6.24 2.7 - - -
6.25 19.7
6.26 5.5 - - -
6.27 2.1 - - -
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6.28 >30
6.29 5.1
6.30 6.0
6.31 2.4
6.32 5.4
Table 4B provides results of combination treatments of the indicated
representative CHD1L
Inhibitors (CHD1Li) with SN38 or Olaparib. Treatments are performed in four
different CRC tumor
organoid types. The concentration of CHD1L inhibitor is varied as indicated.
IC50 for the
combination treatment are generally decreased compared to SN38 and Olaparib
alone. # updated
experimental for improved comparison among compounds, data rounded to one
significant digit
after the decimal point.
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to
Table 4B: Tumor Organoid Cytotoxicity Combination Treatments
\
Co4
Tumor Organoid Cyctotoxicity Combination Treatments
CHD1L
SW620 HCT116 CRC042 CRC042
CRC102 CRC102
Inhibitor
CHD1Li (pM) + CHD1Li (pM) + CHD1Li (pM) +
CHD1Li (pM) + CHD1Li (pM) + CHD1Li (pM) +
SN38 (nM) Olaparib pM SN38 nM Olaparib
pM SN38 nM Olaparib pM
(0 pM)- 356nM; (0 pM) - 377.8 pM; (0 pM)-
52nM; (0 pM) - 134pM; (0 pM) - 340nM; (0 pM)- 202pM;
(3 pM)- 191nM; (5 pM) - 114.4 pM; (14 pM)-
26nM; (22 pM)- 116pM; (20 pM)- 111nM; (22 pM)- 73pM;
6
(4 pM)- 42nM: (6 pM) - 64.7 pM; (18 pM)-
23nM; (25 pM)- 58pM; (22 pM)- 56nM; (25 PM) - 70pM:
(5 pM)- 8nM; (7 pM) - 17.4 pM: (22 pM) -
6nM; (28 pM)- 42pM; (25 pM)- 26nM; (28 pM) - 62pM;
(0 pM)- 356nM;
(2 pM) - 96nM;
6.3
(3 pM)- 6nM;
(4 pM)- 1nM;
(0 pM) - 261.6 pM; (0 pM)- 52nM: (0 pM)
- 134pM; (0 pM) - 340nM; (0 pM)- 202pM;
(0 pM)- 356nM;
6.11 (2.5 pM) 9nM; (2.5 pM) - 300.8 41; (3.5 pM)-
43nM; (4 pM) - 52pM; (6 pM) - 305nM; (6 pM)- 50pM;
-
(3.5 pM)- 16,59 pM: (4.5 pM)- 8nM; (5 pM) -
23pM; (8 pM) - 16nM; (8 pM)- 37pM:
(3 pM)- 8nM: (4.5 pM) - 8.44 pM; (5 pM)-
1nM; (6 pM) - 10pM; (10 pM)- 6nM; (10 pM)- 11pM;
(0 pM)- 356nM;
(8 pM)- 228nM;
6.16
(9 pM)- 197nIVI;
(10 pM) - 224nM;
(0 pM)- 356nM;
6.16 (1.25 pM)- 205nM; _
(1.5 pM)- 90nM;
(1,75 pM)- 5nM;
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FIG. 8B presents a graph of y-H2AX intensity (relative to DMSO) for compound 6
alone, irinotecan
(SN38) alone, and a combination of the two in DLD1 empty vector (EV) cells and
DLD1 (OE)
overexpressing cells. FIG. 8A is a Western Blot showing relative expression of
CHD1L in
DLD1(EV) cells compared to DLD1(0E) cells compared to control expression of cc-
tubulin in these
cells. CHD1L is known to be essential for PARP-1 Mediated DNA Repair, causing
resistance to
DNA damaging chemotherapy [Ahel et al., 2009; Tsuda et al., 2017]. Data in
FIG. 8B demonstrate
CHD1L inhibitor "on target" effects that synergize with SN38 inducing DNA
damage.
FIGs. 9A-9C illustrate the results of synergy studies with exemplary CHD1L
Inhibitors 6, 6.3, 6.9
and 6.11 in SW620 Colorectal Cancer (CRC) Tumor Organoids. SN38 combinations
with 6, and
6.3 are 50-fold, and 150-fold more potent, respectively, than SN38 alone in
killing colon SW620
tumor organoids. SN38 combinations with 6.9 and 6.11 are both over 100-fold
more potent than
SN38 alone. Each of compounds 6, 6.3, 6.9 and 6.11 shows synergism with
irinotecan (and SN38)
for killing SW620 tumor organoids.
Synergy scores for exemplary CHD1L inhibitors where scores are determined as
described in De
Veroli et al. 2016 are provided in Table 5. For interpreting the value of
synergy scores, as
SynergyFinder has normalized input data as percentage inhibition, they can be
directly interpreted
as the proportion of cellular responses that can be attributed to the drug
interactions. (e.g., synergy
score 20 corresponds to 20% of response beyond expectation). According to our
experience, the
synergy scores near 0 gives limited confidence on synergy or antagonism. When
the synergy
score is:
Less than -10: the interaction between two drugs is likely to be antagonism;
From -10 to 10: the interaction between two drugs is likely to be additivity;
Larger than 10: the interaction between two drugs is likely to be synergy.
Table 5: Exemplary Synergy Scores of SN38 with Representative Compounds
LOEWE Synergy Scores (Compound No.(1C50 of Compound))
SN38(nM) Cpd 6 (5pM) Cpd 6.3 (3pM) Cpd 6.11
(3pM)
0.64 -5 4 46
3.2 36 31 54
16 35 36 52
80 45 54 49
400 41 48 48
2000 28 35 36
10000 16 25 25
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FIG. 10 includes a graph of tumor volume (fold) SW620 tumor xenografts as a
function of days (up
to 28 days) of treatment with Compound 6 alone, irinotecan alone or a
combination thereof. The
combination of irinotecan and Compound 6 significantly inhibit colon SW620
tumor xenografts to
almost no tumor volume within 28 days of treatment compared to the single
agent treatment
groups or control.
FIG. 11 includes a graph of tumor volume (fold) SW620 tumor xenografts as a
function of days (up
to 28 days) of treatment with irinotecan alone (1) or a combination of
Compound 6.0 and irinotecan
(2). The combination of irinotecan and Compound 6 significantly inhibits colon
SW620 tumors to
almost no tumor volume beyond the last treatment compared to irinotecan alone.
Within 2-weeks
of the last treatment of irinotecan alone tumor volume rose to above the
volume of the last
treatment, signifying tumor recurrence. In contrast the combination maintained
a lower tumor
volume.
FIG. 12 shows that Compound 6 alone and in combination with irinotecan (4)
significantly
increases the survival of CRC-tumor-bearing mice compared to vehicle (1),
Compound 6 alone (2)
and irinotecan alone (3).
FIG. 13 includes a graph of tumor volume (fold) SW620 tumor xenografts as a
function of days (up
to 20 days) of treatment with Compound 6.11 alone, irinotecan alone or a
combination thereof.
The combination of irinotecan and Compound 6.11 significantly inhibits
colorectal cancer SW620
tumor xenografts compared to irinotecan alone or control.
FIG. 14 includes a graph of tumor volume (fold) SW620 tumor xenografts as a
function of days (up
to 33 days) of treatment with irinotecan alone or a combination of compound
6.11 with irinotecan.
The combination of irinotecan and compound 6.11 significantly inhibits
colorectal SW620 tumors
beyond the last treatment (day 33) compared to irinotecan alone. Eight days
post treatment (Tx
Released), tumor volume with irinotecan treatment alone rose ¨3-fold,
signifying tumor recurrence.
Conversely, tumor volume with treatment of the combination of 6.11 and
irinotecan continued to
drop (by ¨1.5-fold) post treatment. The difference in tumor volume between
treatment with
irinotecan alone and treatment with the combination of 6.11 and irinotecan 8
days post treatment is
3.4-fold.
FIG. 15 shows that Compound 6.11 in combination with irinotecan significantly
increases the
survival of CRC-tumor-bearing mice compared to irinotecan alone and control.
Example 11: Summary of Currently Preferred Structure Activity Relationships
for Inhibitors.
The currently preferred structure activity relationship based on formula I for
CHD1L Inhibitors of
this invention is as follows:
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X
RE; x
(I-1)x
RA A
(LoRH
For the B ring, it is currently preferred the ring is a 6-member aromatic or
fused 6, 6-member
aromatic ring and that both X are N. The B ring optionally contains a fused
ring, which if present,
can contain one or two additional N. Preferred RB (B ring substitution), if
present, include
hydrogen, alkyl and fluoroalkyl groups. In certain embodiments, where x is 1
and L1 is present, and
preferably L1 is ¨CH2-, R5 can be an electronegative group, such as a halogen
and particularly F or
a haloalkyl, particularly CF3-. Preferred RB are hydrogen or C1-C3 alkyl. The
preferred A ring is
optionally substituted phenyl, with unsubstituted phenyl (where RA is
hydrogen) more preferred.
The Rp group is believed to be associated with water solubility, with -
N(R2)(R3) groups generally
preferred and more particularly preferred optionally substituted N-containing
heterocycles, where
R2 and R3 together with the N to which they are attached form a 5- to 8-member
ring which may
contain one or more additional heteroatoms and which may be saturated (no
double bond) or
contain one or more double bonds. RH is believed associated with activity and
potency as well as
metabolic stability. RH is preferably an aromatic group and more particularly
a heteroaromatic
group with ring substitution that stabilizes the aromatic or heteroaronnatic
ring. Preferred Y is NR
with R that is hydrogen more preferred. Preferably x is 0 except as noted
above. Preferred Z is ¨
CO-NH-. Preferred L2 is ¨CH2- or ¨CH2-CH2-. Preferred L1, when present is ¨CH2-
.
In an embodiment, HTS screening for CHD1L identified a phenylamino pyrimidine
pharmacophore
illustrated in formula )0(:
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R4 Ny RN
N
R5
Re
R7
Ri
R10 R8
R9
and salts thereof, where RI-Rs represent hydrogen or optional substituents,
Rio is a moiety
believed to be associated with potency; and RN is a moiety believed to be
associated with
physicochemical properties such as solubility. In embodiments, R5 is a
substituent other than
hydrogen which is believed to be associated with metabolic stability. In
specific embodiments, R5
is a halogen, particularly F or Cl, a 01-03 alkyl group, particularly a methyl
group. In
embodiments, R4 is a substituent other than hydrogen and in particular is a C1-
C3 alkyl group, and
more particularly is a methyl group. In a specific embodiment, R5 is F and R4
is methyl. In
embodiments, R6-R9 are selected from hydrogen, C1-C3-alkyl, halogen, hydroxyl,
C1-C3 alkoxy,
formyl, or C1-C3 acyl. In embodiments, one or two of R6-R9 are moieties other
than hydrogen. In
an embodiment, one of R6-Ro is a halogen, particularly fluorine. In specific
embodiments, all of R6-
Rg are hydrogen. In embodiments, RN is an amino moiety ¨N(R2)(R3). In specific
embodiments, RN
is an optionally substituted heterocyclic group having a 5- to 7- member ring
optionally containing a
second heteroatoms (N, S 0). In embodiments, RN is optionally
substituted pyrrolidin-1-yl,
piperidin-1-yl, azepan-1-yl, piperazin-1-yl, or morpholino. In RN iS
substituted with one substituent
selected from C1-C3 alkyl, formyl, C1-C3 acyl (particularly acetyl), hydroxyl,
halogen (particularly F
or Cl), hydroxyC1-C3 alkyl (particularly ¨CH2-CH2-0H). In embodiments, RN is
unsubstituted
pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, piperazin-1-yl, or morpholino.
In embodiments, Rio is ¨NRy-00-(L2)y-R12 or ¨CO-NRy--(L2)y-R12, where y is 0
or 1 to indicate
the absence of presence of L2 which is an optional 1-6 carbon atom linker
group which linker is
optionally substituted and wherein one or two, carbons of the linker are
optionally replaced with 0,
NH, NRy or S, where Ry is hydrogen or a 1-3 carbon alkyl, and R12 is an aryl
group, cycloalkyl
group, heterocyclic group, or heteroaryl group, each of which is optionally
substituted. IN
embodiments, y is 1. L2 is ¨(CH2)p-, where p is 0-3. In embodiments, R12 is
thiophen-2-yl,
thiophen-3-yl, 4-bromothiophen-2-yl, furany-2-yl, furan-3-yl, pyrrol-2-yl,
pyrrol-3-yl, oxazol-4-yl,
oxazol-5-yl, oxazol-2-yl, indo1-2-yl, indo1-3-yl, benzofuran-2-yl, benzofuran-
3-yl, benzo[b]thiophen-
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2-yl, benzo[b]thiophen-3-yl, isobenzofuran-1-yl, isoindo1-1-yl, or
benzo[c]thiophen-1-yl. In
embodiments, R1 is hydrogen or methyl. In embodiments, R12 is thiophen-2-yl,
furany-2-yl, pyrrol-
2-yl, oxazol-4-yl, indo1-2-yl, benzofuran-2-yl, or benzo[b]thiophen-2-yl. In
embodiments, R12 is
thiophen-2-y1 or indo1-2-yl. In embodiments, Ri is hydrogen or methyl.
Exemplary compounds of the invention are illustrated in Scheme 1. In Scheme 1,
Xis halogen and
preferably Cl or Br. In Scheme 1, R is 01-05 alkyl or cycloalkyl, and
preferably a 01-03 alkyl or
cyclopropyl and more specifically, methyl, ethyl, n-propyl or cyclopropyl.
Exemplary Rp and -N(R2)(R3) groups for any formulas herein are illustrated in
Scheme 2.
Exemplary R12 and RH groups for any formulas herein are illustrated in Scheme
3.
Exemplary B rings for formula I are illustrated in Scheme 4.
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Scheme 1: Exemplary Compounds of Formula I or Formula XX-XXIII
0
0 0 riZ)
II ,) .-=-
.,.,,,,,N.,,)
S¨N
S¨N
II H II H
0 0
1 2
\c)
0 rCf N o
IIs-J
I. s) <N
S¨N
II H H SII¨N---cc
si7"-
0 0
N¨N
3 4
. P 0
---T-I N
S N \
# X.
0
N,H 0,,--------\ 0
7%
5 S N 6
0 H
OH
Nr\l. N..) I
....1\1
C I Y
N,--r-N
0 010 N-'1-1
CI dim N,.
itilli H 7
HN 1 N
H 8 (also
6.3)
15
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Scheme 1 (continued)
N 0
N 0 y
yF../.'yI N
F
0 ,
N, 0
/ \ 0 010 H
S N N N H
/
H
H (6.2) HN 10
OH
r.
r--------N----
ITh
N
N...,...õ)
N, 0
/ 1 0 411 H
140 N,H
N
S N 11 Ji H 12
H HN
N Nõ,) i Y
LT N
F F
- N
, 0 0 -'1-1
/ 1 0 0 N H
N 14
S N 13 Ii H
H HN
N N 0
N N 0
C I Y D c.. ,..-_,,N C N
T,
N
CI N / \ 0 0 N,
H
15 16
S N
H
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Scheme 1 (continued)
OH
..-----.õ.õ..----
N N 0
C I
N N rN
N...,)
NrN C NXr%r,
0 N,
H
I N
H lel 17 / \
S 0
N 40 N.'1-1 18
HN H
OH
r---N
N 0
N
.L,..., Xr) i
0
010 N.'1-1 19 N opi N,H
/ \ 0
I N
H S 20
HN H
N NO
r"=-...,,,.N_ 0
FN
N,H 0 411 N.,.
I N
H 21 S N
H
HN 22
OH
-*=,,,,,,N,.., 0 r'N"--.4'''--
--.-.
F
N,
0 011 H N,
I N
H
(
23 4111 24
,s N -1 LC) H
HN H
134
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Scheme 1 (continued)
OH
r----N'-'---'-'
Ny_N.,J
N
F
O 0 N,
H n N,
H W 411
, N
I H 25
\''..-uN 26
HN H
OH
r------N-----,---- N 10
y
CI
N
F N H HN N,H
O 0 ,
111,
N 27 Nt I H
HN H
28 (6.18)
.f.IN I Y
-,f--N
0 0 NH
0 H 0 NH
N
1 N H
I---- "ss
N
5 H3d N
29 (6.19) 30 (6.20)
NO 0 N 0
-'i.,N
NH NH
0 0 0 0
N N
H H
0
NH / NH
31 (6.21) 32 (6.22)
135
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Scheme 1 (continued)
õr, N
0 01 NH
0 0 NH
N rAN
H N H
Br
NH II /
33 (6.23) 34 (6.24)
N r\D
1 'r
=.i,N1
NH
0 N0 NH 0 0
r-ILN
H
H N
HO N /
NH
35 (6.25) 36 (6.26)
I 1
0 0 NH
0 NH
N riLN0
H N H
N
NH
110 N
37 (6.27) 38 (6.28)
136
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Scheme 1 (continued)
=,.y.1 N
N
0 .,..,...Ny0
N H
H
N
N0
4 0 b S 11 N,H
µS-- H
(--N
F3C 0
39 (6.29)
-..,.Ny.0
1N 0 Y
(.,
. H N N.,
N 0111 H ¨1H
N 0
42H
I HN 0 41 0
r Y I Y
...,,r.N r.N
F
(, ,
IIIP H N
N 0 H --.H
N 0N H
44
1 43
5 HN 0 0
137
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Scheme 1 (continued)
NO
IF--''-rN
H
N 4111 N ,
H
<riH
N 10 N.
H
I 45 46
HN 0 0
F
(-3
01H
N, N,H 1d1
(-1,_. 14111
3 47 S 48
0 0
5
,le,I N INI(N1
111 0
N, N, H 0 kil ip 010 H
I 49
I
HN 0 HN 0
YrNj
F
0 N,H
110 rl 51
I
HN 0
138
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Scheme 1 (continued)
CrIN
( XrXi
N
S
N . H 0 j 11101 Br-CilL 0 N. H Br_a/
N N
H H
52 (6.11) 53
N Ik: ck - NO
1 .1...1
N . H 0 Oil N - H
1 11
''-'-',-===-'-'- -A N N
H H
54 (6.16) 55
rskT,Is0
,,N 0
1 ...'c N
N
N'H N . H
(S) j:IL SI C3 ';---------'µ. 0
N 0
N
H A
56 (6.4) 57 (6.5)
N NO N NO
1 ...., A
N
t.:-.:=,.,=
' 40 -1
_ 9 SI N . H \.:''' 0 N . H
f( -1
ii A A
11N'
58 (6.6) 59 (6.7)
139
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Scheme 1 (continued)
N NO N 0
1 1
9
N.H :.
=-",,.--..-,..
/ = =======-----. 0 40 N.H
-
N..., 'ii N
HU- A H
IiiN---
60 (6.8) 61(6.9)
N NO N Ci
1 2r ri 1
1...fq
H3CO" y
N 0
c)}S 0 N 0 11
1110
N
H H
62 (6.10) 63 (6.12)
_
I \
N 0 1 ;Nii
I..... ,......_
..1..N -1.
--,,,z -0
-1-
........,s;i--
N - = q H
H HN-
64 (6.13) 65 (6.14)
...--,
1 9 rio
NN)
1 1
.rN
0.N
c
N N.H ,S 0 401 - H Br¨ S 0 0
CLõ)/ - L
N N
H H
66 (6.15) 67
140
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Scheme 1 (continued)
r-0 1
(NrXN) 0 N,H
0
0 101 N.H , N
I H
N N
H SENA
68 69
N N 0 n\1
0 0 r\l'H 0 401 NH
N N
H H
, N ---. N
\ / NH \ / NH
N N
70 71
N N r0 N,.õOH
CNI)1; N N,, N,..)
CN;);
0 NH
0
N 0 NH
r-J-L 0
rj-LN
N H
N H
1, 1 IIP 1
72 73
NO
1 r'll
N
0 so NH
0 HN 0 0 NH N -- 1
I
-,.
N CI N
H 74 (6.30) H
75 (6.31)
141
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Scheme 1 (continued)
1 'r
, *
N NH
1 WL)
0 NN NJ
N N
H H
76 77
FN
, * , *
¨t 01 FrrN NO
Br} =
r-- .÷, S 0
CIW 0 H
NN NO
N N
H H
78 79
F-,,---N E.,..,,---
N
_cSkits 0 Ftl--.N NO 0 0 Fr
XN NO
N N
H H
80 81
X
F..,..,-,,,..N N
0 0 N-N NO N-- 1 0 (110
H Nis,1 NO
H
N N
H H
82 83
F., N F,,,,----N
N N NO
H HN ,- 0 0 FrfN NO
N N
x-
H H
84 85
142
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Scheme 1 (continued)
X
FN Fr N
0 lei li.sil N NLD R 0 401 vi N NLD
HN -N ---
N
N
H H
86 87
, * 0 101 NI N NO
0 Ni¨N NO
N ,z N
R-N
N0 H
H
H R
88 89
X
FN F.,J
-'" N
0 101 111- -N NO 0 0 NI N NO
HN ,-
N HN ,,.
N
H
R H
90 91
I
X R
I }
, * e.,- 9
6.....-<õ(...11.. ,N 1:-.>
0 /101 itil --- N N3 4 )>, =-''..-'-ti-k,,,--'
HN ,- \1...õ=<' ir H
N N'
H H
92 93
Fri C F,,,..-.N
0 101 rli N NO 110, , fl,
I ---- N 0 so NN NJ
1 H N NAN
N
H H
94 95
143
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Scheme 1 (continued)
CI
F.,,e....õ-,,N F.,
,,_,...--..N
411 0 0
N--N NO * 0 0 N-.N* No
R N N -ILNJ H N NN H
H H
96 97
CI
F.r.L.N
Fq
4111 0 0
N N NO
N2
0 101 N--'N NO
H
R N N,õAN H HN / N
H H
98 99
F.,.)-..N --" F
N
r,
N 0 FICN NO N " 1 0 101 11 N NO
N CI N
H H
100 101
Fr, F-j-,N
-" N
JJOHNNO l.H N N NO
Br----1---,...N
0 0
102 103
F.,-.....N
N ' I M 11N N NO
H N "
I H 0 ril N NO
N
CI
0 0
104 105
N N 0 N N NO
C :ii C I Ti
NI T N'Th".
N " 1
1 0 0 NH
N " 1
1 0 0 NH
-. -.
CI N CI N
H H
106 107
144
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Scheme 1 (continued)
NN) NN)
1 Y 1
1,.4.NJ N ' 1
1 0 401 NH
N ' I 0 401 NH
CI N N
H H
108 109
rN.---..,õ.0H r..N.--,,..0H
_)N
(NirrscN)
'c N'Nr
1 0 NH
N' , 0
1 AI NH
-,
CI N N
H H
110 111
r(:)
N CI
1 Y
N r.N1
N' 1
1 0 0 NH
NJ NH
1 0 0 NH
--.
CI N N
H H
112 113
N
NH
N' I H 0 NH
CI
0 0
114 115
I rj
NH
0 0 SI
.AN
H
I 116
N õ---
145
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Scheme 1 (continued)
1 Y
.f..1q
0
N 0 NH Br *
/ 1 0 N 41 rli N 0
H
=== N
I S ,-
H
117 (6.32) 118 (6.33)
...õrN 0 0 Br ...,r, N 0
0
(.30 N 0 NH N 40 NH
/ 1 0
S S
H H
119 (6.34) 120 (6.35)
0
NNr Isc -.,.N N,) .,, HN
1
0 OH ..T.-.N
Br Br
/ 1 0 0 NH / 1 0 0 NH
S N S N
H H
121 (6.36) 122 (6.37)
NO NO
I r;ri
,.f,INI
0 0 NH NH
e__3N 0
(--- N
K)LO 0
N
H H H
123 (6.38) 124 (6.39)
(NH
Yrs1)
,k-Br N Br N
A ,
/ 0 0 NH
/ 1 0 4110 ill isr-NNO
S N S N
H H
125 (6.40) 126 (6.41)
146
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Scheme 1 (continued)
N,, NO N 0
I Y
...r.N
cii
el NH
rut
N
NH ral ifio
N N N
H H
127 (6.42) 128 (6.43)
N N'I-
Br ,,..., N
r
.,k
/ 1 0 N 400 N N N B5Th 1 0 411 N N 14 --'-Th
H ,1%1 H
1%L
S S
H H
129 (6.44) 130 (6.45)
-'.---N N---''
Br ., * Br
.,. _.
/ 1 0 41111 [sli-N NO / \ Oil N N---N-
''''''.
H
S N S N
H H
131 (6.46) 132 (6.47)
risl
NN)
Y I Y
..T.,..N ..T.,..-N
Br I Br
/ 1 0 0 NH / \ 0 0 NH
S N S N
H H
133 (6.48) 134 (6.49)
N.,...,--
I Y I Y
r------i- 0 N
N 1,. 0 NH
N.ki-------1 0 N is NH
.,_,N, j.1..,
H H
135 (6.50) 136 (6.51)
147
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Scheme 1 (continued)
NN) -
..,....õ,N..õ.N
1 'r r N
H2
T---=-1 0 0 NH Br¨ s 0 0 NH
hit.,,NA C1-},
N N
H H
137 (6.52) 138 (6.53)
.._,....õ,N.,,,N I--
I / ¨N
--...r. N-N \--
Br¨a)I lei 11 is" Br¨ /õØ,,,)1,
0 0
N NH
H NH2 H
139 (6.54) 140 (6.55)
--1`N
Br-C/S 0 SI F1rN N N I
,...).õ.
ilt, =N-----X
____a_)t.,/,,,S 0 4111 N N N.."1
N H
1-._,NH
H &\1 Br
N
H
141 (6.56) 142 (6.57)
148
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Scheme 1 (continued)
0 CF3 CF3 N
1 rTi
Br_U.( N N NO
H Br¨C-1ILS
N N 0 NH
H H
150 (6.58) 151 (6.59)
CF3
CF3....N 0
I
As
__i,)1,/,_,S 0 40 N NNu.D 9 411) NH
Br H n
N µIsl--.'N
H H H
152 153
CF3
rl'N N 0 0
v. , 0 ri N NO rilS 0 0 N NO
Br¨C..1,,,)1,,...-
µN1 N N
H H H
154 155
09e---\
N
1:(
.-N
N-=-= 9, p
s 0 0 NH Br¨
Br¨ , s
1,..Ø.,_A S 0 4110 1.4"-fkilliC.,....,A/.....-
H
N N
H H
156 157
q
0-,:!D
1 'r
NH
n 9 Si N N Ns
H
C)
N N leN
H H 158 H H 159
149
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Scheme 1 (continued)
0
(IkIA`v
.r.N -..y.N
,n,,,
S 0 NH s 0 Br-0,,A
Br¨C)j.L
N N N 0 NH
H H
160 161
0
rikIATD
N..-NFI2
NN) N N --
,.,õ,..õ..-I
.,r.N ..1.5.N
s 0 0 NH s 0 0 NH
Br-L,).. Br-(LA N N
H H
162 163
0,
µs....--........õ--
r-- sisl- b
'rirNI)
,N
1 ,k
Br-}.
N
0 0 NH S 0 .
Br¨a.).1,/,...- N N N-N."1
H ,Isli(A
N
H H 0
164 165
I ,) N_
Br_CSK)01, 0 N NN] H I ,r(c)
B S 0 0 NNNI..õõN r¨Cc}L/
H
N
H 0 H
166 167
0 N
..9.,
S 0 0 N N N-Th S 0 IS Fisi N NO
Br-)L L,,,N, P Br
N ,S,..,,,-,-, ) N
H 0' H
168 169 (6.60)
150
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Scheme 1 (continued)
N 0
(,r7r, 1 'r
H N
NH
___C)}L,/,,,S 0 41110 S 0 4110 NyNH
Br 0 Br¨A/,-=
0
N N
H H
170 171
NN 0
I Y
.1...N
0 NH
s 0 0 NH 0
Br---CT A NAN
N N 5 H H Br¨CH H
172 173
H H
1 *r 1
-,,r--N 0 --y-N 0
s 0 0 NH s 0 0 NH
Br¨ 1t, Br¨C1-7.},
N N
H H
174 175
H
Br\ f:IL
N N N NN H2
--- )-rsi s o 0
S 0 (.1 ..,.._ _NH CL.,).L..- H H
N--1*/* N ¨ 2 Br¨ N
H H H
176 177
151
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Scheme 2 (Exemplary Rp and -N(R2)(R3) groups)
1--- NO 1--- NO-R
)- 1-N/\ ) I-N/ _____________________________________________________________
R
\ ___________________________________________________________________________
-
RN 1 RN2 RN3 RN4
0 0 R
/ _______________________________________________________________________ 1
R \ ___________ \
R
RN5 RN6 N7 RN8
R = hydrogen, alkyl, OH, acyl, acyloxy, alkoxycarbonyl, carboxyl, halogen,
trifluormethyl,
hydroxalkyl, -CH2CH2-0H
HO ___
/ \N / N
1Nr¨N
N-R
1---N\
/ \ _____
/
RO
RN9 RN10, RN11,
R = alkyl, -COalkyl, -CONH2, R = H,
alkyl, acyl, OH,
-CONH(alkyl), -CON(alkyl)2 SO2alkyl
1----N/ \ /\ F_ _N 0/ \
Y 1---N 0
\ ___________________________ / \ ______ /
RN12, Y = S, SO2 RN13 RN14,
R= alkyl, acyl, acyloxy,
alkylcarbonyl, carboxyl,
OH, hydroxyalkyl
152
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Scheme 2 (continued)
--"----N,,--NR
RN16,
R = H, alkyl, acyl, acyloxy, RN16,
R = H, alkyl, acyl, acyloxy,
alkoxycarbonyl, carboxyl,
alkoxycarbonyl, carboxyl,
OH, hydroxyalkyl
OH, hydroxyalkyl
/ \N ________________________
1---N
RN17 RN18
R = H, alkyl, OH, hydroxyalkyl,
_______________ N o / \ / _.--\ R
¨ \ acyl, acyloxy, alkoxycarbonyl,
N¨N carboxyl
1
RN19
E-N/ \N _______________________________________________________ ( N
N-N N-R
NR
RN20, R = H, alkyl, OH, acyl, alkoxycarbonyl, RN21, R = H, alkyl, OH,
acyl,
S02-alkyl alkoxycarbonyl, S02-alkyl
/ \ / \ R
/ \
1¨N N¨N
\ ___________________ / \ _________ / \ ___ /
RN22, Y = S, SO2 RN23, R = H, alkyl, acyl, acyloxy,
alkoxycarbonyl, carboxyl, OH, hydroxyalkyl
1--N/ _______________ \ _,..---.........-----.,..,..
\ ___________________ /N
RN24, R = H, alkyl, acyl, OH,
R hydroxyalkyl, S02-alkyl
153
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Scheme 2 (continued)
RN25,
R = H, alkyl, acyl, acyloxy,
alkoxycarbonyl, carboxyl, OH,
hydroyxalkyl
is¨NO
11-\11¨<1
O
RN26 RN27 RN28 R = H, alkyl,
acyl,
acyloxy, alkoxycarbonyl,
carboxyl, OH,
__________________________ \R
_______________________________________________________________________________
_ k R
______________ 111 ___ (
RN3o RN31
RN29
R = H, alkyl, acyl, acyloxy, alkoxycarbonyl, carboxyl, halogen, OH,
hydroxyalkyl
H NRR
N
_______________________ N m is 1-6
RN32 R = H, alkyl, acyl, RN33 m = 1-6, each RN34
acyloxy, alkoxycarbonyl, R independently is
carboxyl, OH, hydroxyalkyl H, alkyl or aryl
154
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Scheme 2 (continued)
0
0 0 li
_ .......s
/ ----Th R
N N NRR \-N" 1 __ N I
m R n
R H \(CH2)r
RN35 m = 1-6, each RN36 R is H, alkyl, RN37 r is 1-6, R
is H, alkyl, acyl,
R independently is aryl, heterocyclyl acyloxy, halogen,
OH,
H, alkyl or aryl hydroxyalkyl
0 0
(D
0
/
---S--... S ___
1 _____________ N 1 ___________ N/x )
\,-----
RN38 RN39
155
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Scheme 3: Exemplary R12 and RH Groups
(S-21/4. ) (0,
S 0
R12-1 R12-2 R12-3 R12-4
R'
1
R'N..-N, A R'\ ...,<-
\,..N
1 1 .=,,,.-------' N
R R R12-7
R12-5, R12-6, R is H or alkyl
R is H, alkyl R is H or alkyl R' is H, alkyl,
acyl, halogen
R is H, alkyl, acyl, halogen R' is H, alkyl, acyl, halogen
0
R'
1 I"
0 NR NR
)
R
R12-7 R12-8 0 R12-9 0
R is H or alkyl R is H or alkyl R is H or alkyl
c
R..4.,,,-...-..='...,,..., --N\ 7 ¨1
R.'"........--N\ /s"----(CF12)P-1
1 i 1
N --;;----N ==.,... õfj------.N
N i
R12-10, R12-11,
R is H or alkyl, R is H or alkyl,
R. is H, alkyl, acyl, halogen R' is H,alkyl, acyl, halogen,
p is 1-5
H(CH2)p H(CH2)P =
H(CH2)p =
MrCF3 0"....R
F3C
R12-12, p is 0-3 R12-13, p is 0-3 R12-14,
R is H, alkyl, phenyl, -COH,
acyl
p is 0-3
156
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Scheme 3 (continued)
F(CI-12)P
1
0 0 /\ R
0
R12-15, R12-16,
R is H, alkyl, phenyl, acyl R is H, alky. phenyl, -
COH, acyl
p is 0-3 p is 0-3
0
1-(C1-12)12.,,
H(CH2)P
1 -......,./...\NR
N 1
R
R12-17, R12-18,
R is H, alkyl, acyl, phenyl R is H, alkyl
p is 0-3 p is 0-3
R'
1¨(CH2)1D/1 "**.*-
1
rl I
0
)-----R 0 )
R12-19 R12-20 N-......._
N
R is H, alkyl N R is H, alkyl
R is H, alkyl, acyl, halogen R' is H, alkyl, acyl,
halogen
p is 0-3 p is 0-3
0 0
H(CH2)1
H(CH2)p___,../\ NR NR
1 1
R' -,....,,.....,...0
R12-21 \ / R12-22
\\ )-----R.
R is H or alkyl
R is H, or alkyl
p is 0-3
p is 0-3
157
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Scheme 3 (continued)
----- -----
si CS S s'ICO 0
----..._
R12-23 R12-24 R12-25 R12-26
IsiNc.N5 NR ------
I
/ 1 / 1-EN .0- NR
---..., NR
R12-27 R12-28 R12-29 R12-30
R is H, or alkyl R is H, or alkyl R is H or
alkyl R is H or alkyl
\---------.\..r.\\ H(CH2)1?,..õ.--"o's,,s_
fr\O 0 N 1 ___ 0 ii
.----- --zzi N _____ N
N--.-:,.--/
R12-31 R12-32 R12-33 R12-34
p is 0, 1 or 2 p is 0, 1
or 2
(CH2)p-1 IR'
RT........_____
R'..,.._
(CH2)p-i
'..--.,./"------ N I -'\,= __ 0
R12-35 R12-36 R12-37
R is H or alkyl R' is H, alkyl, acyl R' is H,
alkyl, acyl
R' is H, alkyl, acyl or halogen or halogen
or halogen p is 0, 1 or 2 p is 0, 1 or 2
p is 0, 1 or 2
(CH2)p-1 R' (CH2)p-
1
IR___
1)- (CH2)p-1 .-IS\----....'s=-..--;,-------(,\---
..------ \
1 .-.,----- /...,_
NR
R12-38 R12-40
R12-39
R' is H, alkyl, acyl R is H or alky
R is H, alkyl, acyl
or halogen R' is H, alkyl,
acyl
l haogen
p is 0, 1 or 2 or or halogen
p is 0, 1 or 2 p is 0, 1 or 2
158
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Scheme 3 (continued)
(CH2)P4 (CH2)P4
*.-'¨'.-----s---i---c
0 S
j------
R12-41 R12-42
R' is H, alkyl, acyl R' is H, alkyl, acyl
or halogen or halogen
p is 0, 1 or 2 p is 0, 1 or 2
''''''..',=*'-''''''..-1 27...1'' 0 `._
I 1 1
R R' R' /
R12-43, R12-44, R12-45,
R' is H, alky, acyl, halogen R' is H, alky, acyl, halogen R' is H,
alky, acyl, halogen
1 1
N1---.-.-----N N"-------N N-.--'---N
H H H
R12-46, R12-47, R12-48
R' is H, alky, acyl, halogen R' is H, alky, acyl, halogen R' is H,
alky, acyl, halogen
1 1
1
N
R' N R, ----- ---- R,, N
R12-49, R12-50, R12-51,
R' is H, alky, acyl, halogen R' is H, alky, acyl, halogen R' is H, alky, acyl,
halogen
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Scheme 3 (continued)
*MAW,
N '''...-'=''.= N 1 N
I I
R'
A.N ke YNI
R' R'
R2-52, R12-53, R12-54,
R' is H, alky, acyl, halogen R' is H, alky, acyl, halogen R' is H, alky,
acyl, halogen
R' Ri
- ...., , . - -. .. , , , . / --===== - , .,., ,.. / - " '.-,,,,,- = , ,
i s sY= k _. . -\.- -- - =-======,= . - =,'''. .. .' n n
1 1 10
R' R'
R12-55, R12-56, R12-57, R12-58,
R' is H, alky, acyl, R' is H, alky, acyl, R' is H,
alky, acyl, R' is H, alky, acyl,
halogen halogen halogen halogen
~AWN
R' R ' \ / \ . - " - = , ) %,- .
/ \ ,
- ' ' 4. - ..-k='/ - . . \- - k =`2, : 1 . ." ' ) 1 - -
1
-,___,.=---,,,,,- N -,- N R,/
R'
R12-59, R12-60, R12-61, R12-62
R' is H, alky, acyl, R' is H, alky, R' is H, alky, R'
is H, alky, acyl,
halogen acyl, halogen acyl, Halogen
20 halogen
.¨õ..
G
N,.....,,,...N...,.,,,\. N,...-.......k.N....., ....õ...--,....,..
.....A,,,... N.,... ...........õ.......õ...R.....,,1
N ft R'
. /..,,%\.N%--%"-,..N*----.
R " R' N
R12-64,
R12-63, R12-66, R12-65,
R' is H, alky, acyl,
R' is H, alky, acyl, R' is H, alky, acyl,
R' is H, alky, acyl,
halogen halogen halogen halogen
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Scheme 3 (continued)
R' R' '0 R' 0
R12-67, R12-68, R12-69,
R' is H, alky, acyl, R' is H, alky, acyl, R' is H, alky,
acyl,
halogen halogen halogen
?1/4
(CH2)p
Rs
Rs X11
Rs Xi
R12-70, R12-71,
X11 is CH or N; p is 0, 1 or 2; X11 is CH or N; X10 is CR or N;
R is hydrogen, C1-C6 alkyl, 04-07 p is 0, 1 0r2;
cycloalkylalkyl, -S02-R', phenyl, or R is hydrogen, 01-06 alkyl, C4-
C7,
benzyl; cycloalkylalkyl, -S02-R',
phenyl, or
R' is hydrogen, 01-06 alkyl, C4-C7, benzyl;
cycloalkylalkyl, phenyl, or benzyl; R' is hydrogen, C1-06 alkyl, 04-
07,
each Rs, independently, is hydrogen, cycloalkylalkyl, phenyl, or
benzyl;
halogen, hydroxide, 01-06 alkyl, each Rs, independently, is
04-C7cycloalkylalkyl, phenyl or hydrogen,
benzyl or C1-03 alkoxide halogen, hydroxide, 01-06
alkyl,
C4-C7cycloalkylalkyl, phenyl or
benzyl or 01-03 alkoxide
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Scheme 3 (continued)
I I 0
=-=.,, N
N CI N H
R12-72 R12-73 R12-74
2\ 2µ
(CHAp (CH2)p
Rs Rs
--.-- / /
I I
-....,
Rs
X11 N..,...... ...,....,õ
Rs N X ^
R12-75 R12-76
R12-75, R12-76,
X is halogen, particularly Cl and Br; X is halogen, particularly Cl
and Br;
p is 0, 1 or 2; p is 0, 1 or 2; X11 is CH or N;
each Rs, independently, is hydrogen, each Rs, independently, is
hydrogen,
halogen, hydroxide, C1-06 alkyl, halogen, hydroxide, C1-C6
alkyl,
C3-C7-cycloalkylalkyl, phenyl or C3-C7-cycloalkylalkyl, phenyl or
benzyl or C1-C3 alkoxide, and benzyl or C1-03 alkoxide, and
particularly each Rs is independently particularly each Rs is
independently
H or C1-C3 alkyl or 03-cycloalkyl H or C1-03 alkyl or 03-
cycloalkyl
Rs 2\ R12-77,
p is 0, 1 or 2; Xii is CH or N;
(CH2)p each Rs, independently, is
hydrogen,
Rs Rs halogen, hydroxide, 01-06
alkyl,
I 03-07-cycloalkylalkyl, phenyl
or
benzyl or 01-03 alkoxide, and
Rs....:...- .,...":õ... õ,..."-Rs particularly each Rs is
independently
X11 N H or C1-03 alkyl or C3-
cycloalkyl or
halogen. Halogen is particularly Cl or
R12-77 Br.
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Scheme 3 (continued)
R12-78,
p is 0, 1 or 2;
(CH2)p R is hydrogen or C1-C4 alkyl or
Rs cycloalkyl;
each Rs, independently, is hydrogen,
0 halogen, hydroxide, C1-C6 alkyl,
C3-C7-cycloalkylalkyl, phenyl or
Rs benzyl or C1-C3 alkoxide, and
particularly each Rs is independently
H or C1-C3 alkyl or C3-cycloalkyl or
R12-78 halogen. Halogen is particularly Cl
or
Br. R is particularly hydrogen or
methyl.
X Br
S)/
R12-79
R12-80 R12-81
X = halogen
N,NR
N-cr.
R12-82 R12-83 R12-84
R = hydrogen, alkyl R = hydrogen, alkyl
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Scheme 4: Exemplary B rings for formula I
X3
RB#B
Xq 2X RBI, which is bonded to Y or Rp at the
indicated
positions, where X' and X2 are selected from CH and
N and at least one of X1 and X2 is N, X3-X6 are
JVVVVVV.
selected from CH2, CH, 0, S, N, and NH and the B
ring is saturated, partially unsaturated or aromatic
dependent upon choice of X3-X6, and RB represents
optional substitution as defined for formula I at ring
carbons and or nitrogens.
RB N BN,\
N
N
VVVVVW
RB2 RB3
/\\
I
DD I I
RB
N
../VVVVVIP VIOVW/V.
RB4 RB5
N
RB RB2-RB6, which are bonded to Y or Rp
at the
N indicated positions, and RB represents optional
substitution as defined for formula I at rina carbons
.nrituvuv. RB6
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Scheme 4 (continued)
RB R RB Rp
p
--'\----- '...\----- N'-'-....../'
1 1 RB
R
N ==,_ N N N1 ,,,,,,
N
RB7 RB8 RB9
RB N
N
N
1 II ________________________________________________________ Rp
Ri3 N.,....,..5/;.,-..õN>
N H
...vv.
RB10 RB11
RB
\ ./.......,õ,õ.õ.õ,,,Rp
If il
RB 1
N N .,...,., N
N -\_/
RB12 RB13 RB14
CF31\1,,Rp .õ.,.,---,...,,..Ap CF3,.,,Rp
1 I 1
-.,.N N N N._...,-,N
.\./==
JINN!~
RB15 RB16 RB17
RB7-RB17, which are bonded to Y at the indicated position, and RB represents
optional substitution as defined for formula I at ring carbons or at
specifically
indicated carbons
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Example 12: Exemplary Synthetic Methods
Compounds of Formula XX ¨XXIII as well as many other compounds of this
invention are
prepared, for example, by the method illustrated in Scheme 5, where variables
are as defined
above. This three-step synthesis starts with selective aromatic nucleophilic
substitution on the 4-
position of a 2,4-dichloro-pyrimidine A (e.g., 2,4-dichloro-6-
methylpyrimidine, where R4 is methyl or
2,4-dichloro-5-fluoropyrimidine, where R5 is fluorine) with a p-
phenylenediamine B to form the
intermediate C. Exemplary reaction conditions are shown in Scheme 5 where
reactants are added
with trimethylamine to ice cold ethanol and stirred at rt for 15 h. [Kumar et
al., 2014; Odingo et al.,
2014]. Chlorinated intermediate C is then reacted with any amine HNR2R3 D by
amination to
generate intermediate E. Exemplary annination conditions are shown in Scheme
5, where
reactants are reacted in DMF in the presence of K2CO3 at elevated temperature.
Step three
couples the R10 group employing acid F to intermediate E. Various known
synthetic methods can
be employed to introduce a selected Rio group, for example, cross coupling,
click chemistry or
substation reactions (e.g., SN2, aromatic, electrophilic) [Li et al., 2014a;
Li et al., 2014b; LaBarbera
et al., 2007]. Scheme 5 illustrates coupling of the amine group of E with a
selected carboxylic acid
F to form R10 which is ¨NH-CO-Ri2 in compound G. Exemplary R12 are aryl, aryl-
substituted alkyl,
heteroaryl and heteroaryl-substituted alkyl. Exemplary coupling conditions are
illustrated in
Scheme 5, where coupling proceeds in the presence of propylphosphonic
anhydride (T3P) and
triethyamine at room temperature to form the desired compound G. The
illustrated method has
been employed, for example to prepare compound 6, and compound 8 (see, Scheme
6).
Various substituted starting materials A, B, D and F are commercially
available or can be prepared
using known methods. In embodiments, aniline derivatives already substituted
with R10 (B') can be
used in place of p-phenylenediamine derivatives B to form a corresponding R10-
substituted
intermediate C'. Carrying out step 2 of the illustrated reaction, by reacting
intermediate C' with D
will result in desired corresponding compound G' (where R10 replaces R12-CO-NH-
). As will be
appreciated by one of ordinary skill in the art, it may be useful or necessary
to protect certain
groups in the starting materials or intermediates during reactions shown to
prevent undesired side-
reactions. For example, ring N in reactants F may be protected with
appropriate amine protecting
groups. Use of appropriate protecting groups is generally routine in the art.
A variety of primary or
secondary amines (D) are commercially available or can be prepared by well-
known methods.
Alternatively, chlorinated intermediate C can be reacted with an appropriate
nucleophile to add a
selected ¨NR2R3 group at the 4-chloro position. For example, D can be a cyclic
amine such as
pyrrolidine. As another possible alternative, Suzuki coupling may be used to
install an amine
containing group by C-C bond formation [Li et al., 2014a]. As another possible
alternative,
Buchwald-Hartwig cross coupling can be used to form carbon and amine bonds in
such
intermediates.
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R6
R7 401 NH2
I R4j;ICI H2N R8 NI
1\1 Rg R7 N.H
Rg
TEA, Et0H, it 15h
A CI H2N R8 c
R9
HNR2R3
K2CO3, DMF, 80 C, 8 h
I N--T"'NR2R3
0
N,H R12-A-OH N,
0 11101 H
H2N TEA, T3P, DCM, it, 15 h R12 N
CI
R6
I I
R7 401 NH2
R7 N. C' G'
.H
R10 R8 H N
R9 R10 R8 D
IR9
Scheme 5
Detailed Synthesis of Compounds 6 and 8 (Scheme 6)
N-(4-aminophenyI)-2-chloro-6-methyl-pyrimidin-4-amine (102). To 0.5 g (3.06
mmol) of 2,4-
dichloro-6-methylpyrmidine (100) dissolved in 10 mL of ethanol at 0 C were
added 513.7 pL (1.2
equivalents, 372.5 mg, 3.68 mmol) of triethyl amine (TEA), and 330.5 mg (3.06
mmol) of p-
phenylenediamine (101). The reaction mixture was warmed to room temperature
and stirred at that
temperature overnight. The solvents were removed in vacuo and the resulting
residue was
chromatographed on silica gel using 40% hexane in ethyl acetate as the eluent
to afford 500 mg
(70% yield) of the pure product 102).1H NMR: (400 MHz, CDCI3): 0 7.19 (broad
s, 1H), 7.02 (d, J
= 8.8 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 6.18 (s, 1H), 3.77 (broad s, 2H),
2.26 (s, 3H).
N-(4-aminopheny1)-6-methyl-2-(pyrolidin-1-y1)pyrimidin-4-amine (104). To 1.2 g
of N-(4-
aminophenyI)-2-chloro-6-methyl-pyrimidin-4-amine (102) dissolved in 120 mL of
DMF were added
777.3 mg (5.62 mmol) of potassium carbonate and 3.63 mg (4.12 mL, 51.1 mmol)
of pyrrolidine
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(103) at room temperature. The reaction mixture was heated 80 C for 8 h. The
reaction was
cooled to room temperature and diluted with water. The product was extracted
with ethyl acetate (3
x 100 mL). The organic layers were combined and washed with brine, followed by
drying over
Na2SO4, filtered and concentrated to give an oily crude product that was
chromatographed on silica
gel using 10% methanol in DCM (with drops of TEA) to give 1.31 g (96% yield)
of the pure product
(104). 1H NMR: (400 MHz, CDCI3): 6 7.11 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4
Hz, 2H), 6.25 (broad
s, 1H), 5.68 (s, 1H), 3.63 (broad s, 2H), 3.56 (t, J = 6.8 Hz, 4H), 2.19 (s,
3H), 1.93 (t, J = 6.8 Hz,
4H).
N-(44(6-methyl-2-(pyrrolidin-1-yl)pyrimidin-4-yl)amino)pheny1)-2-(thiophen-2-
y1)acetamide (6). To
700 mg (2.60 mmol) of N-(4-aminophenyI)-6-methyl-2-(pyrolidin-1-yl)pyrimidin-4-
amine (104) in 15
mL were added 406 mg (2.86 mmol) of 2-thiopheneacetic acid (105), 906.8 pL
(657.5 mg, 6.50
mmol) of TEA, and 3.06 mL (1.65 mg, 5.20 mmol) of T3P (50% weight solution in
ethyl acetate) at
0 C. The mixture was warmed to room temperature and stirred for 15 h. The
reaction was
quenched by gradual addition of water, and the product was extracted with DCM
(3 x 150 mL),
followed by washing with brine. The organic layers were combined, dried over
Na2SO4, filtered and
concentrated under reduced pressure to give a crude product that was purified
using silica gel and
10% methanol in DCM to give 864.5 mg (84% yield) of the pure product (6). 1H
NMR: (400 MHz,
DMSO-d6): 6 10.07 (s, 1H), 9.06 (broad s, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.49
(d, J = 9.2 Hz, 2H),
7.39 - 7.37 (m, 1H), 6.98 - 6.96 (m, 2H), 3.84(s, 2H), 3.47(s, 4H), 2.13(s,
3H), 1.89 (t, J = 6.6
Hz, 4H). HPLC: 98% pure.
Boc-protected indole-3-caboxylic acid 106-boc was used in a peptide coupling
methodology with
compound 104 in the presence of T3P and TEA to achieve the synthesis of boc-
protected indole
derivative 8-boc, which was converted to the indole derivative 8 in good yield
via TFA deprotection
of the boc-protecting group. Note that the boc protecting group is -000-t-
butylK2Co3,KI, Et0H
N-(44[6-Methyl-2-(1-pyrrolidiny1)-4-pyrimidinyipmino)phenyl)-1-{[(2-Methyl-2-
propanyi)oxylearbonyll-lH-indole-3-carboxamide (8-boc). To 80 mg (0.297 mmol)
of
compound 104 in 8 mL DCM, were added 85.36 mg (0.3267 mmol of boc-protected
indole-3-
carboxylic acid (106-boc), 103.6 pL (75.13 mg, 0.742 mmol) of TEA, 350 pL (189
mg, 0.594 mmol)
of T3P at 0 C. The mixture was warmed to room temperature and stirred for 20
h. The reaction
was quenched by gradual addition of water, and the product was extracted with
DCM (3 x 50 mL),
followed by washing with brine. The organic layers were combined, dried over
Na2SO4, filtered and
concentrated under reduced pressure to give a crude product that was purified
using silica gel and
10% methanol in DCM to give 76 mg (50% yield) of the pure product (8-boc). 1H
NMR: (400 MHz,
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CDCI3): 58.28 (broad s, 1H), 8.24 - 8.13 (m, 3H), 7.57 (q, J= 4.8, 8.8 Hz,
4H), 7.40 -7.31 m, 3H),
5.92 (s, 1H), 3.54 (s, 4H), 2.23 (s, 3H), 1.86 (s, 4H), 1.66 (s, 9H).
N-(44(6-Methyl-2-(1-pyrrolidinyl)-4-pyrimidinyi]amino)phenyi)-1H-indole-3-
carboxamide (8).
70 mg (0.136 mmol) of N-(4-{[6-Methyl-2-(1-pyrrolidinyl)-4-
pyrimiclinyl]amino}phehyl)-11-i-indole-3-
carboxamide (8-boc) was dissolved in 25% TFA in DCM (5 mL). The solution was
stirred for 3 h at
room temperature. The solvents were removed under reduced pressure and the
crude product was
purified using silica gel and 10% methanol in DCM to give 43.78 mg (78 %
yield) of the pure
product. 1H NMR: (400 MHz, DMSO-d6): 511.7 (s, 1H), 9.65 (s, 1H),9.09 (s, 1H),
8.28 (broad s,
1H), 8.20(d, J= 7.6 Hz, 1H), 7.67(s, 4H), 7.47(d, J= 8.0 Hz, 1H), 7.20 -7.12
(m, 2H), 5.88(s,
1H), 3.50 (s, 4H), 2.14 (s, 3H), 1.91 (s, 4H).
401 NH2 NyCIm
NCI c
101 I m)
H2N 103 I J.
1 N
TEA, Et0H, rt, 15 h N-H K2CO3, DMF, 80 C N,H
CI 70% = 102 96%
100 H2N
H2N IP 104
0 OH 0 OH
OR
COMPOUNDS 6 or 8
105 N-boc 106-boc
TEA,T3P, DCM, rt, 15 h
+ deprotection of 8-boc with 25% TFA in DCM
Scheme 6
Scheme 7 illustrates an alternative method of synthesis optimized for yield of
compound 6. In this
method, a t-butyl protected carbamate, for example, compound 35 is reacted
with a selected
aromatic carboxylic acid, for example, compound 36 to form a protected
carbamate intermediate,
for example, compound 37. The intermediate is deprotected as known in the art,
for example with
trifluoroacetic acid (TFA) and the deprotected carbamate is reacted with a
chlorinated heterocyclic
group carrying a primary or secondary amine group (e.g., a pyrrolidinyl
group), for example,
compound 38 to form the desired compound of Formula XX, for example, compound
6. This
method can also be employed to prepare various compounds of formula )0( by
selection of starting
aromatic carboxylic acids and chlorinated heterocyclic compound carrying a
primary of secondary
amine group.
In Scheme 7, reagents employed for synthesis of compound 6 are shown, where in
the first
reaction DCC is N,N'-dicyclohexylcarbondiimide, DMAP is dimethylaminopyridine
and the solvent
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is DCM dichloromethane. In the second reaction, after TEA deprotection,
potassium carbonate,
and potassium iodide in ethanol is employed. One of ordinary skill in the art
can readily adapt the
reagents and reaction conditions employed to prepare desired compounds of
formula )0(.
101-boc
NH-boc
NH-boc DCC, DMAP, Dry DCM
Various aromatic/heteroaromtic
H2N carboxylic acids, e.g., H 107-
boc
0 OH 1. TEA
Deprotection
/ 2. K2CO3, KI, Et0H
\S
105
\
N
108
CI
I J.
sy
N,
0 H
/S 6
Scheme 7
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Example 13: Biological Evaluation and Comparison of Inhibitors of Oncogenic
CHD1L
CHD1L is unique from other chromatin rennodelers and has a diverse repertoire
of cellular
functions. (Xiong et al., 2021) CHD1L is essential for PARP-mediated DNA
repair and knockdown
of CHD1L sensitizes tumor cells to DNA damaging agents. (Ahel et al., 2009)
Two recent reports
validate CHD1L as significant factor promoting drug resistance to PARP
inhibitors via CHD1L
mediated nucleosome sliding, alleviating PARP trapping. (Verma et al., 2021;
Juhasz et al., 2020)
Knockout of CHD1L is reported to sensitize BRCA1/2 mutant HR-deficient tumor
cells to PARP
inhibition causing cell death in vitro and loss of tumor growth with increased
survival in vivo.
(Verma et al., 2021; Juhasz et al., 2020)
In an aspect herein, we show that CHD1L is a required component of the TCF/LEF-
transcription
factor complex (denoted henceforth as TCF-transcription) (see also, Abbott et
al., 2020), which is
linked as a driver of GI cancers and many other cancers. (van de Wetering, et
al., 2002; Ram
Makena et al., 2019; Bathe et al., 2002; He et al., 2020; Polakis, 2012b;
Clevers et al., 2006) We
have determined this complex to be a master regulator of epithelial-
mesenchymal transition (EMT)
that promotes epithelial-nnesenchynnal plasticity (EMP). (Esquer et al., 2021;
Zhou et al., 2016)
Others have confirmed this. (Yang et al., 2020; Sanchez-TillO et al., 2011;
Kroger et al., 2019) In
particular, we demonstrated the TCF-transcription is upregulated in isolated
quasi-mesenchynnal
cell phenotypes compared to other EMT phenotypes, promoting increased cancer
stem cell (CSC)
stemness and invasiveness. (Esquer et al., 2021). Our work suggested that
targeted small
molecule inhibitors of CHD1L can provide an effective therapeutic strategy to
treat CRC and other
cancers.
Herein, we describe the high-throughput screen (HTS) drug discovery and hit-to-
lead validation of
the first-in-class CHD1L inhibitors (CHD1Li). (See also, Abbott et al., 2020)
In this example, we
provide additional description of the medicinal chemistry optimization of
compound 6.0, its
biological evaluation, and structure activity relationship (SAR) of certain
the CHD1L inhibitors
structurally related to compound 6Ø In addition, we demonstrate that analog
6.11 displays
improved pharmacokinetics compared to 6.0, including oral bioavailability and
in vivo antitumor
efficacy against CRC HCT116 tumor xenografts.
In this example, we describe the synthesis of compound 6.0 and optimize the
chemistry to efficiently
prepare analogs for ligand-based drug design. The synthesis began employing
commercially
available starting material including p-phenylenediamine (1) and
dichloropyrimidine analogs (2.1-
2.3) (Scheme 8). Compounds 1 and 2 (Scheme 8) were reacted in the presence of
triethylamine to
obtain a selective nucleophilic aromatic substitution, providing intermediates
3.1-3.3 (Scheme 8) in
good yield ranging from 70-83%. A second nucleophilic aromatic substitution
with pyrrolidine
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afforded the core pyrimidine pharmacophore of 6Ø Next, using
propanephosphonic acid anhydride
(T3P), commercially available thiophene (5) (Scheme 8) was coupled to provide
6.0, 6.1, and 6.2
with yields ranging from 77-84%. Analog 6.1 was reacted with methyl iodide to
provide analog 6.4.
(Chemical structures of compounds 6.0-6.4, and 6.11-6.14 are found in Scheme
1.) This chemistry
provided several CHD1L inhibitor analogs to investigate the structure activity
relationship (SAR)
around the pyrimidine ring. Initially, we also utilized this synthetic
approach to modify the thiophene
aromatic ring coupled as amides to the phenylenediamine ring.
The synthetic approach to produce 6.3 as shown in Scheme 8 gave low yields and
difficulty in
purification. Therefore, we further optimized the synthesis starting with a
tert-butoxycarbonyl
(BOO) protected phenylenediamine, which resulted in a significant increase in
purity of the desired
substitution at the 4-position of the pyrimidine ring, facilitating the
pyrrolidine substitution in the 2-
position. Unfortunately, after BOO deprotection the challenges persisted with
peptide coupling of
the 3-indole carboxylic acid to produce 6.3. However, increasing the carbon
spacer between the
aromatic rings and the carboxylic acid functionality allowed for efficient
peptide coupling, leading to
the optimized syntheses of CHD1Li (Schemes 9A and 9B). In Schemes 9A and 9B,
we utilized
BOO protecting groups to facilitate derivatization of the R4 group therein
with various aromatic
groups, including thiophene, indole, azaindole, benzimidazole, and quinoline
rings (Scheme 9A). In
addition, we substituted pyrrolidine for morpholine amine rings in the R3
group therein. Finally, to
investigate the necessity of the aniline linkage of 6.0, we generated ether
linked analogs of 6.0
(Scheme 9B). The methods of Scheme 9A and 9B produced analogs of 6.0,
including among
others 6.5 -6.33 (see Scheme 1).
Example 13: Synthetic Examples (compound number in the following paragraphs
refer to Schemes
8, 9A and 9B)
N-(4-aminopheny0-2-chloro-6-methyl-pyrimidin-4-amine (3.1). To 0.5 g (3.06
mmol) of 2,4-
dichloro-6-methylpyrmidine (2) dissolved in 10 mL of ethanol at 0 C were
added 513.7 pL (1.2
equivalents, 372.5 mg, 3.68 mmol) of triethyl amine, and 330.5 mg (3.06 mmol)
of p-
phenylenediamine (1). The reaction mixture was warm to room temperature and
stirred overnight.
The solvents were removed under reduced pressure and the resulting residue was
chromatographed
on silica gel using 40 % hexane in ethyl acetate as the eluent to afford 500
mg (70 % yield) of the
3.1. Rf = 0.40; m.p. 157-159 C; 1H NMR (400 MHz, CDCI3) 6 7.507 (s, N-H),
7.003-7.025 (d, J=8.6
Hz, 2H), 6.670-6.691 (d, J=8.6 Hz, 2H), 6.162 (s, 1H), 3.777 (s, N-H, 2H),
2.239 (s, 3H); 130-NMR
(100 MHz, CDC13); 168.384, 164.430, 160.118, 145.450, 127.560, 126.906,
115.827, 100.182,
23.936; IR (neat) umax 33214.14, 1590.07, 1506.88, 1424.41, 1214.54, 1028.66,
970.00, 905.78,
826.69, 757.43, 547.51, 510.10; ESI-HRMS [M+H] calculated for C11H11CIN4,
234.07, found
235.0735.
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N-(2-chloro-5-fluoro-6-methylpyrimidin-4-yObenzene-1,4-diamine (3.2). 2,4-
dichloro-5-fluoro-6-
methylpyrimidine (2.2) (250 mg, 1.381 mmol, 1.0 equiv) was dissolved in
ethanol (10 mL) and
cooled in an ice bath. Triethyl amine (231 pL, 1.657 mmol, 1.2 equiv) and p-
phenylenediamine (1)
(324.1 mg, 1.381 mmol, 1.0 equiv) were added and the reaction was allowed to
warm to RT and
stir for 15h. The solvent was removed under reduced pressure and the crude
mixture was purified
via column chromatography using 60% ethyl acetate in Hexanes to provide 3.2
(290 mg, 83%
yield) as a dark yellow solid. TLC (60% ethyl acetate in hexanes), Rf = 0.40;
m.p. 157-159 C; 1H
NMR (400 MHz, CD013) 67.313-7.335 (d, J=8.7 Hz, 2H), 6.746 (s, 1H), 6.674-
6.695 (d, J=8.7 Hz,
2H), 3.667 (s, N-H, 2H), 2.360-2.376 (d, J=3.0 Hz, 3H); 13C-NMR (100 MHz,
CDC13); 153.422,
151.036, 150.891, 150.742, 144.413, 143.984, 141.895, 128.176, 123.111,
115.638, 17.025; IR
(neat) umax 3328.86, 1616.04, 1507.65, 1281.35, 829.94, 830.88, 624.12,
562.34, 511.93; ES1-
HRMS [M+H] calculated for C11H13C1FN4, 252.06, found 253.0640.
N-(2-chloro-5-fluoropyrimidin-4-yObenzene-1,4-diamine (3.3). 2,4-dichloro-5-
fluoropyrimidine
(2.3) (500 mg, 2.995 mmol, 1.0 equiv) was dissolved in Et0H (20 mL) and cooled
in an ice bath.
Triethyl amine (501.57 pL, 3.593 mmol, 1.2 equiv) and p-phenylenediamine (1)
(323.88 mg, 2.995
mmol, 1.0 equiv) were added and the reaction was allowed to warm to RT and
stir for 8h. The solvent
was removed under reduced pressure and the crude mixture was purified via
column
chromatography using 60% ethyl acetate in Hexanes to provide 3.3 (544 mg, 76%
yield) as a tan
solid. TLC (60% ethyl acetate in hexanes), Rf = 0.36; m.p. 155-157 C; 1H NMR
(400 MHz, CDCI3)
6 7.981-7.988 (d, J=2.7 Hz, 1H), 7.340-7.361 (d, J=8.7 Hz, 2H), 6.801 (s, N-
H), 6.692-6.714 (d, J=8.7
Hz, 2H), 3.691 (s, N-H, 2H); 13C-NMR (100 MHz, 0D013)154.642, 151.535,
151.433, 146.62,
144.223, 143.900, 140.533, 140.331, 127.753, 123.174, 115.620; IR (neat) umax
3014.43, 1627.78,
1580.69, 1506.69, 1323.90, 1235.19, 946.92, 816.77, 746.87, 690.44, 641.61,
592.67, 514.39,
430.10; ES1-HRMS [M+H]* calculated for C10H8C1FN4, 238.04, found 239.0484.
N-(6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-yObenzene-1,4-diamine (4.1). 3.1 was
dissolved in 5
mL of DCM and treated with 5 mL of TFA at 0 C, resulting in a red colored
solution. The reaction
was warmed to RT and allowed to stir for 3h. The reaction was concentrated and
redissolved in
10% methanol and DCM, then washed with bicarb and water. The organic later was
dried over
sodium sulfate and concentrated, purified via column chromatography using 10%
methanol in DCM
to produce 4.1 (2.09 g, 67% over two steps) as an orange solid. TLC (5%
methanol/dichloromethane), R= 0.18; m.p. 190-192 C; 1H NMR (400 MHz, CD0I3)
67.105-7.127
(d, J=8.6 Hz, 2H), 6.666-6.687 (d, J=8.6 Hz, 2H), 6.190 (s, N-H), 5.680 (s,
1H), 3.542-3.575 (m,
4H), 2.188 (s, 3H), 1.920-1.953 (m, 4H); 13C-NMR (100 MHz, CDCI3) 192.975,
162.718, 160.872,
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143.621, 130.275, 125.357, 115.779, 91.608, 46.674, 25.689, 24.546; IR (neat)
uma,1755.53,
1658.71, 1612.71, 1548.12, 1504.15, 1403.49, 1251.05, 1138.16, 890.44, 696.38,
517.52; ESI-
HRMS [M+H]0 calculated for 015H19N5, 269.13, found 270.1700.
N-(2-chloro-5-fluoro-6-methylpyrimidin-4-yObenzene-1,4-diamine (4.2). 3.2 (260
mg, 1.029
mmol, 1.0 equiv) was dissolved in DMF (29 mL) and treated with potassium
carbonate (156.4 mg,
1.132 mmol, 1.1 equiv) and pyrrolidine (422.5 pL, 5.145 mmol, 5.0 equiv). The
reaction was heated
to 80 C for 8h then diluted with ethyl acetate and washed with water and a 5%
lithium chloride
solution. The organic layer was dried over sodium sulfate, concentrated under
reduced pressure,
then purified via column chromatography using 10% methanol in ethyl acetate,
to provide 4.2 (243
mg, 82% yield) as a brown solid. TLC (10% methanol/ethyl acetate), Rf = 0.57;
m.p. 180-182 C;
1H NMR (400 MHz, CDCI3) 6 7.456-7.478 (d, J=8.6 Hz, 2H), 6.663-6.685 (d, J=8.6
Hz, 2H), 6.436
(s, N-H), 3.497-3.530 (m, 4H), 2.267-2.274 (d, J=2.9 Hz, 3H), 1.914-1.947 (m,
4H); 13C-NMR (100
MHz, CDC13)156.073, 149.566, 149.460, 149.193, 149.062, 142.081, 139.428,
139.056, 130.876,
121.623, 115.600, 47.019, 25.812, 17.361; IR (neat) umax 3185.05, 1600.39,
1506.17, 1444.25,
1238.75, 826.91, 762.06, 509.83; ESI-HRMS [M+H] calculated for 015H18FN5,
287.15, found
288.1606.
N-(5-fluoro-2-(pyrrolidin-1-yl)pyrimidin-4-yl)benzene-1,4-diamine (4.3). 3.3
(310 mg, 1.30
mmol, 1.0 equiv) was dissolved in DMF (36 mL) and treated with potassium
carbonate (197.6 mg,
1.43 mmol, 1.1 equiv) and pyrrolidine (533.8 pL, 6.5 mmol, 5.0 equiv). The
reaction was heated to
80 00 for 8h then diluted with ethyl acetate and washed with water and brine.
The organic layer
was dried over Na2SO4 and concentrated under reduced pressure to provide 4.3
as a dark yellow
solid, which was carried on crude. TLC (10% methanol/dichloromethane), Rf=
0.59; m.p. 179-181
C; 1H NMR (400 MHz, CDCI3) 67.846-7.855 (d, J=3.7 Hz, 1H), 7.458-7.479 (d,
J=8.7 Hz, 2H),
6.671-6.693 (d, J=8.7 Hz, 2H), 6.494 (s, N-H), 3.497-3.530 (m, 4H), 1.934-
1.967 (m, 4H); 13C-NMR
(100 MHz, 0D013)156.889, 149.984, 149.886, 142.406, 141.214, 139.910, 139.717,
138.808,
130.346, 122.189, 121.827, 115.572, 47.026, 37.755, 25.808; IR (neat)l)mõ
3388.87, 1598.56,
1568.68, 1500.63, 1447.20, 1227.27, 930.93, 831.66, 763.87, 496.87; ESI-HRMS
[M+H]
calculated for 0141-116FN5, 273.14, found 274.1450.
N-(44(6-methy1-2-(1-pyrrolidiny0-4-pyrimidiny0amino)pheny1)-2-(2-
thienyl)acetamide (6.0)
4.1 (262.0 mg, 0.973 mmol, 1.0 equiv) was dissolved in DCM (40 mL, anhydrous)
then treated with
5.1 (145.3 mg, 1.02 mmol, 1.05 equiv), DMAP (118.9 mg, 0.973 mmol, 1.0 equiv),
and then DCC
(251 mg, 1.22 mmol, 1.25 equiv) under nitrogen. The reaction was allowed to
stir for 8h then the
material was concentrated onto silica gel and purified via column
chromatography using 1:1 ethyl
acetate:dichloromethane and 3 % methanol to provide compound 6.0 (302.8 mg,
79% yield) as a
yellow solid). TLC (10% methanol/ethyl acetate), Rf= 0.49; m.p. 186-188 C; 1H
NMR (400 MHz,
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CDCI3) 6 7.611 (s, N-H), 7.389(s, 4H), 7.273-7.289 (dd, 1H), 7.012-7.032 (m,
2H), 6.600 (s, N-H),
5.762 (s, 1H), 3.919 (s, 2H), 3.537-3.570 (m, 4H), 2.204 (s, 3H), 1.904-1.938
(s, 4H); 13C-NMR
(100 MHz, CDC13); 167.993, 166.584, 161.249, 160.656, 136.446, 135.883,
132.826, 127.768,
127.625, 126.049, 121.643, 120.996, 92.885, 46.727, 38.504, 25.626, 24.380; IR
(neat) uma,
2862.14, 1572.75, 1500.37, 1398.29, 1330.32, 1226.59, 1169.02, 830.35, 782.81,
681.54, 513.32;
ESI-HRMS [M+H] calculated for 0211-123N50S, 393.16, found 394.1680.
N-(4-((5-fluoro-6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny0-2-
(thiophen-2-
yOacetamide (6.1). 4.2 (450 mg, 1.566 mmol, 1.0 equiv) was dissolved in DCM
(15.0 mL,
anhydrous) and treated with 2-thiopheneacetic acid (5.1) (244.9 mg, 1.723
mmol, 1.1 equiv), and
triethylamine (546.4 pL, 3.915 mmol, 2.5 equiv). The reaction was allowed to
stir for 5 min. then
propanephosphonic acid anhydride (1.85 mL, 3.132 mmol, 2.0 equiv) was added
and the reaction
was allowed to stir for 15h. The reaction was then quenched with ice water and
extracted with
DCM (x3). The organic layer was dried over sodium sulfate and concentrated
under reduced
pressure and purified via column chromatography using 10% methanol in ethyl
acetate to provide
6.1 as a yellow solid (529 mg, 82% yield). TLC (10% methanol/ethyl acetate),
Rf = 0.42; m.p. 245-
247 C; 1H NMR (400 MHz, DMSO-d6) 6 10.093 (s, N-H), 8.950 (s, N-H), 7.752-
7.774 (d, J=8.9
Hz, 2H), 7.482-7.504 (d, J=8.9 Hz, 2H), 7.375-7.391 (dd, 1H), 6.963-6.982 (m,
2H) 3.844 (s, 2H),
3.391-3.423 (m, 4H), 2.183-2.190 (d, J=2.9 Hz, 3H), 1.860-1.892 (m, 4H); 130-
NMR (100 MHz,
DMSO-d6); 167.579, 155.240, 148.710, 149.054, 138.597, 137.281, 136.205,
135.432, 133.499,
126.622, 126.211, 124.986, 120.444, 119.297, 46.517, 37.493, 25.128, 17.109;
IR (neat) max
3256.79, 1654.91, 1621.65, 1587.57, 1501.62, 1417.54, 1292.95, 1226.40,
826.57, 689.26,
548.39, 512.40; ESI-HRMS [m+H] calculated for 021 H22FN50S, 411.5, found
412.1587.
N-(44(5-fluoro-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny0-2-(thiophen-2-
yOacetamide
(6.2). 4.1 (50.0 mg, 0.183 mmol, 1.0 equiv) was dissolved in DCM (15.0 mL,
anhydrous) and
treated with 2-thiopheneacetic acid (5.1) (28.6 mg, 0.201 mmol, 1.1 equiv),
and triethylamine (63.9
pL, 0.458 mmol, 2.5 equiv). The reaction was allowed to stir for 5 min., then
propanephosphonic
acid anhydride (0.194 mL, 0.366 mmol, 2.0 equiv) was added and the reaction
was allowed to stir
for 15h. The reaction was then quenched with ice water and extracted with DCM
(x3). The organic
layer was dried over sodium sulfate and concentrated under reduced pressure
and purified via
column chromatography using 10% methanol in ethyl acetate to provide 6.1 as a
yellow solid (56
mg, 77% yield). TLC (10% methanol/ethyl acetate), R. = 0.61; m.p. 245-247 'C;
1H NMR (400
MHz, CD30D) 6 10.112 (s, N-H), 9.110 (s, N-H), 7.950-7.960 (d, J=3.9 Hz, 1H),
7.766-7.789 (d,
J=8.9 Hz, 2H), 7.499-7.522 (d, J=8.9 Hz, 2H), 7.377-7.393 (dd, 1H), 6.964-
6.983 (m, 2H) 3.848 (s,
2H), 3.402-3.434 (m, 4H), 1.874-1.907 (m, 4H); 13C-NMR (100 MHz, CD30D);
167.617, 156.244,
149.151, 149.046, 140.693, 140.541, 140.350, 138.276, 137.262, 135.08,
133.735, 126.627,
126.223, 124.994, 120.588, 119.306, 46.568, 37.498, 25.115; IR (neat) umax
3256.79, 1654.91,
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1621.65, 1587.57, 1501.62, 1417.54, 1292.95, 1226.40, 826.57, 689.26, 548.39,
512.40; ESI-
HRMS [M+H] calculated for 0201-120FN50S, 397.47, found 398.1431.
N-(44(5-fluoro-6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0(methyl)amino)pheny1)-
2-(thiophen-
2-yOacetamide (6.3). 6.1 (13 mg, 0.0316 mmol, 1.0 equiv) was dissolved in THF
(1.0 mL,
anhydrous) and treated with sodium hydride (1.52 mg, 0.0379 mmol, 1.2 equiv)
at 0 C under
nitrogen. The reaction was allowed to stir for 10 min. then iodomethane (3.0
pL, 0.047 mmol, 1.5
equiv) was added and the reaction was allowed to stir for 15h. The reaction
was then quenched
with ice water and extracted with ethyl acetate (x3). The organic layer was
dried over sodium
sulfate and concentrated under reduced pressure and purified via 1000mm prep
plate using 3%
methanol in dichloromethane to provide 6.3 as a yellow oil (6.8 mg, 44 %
yield). TLC (5 %
methanol/ dichloromethane), Rf = 0.62; m.p. 178-180 C; 1H NMR (400 MHz,
CD0I3) 6 7.783-7.806
(d, J=8.7 Hz, 2H), 7.154-7.166 (dd, 1H), 7.118-7.139 (d, J=8.7 Hz, 2H), 6.875-
6.897 (m, 1H),
6.715-6.736 (m, 1H), 3.674 (s, 2H), 3.547-3.580 (m, 4H), 3.284 (s, 3H), 2.311
(s. 3H), 1.967-1.991
(m, 4H); 13C-NMR (100 MHz, CDCI3); 170.285, 155.868, 148.927, 139.213,
138.053, 137.039,
127.982, 126.533, 126.362, 126.270, 125.259, 124.801, 120.274, 47.142, 37.865,
35.280, 25.822,
17.541; IR (neat) umax 1583.85, 1508.03, 1441.51, 1231.31, 910.69, 729.28; ESI-
HRMS [M+H]
calculated for 022H24FN50S, 425.17, found 426.1741.
N-(44(6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny1)-1H-indole-3-
carboxamide
(6.3). Indole-3-carboxylic acid (11.6 mg, 0.0722 mmol, 1.2 equiv) was slurried
with DIPEA (12.6
pL, 0.0722 mmol, 1.2 equiv) in DMF (anhydrous, 0.5 mL) and was treated with
Hbtu (27.4 mg,
0.0722 mmol, 1.2 equiv) in DMF (anhydrous, 0.2 mL). The reaction was allowed
to stir for 15 min
at RT then 4.1 (16.2 mg, 0.0602 mmol, 1.0 equiv) in DMF (anhydrous, 0.2 mL)
was added
dropwise and the reaction was allowed to continue stirring for another 8h at
RT. The reaction was
diluted with DCM and washed with water and brine. The organic layer was dried
over sodium
sulfate and concentrated onto silica gel. Purification via column
chromatography using 1:1 ethyl
acetate/dichloromethane and 3% methanol produced the desired product 6.3 as a
white solid (7.5
mg, 25% yield). TLC (3% methanol/ dichloromethane), Rf = 0.20; m.p. 182-184
00; 1H NMR (400
MHz, CDCI3) 6 8.171-8.193 (d, J=7.7 Hz, 1H), 7.973 (s, 1H), 7.896 (s, N-H),
7.662 (s, 4H), 7.440-
7.460 (d, J=7.7 Hz, 1H), 7.155-7.238 (m, 2H), 5.963 (s, 1H), 3.574-3.594 (m,
4H), 2.274 (s, 3H),
2.022-2.050 (m, 4H); 130-NMR (100 MHz, CDCI3); 166.616, 164.856, 162.165,
138.153, 136.427,
135.951, 129.349, 127.596, 123.682, 122.418, 122.314, 122.137, 122.074,
112.812, 112.005,
97.046, 79.467, 36.944, 31.641, 26.260, 20.453; IR (neat) umõ 1505.51,
1232.99, 1176.73, 833.69,
743.65, 552.85; ESI- HRMS [M+H]* calculated for 024H24N60, 412.2, found
413.2066.
tert-butyl (4((2-chloro-6-methylpyrimidin-4-yl)amino)phenyOcarbamate (8). 2,4-
dichloro-6-
methylpyrimidine (2.1) (2.36g, 0.0145 mol, 1.05 equiv) was dissolved in 30 mL
of absolute ethanol
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and cooled with an ice bath and triethyl amine (2.5 mL, 0.0179 mol, 1.3 equiv)
was added. ten'-
butyl (4-aminophenyl)carbamate (2.879, 0.0138 mol, 1.0 equiv) was dissolved in
15 mL of absolute
ethanol and transferred to an additional funnel. The aniline was added
dropwise and the reaction
was allowed to warm to RT. After 24h, the reaction was heated to 40 C until
the reaction was
complete. The solvent was removed under reduced pressure and purified via
column
chromatography using 5-50% ethyl acetate in hexanes to produce 8 (3.88g, 80%)
as an orange
solid. TLC (20% ethyl acetate/dichloromethane), Rf= 0.49; m.p. 109-111 C; 1H
NMR (400 MHz,
CD30D) 67.371-7.437 (m, 4H), 6.446 (s, 1H), 2.281 (s, 3H), 1.513 (s, 9H); 13C-
NMR (100 MHz,
CD30D) 168.015, 163.907, 160.978, 155.334, 137.049, 134.613, 123.140, 120.421,
80.832,
54.787, 28.713, 23.136; IR (neat) umõ 1723.38, 1591.78, 1518.26, 1398.75,
1310.28, 1221.08,
1152.21, 1024.67, 970.18, 910.69, 835.64, 735.57, 515.54; ESI-HRMS [m+H]
calculated for
016H19C1N402, 334.12, found 335.1257.
tert-butyl (4((6-methy1-2-(pyrrolidin-1-yl)pyrimidin-4-y1)
amino)phenyl)carbamate (9.1). 8
(3.88 g, 0.0116 mol, 1.0 equiv) was dissolved in 25 mL anhydrous DMF.
Potassium Carbonate
(2.08 g, 0.015 mol, 1.3 equiv) was added followed pyrrolidine (4.85 mL, 0.0581
mol, 5.0 equiv) and
the reaction was heated to 80 "C for 8h. The reaction was then diluted with
ethyl acetate and
washed with water then brine. The organic layer was dried over sodium sulfate
and concentrated
under reduced vacuum, resulting in an orange solid. The crude material was run
through a plug of
silica gel with 5% methanol in DCM, and concentrated to produce carbamate 9.1
as a yellow solid.
TLC (3% methanol/dichloromethane), Rf = 0.28; m.p. 118-120 C; 1H NMR (400
MHz, CDCI3) 6
7.303-7.352 (m, 4H), 6.428 (s, N-H), 6.294 (s, N-H), 5.767 (s, 1H), 3.532-
3.609 (m, 4H), 2.219 (s,
3H), 1.928-1.961 (m, 4H), 1.520 (s, 9H); 13C-NMR (100 MHz, CDCI3) 166.567,
161.606, 160.709,
153.112, 134.881, 134.083, 122.437, 119.567, 92.520, 80.474, 46.660, 28.447,
25.602, 24.318; IR
(neat) umax 2971.67, 1718.69, 1569.84, 1505.67, 1399.06, 1227.56, 1155.05,
1050.05, 750.41,
515.91; ESI-HRMS [M+H]* calculated for C20H27H502, 369.22, found 370.2225.
tert-butyl (4((6-methy1-2-morpholinopyrimidin-4-ypamino)phenyOcarbamate (9.2).
8 (199.0
mg, 0.594 mmol, 1.0 equiv) was dissolved in acetone (3.4 mL) and cooled with
an ice bath.
Sodium carbonate (69.3 mg, 0.653 mmol, 1.1 equiv) was added followed by
morpholine (53.0 pL,
0.612 mmol, 1.03 equiv) in 1.0 mL of acetone, dropwise. The ice bath was
removed and the
reaction was heated to 80 00 for 8h. The reaction was diluted with ethyl
acetate, then washed with
water and brine. The organic layer was dried over sodium sulfate, concentrated
under reduced
pressure, then purified via column chromatography using 1% methanol in
dichloromethane to
produce 9.2 (122.88 mg, 54% yield) as a white solid. TLC (5% acetone in
dichloromethane), Rf=
0.18; m.p. 226-228 C; 1H NMR (400 MHz, CDCI3) 6 7.317-7.349 (m, 2H), 7.243-
7.264 (m, 2H),
6.465 (s, N-H), 6.314 (s, N-H), 5.816 (s, 1H), 3.742-3.765 (m, 8H), 2.203 (s,
3H), 1.520 (s, 9H);
13C-NMR (100 MHz, CDC13) 167.048, 162.072, 161.944, 153.257, 134.747, 134.264,
123.308,
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119.610, 93.338, 80.766, 67.148, 44.511, 28.500, 24.474; IR (neat) 1),õ
2947.41, 1702.51,
1579.06, 1489.70, 1357.90, 1230.06, 1155.92, 1004.35, 811.14, 746.29, 516.15;
ESI-HRMS
[M+H] calculated for C201-127N603, 385.21, found 386.2170.
241 H-indo1-3-y1)-N-(4-((6-methyl-2-(pyrrolidin-1-yOpyrimidin-4-
yOarnino)phenyOacetarnide
(6.5). 4.1 (70.0 mg, 0.260 mmol, 1.0 equiv) was dissolved in anhydrous DCM
(1.5 mL) then treated
with DMAP (31.8 mg, 0.260 mmol, 1.0 equiv), DCC (67.1 mg, 0.325 mmol, 1.25
equiv), and 2-(1H-
indo1-3-yl)acetic acid (47.8 mg, 0.273 mmol, 1.05 equiv). The reaction was
allowed to stir for 12h at
room temperature. Upon completion, the reaction was filtered through a pad of
celite and
concentrated on to silica gel. Purification via column chromatography using
1:1 ethyl
acetate/dichloromethane and 3% methanol produced the desired product 6.5 (92.0
mg, 83 % yield)
as a white solid. TLC (3% methanol/dichloromethane), Rf = 0.24; m.p. 197-199
C; 1H NMR (400
MHz, CD30D) 6 7.894 (s, N-H), 7.603-7.638 (m, 3H), 7.417-7.440 (d, J=8.9 Hz,
2H), 7.347-7.367
(d, J=8.2 Hz, 1H), 7.226 (s, 1H), 7.109 (t, 1H), 7.028 (t, 1H), 5.831 (s, 1H),
3.807 (s, 2H), 3.527-
3.560 (m, 4H), 2.187 (s, 3H), 1.969-1.978 (m, 4H); 13C-NMR (100 MHz, CD30D)
172.991, 162.612,
161.617, 138.384, 138.140, 134.053, 128.607, 124.802, 122.560, 121.985,
121.160, 119.975,
119.411, 112.328, 109.563, 95.337, 79.466, 47.804, 34.895, 26.455, 23.433; IR
(neat) uma,
1503.05, 1401.52, 1202.57, 828.19, 738.45, 512.00; ESI-HRMS [M+H] calculated
for C28H26N60,
426.22, found 427.2220.
341 H-indo1-3-y1)-N-(446-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)phenyl)
propanamide (6.6). 4.1 (30.1 mg, 0.112 mmol, 1.0 equiv) was dissolved in
anhydrous DCM (1.5
mL) then treated with DMAP (13.7 mg, 0.112 mmol, 1.0 equiv), DCC (28.9 mg,
0.14 mmol, 1.25
equiv), and 3-(1H-indo1-3-yl)propanoic acid (23.3 mg, 0.123 mmol, 1.1 equiv).
The reaction was
allowed to stir for 12h at room temperature. Upon completion, the reaction was
filtered through a
pad of celite and concentrated on to silica gel. Purification via column
chromatography using 1:1
ethyl acetate/dichloromethane and 3% methanol produced the desired product 6.6
(38.3 mg, 78 %
yield) as a light orange solid. TLC (3% methanol/dichloromethane), Rf= 0.20;
m.p. 89-90 C; 1H
NMR (400 MHz, CD30D) 6 7.888 (s, N-H), 7.558-7.629 (m, 3H), 7.388-7.410 (d,
J=8.9 Hz, 2H),
7.306-7.327 (d, J=8.0 Hz, 1H), 7.061-7.097 (m, 2H), 6.979-7.015 (t, 1H), 5.834
(s, 1H), 3.529-
3.562 (m, 4H), 3.127-3.165 (m, 2H), 2.700-2.738 (t, 2H), 2.190 (s, 3H), 1.947-
1.980 (m, 4H); 13C-
NMR (100 MHz, CD30D) 174.149, 166.029, 162.608, 161.578, 138.254, 138.174,
134.099,
128.543, 122.992, 122.286, 121.974, 121.142, 119.538, 119.330, 115.073,
112.169, 95.353,
79.460, 47.808, 39.114, 26.452, 23.426, 22.627; IR (neat) umõ 1570.80,
1503.13, 1399.39,
1227.17, 791.94, 738.86, 514.31; ESI-HRMS [m+H] calculated for C26H28N60,
440.23, found
441.2382.
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441 H-indo1-3-y1)-N-(4-((6-methyl-2-(pyrrolidin-1-y1)pyrimidin-4-
y0amino)pheny0 butanamide
(6.7). 4.1 (26.7 mg, 0.0992 mmol, 1.0 equiv) was dissolved in anhydrous DCM
(1.5 mL) then
treated with DMAP (12.1 mg, 0.0992 mmol, 1.0 equiv), DCC (25.6 mg, 0.124 mmol,
1.25 equiv),
and 4-(1H-indo1-3-yl)butanoic acid (20.2 mg, 0.109 mmol, 1.1 equiv). The
reaction was allowed to
stir for 12h at room temperature. Upon completion, the reaction was filtered
through a pad of celite
and concentrated on to silica gel. Purification via column chromatography
using 1:1 ethyl
acetate/dichloromethane and 3% methanol produced the desired product 6.7 (43
mg, 95% yield)
as a light orange solid. TLC (5% methanol/dichloromethane), Rf= 0.33; m.p. 157-
159 C; 1H NMR
(400 MHz, CD30D) 87.656 (s, N-H), 7.404-7.426 (d, J=8.9 Hz, 2H), 7.314-7.334
(d, J=7.9 Hz, 1H),
7.212-7.234 (d, J=8.9 Hz, 2H), 7.090-7.110 (d, J=8.0 Hz, 1H), 6.750-6.875 (m,
3H), 5.609 (s, 1H),
3.250-3.350 (m, 4H), 2.616 (t, 2H), 2.188 (t, 2H), 1.967 (s, 3H), 1.877 (m,
2H), 1.660-1.750 (m,
4H); 130-NMR (100 MHz, CD30D) 174.403, 165.762, 162.519, 161.371, 138.179,
138.136,
134.163, 128.769, 122.968, 122.176, 121.839, 121.154, 119.419, 119.384,
115.626, 112.132,
95.418, 79.435, 47.780, 37.599, 28.738, 27.761, 26.403, 25.708, 23.363; IR
(neat) lima, 2861.61,
1570.45, 1503.18, 1398.69, 1229.00, 785.54, 738.42, 511.98; ESI-HRMS [M+H]
calculated for
02+1301\160, 454.25, found 455.2534.
(E)-3-(1H-indo1-3-y1)-N-(4-((6-methyl-2-(pyrrolidin-1-yOpyrimidin-4-
y0amino)pheny1)-
aciyiamide (6.8). 4.1 (19.0 mg, 0.0706 mmol, 1.0 equiv) was dissolved in
anhydrous DCM (1.5
mL) then treated with (E)-3-(1H-indo1-3-yl)acrylic acid (13.2 mg, 0.0706 mmol,
1.0 equiv), HBTU
(34.8 mg, 0.0918 mmol, 1.3 equiv), and DIPEA (25.0 mL, 0.141 mmol, 1.1 equiv).
The reaction
was allowed to stir for 12h at room temperature. Upon completion, the reaction
was diluted with
ethyl acetate and washed with water and brine. The organic layer was dried
over sodium sulfate
and concentrated, then purified via column chromatography using 0-10% methanol
in
dichloronnethane to produce the desired product 6.8 (20 mg, 63 % yield) as a
yellow oil. TLC (3%
methanol/dichloromethane, run twice), Rf= 0.33; m.p. 188-190 C; 1H NMR (400
MHz, CD30D) 6
7.918-7.978 (d, J=7.9 Hz, 1H), 7.860-7.899 (d, J=15.6 Hz, 1H), 7.669 (s, 4H),
7.626 (s, 1H), 7.436-
7.455 (d, J=7.9 Hz, 1H), 7.176-7.252 (m, 2H), 6.726-6.765 (d, J=15.6 Hz, 1H),
5.986 (s, 1H),
3.539-3.630 (bs, 4H), 2.296 (s, 3H), 2.072 (bs, 4H); 13C-NMR (100 MHz, CD30D)
168.323,
162.306, 139.243, 137.042, 135.967, 135.664, 131.257, 126.624, 123.692,
121.838, 121.798,
121.453, 121.117, 116.081, 114.168, 113.084, 96.286, 79.465, 36.938, 31.638
26.347, 21.908; IR
(neat) 0ma, 3107.31, 1581.55, 1508.82, 1403.71, 1343.98, 1241.42, 1180.48,
829.45, 743.93; ESI-
HRMS [m+H]* calculated for C26H26N60, 438.22, found 439.2223.
1H-pyrrolo12,3-131pyridine-3-carboxylic acid N-(44(6-methy1-2-(pyrrolidin-1-
yOpyrimidin-4-
y0amino)phenyl)- 1H-pyrrolo[2,3-13.1pyridine-3-carboxamide (6.9). 4.1 (13.2
mg, 0.049 mmol,
1.0 equiv) was dissolved in anhydrous DFM (0.5 mL) then treated with 1H-
pyrrolo[2,3-b]pyridine-3-
carboxylic acid (8.0 mg, 0.049 mmol, 1.0 equiv), HBTU (21.4 mg, 0.056 mmol,
1.15 equiv), and
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DIPEA (9.8 mL, 0.056 mmol, 1.15 equiv). The reaction was allowed to stir for
12h at room
temperature. Upon completion, the reaction was diluted with ethyl acetate and
washed with water
and brine. The organic layer was dried over sodium sulfate and concentrated,
then purified via
column chromatography using 0-10% methanol in dichloromethane to produce the
desired product
6.9 (17.4 mg, 86 % yield) as a yellow solid. TLC (3% methanol / dichloro-
methane), Rf= 0.24
(3%methanol/dicloromethane); m.p. 295-297 C; 1H NMR (400 MHz, CD30D) 6 2.234
(s, N-H),
8.482-8.505 (m, 1H), 8.440 (s, 1H), 8.296-8.311 (m, 1H), 7.692 (s, 4H), 7-195-
7.226 (dd, 1H),
5.926 (s, 1H), 3.509 (bs, 4H), 2.177 (s, 3H), 1.924 (bs, 4H); 130-NMR (100
MHz, CD0I3) 162.263,
160.458, 148.461, 143.662, 129.324, 128.763, 128.564, 120.305, 120.199,
120.197, 119.935,
118.752, 117.094, 109.395, 99.522, 46.569, 24.964; IR (neat) umõ3297.80,
1661.79, 1576.88,
1501.18, 1450.36, 1338.97, 1221.50, 1012.59, 825.14, 689.68, 596.71, 510.91;
ESI-HRMS [M+H]
calculated for 023H23N70, 413.20, found 414.2022.
N-(4-((5-methoxy-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny1)-2-(thiophen-2-
y1)-acetamide
(6.10). The synthesis of 6.10 utilized Scheme 8 methodology and the following
starting materials.
2,4-dichloro-5-methoxypyrimidine (13) (116.7 mg, 0.652 mmol, 1.0 equiv) was
dissolved in ethanol
(3 mL) and cooled to 000, then treated with triethyl amine (109.1 L, 0.782
mmol, 1.2 equiv) and 1
(70.5 mg, 0.652 mmol, 1.0 equiv). The reaction was allowed to warm to RT and
stir for 8h then
concentrated and purified via column chromatography using 5% methanol in
dichloronnethane to
provide (14) (154.0 mg, 94% yield) as a white solid. TLC (7%
methanol/dichloromethane), Rf =
0.19; m.p. 167-169 00; 1H NMR (400 MHz, CDCI3) d 7.644 (s, 1H), 7.412-7.434
(d, J=8.7 Hz, 2H),
7.089 (s, N-H), 6.691-6.713 (d, J=8.7 Hz, 2H), 3.944 (s, 3H), 3.631(s, N-H,
2H); 130-NMR (100
MHz, CDCI3) 152.720, 151.597, 143.403, 139.311, 133.572, 129.027, 122.385,
115.666, 56.372;
IR (neat) 1)max 3332.41, 1608.05, 1575.91, 1509.54, 1461.05, 1335.49, 1254.70,
1003.45, 961.94,
833.83, 756.01, 634.82, 553.64, 515.78, 459.09; ESI-HRMS [M+H] calculated for
Cii Hi iCIN40,
250.06, found 251.0685.
2-(4-bromothiophen-2-y1)-N-(44(6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-
y0amino)pheny1)-
acetamide (6.11). 4.1 (2.33 g, 0.00866 mol, 1.0 equiv) was dissolved in
anhydrous DCM (100 mL)
then treated with DMAP (1.06 g, 0.00866 mol, 1.0 equiv), DCC (2.23 g, 0.0108
mol, 1.25 equiv),
and 2-(4-bromothiophen-2-yl)acetic acid (2.11 g, 0.00952 mol, 1.1 equiv). The
reaction was
allowed to stir for 24h at room temperature. Upon completion, the reaction was
filtered through a
pad of celite and concentrated on to silica gel. Purification via column
chromatography using 1:1
ethyl acetate/dichloromethane and 3% methanol produced the desired product
6.11 (3.76 mg,
brown solid) in 92% yield. TLC (8% methanol/dichloromethane), Rf = 0.42; m.p.
195-197 00; 1H
NMR (400 MHz, CDCI3) 37.386-7.434 (m, 4H), 7.325 (s, N-H), 7.197 (s, 1H),
6.957 (s, 1H), 6.370
(s, N-H), 5.777 (s, 1H), 3.885 (s, 2H), 3.547-3.580 (m, 4H), 2.224 (s, 3H),
1.925-1.959 (m, 4H);
13C-NMR (100 MHz, CDC13) 166.809, 161.075, 137.195, 136.470, 132.471, 130,052,
123.159,
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121.889, 121.607, 120.978, 109.899, 99.980, 92.628, 46.618, 38.423, 25.531,
24.357; IR (neat)
um, 2862.68, 1568.07, 1501.64, 1400.13, 1333.38, 1231.08, 785.83, 563.89; ESI-
HRMS [M+H]
calculated for C21H22BrN50S, 471.07, found 472.0787.
N-(44(2-chloro-6-methylpyrimidin-4-yl)oxy)pheny1)-2-(thiophen-2-yOacetamide
(6.12).
11.1 (63.0 mg, 0.27 mool, 1.0 equiv) was dissolved in ethanol (absolute, 3 mL)
then treated with
2.1 (44.1 mg, 0.270 mmol, 1.0 equiv), potassium carbonate (44.8 mg, 0.324
mmol, 1.2 equiv) then
a crystal of KI. The reaction was allowed to stir at RT for 8h, which was then
concentrated onto
silica gel and purified via column chromatography using 0-3% methanol in
dichloromethane to
produce 6.12 (92.8 mg, 96%) was found as an off-white solid. TLC (3%
methanol/dichloromethane), Rf = 0.45; m.p. 176-177 C; 1H NMR (400 MHz, CDCI3)
LILII7.566 (s,
N-H), 7.476-7.512 (d, J=8.9 Hz, 2H), 7.287-7.304 (dd, 1H), 7.018-7.063 (m,
4H), 6.564 (s, 1H),
3.937 (s, 2H), 2.453 (s, 3H); 13C-NMR (100 MHz, CDCI3) 171.760, 170.920,
168.118, 160.102,
148.396, 135.564, 135.517, 127.912, 127.728, 126.211, 122.103, 121.936,
121.570, 121.238,
115.629, 105.169, 38.549, 24.044; IR (neat) uma, 1655.62, 1578.47, 1507.89,
1323.18, 1207.23,
846.34, 793.77, 690.66, 481.83; ESI-HRMS [M+H] calculated for C17H14CIN302S,
359.05, found
360.0552.
N-(4-((6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0oxy)pheny0-2-(thiophen-2-
yOacetamide
(6.13). 6.12 (44.3 mg, 0.123 mmol, 1.0 equiv) was dissolved in DMF (anhydrous,
1 mL) and then
treated with potassium carbonate (20.3 mg, 0.148 mmol, 1.2 equiv) and
pyrrolidine (20.6 mL, 0.247
mmol, 2.0 equiv). The mixture was heated to 80 C for 8h, then diluted in
dichloromethane and
washed with water then brine. The organic layer was dried over sodium sulfate,
concentrated then
purified via column chromatography using 30-60 % ethyl acetate in hexanes to
provide 6.13 (8.5
mg, 18% yield) as a white solid. TLC (3% methanol/dichloromethane), Rf = 0.43;
m.p. 187-189 C;
1H NMR (400 MHz, CDCI3) 6 7.438-7.460 (d, J= 8.9 Hz, 2H), 7.310-7.350 (m, 1H),
7.049-7.102 (m,
3H), 5.727 (s, 1H), 3.960 (s, 2H), 3.484 (bs, 4H), 2.258 (s, 3H), 1.882-1.936
(m, 4H); 130-NMR
(100 MHz, CDC13) 170.292, 169.595, 167.758, 160.509, 149.532, 135.530,
134.331, 127.895,
127.678, 126.174, 122.203, 120.974, 93.152, 46.590, 38.484, 25.418, 24.404; IR
(neat) umax
1655.18, 1575.53, 1503.62, 1330.12, 1204.97, 961.26, 844.36, 792.73, 688.69,
567.34, 519.75,
480.84; ESI-HRMS [M+H] calculated for 017H14CIN302S, 394.15, found 395.1522.
N-(44(6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0oxy)pheny0-1H-indole-3-
carboxamide (6.14).
11.2 (9.0 mg, 0.024 mmol, 1.0 equiv) was dissolved in 1.0 mL anhydrous DMF.
Potassium
Carbonate (4.3 mg, 0.0312 mol, 1.3 equiv) was added followed pyrroldine (10
mL, 0.119 mmol, 5.0
equiv) and the reaction was heated to 80 'C for 8h. The reaction was then
diluted with ethyl
acetate and washed with water then brine. The organic layer was dried over
sodium sulfate and
concentrated under reduced vacuum, resulting in an orange solid. The crude
material was run
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through a plug of silica gel with 5% methanol in dichloromethane and
concentrated to produce 6.14
as a yellow solid. TLC (3% nnethanol/dichloronnethane), Rf= 0.20; m.p. 172-174
00; 1H NMR (400
MHz, CDCI3) 6 8.710 (s, N-H), 8.054-8.094 (m, 1H), 7.882-7.889 (d, J=4.0 Hz,
1H), 7.733 (s, N-H),
7.667-7.689 (d, J=8.9 Hz, 2H), 7.457-7.500 (m, 1H), 7.300-7.343 (m, 2H), 7.162-
7.184 (d, J= 8.9
Hz, 2H), 5.778 (s, 1H), 3.522 (bs, 4H), 2.281 (s, 3H), 1.909-1.943 (m, 4H);
130-NMR (100 MHz,
CDCI3) 170.631, 169.798, 163.563, 149.229, 136.539, 135.440, 128.389, 124.854,
123.429,
122.408, 122.143, 121.465, 120.113, 112.729, 112.252, 93.360, 60.557, 46.804,
25.581, 24.551,
21.214, 14.348; IR (neat) umax 3325.82, 1575.09, 1506.16, 1425.63, 1248.26,
1094.00, 949.73,
808.19, 775.56, 487.07; ESI-HRMS [M+H] calculated for C24H23N502, 413.19,
found 414.1908.
N-(44(6-methy1-2-morpholinopyrimidin-4-yl)amino)pheny1)-2-(thiophen-2-
y1)acetamide (6.15).
4.1 (17.5 mg, 0.061 mmol, 1.0 equiv) was dissolved in anhydrous DCM (1.5 mL)
then treated with
DMAP (7.5 mg, 0.061 mmol, 1.0 equiv), DCC (19.9 mg, 0.0763 mmol, 1.25 equiv),
and 5.1 (9.1
mg, 0.064 mmol, 1.05 equiv). The reaction was allowed to stir for 12h at room
temperature. Upon
completion, the reaction was filtered through a pad of celite and concentrated
on to silica gel.
Purification via column chromatography using 1:1 ethyl acetate/dichloromethane
and 3% methanol
produced the desired product 6.15 (14 mg, 56% yield) as a dark solid. TLC (5%
Methanol/dichloromethane), Rf = 0.60; m.p. 196-198 C; 1H NMR (400 MHz, CD30D)
67.454-
7.522 (q, 4H), 7.263-7.277 (d, J=5.7 Hz, 1H), 6.995 (bs, 1H), 6.952-6.974 (t,
1H), 5.907 (s, 1H),
3.870 (s, 2H), 3.723 (s, 8H), 2.190 (s, 3H); 13C-NMR (100 MHz, CD30D) 170.716,
166.644,
163.165, 162.779, 137.897, 134.192, 127.716, 127.482, 125.742, 121.808,
121.653, 96.230,
67.886, 45.856, 38.672, 23.833; IR (neat) umax 2842.93 1654.21, 1579.80,
1545.32, 1486.92,
1439.58, 1398.31, 1354.51, 1232.28, 1104.29, 993.25, 830.14, 786.59, 696.36,
483.49; ESI-
HRMS [M+H]* calculated for C21 H23N502S, 409.16, found 410.1630.
N-(44(6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny1)-2-(naphthalen-1-
yOacetamide
(6.16). 4.1 (33.6 mg, 0.125 mmol, 1.0 equiv) was dissolved in anhydrous DCM
(1.5 mL) then
treated with DMAP (15.3 mg, 0.125 mmol, 1.0 equiv), DCC (32.2 mg, 0.156 mmol,
1.25 equiv), and
2-(naphthalen-1-yl)acetic acid (25.6 mg, 0.137 mmol, 1.1 equiv). The reaction
was allowed to stir
for 12h at room temperature. Upon completion, the reaction was filtered
through a pad of celite and
concentrated on to silica gel. Purification via column chromatography using
1:1 ethyl
acetate/dichloromethane and 3% methanol produced the desired product 6.16 (22
mg, 40% yield)
as an off white solid. TLC (3% methanol/dichloromethane, run twice), Rf = 0.5;
m.p. 275-278 C; 1H
NMR (400 MHz, DMSO-d6) 6 10.168 (s, N-H, 1H), 8.946 (s, N-H, 1H), 8.134-8.154
(d, J=8.1 Hz,
1H), 7.926-7.948 (d, J=7.9 Hz, 1H), 7.830-7.855 (dd, 1H), 7.609-7.631 (d,
J=8.9 Hz 1H), 7.457-
7.582 (m, 6H), 5.824 (s, 1H), 4.121 (s, 2H), 3.435-3.467 (m, 4H), 2.113 (s,
3H), 1.864-1.896 (m,
4H); 13C NMR (100 MHz, DMS0-&) 168.533, 164.598, 160.633, 159.945, 136.439,
133.358,
132.978, 132.628, 131.997, 128.411, 127.776, 127.161, 126.065, 125.671,
125.539, 124.213,
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119.633, 119.483, 93.799, 46.185, 40.620, 25.019, 23.938; IR (neat) 1),õ
1660.02, 1575.10,
1503.95, 1401.14, 1227.15, 787.35, 558.68; ESI-HRMS [M+H] calculated for
C27H27N50, 437.22,
found 438.2273.
2-(6-chloro-1H-indo1-3-y0-N-(44(6-methyl-2-(pyrrolidin-1-yOpyrimidin-4-
yOamino)phenyOacetamide (6.18). 4.1 (25.8 mg, 0.096 mmol, 1.0 equiv) was
dissolved in
dichloromethane, anhydrous (1.5 mL) and treated with DMAP (12.8 mg, 0.105
mmol, 1.1 equiv),
DCC (24.8 mg, 0.12 mmol, 1.25 equiv) and 2-(6-chloro-1H-indo1-3-yl)acetic acid
(22.1 mg, 0.105
mmol, 1.0 equiv). The vial was purged with nitrogen and allowed to stir for
24h at RT. The solution
was filtered through a pad of celite and concentrated on to silica gel.
Purification via column
chromatography using 1:1 ethyl acetate/dichloromethane and 3% methanol
provided 6.18 (25.4
mg, 57% yield) as an off white solid. TLC (2% Methanol, 20% acetone, 78%
dichloromethane), Rf
= 0.57; m.p. 146-148 C; 1H NMR (400 MHz, CD30D) 6 7.613-7.651 (d, J=8.9 Hz,
2H), 7.557-7.578
(d, J=8.5 Hz, 1H), 7.427-7.449 (d, J=8.9 Hz, 2H), 7.348-7.360 (d, J=2.0 Hz,
1H), 7.242 (s, 1H),
6.994-7.019 (dd, 1H), 5.825(s, 1H), 3.784(s, 2H), 3.529-3.563(m, 4H), 2.189(s,
3H), 1.949-1.983
(m, 4H); 13C-NMR (100 MHz, CD30D) 172.615, 162.616, 159.239, 138.467, 138.412,
134.068,
128.481, 127.362, 125.759, 121.970, 121.184, 120.604, 120.468, 112.127,
110.057, 95.356,
76.019, 47.820, 34.666, 26.458, 23.418; IR (neat) uma, 1660.51, 1571.67,
1505.47, 1399.30,
795.55; ESI-HRMS [M+H] calculated for C25H25CIN60, 460.18, found 461.1834.
1-methyl-N-(44(6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny0-1H-indole-
3-
carboxamide (6.19). 4.1 (60 mg, 0.223 mmol, 1.0 equiv) was dissolved in
dichloromethane,
anhydrous (1.5 mL) and treated with DMAP (30 mg, 0.245 mmol, 1.1 equiv), DCC
(57.5 mg, 0.279
mmol, 1.25 equiv) and 1-methyl-1H-indole-3-carboxylic acid (39 mg, 0.223 mmol,
1.0 equiv). The
vial was purged with nitrogen and allowed to stir for 24h at RT. The solution
was filtered through a
pad of celite and concentrated on to silica gel. Purification via column
chromatography using 1:1
ethyl acetate/dichloromethane and 3% methanol provided 6.19 (23.6 mg, 25%
yield) as a light
yellow solid. TLC (5% methanol/dichloromethane, run twice), Rf = 0.58; m.p.
262-264 'C; 1H NMR
(400 MHz, CD30D) 6 8.176-8.196 (d, J=8.1 Hz, 1H), 7.945 (s, 1H), 7.668-7.690
(d, J=8.9 Hz, 2H),
7.560-7.582 (d, J=8.9 Hz, 2H), 7.425-7.445 (d, J=8.1 Hz, 1H), 7.250-7.291 (t,
1H), 7.187-7.227 (t,
1H), 5.857 (s, 1H), 3.855 (s, 3H), 3.544-3.577(m, 4H), 2.201 (s, 3H), 1.958-
1.991 (m, 4H); 13C-
NMR (100 MHz, CD30D) 166.950, 165.212, 162.641, 161.568, 138.775, 138.012,
134.530,
133.132, 128.188, 123.729, 122.537, 122.328, 121.277, 111.161, 110.925,
95.376, 79.464,
47.819, 36.937, 33.480, 26.463, 23.422; IR (neat) um, 3307.96, 1571.45,
1502.60, 1399.70,
1227.73, 1100.50, 741.44; ESI-HRMS [M+H] calculated for C25H26N60, 426.22,
found 427.2227.
N-(44(6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny0-2-(1H-pyrrolo12,3-
blpyridin-3-
yOacetamide (6.20) 4.1 (25.2 mg, 0.0936 mmol, 1.0 equiv) was dissolved in
dichloroemethane,
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anhydrous (1.5 mL) and treated with DMAP (12.6 mg, 0.103 mmol, 1.1 equiv), DCC
(24.1 mg,
0.117 mmol, 1.25 equiv) and 2-(1H-pyrrolo[2,3-b]pyridin-3-yl)acetic acid (39
mg, 0.223 mmol, 1.0
equiv). The vial was purged with nitrogen and allowed to stir for 24h at RT.
The solution was
filtered through a pad of celite and concentrated on to silica gel.
Purification via column
chromatography using 1:1 ethyl acetate/dichloromethane and 3% methanol
provided 6.20 (36.0
mg, 90% yield) as a white solid. TLC (20% acetone, 2% methanol, 78%
dichloromethane, run
twice), Rf = 0.35; m.p. 236-238 C; 1H NMR (400 MHz, CD30D) 38.175-8.191 (d,
J=5.1 Hz, 1H),
8.080-8.104 (d, J=7.9 Hz, 1H), 7.831 (s, 1H), 7.610-7.648 (d, J=9.0 Hz, 2H),
7.441-7.479 (d, J=9.0
Hz, 2H), 7.092-7.124 (dd, 1H), 5.849 (s, 1H), 3.818 (s, 2H), 3.545-3.578 (m,
4H), 2.204 (s, 3H),
1.965-1.999 (m, 4H); 13C-NMR (100 MHz, CD30D) 172.217, 162.578, 149.416,
143.291, 138.215,
134.262, 129.094, 125.774, 121.924, 121.851, 121.317, 116.466, 109.195,
101.393, 95.543,
47.878, 34.712, 26.440, 23.082; IR (neat) um, 2864.93, 1572.18, 1502.48,
1398.08, 1229.57,
753.09, 515.55; ESI- HRMS [M+H] calculated for C241-126N70, 427.21, found
428.2178.
2-(2-methy1-1H-indo1-3-y0-N-(44(6-methyl-2-(pyrrolidin-1-yOpyrimidin-4-
yOamino)phenyOacetamide (6.21). 4.1 (20.8 mg, 0.0773 mmol, 1.0 equiv) was
dissolved in
dichloromethane, anhydrous (1.5 mL) and treated with DMAP (9.44 mg, 0.0773
mmol, 1.1 equiv),
DCC (19.9 mg, 0.0966 mmol, 1.25 equiv) and 2-(2-methy1-1H-indo1-3-y1)acetic
acid (15.4 mg,
0.0773 mmol, 1.0 equiv). The vial was purged with nitrogen and allowed to stir
for 24h at RT. The
solution was filtered through a pad of celite and concentrated on to silica
gel. Purification via
column chromatography using 1:1 ethyl acetate/dichloromethane and 3% methanol
provided 6.21
(24.1 mg, 73.3% yield) as a white solid. TLC (3% methanol/dichloromethane), Rf
= 0.42; m.p. 142-
144 C; 1H NMR (400 MHz, CD30D) 6 7.592-7.614 (d, J=8.9 Hz, 2H), 7.471-7.491
(d, J=7.6 Hz,
1H), 7.383-7.405 (d, J=8.9 Hz, 2H), 7.240-7.258 (d, J=7.6 Hz, 1H), 6.947-7.038
(m, 2H), 5.802 (s,
1H), 3.739 (s, 2H), 3.492-3.525 (m, 4H), 2.417 (s, 3H), 2.168 (s, 3H), 1.908-
1.941 (m, 4H); 13C-
NMR (100 MHz, CD30D). 173.092, 165.696, 162.506, 161.309, 138.322, 137.089,
134.671,
133.965, 129.856, 122.078, 121.634, 121.134, 119.853, 118.485, 111.417,
105.226, 95.441,
47.791, 33.567, 26.409, 23.316, 11.539; IR (neat) umax 1571.52, 1505.98,
1399.07, 741.35; ESI-
HRMS [m+H] calculated for C26H28N60, 440.23, found 441.2381.
2-(5-methoxy-1H-indo1-3-y0-N-(44(6-methy1-2-(pyrroliclin-1-yOpyrimidin-4-
y0amino)pheny0
acetamide (6.22). 4.1 (21.6 mg, 0.0802 mmol, 1.0 equiv) was dissolved in
dichloromethane,
anhydrous (1.5 mL) and treated with DMAP (9.80 mg, 0.0802 mmol, 1.1 equiv),
DCC (20.7 mg,
0.100 mmol, 1.25 equiv) and 2-(5-methoxp1H-indo1-3-yl)acetic acid (17.3 mg,
0.0843 mmol, 1.0
equiv). The vial was purged with nitrogen and allowed to stir for 24h at RT.
The solution was
filtered through a pad of celite and concentrated on to silica gel.
Purification via column
chromatography using 1:1 ethyl acetate/dichloromethane and 3% methanol
provided 6.22 (15.5
mg, 44% yield) as a white solid. TLC (3% methanol/dichloromethane), Rf = 0.55;
m.p. 167-169 C;
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1H NMR (400 MHz, CD30D) 6 7.621-7.643 (d, J=8.9 Hz, 2H), 7.429-7.451 (d, J=8.9
Hz, 2H),
7.237-7.259 (d, J=8.7 Hz, 1H), 7.199 (s, 1H), 7.118-7.124 (d, J=2.0 Hz 1H),
6.760-6.788 (dd, 1H),
5.844 (s, 1H), 3.802 (s, 3H), 3.775 (s, 2H), 3.552 (bm, 4H), 2.195 (s, 3H),
1.974 (bm, 4H). 13C-
NMR (100 MHz, CD30D); 173.054, 162.612, 155.223, 137.780, 134.158, 133.356,
128.899,
125.518, 121.988, 121.257, 113.029, 112.906, 109.361, 101.367, 95.418, 79.464,
56.255, 47.828,
35.031, 26.454, 23.724, 20.049; IR (neat) urr,õ 2864.55, 1573.52, 1504.15,
1398.88, 1224.02,
790.75, 513.02; ESI- HRMS [M+H] calculated for 026H28N602, 456.23, found
457.2329.
2-(5-bromo-1H-indo1-3-y1)-N-(446-methy1-2-(pyrrolidin-1-yl)pyrimidin-4-
yl)amino)phenyl)
acetamide (6.23). 4.1 (17.0 mg, 0.0632 mmol, 1.0 equiv) was dissolved in
dichloromethane,
anhydrous (1.5 mL) and treated with DMAP (7.72 mg, 0.0632 mmol, 1.1 equiv),
DCC (16.3 mg,
0.079 mmol, 1.25 equiv) and 2-(5-bromo-1H-indo1-3-yl)acetic acid (17.7 mg,
0.0695 mmol, 1.0
equiv). The vial was purged with nitrogen and allowed to stir for 24h at RT.
The solution was
filtered through a pad of celite and concentrated on to silica gel.
Purification via column
chromatography using 1:1 ethyl acetate/dichlorornethane and 3% methanol
provided 6.23 (30.7
mg, 99% yield) as a white solid. TLC (5% methanol/dichloromethane), Rf = 0.47;
m.p. 151-153 C;
1H NMR (400 MHz, CD30D) 6 7.899 (s, 1H), 7.784-7.788 (d, J=1.9 Hz, 1H), 7.630-
7.652 (d, J=8.9
Hz, 2H), 7.436-7.459(d, J=8.9 Hz 2H), 7.264-7.272(m, 2H), 7.183-7.210 (dd,
1H), 5.843 (s, 1H),
3.769 (s, 2H), 3.518-3.598 (m, 4H), 2.193 (s, 3H), 1.948-2.003 (m, 4H); 13C-
NMR (100 MHz,
CD30D) 172.565, 162.617, 161.530, 155.127, 138.382, 136.716, 134.086, 130.445,
126.290,
125.261, 122.170, 122.017, 121.233, 113.973, 113.149, 109.537, 95.377, 47.823,
34.618, 26.455,
23.383; IR (neat) umõ 2862.30, 1570.94, 1504.59, 1399.41, 1225.39, 789.49,
513.22; ESI- HRMS
[M+H] calculated for C25H25BrN60, 504.13, found 505.1327.
241 H-indo1-1-y0-N-(446-methyl-2-(pyrrolidin-1-yOpyrimidin-4-
y0amino)phenyOacetamide
(6.24). 4.1 (16.6 mg, 0.0617 mmol, 1.0 equiv) was dissolved in
dichloromethane, anhydrous (1.5
mL) and treated with DMAP (7.5 mg, 0.0617 mmol, 1.1 equiv), DCC (16.0 mg,
0.077 mmol, 1.25
equiv) and 2-(1H-indo1-1-yl)acetic acid (11.3 mg, 0.0648 mmol, 1.0 equiv). The
vial was purged
with nitrogen and allowed to stir for 24h at RT. The solution was filtered
through a pad of celite and
concentrated on to silica gel. Purification via column chromatography using
1:1 ethyl
acetate/dichloromethane and 3% methanol provided 6.24 (12.0 mg, 47% yield) as
an off white
solid. TLC (3% methanol/dichloromethane), Rf= 0.32; m.p. 275-277 00;1H NMR
(400 MHz,
CD30D) 67.111-7.130 (d, J=7.8 Hz, 1H), 6.875-6.899 (d, J=8.9 Hz, 2H), 6.756-
6.825 (m, 4H),
6.708 (t, 1H), 6.599-6.650 (m, 2H), 6.083-6.091 (d, J=3.2 Hz, 1H), 5.230 (s,
1H), 4.381 (s, 2H),
2.960-2.993 (m, 3H), 1.642 (s, 3H), 1.377-1.410 (m, 4H); 13C-NMR (100 MHz,
CD30D) 173.133,
162.616, 138.337, 136.690, 136.060, 134.116, 128.780, 124.921, 123.207,
122.022, 121.234,
117.835, 112.158, 109.187, 95.39434.965, 30.173, 26.451, 23.333, 17.153; IR
(neat) um.
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1668.49, 1576.69, 1502.37, 1400.50, 1330.94, 1227.86, 794.97, 735.87, 568.62,
509.47; ESI-
HRMS [M+H] calculated for 025H26N60, 426.22, found 427.2224.
2-(5-hydroxy-1H-indo1-3-y1)-1V-(446-methyl-2-(pyrrolidin-1-yOpyrimidin-4-
y0amino)phenyl)
acetamide (6.25). 4.1 (44.2 mg, 0.164 mmol, 1.0 equiv) was dissolved in
dichloromethane,
anhydrous (1.5 mL) and treated with DMAP (20.0 mg, 0.164 mmol, 1.0 equiv), DCC
(42.3 mg,
0.205 mmol, 1.25 equiv) and 2-(5-hydroxy-1H-indo1-3-yl)acetic acid (33.0 mg,
0.173 mmol, 1.0
equiv). The vial was purged with nitrogen and allowed to stir for 24h at RT.
The solution was
filtered through a pad of celite and concentrated on to silica gel.
Purification via column
chromatography using 1:1 ethyl acetate/dichloromethane and 3% methanol
provided 6.25 (15.5
mg, 21% yield) as a white solid. TLC (5% methanol/dichloromethane), Rf = 0.28;
m.p. 196-198 C;
1H NMR (400 MHz, CD30D) 6 7.614-7.636 (d, J=8.9 Hz, 2H), 7.445-7.467 (d, J=8.9
Hz, 2H),
7.173-7.203 (m, 2H), 6.983-6.988 (d, J=3.2 Hz, 1H), 6.675-6.703 (dd, 1H),
5.877 (s, 1H), 3.746 (s,
2H), 3.547-3.581 (m, 4H), 2.221 (s, 3H), 1.961-2.025 (m, 4H); 13C-NMR (100
MHz, CD30D)
173.106, 162.529, 151.451, 151.370, 134.650, 133.095, 129.301, 125.657,
125.397, 121.978,
121.555, 112.809, 112.696, 108.727, 103.674, 103.594, 95.786, 52.325, 35.009,
31.977, 26.407;
IR (neat) um, 3258.66, 1578.16, 1506.67, 1397.36, 1228.60, 793.25; ESI-HRMS
[M+H] calculated
for C25H26N602, 442.21, found 443.2172.
2-(3-(cyclopropylmethyl)-1H-indol-1-y1)-N-(44(6-methyl-2-(pyrrolidin-1-
yl)pyrimidin-4-
y0amino)phenyOacetamide (6.26). 4.1 (17.0 mg, 0.0632 mmol, 1.0 equiv) was
dissolved in
dichloromethane, anhydrous (1.5 mL) and treated with DMAP (7.72 mg, 0.0632
mmol, 1.1 equiv),
DCC (16.3 mg, 0.079 mmol, 1.25 equiv) and 2-(3-(cyclopropylmethyl)-1H-indo1-1-
y1)acetic acid
(16.1 mg, 0.0663 mmol, 1.0 equiv). The vial was purged with nitrogen and
allowed to stir for 24h at
RT. The solution was filtered through a pad of celite and concentrated on to
silica gel. Purification
via column chromatography using 1:1 ethyl acetate/dichloromethane and 3%
methanol provided
6.26 (23.7 mg, 78% yield) as an off white solid. TLC (3%
methanol/dichloromethane), Rf = 0.27;
m.p. 275-277 C; 1H NMR (400 MHz, CD30D) (38.311 (s, 1H), 8.270-8.289 (d,
J=7.8 Hz, 1H),
7.780 (s, 1H), 7.625-7.647 (d, J= 8.9 Hz, 2H), 7.517-7.539 (d, J=8.9 Hz, 2H),
7.425-7.444 (d, J=
7.8 Hz, 1H), 7.218-7.305 (m, 2H), 5.918 (s, 1H), 5.109 (s, 2H), 3.554-3.586
(m, 4H), 2.605-2.667
(m, 1H), 2.250 (s, 3H), 1.991-2.024 (m, 4H), 1.956(s, 2H), 1.121-1.158 (m,
2H), 0.949-0.995 (m,
2H); 13C-NMR (100 MHz, CD30D) 197.917, 166.991, 162.055, 139.009, 138.658,
138.587,
137.137, 134.355, 127.271, 124.469, 123.493, 123.177, 121.899, 121.535,
118.579, 110.702,
96.389, 49.285, 50.561, 26.188, 22.943, 21.585, 18.662, 10.523; IR (neat) uma,
2009.39, 1508.43,
1386.73, 1167.26, 1065.42, 946.58, 744.69; ESI-HRMS [m+H]* calculated for
C29H32N602, 494.20,
found 495.2484.
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2-(5-ethy1-1H-indo1-3-y1)-N-(446-methyl-2-(pyrrolidin-1-yOpyrimidin-4-
y0amino)phenyl)
acetamide (6.27). 4.1 (15.0 mg, 0.056 mmol, 1.0 equiv) was dissolved in
dichloronnethane,
anhydrous (1.5 mL) and treated with DMAP (6.84 mg, 0.056 mmol, 1.1 equiv), DCC
(14.4 mg,
0.070 mmol, 1.25 equiv) and 245-ethyl-I H-indo1-3-yl)acetic acid (11.9 mg,
0.0585 mmol, 1.0
equiv). The vial was purged with nitrogen and allowed to stir for 24h at RT.
The solution was
filtered through a pad of celite and concentrated on to silica gel.
Purification via column
chromatography using 1:1 ethyl acetate/dichloromethane and 3% methanol
provided 6.27 (24.8
mg, 98% yield) as an off white solid. TLC (3% methanol/dichloromethane), Rf =
0.30; m.p. 177-179
C; 1H NMR (400 MHz, CD30D) 6 7.617-7.639 (d, J=8.9 Hz, 2H), 7.417-7.439 (m,
3H), 7.257-
7.278 (d, J=8.9 Hz, 1H), 7.189 (s, 1H), 6.972-6.996 (d, J=8.4 Hz 1H), 5.838
(s, 1H), 3.789 (s, 2H),
3.530-3.563 (m, 4H), 2.685-2.742 (q, 2H), 2.191 (s, 3H), 1.950-1.984 (m, 4H),
1.253 (t, 3H); 13C-
NMR (100 MHz, CD30D) 173.133, 162.616, 138.337, 138.332, 136.690, 136.060,
134.116,
128.780, 124.921, 123.207, 122.022, 121.234, 117.835, 112.158, 109.187,
95.394, 47.829,
34.965, 30.173, 26.452, 23.333, 17.153; IR (neat) Umax 2957.96, 1504.91,
1399.77, 1253.22,
803.62; ESI- HRMS [M+H] calculated for 027H30N60, 454.25, found 455.2535.
2-(1H-benzordlimidazole-1-y1)-N-(446-methyl-2-(pyrrolidin-1-yOpyrimidin-4-
Aamino)phenyl)
acetamide (6.28). 4.1 (15.6 mg, 0.0580 mmol, 1.0 equiv) was dissolved in
dichloromethane,
anhydrous (1.5 mL) and treated with DMAP (7.1 mg, 0.0580 mmol, 1.1 equiv), DCC
(15.0 mg,
0.0725 mmol, 1.25 equiv) and 2-(1H-benzo[d]imidazol-1-yl)acetic acid (10.7 mg,
0.0609 mmol, 1.0
equiv). The vial was purged with nitrogen and allowed to stir for 24h at RT.
The solution was
filtered through a pad of celite and concentrated on to silica gel.
Purification via column
chromatography using 1:1 ethyl acetate/dichloromethane and 3% methanol
provided 6.28 (16.9
mg, 68% yld) as a white solid. TLC (3% methanol/dichloromethane), Rf = 0.27;
m.p. 175-177 00; 1H
NMR (400 MHz, DMSO-d6) 6 10.368 (s, 1H), 8.229 (s, 1H), 7.641-7.678 (m, 3H),
7.486-7.542 (m,
3H), 7.190-7.270 (m, 2H), 5.857 (s, 1H), 5.148 (s, 2H), 3.446-3.505 (m, 4H),
2.135 (s, 3H), 1.885-
1.916 (m, 4H); 13C-NMR (100 MHz, DMSO-d8) 179.572, 164.927, 144.989, 143.167,
134.379,
122.336, 121.465, 119.673, 119.323, 110.280, 72.465, 63.055, 48.568, 47.275,
46.295, 24.955; IR
(neat) urna, 3093.18, 1581.23, 1502.31, 1403.46, 1298.11, 1230.31, 1175.88,
835.05, 738.59; ESI-
HRMS [m+H]* calculated for C24H25N70, 427.21, found 428.2177.
N-(44(6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny1)-2-(144-
(trifluoromethyl)
phenyl)sulfony1)-1H-indo1-3-yl)acetamide (6.29). 4.1 (15.6 mg, 0.0580 mmol,
1.0 equiv) was
dissolved in dichloromethane, anhydrous (1.5 mL) and treated with DMAP (7.1
mg, 0.0580 mmol,
1.1 equiv), DCC (15.0 mg, 0.0725 mmol, 1.25 equiv) and 2-(1-((4-
(trifluoromethyl)phenyl)sulfonyl-
H-indo1-3-y1) acetic acid (117, 1.0 equiv). The vial was purged with nitrogen
and allowed to stir for
24h at RT. The solution was filtered through a pad of celite and concentrated
on to silica gel.
Purification via column chromatography using 1:1 ethyl acetate/dichloromethane
and 3% methanol
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provided 6.29 (16.9 mg, 68% yield) as a white solid. TLC (3%
methanol/dichloromethane), Rf =
0.47; m.p. 230-232 00; 1H NMR (400 MHz, CD30D) 6 8.102-8.123 (d, J=8.4 Hz,
2H), 7.989-8.010
(d, J=8.4 Hz, 1H), 7.793-7.814 (d, J=8.4 Hz, 1H), 7.612-7.676 (m, 4H), 7.449-
7.471 (d, J=8.9 Hz,
2H), 7.344-7.383 (t, 1H), 7.264-7.302 (t, 1H), 5.878 (s, 1H), 3.787 (s, 2H),
3.554-3.586 (m, 4H),
2.219 (s, 3H), 1.978-2.010 (m, 4H); 130-NMR (100 MHz, CD30D) 170.699, 162.550,
142.731,
136.537, 136.446, 136.116, 132.265, 128.837, 127.658, 126.295, 126.000,
124.954, 121.830,
121.473, 121.061, 119.402, 114.738, 101.399, 79.474, 47.965, 33.994, 26.429;
IR (neat) umõ
1649.02, 1577.42, 1505.48, 1400.80, 1319.87, 1169.71, 1126.77, 1059.29,
830.21, 784.74,
713.73, 607.93, 558.38, 427.28; ESI-HRMS [M+H]* calculated for 032H29F3N603S,
634.2, found
635.2022.
Synthesis of intermediate 2-(1((4-(trifluoromethyl)phenyl)sulfonyl-H-indo1-3-
y1) acetic acid
(117) is shown in Scheme 10.
0
..,i? 00 3,
TRAH2SO4, SO% #(0143,....
11 .1 Phfole
vc --- /
I
N
7. =
Fz-C
116
d:oxa1e12N NaOH N
µS-=
\N
Fz,C.
117
Scheme 10
Methyl 2-(1-((4-(trifluoronnethyl)phenyl)sulfony1)-1H-indo1-3-yl)acetate (116)
37.3 mg, 0.0939 rinnnol)
was dissolved in dioxane (1 mL)/water (0.5 mL) then treated with 2N NaOH
aqueous solution (0.5
mL) and the reaction was allowed to stir for 0.5 h. The reaction was quenched
with 1N HCI until the
pH was around 4. The aqueous layer was extracted with ethyl acetate, dried
over sodium sulfate,
to obtain 117 as an off white solid (31.3 mg, 87% yield). TLC (3% methanol/
dichloromethane), Rf =
0.69; m.p. 196-198 C; 1H NMR (400 MHz, CD30D) d 8.092 (d, J=8.4 Hz, 2H),
7.964-7.984 (d, J=
8.4 Hz, 1H), 7.782-7.803 (d, J=8.4 Hz, 2H), 7.640 (s, 1H), 7.542-7.562 (d, J=
7.8 Hz, 1H), 7.315-
7.355 (t, 1H), 7.232-7.271 (t, 1H), 3.682 (s, 2H); 130-NMR (100 MHz, CD30D)
175.390, 142.801,
136.448, 136.373, 136.044, 132.443, 128.792, 127.601, 126.092, 125.838,
124.780, 121.131,
188
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119.441, 114.592, 32.205; IR (neat) umax 1690.56, 1272.57, 1318.67, 1170.49,
1121.30, 1057.78,
979.30, 837.70, 749.36, 711.52, 640.54, 603.04, 557.08, 426.66; ESI-HRMS
[M+H]+ calculated for
C17H12F3NO4S, 383.3412, found 384.0497.
Synthesis of intermediate Methyl 2-(1-((4-(trifluoromethyl)phenyl)sulfony1)-1H-
indo1-3-yl)acetate
(116). Methyl 2-(1Hindo1-3-yl)acetate (54.8 mg, 0.290 mmol, 1.0 equiv), 4-
(trifluoromethyl)benzenesulfonyl chloride (85.1 mg, 0.348 mmol, 1.2 equiv),
and
tetrabutylammonium hydrogen sulfate (9.85 mg, 0.029 mmol, 0.1 equiv) were
dissolved in toluene
(2 mL) and cooled in an ice bath (Scheme 10). A 50% potassium hydroxide
solution (400 mL) was
added dropwise and the reaction was allowed to stir for 8h with vigorous
stirring. The reaction was
diluted with ethyl acetate and washed with water and brine. The organic layer
was dried over
sodium sulfate, concentrated under reduced pressure, and purified via column
chromatography
using 5% acetone in dichloromethane to produce 16 (37.3 mg, 32% yield) as a
clear oil. TLC (5%
acetone/dichloromethane), Rf = 0.62; m.p. 94-96 C; 1HNMR (400 MHz, CDCI3) d
7.976-8.011 (m,
3H), 7.689 (d, J= 8.5 Hz, 2H), 7.579 (s, 1H), 7.502-7.512 (d, J= 7.8 Hz, 1H),
7.338-7.380 (m, 1H),
7.265-7.305 (m, 1H), 3.714 (s, 3H); 13C-NMR (100 MHz, CDC13) 170.919, 141.610,
135.971,
135.309, 135.086, 130.706, 127.445, 126.620, 125.465, 124.601, 123.953,
119.910, 116.313,
113.709, 52.364, 30.816; 19F-NMR (100 MHz, CD0I3) - 63.358; IR (neat) vnia,
1442.28, 1737.13,
373.50, 1316.00, 1251.99, 1168.40, 1123.27, 1058.93, 1010.33, 976.94, 839.25,
749.05, 708.96,
605.14, 557.41, 425.79; ESI-HRMS [M+H]+ calculated for 018H14F3N04S, 397.3682,
found
398.0655.
N-(4-((6-methy1-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny1)-2-(2-oxoindolin-
3-y1)
acetamide (6.30). 4.1 (22.0 mg, 0.0817 mmol, 1.0 equiv) was dissolved in
dichloromethane,
anhydrous (1.5 mL) and treated with HBTU (40.3 mg, 0.106 mmol, 1. equiv), 2-(2-
oxoindolin-3-
yl)acetic acid (17.2 mg, 0.090 mmol, 1.1 equiv) and then DIPEA (28.3 mL, 0.163
mmol, 1.0 equiv).
The vial was purged with nitrogen and allowed to stir for 24h at RT. The
solution was concentrated
on to silica gel and purified via column chromatography using 5-20% methanol
in dichloromethane
to provide 6.30 (16.0 mg, 45% yield) as a white solid. TLC (3%
methanol/dichloromethane), R=
0.27; m.p. 170-172 C; 1H NMR (400 MHz, CD30D) 6 7.626-7.648 (d, J=8.9 Hz,
2H), 7.484-7.506
(d, J=8.9 Hz, 2H), 7.192-7.252 (q, 2H), 6.961-6.999 (t, 1H), 6.907-6.926 (d,
J=7.8 Hz, 1H), 5.952
(s, 1H), 3.882-3.915 (m, 1H), 3.581-3.654(m, 4H), 3.060-3.111 (dd, 1H), 2.746-
2.818 (m, 1H),
2.277 (s, 3H), 2.036 (m, 4H); 130-NMR (100 MHz, CD30D) 181.633, 170.864,
162.145, 143.659,
136.567, 135.491, 130.594, 129.266, 125.084, 123.288, 122.212, 121.651,
110.921, 96.940,
55.830, 38.068, 36.945, 31.639, 26.245, 20.701, 13.165; IR (neat) Urnax
3323.06, 1571.06, 1502.57,
1335.88, 1231.30, 816.45, 663.70, 520.32; ESI- HRMS [M+H] calculated for
C25H26N602, 442.21,
found 443.2175.
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N-(44(6-methyl-2-(pyrrolidin-1-yOpyrimidin-4-y0amino)pheny1)-2-(144-
(trifluoromethyl)
phenyl)sulfony1)-1H-indo1-3-yl)acetamide (6.31). 4.1 (62.0 mg, 0.230 mmol, 1.0
equiv) was
dissolved in dichloromethane, anhydrous (6.0 mL) and treated with DMAP (28.1
mg, 0.230 mmol,
1.1 equiv), DCC (59.3 mg, 0.288 mmol, 1.25 equiv) and 2-(2-chloroquinolin-4-
yl)acetic acid (56.0
mg, 0.253 mmol, 1.0 equiv). The vial was purged with nitrogen and allowed to
stir for 24h at RT.
The solution was filtered through a pad of celite and concentrated on to
silica gel. Purification via
column chromatography using 1:1 ethyl acetate/dichloromethane and 3% methanol
provided 6.31
(13.5 mg, 57% yield) as a white solid. TLC (3% methanol/dichloromethane), Rf =
0.58; m.p. 260-
262 C; 1H NMR (400 MHz, CDCI3) 88.199-8.220 (d, J=8.5 Hz, 1H), 7.976-7.997
(d, J=8.5 Hz, 1H),
7.798-7.839 (t, 1H), 7.658-7.710 (m, 3H), 7.553 (s, 1H), 7.475-7.497 (d, J=8.9
Hz, 2H), 5.859 (s,
1H), 4.243 (s, 2H), 3.558-3.591 (m, 4H), 2.214 (s, 3H), 1.976-2.009 (m, 4H);
13C-NMR (100 MHz,
0DC13) 167.354, 165.894, 161.314, 160.216, 150.517, 147.781, 145.224, 136.193,
133.217,
130.757, 128.675, 127.402, 126.491, 124.017, 123.331, 121.492, 120.797,
116.000, 93.281,
46.653, 40.236, 25.483, 23.457; IR (neat) uma, 1755.53, 1658.71, 1612.71,
1548.21, 1504.15,
1403.49, 1251.05, 1138.16, 890.44, 696.38, 517.52 ESI-HRMS [M+H] calculated
for C26H25CIN60,
472.18, found 473.1834.
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LO
CO
Yj
Cn
N CI
Rih1
NH2 R1,1,C1
R271*14,1 ., 7
H2N S
CD
+ R2 ¨ Triethyl Amine,
NH Pyrrolidine R2rN o 3
NH R4AOH
CICo
Et0H, 0 C -> RI H2N K2CO3,
DMF, 80 C
H2N
1 2.1, Ri = Me; R2 = H 3.1, Ri = Me; R2 =
H 4.1, Ri = Me; R2 = H 5.1, R = Methyl Thiophene
(1)
2.2, = Me; R2 = F 3.2, Ri = Me; R2 = F
4.2, Ri = Me; R2 = F 5.2, R = 3-Indole
2.3, Ri = H; R2 = F 3.3, = H; R2 = F
4.3, Ri = H; R2 = F
CD
Co.
N
6.0, Ri = Me; R2 = H; R3 = H; R4 = Methyl Thiophene
C2F,
13P R2XN 6.1, Ri =
Me; R2 = F; R3 = H; R4 = Methyl Thiophene
o
6.2, Ri = H; R2 = F; R3= H; R4 = Methyl Thiophene
NaH, Mel,
THF, 0 C
3
Et3N, DCM 0 = 3 6.4, Ri =
Me; R2 = F; R3 = Me; R4 = Methyl Thlophene--. -o
R4AN 6.3, Ri = Me; R2 = H; R3 = H; 1:14 = 3-Indole 0
a
a
ts.)
n
>
o
u..
r.,
1..
u,
...
LO
CO
r,
0
r,
Y
r,
,
(1)
C,
N CI
7 0
il,,R3
A 1 'r
.
1 1 3 N
ii& NH2 1,.....,C1 Triethyl Amine,
1,N Pyrrolidine or Morpholine , cN
0 I I
0 N
>OAN iglr + . N _AI.
_
0
=a-,
Et0H, 0 C -> RI 2'
K2CO3, DMF, 80 C 0 lib NH co ui
H CI l't 0 6 NH
H
0
H
7 2.1 B
9.1, R3 = Pyrrolidine 0.
co
9.2, R3 = Morpholine
P
H
Nr, R3
D"
.,N R3
I "r 1 1 CD
TFA
N 0 . N o
_Jo,- I- R4OH -a
=s'
_s....
NH
0 di NH
m
DCM, 0 C to rt. I. DCC/DCM
Ri
or
Cl)
H2N cp_ HBTU/DMF 1:14)L
HN W
cn
4.1, R3 = Pyrrolidine
Analogs of 6
=
4.4, R3 = Morpholine
m-
1¨k
CD
Cr,
N
CD
(I'
B
r,:,:,,,,, R3 Ca
OH \cly-CI
I
r N 0
CD
0 0
o
+ A 1) DCC, DMAP, DCM 0
¨)... õit, 40 + 1 1
r N 1)
K2CO3, KI, Et0H
_______________________________________________________________________________
___ 1//0 rill 0 P.
fp
0 R4 OH 2) TFA, DCM R4 N 2)
Pyrrolidine, K2CO3, DMF 0 5.
H2N H CI
A 4V cr,
R4 N b
H Cl)
10 11.1, R4 = 2-methyl thiophene 2.1 12, R3 = CI; R4 =3-
Indole =
LI)
11.2, R4 = 3-indole
6.12, R3= CI; R4 = Methyl Thiophene 5
co
6.13, R3= Pyrrolidine; R4 = Methyl Thiophene pt,
6.14, R3= Pyrrolidine; R4 = 3-Indole
ro
n
.t.!
Cl)
N
0
N
N
e---
.6.
.6.
-4
.6.
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Example 14: Additional Synthetic Examples
Exemplary Synthesis of Benzyl Analogues of Compound 6
Scheme 10 illustrated an exemplary synthesis of benzyl analogues of compound
6. The
compound numbers below refer to the compound numbers in Scheme 10.
Scheme 10
40 NH2
+ N Et3N N
H2N AcCN, rt, 16= + N CI CI
N so
CI N CI
H2N
NH2
1 2 3 4
BrLCOOH
5
DMAP, DCC, DCM, 8 hrs
N
0= NNCI or _S 0 N N CI
Br¨Cc).L.
6 7
.....
8
DIPEA, n-BuOH,
120 C, 16 h, DMSO,
NaHCO3, rt ,10 min
or
N N(\ fNNN = 0 S \
Br /___S 0
===
..... =
Br
6.33, 6.44, 6.46 6.41,
6.45, 6.47
Synthesis of Intermediates 3 and 4 (Scheme 10)
1.5 g of 4-aminobenzylamine, 1, (12.28 mmol) was added to 25 mL acetonitrile
solution of 2,4-
dichloro-6-methylpyrimidine, 2, (2.0 g, 1 eq) and triethylamine (3.43 mL, 2
eq). The resulting
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solution was stirred at room temperature overnight, then diluted with water
and saturated sodium
bicarbonate solution followed by extraction with ethyl acetate. The combined
organic layers were
dried over anhydrous sodium sulfate, concentrated onto silica gel and purified
with Teledyne
Combiflash chromatography system to afford the 4-position isomer, 3, (orange
yellow solid, 1.84 g,
60.3%) and 2-position isomer, 4, (pale yellow solid, 0.66 g, 5.4%).
Synthesis of intermediates 6 and 7 (Scheme 10)
To a slurry of 3 (500 mg) and DMAP (1.1 eq) in freshly dried DCM (50 mL), was
added DCC (1.2
eq) and 4-bromothiophene acetic acid, 5, (1.2.eq). After stirring at room
temperature for 8 h, the
reaction mixture was filtered through a celite pad and concentrated on silica
gel. Purification via
silica gel flash chromatography gave 6 as brown solid in quantitative yield.
The reaction protocol
was repeated to obtain 7 as beige solid in quantitative yield.
Synthesis of CHD1Li 6.33, 6.40, 6.41, 6.44-6.47 and 6.57 (Scheme 10)
6 or 7 (150 mg) were each treated with 3 mL n-BuOH and DIPEA (3 eq) then
stirred at room
temperature for few minutes. The corresponding cyclic 2 amine was added and
the reaction
mixture was refluxed at 120 C overnight, then concentrated in vacuo to
dryness. The residue was
dissolved in ethyl acetate and basified with saturated sodium bicarbonate
solution. The combined
ethyl acetate extracts were dried over anhydrous sodium sulfate, concentrated
on silica gel and
purified via silica gel flash chromatography. Subsequently, the brown solid
products were dissolved
in DMSO and treated with saturated sodium bicarbonate solution to give an off
white precipitate.
Which was collected by filtration to afford the desired CHD1Li.
Exemplary Synthesis of Triazolopyridine Analogues of Compound 6.11
Scheme 11 illustrates an exemplary synthesis of triazolopyrimidine analogues
of compound 6.11.
Compound numbers referenced below refer to those in Scheme 11.
Scheme 12:
N-N 0 0 AcOH POCI3
\\_
poi
H2N¨N¨NH2 \/>¨N H2 )-L,}1.
pi-N 2
H9 90 C, 6 h
11 115 C, 2 h
10 CI12
NH2
DCC, DMAP
Boc.N Br COOH
DCM, r.t, 7 h, 90% Br¨GN N Boc
13 5 14
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TFA
Am NH
Br-C&..A N
DCM, 0 C to r.t, 4 h, 7513/0
NH2 BOC20 H NH2
H2N THF, r.t, 3 h, 95(7/0 Boc-N
1 16
Br-C-1---SCOOH
5 0 N Boc
-
DCC, DMAP, DCM, N
r.t, 12 h 87%
17
TFA S 0 NH2
N
DCM, r.t, 4 h, 87%
18
5
Br¨s NH
"Pj
NN Et3N, DMF
+ or 90 C,18 h, 55-
60%
CI
12 0 NH2
18
Nr4/>-NH
NN
2
Br-Cs 0 NH c5 0 N NN
IN Br-C)).LN 11
NH2
6.53 6.54
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Synthesis of Intermediate 12 (Scheme 11)
1.09 of triazole 9 and 1.28 mL of ethyl acetoacetate 10 were added to 30 nriL
acetic acid and refluxed
at 130 C for 6 h. The reaction mixture was diluted with ethanol and kept at -
20 C overnight. The
resulting white precipitate was filtered in vacuo to afford 11 in quantitative
yield. Subsequently, 11
(0.7 g) was added in portions to 5 mL POCI3 at 0 C. After stirring at this
temperature for few minutes,
the mixture was refluxed at 115 C for 2 h then concentrated in vacuo to
dryness. The brick red
residue was re-dissolved in DCM, basified with saturated NaHCO3 and filtered
in vacuo to give 0.51
g of 12 as an orange solid.
Synthesis of intermediate 15 (Scheme 11)
The required acetamide intermediate 14 was synthesized in 90% yield as a beige
solid following the
synthetic procedure for 7. Subsequent boc-deprotection with DCM-TFA (1:1)
solution afforded 15 in
75% yield as a brown solid.
Synthesis of intermediate 18 (Scheme 11)
A solution of 1 (1.0 mL) in dry THF was treated with dropwise addition of boc-
anhydride (1 eq) pre-
dissolved in dry THF. The reaction mixture was stirred under nitrogen for 2 h
then concentrated to
dryness under vacuum. The resulting yellow solid was washed with CHCI3-hexane
(1:2) to give 16
in quantitative yield. Analogous amide coupling and boc-deprotection as in 15
above afforded 18 in
87% yield as an off-white solid.
Synthesis of 6.53 and 6.54 (Scheme 11)
70 mg of 12 was added to a DMF solution (5 mL) of 15 or 16 (1 eq) containing
triethylamine (2 eq).
The reaction mixture was heated at 90 C for 18 h then poured onto crushed ice,
stirred and filtered
in vacuo to obtain crude products as brown solids. Purification with silica
gel Combiflash
chromatography afforded pure 6.53 and 6.54 as off-white solids in 55 and 60%
yields, respectively.
Scheme 12 illustrates exemplary synthesis of compounds 6.55 and 6.56.
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SCHEME 12:
N.CN
AcCN N-N
cN:1/> 111"21-1IA 4.1-1 "ir.,
2%.1
¨ itC
70 C, 7 h, 87%FI2NsIS17
19 8 20 21
0 0
}.}LC*
N
/>¨N P OC 3
- N
AcOH, 90 C, 6 h, 115 C, 2 h, 78%
0 CI
22 23
s 0 NH2
Br¨ /c,_N =
Et3N, DMF
or 90 C,18 h, 55-60%
N-N
CI
23 S NO 40 H2
N
18
N
),
NH NS 0 410N N
Br¨C1,N H
5 6.55 6.56
In the following description, compound numbers refer to Scheme 12.
Synthesis of Intermediate 22
A solution of 1.024 g (7 mmol) of dimethyl cyanocarbonimidodithioate 10 and
pyrrolidine (1.0 eq) in
10 acetonitrile (5 mL) were refluxed at 85 C for 2 h. Then, hydrazine
monohydrate (1.5 eq) was added
and refluxing continued for 5 h. After cooling to room temperature, the
resulting pale pink precipitate
was filtered in vacuo while washing with cold diethyl ether to obtain 21 in
quantitative yield.
Subsequently, 500 mg of 21 in acetic acid (7 mL) was treated with 10 (1 eq)
and refluxed at 90 C
for 6 h. The resulting precipitate was filtered while washing with cold
diethyl ether to give 22 as an
15 off-white solid in 89% yield. The conversion of 22 to 23 achieved in 78%
yield using the same
procedure as in 12 above.
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Synthesis of 6.55 and 6.56
6.55 and 6.56 were synthesized according the method described for 6.53 and
6.54 above.
Synthesis of 1,3-propanesultam analogue (Scheme 13)
In the following description, compound numbers refer to Scheme 13. To a 5 mL
acetonitrile solution
of boc-protected p-phenylenediamine (250 mg, 1.2 mmol) and Et3N (2 eq), 2,4-
dichloro-6-
methylpyrimidine was added at RT and the reaction mixture was stirred for 12
h. Routine workup
with water, saturated NaHCO3, and extraction with DCM, followed by
purification with flash column
chromatography using Et0Ac-hexane afforded intermediate 1 as an off-white
solid in excellent yield
(90%). Subsequently, 1 (200 mg, 0.597 mmol) was dissolved in 5 mL dioxane and
treated with
CsCO3 (3 eq), Pd2(dba)3 (0.5 eq) and xantphos (1.5 eq) at RT. After refluxing
at 110 C for 12 h, the
reaction mixture was filtered through a pad of celite and concentrated on
silica gel for flash
chromatography using Et0Ac-hexane. The boc-protected product was redissolved
in 5 mL DCM,
treated with 5 mL TFA and stirred at RT for 3 h. Routine workup with Et0Ac and
purification via flash
chromatography using Et0Ac-hexane gave intermediate 2 as a beige solid in 40%
yield over 2 steps.
Finally, to a solution of 2 (50 mg, 0.157 mmol) and DMAP (1.2 eq) in freshly
dried DCM, DCC (1.2
eq) and 4-bromothiophene acetic (1.2 eq) were added simultaneously. After
stirring at RT for 12 h,
the reaction mixture was filtered through a pad of celite and concentrated on
silica gel. Flash
chromatography using methanol-chloroform afforded the desired CHD1Li (3/158)
in 60% yield as an
off-white solid.
Scheme 13:
C\0
N CI N
N Cs2CO3, Pd214(dba)3,
xantphos N
ci H2 I .N
dioxane, 110 C, 10 h
Boo-N AcCN, Et3N, 40 NH
TEA, DCM, r.t, 3 h
r.t., 12 h Boo.N
140 NH
H2N
1
2
Br¨a..õ-COOH N
DCC, DMAP, DCM, it, 12 h
Br¨00 NH
3/158
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Exemplary Synthesis of amide and urea analogues (Scheme 14)
In the following, compound numbers are referenced to Scheme 14.
Compound 6
2-chloro-6-methylpyrimidin-4-amine (500 mg, 3.48 mmol) and pyrrolidine (3 eq)
were added
simultaneously to a flask containing K2003 (1.05 eq) and DMF (2 mL). After
refluxing at 75 C for 8
h, the reaction mixture was poured into crushed ice and stirred vigorously.
The resulting precipitate
was collected via vacuum filtration to give intermediate 4 as an off-white
solid in 76% yield.
Thereafter, to a stirring solution of 4 (150 mg, 0.842 mmol), triethylamine
(1.2 eq) and DMAP (1.0
eq) in freshly dried DCM at RT, 4-nitrobenzoyl chloride (1.0 eq) was added and
stirring continued for
16 h. The mixture was then poured into water and basified with saturated
NaHCO3 solution. The
aqueous layer was extracted with DCM and combined organic extracts were dried
over anhydrous
Na2SO4 and concentrated on silica gel. Flash chromatography with Et0Ac-hexane
gave the nitro
precursor 0f5 as a yellow solid. 100 mg (0.306 mmol) of this nitro precursor
in 8 mL Et0H was added
to a mixture of Fe powder (7 eq) and NH4CI (3.5 eq) in 2 mL water. The
reaction mixture was refluxed
at 80 C for 3 h, filtered through a pad of celite and concentrated to dryness
in vacuo. The residue
was suspended in ethyl acetate and extracted with water. The ethyl acetate
layer was dried over
anhydrous Na2SO4 and concentrated in vacuo to afford crude intermediate 5 as a
beige solid. 5 was
treated with 4-bromothiophene acetic akin to 3 above to afford 6 as an off-
white solid.
Scheme 14
0
Cl
I.
NyCl11101 r%
N
I N
Et3N, DCM, rt, 16 h
--N
K2CO3, DMF, ike/NH4CI, Et0H:H20
NH2 75 C, 8 h NH2 80 C, 3 h
4
x,--N
õ.)--N
Br¨a__,COOH
NN
N
DCC, DMAP, DCM, rt 16 h c).(
140
H2N
5 6
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Compound 8 (Scheme 15)
In the following description, compound numbers refer to Scheme 15. Compound 8
was synthesized
using the protocol for compound 6 synthesis. The required acid chloride was
prepared by refluxing
250 mg of 4-nitrophenyl acetic acid in excess thionyl chloride.
Scheme 15:
02N so 0 =SOCl2, 70 C, 6
h 02N 401 0
OH ININ
4 7
NH2
Et3N, DCM, rt, 8 h
i. Fe/NH4CI, Et0H:H20
80 C, 3 h %.--LN
II
___________________________________ Br--CIThcio 0
N-N
Br¨ecCOOH
DCC, DMAP, DCM, rt 10 h 8
Compound 10 (Scheme 16)
In the following description, compound numbers refer to Scheme 16. Compound 10
was synthesized
as an off-white solid using the protocol for compound 6 synthesis. The
required nitro precursor of 9
was prepared as follows; intermediate 4 (150 mg, 0.842 mmol), 4-nitrophenyl
isocyanate (1.0 eq)
and triethylamine (3 eq) were refluxed in 5 mL dioxane at 110 C for 16 h.
After cooling to RT, the
resulting precipitate was filtered while washing with 5mL cold diethyl ether
to afford the desired nitro
precursor as a yellow solid.
Scheme 16:
401 NCO
i. 02N
Et3N,
'r dioxane,
110 C 16 h
N NH
NH2 ii= Fe/NH4CI, Et0H:H20 0
4 80 C, 3 h H2N
9
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N
DCC, DMAP, DCM, rt, 16 h 40 N NH
0 -for
Compound 13 (Scheme 17)
The urea intermediate 11 was prepared as described for 9 while its conversion
to 12 was achieved
using the procedure for intermediate 4. Thereafter, 80 mg of 12 (0.177 mmol)
in 3 mL n-BuOH was
5 treated with pyrrolidine (3 eq) and refluxed at 120 C for 16 h. The
reaction mixture was concentrated
in vacuo, redissolved in ethyl acetate and basified with saturated NaHCO3
solution. The aqueous
layer was extracted with Et0Ac and the combined organic extracts were dried
over anhydrous
Na2SO4. Flash chromatography purification afforded 13 as beige solid.
10 Scheme 17:
lb NCO
0
1. N 2 Et3N,
dioxane, 0 NH2
1 1 0 C 8 h
Br----CN
S \ s H
Fe/NH4CI, Et0H:H20 H
80 C, 3 h
11
N Cl
r\ =rj(CI
Cl
K2CO3, DMF, 0 NH
n-BuOH,
75 C, 16 h N N Dl PEA,
Br-- H H 120 C, 16 h
12
=-=N
0 NH
NAN
\ s H H
13
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Example 15: In vitro Biological Evaluation of CHD1L Inhibitors.
CHD1L inhibitors were assessed for their ability to inhibit the recombinant
catalytic domain of
CHD1L (cat-CHD1L). (See Abbott et al., 2020) The results of these studies
demonstrate feasibility
of designing drugs based on the pharmacophore of compound 6.0 (Figures 16A and
16B).
Notably, the more potent CHD1L inhibitors were compounds 6.5, 6.11, 6.16,
6.18, 6.21, and 6.31
(Figure 16A), which also displayed similar increases in cytotoxic potency in
SW620 parental tumor
organoids compared to 6.0 (Figure 1B). Structures of compounds 6.5, 6.11,
6.16, 6.18, 6.21, and
6.31 are found in Scheme 1.
A novel fluorescent EMT dual-reporter suitable for 2D and 3D high-content
imaging has been
developed that is an effective tool to measure EMP in real time while
simultaneously tracking the
spectrum of EMT cellular phenotypes. (See also Zhou et al., 2016) EMT
phenotypes can be isolated
by FACS based on dual-reporter fluorescence and interrogated as individual
cell populations in long-
term culture, including stable xE/xM (RFP¨/GFP¨), and quasi-EMT populations E
(RFP+), E/M
(RFP+/GFP+), and M (GFP+). Isolated EMT phenotypes display unequivocal
differences
morphologically and metabolically, and these phenotypic differences are driven
by TCF-
transcription. The correlation between cytotoxicity and CHD1L inhibition are
consistent with the
inhibition of CHD1L mediated TCF-transcription in CRC. (Esquer et al., 2021;
Abbott et al., 2020) In
particular, we demonstrate that the more tumorigenic CSC quasi-M-phenotype has
upregulated
TCF-transcription compared to the other EMT phenotypes (Figure 17A). Thus, we
treated M-
phenotype 5W620 and HCT116 cells with CHD1L inhibitors and observed a dose-
dependent
decrease in TCF-transcription with all listed compounds with inhibition
concentration 50% (IC5o)
values in the low micromolar concentration (Figures 17B and 17C,
respectively).
CHD1Li cytotoxicity was then measured in both cell line (SW620 and HCT116) and
patient cell
derived (CRC042 and CRC102) tumor organoids (Figures 18A-18D). CHD1L
inhibitors displayed
potent antitumor activity in 5W620 and HCT116 M-Phenotype tumor organoids,
inhibiting cell
viability at low nnicromolar IC50 values (Figure 18A and 18B). Likewise,
CHD1Li had potent
cytotoxicity against CRC042 and CRC102 patient tumor organoids (Figure 18C and
18D). These
results underscore the potent antitumor activity of CHD1Li in a variety of CRC
cell models, including
CRC patient tumor organoids.
CHD1L inhibitors were then evaluated for their ability to inhibit EMT and/or
induce mesenchymal-
epithelial transition (MET, i.e. reverse EMT) by simultaneously measuring the
fluorescent signal of
the EMT dual-reporter (VimPro-GFP and EcadPro-RFP), using the high-content
imaging
methodology previously described. (Zhou et al., 2016) Indeed, CHD1L inhibitors
prevent EMT and
induce MET in 5W620 and HCT116 M-Phenotype tumor organoids characterized by
dose
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dependent downregulation of vimentin with concomitant upregulation of E-
cadherin expression
(Figures 19A-19E). To quantify E-cadherin upregulation, resulting from CHD1Li,
we generated a
non-linear regression model to determine the dose at which a 2-fold increase
of RFP fluorescent
signal occurs. Representative dual-reporter M-phenotype HCT116 tumor organoid
images treated
with compound 6.5 are shown (Figure 19E). A marked down-regulation of the
VimPro-GFP is
observed, while an upregulation of EcadPro-RFP is noted as the treatment dose
increases and is
consistent with our previous results of CHD1Li 6.0 induced MET. (See Abbott et
al., 2020)
TCF-driven EMT is linked as a mechanism enabling mesenchymal cells with
increased CSC traits
including self-renewal, resistance to apoptosis, and increased metastatic
potential. (Scheel et al.,
2012; Chaffer et al., 2016) This fact is consistent with our results that
isolated M-phenotype tumor
cells have significant increased CSC stemness. (See also Esquer et al., 2021)
We have shown that
compound 6.0 significantly inhibits CSC stemness (See also Abbott et al.,
2020) based on the
clonogenic colony formation assay. (Esquer et al., 2020; Franken et al., 2006)
Thus, we evaluated
CHD1L inhibitors for their ability to inhibit CSC sternness in SW620 and
HCT116 M-Phenotype cells,
using a high-content imaging. (Esquer et al., 2020) CHD1L inhibitors
effectively inhibited colony
formation over a low M to nM range (Figures 20A and 20B). CHD1L inhibitor
6.31 was the most
potent of the compounds assessed with an IC50 value of 300 nM in SW620 cells
and 200 nM in
HCT116 cells. CHD1L inhibitors prove to be effective antitumor agents that
prevent CHD1L-
mediated TCF-transcription that in turn inhibits EMT and induces MET,
resulting in loss of CSC
stemness while promoting cytotoxicity to tumor cells.
Example 16: In vivo Biological Evaluation of CHD1L Inhibitors.
We described that CHD1Li 6.0 has a good in vivo disposition, including a
plasma half-life of 3 h in
mice. (See above, also Abbott et al., 2020) We considered that the half-life
of 6.0 may be
detrimentally affected due to liver metabolism of the thiophene ring.
Thiophene rings may form
reactive metabolites (e.g., by S-oxidation), and substituted thiophenes are
generally more stable to
liver metabolizing enzymes. (Gramec et al., 2014) Moreover, 6.0 does not
display any liver toxicity
when treating mice at a maximum tolerated dose of 50 mg/kg by intraperitoneal
(i.p.) administration
daily over five days. (See also, Abbott et al., 2020) Thus, thiophene reactive
metabolites leading to
toxicity does not appear to be a limiting adverse effect. Prior to conducting
in vivo studies with CHD1L
inhibitors, we conducted in vitro mouse microsomal stability studies with
select compound, including
6.0, 6.4, 6.5, 6.11, and 6.31. CHD1L inhibitor 6.11 proved to be the most
metabolically stable of
these compounds when exposed to nnicrosomes with a 2-fold longer half-life of
130.3 minutes
compared to 67.2 minutes for compound 6Ø Therefore, 6.11 was prioritized for
in vivo evaluation.
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As described in more detail above, using CD-1 mice, we administered 6.11 by
i.p. injection at a dose
of 50 mg/kg and assessed the pharmacokinetics (PK) of 6.11, including
elimination half-life (t1i2k)
from plasma, and liver and fat tissues. The t112k of 6.11 in the plasma and
tissues is 8 h, which is a
2.7-fold longer half-life than 6Ø Next, using the same dose, we assessed the
oral bioavailability of
6.11 after oral gavage (p.o.) and found that 6.11 is oral bioavailable with
44% uptake in the plasma
and a t112kof 8 h (Figure 6B). The in vivo half-life 6.11 is consistent with
its in vitro microsomal stability,
indicating that the bromothiophene moiety of 6.11 significantly improves the
in vivo PK by increasing
its stability to liver enzymes compared to 6Ø
Example 17: Experimental Methods
General Experimental Methods. All commercial chemicals were used as supplied
unless
otherwise stated. All solvents used were dried and distilled using standard
procedures. Thin layer
chromatography (TLC) was performed using Aluminum backed plates coated with
60A Silica gel
F254 (Sorbent Technologies, Norcross, GA, USA). Plates were visualized using a
UV lamp (Amax
= 254 nm). Column chromatography was carried out using 230-400 mesh 60A silica
gel or using a
Teledyne Isco Combiflash next gen 300+ chromatography system with high
performance gold
columns. NMR spectra were recorded on a Bruker Avance III 400 (1H 400 MHz, 130
100 MHz). All
chemical shifts are recorded in parts per million (ppm), referenced to
residual solvent frequencies
(1H NMR: Me4Si = 0, CDCI3= 7.26, D20 = 4.79, CD3OD = 4.87 or 3.31, DMSO-d6 =
2.50, Acetone-
d6 = 2.05 and 13C NMR: CDCI3 = 77.16; CD3OD = 49.0, DMSO-d6 = 39.5, Acetone-d6
= 29.9
Coupling constants (J) values are expressed in hertz (Hz). The following
splitting abbreviations
were used: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m =
multiplet, br = broad, dd
= doublet of doublets, dt = doublet of triplets, td = triplet of doublets.
Melting points (m.p.) were
determined using a Stuart melting point apparatus (SMP20). Infrared (IR)
spectra were recorded
on a Bruker ALPHA platinum ATR (oils and solids were examined neat). Compounds
purity
95%) was measured using a Shimadzu prominence HPLC equipped with a photodiode
array
detector (PDA) and LunaR Omega Polar C18 column (5 jam, 100 A, 250 mm x4.6
mm). Using a
flow rate of 0.525 mllmin, compounds were eluted with a gradient of water/
methanol, with
0.1%TFA in water/ 0.1%TFA in methanol over 0 to 25 min. High resolution mass
spectrometry
(HRMS) were recorded using Q Exactive mass spectrometer (ThermoFisher, San
Jose, CA)
operated independently in positive or negative ion mode, scanning in full MS
mode (2 pscans) from
150 to 1500 m/z at 140,000 resolution, with 4 kV spray voltage, 45 sheath gas,
15 auxiliary gas.
Acquired data were then converted from raw to mzXML file format using Mass
Matrix (Cleveland,
OH).
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CHD1L Enzyme ATPase Assay. CHD1L enzyme inhibition assay was performed as
described
previously (Abbott et al., 2020). All reactions were carried out using low
volume nonbinding surface
384-well plates (Corning Inc., Corning, NY). 800 nM cat-CHD1L, 200 nM
mononucleosome (Active
Motif, Carlsbad, CA), and various concentrations of inhibitors were
preincubated at 37 C for 10 min
in lx buffer containing 50 mmol/L Iris pH 7.5, 50 mmol/L NaCI, 5 mmol/L MgCl2,
2 mmol/L DTT,
and 5% glycerol. The reaction was initiated by the addition of 10 pmol/L ATP
(New England
Biolabs, Ipswich, MA) to a total volume of 10 pL and incubated at 37 C for 1
hour. ATPase activity
was measured by adding 500 nmol/L of Phosphate Sensor (ThermoFisher, Waltham,
MA)
measuring excitation (430 nm) and emission (450 nm) immediately on an Envision
plate reader
(PerkinElmer, Waltham, MA). Background signal was determined by using all
assay components
excluding the enzyme.
Cell lines. Cell lines were purchased directly from ATCC and used as
indicated. Engineered cell
lines previously reported were STR profiled for authenticity. All cell lines
were tested for bacterial
and mycoplasma contamination before use. Deidentified patient sample cells
were obtained from
the CU Cancer Center GI tissue bank.
Cell Culture. SW620 and HCT116 cell lines were obtained from American Type
Culture Collection
(ATCC) (Manassas, VA) and grown in RPM 1-1640 media supplemented with 5% fetal
bovine
serum (FBS) in a humidified incubator at 37 C and 5% CO2. Cells were expanded
in 10 cm2 tissue
culture-treated dishes (ThermoFisher) following ATCC protocols. Epithelial-
Mesenchymal transition
(EMT) dual reporter cell lines (SW620 and HCT116 E, E/M, and M) were
generated, characterized,
and maintained as previously outlined (PM ID: 33742123). Both wildtype and
dual reporter cell lines
were harvested and prepared for experiments by aspirating media, washing with
10 mL PBS,
detaching with 1 mL of Trypsin-EDTA at 0.25% (ThermoFisher), and neutralizing
with 4 mL of
complete growth medium. Cells were counted using a Bio-Rad TC20 automated cell
counter (Bio-
Rad, Hercules, CA) by Trypan Blue (1:1) live/dead cell exclusion.
Cell Line Tumor Organoid Culture. Tumor organoids were prepared by plating at
2,000 cells/well
into CellCarrier Spheroid Ultra-Low Attachment (ULA) 96-well plates (Cat. No.
6055330,
PerkinElmer) or Clear Round Bottom ULA 96-well plates (Cat. No. 7007,
Corning). Briefly, the
plated cells were centrifuged at 1,000 RPM to promote cellular aggregation,
afterwards a final
concentration of 2% Matrigel (Corning) was added, and the plates were placed
in a 37 C and 5%
CO2 humidified incubator for 72 h to reach proper tumor organoid structure.
Tumor organoids were
then treated with CHD1Li compounds for an additional 72 h in dose response
assays and analyzed
for EMT reversal and cytotoxicity.
Patient Cell Tumor Organoid Culture. CRC048 and CRC102 patient cell samples
were
expanded and cultured as PDTOs using reported methodologies and reagents.
(Drost et al., 2016;
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Sato et al., 2011) Briefly, patient cells were washed with PBS, digested with
TrypLE, and filtered
through 100 pm cell strainer before used in 3D cell culture. Patient cells
were seeded at 5,000 cells
per well in 96-well plates, coated with 2% Matrigel, and allowed to self-
assemble as PDTOs over
72 h.
Tumor Organoid Cytotoxicity. CHD1Li cytotoxicity was assessed using CellTiter
Glo 3D (Cat.
No. G9681, Promega, Madison, WI). Tumor organoids were manually transferred
from Clear
Round Bottom ULA 96-well plates to Corning 96-or 384-well solid bottom white
plates (Cat. No.
655083, Greiner Bio-One, Monroe, NC). CellTiter Glo 3D was added at a 1:1
ratio, incubated for 45
min at room temperature on an orbital shaker at 450 RPM. Finally, luminescence
was quantified
using an Envision Plate Reader (PerkinElmer). Cell cytotoxicity was normalized
to 0.5% DMSO as
vehicle control.
3D High-Content Imaging and Analysis of EMT Dual-Reporter Activity. Tumor
organoids were
imaged using the Opera Phenix high-content screening system (PerkinElmer) in
confocal mode
utilizing a 10x air objective (NA 0.3). The following excitation and emission
(Ex/Em) wavelengths
were employed: RFP (561/570-630) and GFP (488/500-550). Organoids were also
imaged in
Brightfield to segment and perform high-content analysis of dual reporter
specific fluorescence
intensity. RFP fluorescent signal correlates with E-Cadherin promoter
activity, while GFP
fluorescent signal is correlates with vimentin promoter activity. Both
fluorescent signals were
quantified and normalized as previously described. (Esquer, et al., 2021)
TCF-transcriptional Reporter Assay. Assay has been adapted and modified from
(Esquer et al,
2021; Zhou et al., 2016; Yang et al., 2020) SW620 cells were plated into
duplicate 96-well plates at
20,000 cells/well (HCT116 at 10,000 cells/well), one white solid bottom plate
was used to assess
TCF-transcriptional activity and one clear plate was used to measure total
protein by BCA assay
for normalization purpose. Cell lines were allowed to attach overnight and
then were transiently
transfected with TOPflash plasmid (Millipore, Billerica, MA) using TransIT-LT1
transfection reagent
(Mirus Bio, Madison, WI) for 72 h. Afterwards, cells from the white solid
bottom plate were carefully
washed with PBS and a 1:1 ratio of PBS: One-Glo Luciferase Assay System
(Promega) was
added, incubated for 10 min, and luminescence was quantified on Envision plate
reader
(PerkinElmer) for TCF-activity. As a control, cells from the clear plate were
lysed with Mammalian
Cell Lysis Buffer (Promega) and the total protein from each well was
quantified with BCA assay
(ThermoFisher).
Clonogenic Assay. Cancer stem cell colony formation after CHD1Li treatment was
assessed as
previously described (Esquer et al., 2020). In brief, HCT116 cells were plated
at an optimal cell
concentration to avoid colonies merging over a growth period of 7-10 days.
5W620 and HCT116
M-Phenotype cells were plated at 200 and 75 cells/well, respectively, into 96-
well Clear CellStar
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black plates (Greiner Bio-One). Fresh media and drug treatments were
replenished every three
days. Images were acquired using the Opera Phenix HCS system and colony
number, area (4m2),
and confluence (j.inn2) were quantified and analyzed using the Harmony
software. Experiments
were replicated three times (n = 3 for each condition).
In vivo PK studies. The PK studies were conducted using our previously
published methods
(Abraham et al., 2019) where CHD1Li 6.11 was administered by oral gavage
(p.o.) at a dose of 50
mg/kg in a vehicle of 100% DMSO.
Statistical Analysis
All statistical analyses were performed using GraphPad Prism v9.0 (GraphPad
Software Inc., La
Jolla, CA). The data were collected using experimental replicates unless
otherwise noted. All P-value
significant is represented as*, p<0.05; **, p<0.01; ***, p<0.001; ****,
p<0.0001.
Abbreviations
Chromodomain Helicase DNA Binding Protein 1 Like (CHD1L), also known as
Amplified in Liver
Cancer 1 (ALC1); T cell factor/lymphoid enhancer factor (TCF/LEF); epithelial-
mesenchymal
transition (EMT), mesenchymal-epithelial transition (MET); epithelial-
mesenchymal plasticity (EMP);
cancer stem cell (CSC); gastrointestinal (GI); colorectal cancer (CRC).
Example 18: In Vivo Anti-Tumor Activity of Compound 6.11 Administered by Oral
Gavage
11-week-old athymic nude mice (35) where inoculated in the flanks (Flk) with
isolated SW620 EMT
dual-reporter quasi-nnesenchynnal cells (GFP+). (Esquer et al., 2021) Five
days after cell injection,
injected mice were randomized into three groups for treatment (Tx). Group 1
(12 mice) received
control treatment of gavage vehicle (10% DMSO, 90% PEG 400 (polyethylene
glycol 400) by
volume). Group 2 (11 mice) received oral gavage of compound 6.11 at 75 mg/kg
dissolved in
vehicle. Group 3(12 mice) received oral gavage of compound 6.11 at 125 mg/kg
dissolved in
vehicle. Mice were treated once a day, 5 days a week PO (via oral gavage).
Mice were weighed
and tumor volume assessed twice a week by caliper measurement as illustrated
in Figs. 21A and
21B. At sacrifice, tumors were weighed and measured. In addition, gross
observations on
condition of mice were made and samples of plasma, tumors, liver spleen and
kidneys were
collected for further assessment, e.g., drug quantification and toxicity
assessment.
Fig. 21A is a graph of tumor volume (mm3) starting at 3 days after treatment
was initiated. This
graph shows a significant dose dependent decrease in tumor volume on oral
treatment of mice
with compound 6.11 over 27 days of treatment. Figure 21B is a graph of average
mouse body
weight (grams) by treatment group as a function of days after treatment was
initiated. This graph
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indicates no significant difference in body weight among the mice of the three
treatment groups.
Body weight loss is a general measure of treatment toxicity.
All animal studies were conducted in accordance with the animal protocol
procedures approved by
the Institutional Animal Care and Use Committee (IACUC) at the University of
Colorado Denver
Anschutz Medical Campus (Aurora, CO).
Example 19: Summary Table of Selected Biological Activity of CHD1L Inhibitors
Table 6 provides a summary of Cat-CHD1L activity, 3D cytotocxicity data and
microsomal stability
data for certain CHD1L inhibitors described herein. Methods for measuring
these biological
activities are described in the Examples above. See also Abbott et al., 2020
and Prigaro et al.,
2022 for additional detail and description of methods for assessing biological
activity.
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Table 6: Biological Activity Table
cat- 30 Cytotoxicity Microsomal stability
CHD1L 72hr IC50 (pM)
CHD1Li inhibition
IC50 (IJM)
SW620 HCT116 Human t1/2 (min) Mouse t1/2
(min)
6.33 0.3 4.0 11.6 38.24 75.98
6.41 0.5 13.5 21.3 171.32 62.03
6.46 0.7 12.5 8.3
6.47 0.8 13.8 9.3
6.18 1.0 1.5 3.0 88.09 141.06
6.21 1.0 1.7 NA 43.78 47.76
6.31 1.0 2.4 NA
6.59 1.0 14.8 11.90
6.44 1.2 22.7 14.0
6.5 1.2 1.2 2.2 70.19
6.16 1.3 1.5 2.4 59.52 55.51
6.11 1.3 2.6 3.5 262.95 57.22
6.58 1.4 2.2 7.8
6.45 1.4 27.2 10.6
6.35 1.8 8.6 NA
6.38 2.1 6.1 7.6 199.01 96.62
6.49 2.3 17.0 14.7
6.57 2.4 14.6 10.8
6.56 2.5 >40 >40
6.34 3.0 >20 NA
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Table 6 (continued)
cat- 30 Cytotoxicity Microsomal stability
CHD1L 72hr IC50 (pM)
CHD1Li inhibition
IC50 OM
SW620 HCT116 Human t1/2 (min) Mouse t1/2
(min)
6.54 3.5 >40 >40
6.48 4.5 21.8 7.6
6.51 6.0 40.7 15.1
6.43 10.3 0.0 19.1
6.40 11.4 10.5 4.8
6.42 17.7 0.0 20.9
6.55 18.7 >40 >40
6.39 >20 14.2 10.8 88.64 70.48
6.36 >20 >20 NA
6.50 >20 >40 26.2
6.52 >20 >20 >20
6.53 >20 >40 >40
6.20 NA 2.2 NA
6.24 NA 3.0 NA
6.27 NA 3.0 NA
6.3 NA 3.6 3.7
6.8 NA 4.3 6.8
6.26 NA 4.7 NA
6.29 NA 5.4 NA
6.17 NA 5.5 5.6
6.30 NA 6.1 NA
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Table 6 (continued)
cat- 30 Cytotoxicity Microsomal stability
CHD1L 72hr IC50 (pM)
CHD1Li inhibition
IC50 OM
SW620 HCT116 Human t1/2 (min) Mouse t1/2
(min)
6.19 NA 7.7 >40
6.9 NA 8.3 19.7
6.32 NA 5.0 5.0
6.14 NA 11.1 15.3
6.22 NA 11.3 NA
6.7 NA 13.0 17.6
6.15 NA 13.1 19.7
6.25 NA 17.6 NA
6.10 NA 24.1 >40
6.12 NA >20 >40
6.13 NA >20 >40
6.1 NA >40 >40
6.2 NA >40 NA
6.4 NA >40 >40
6.6 NA >40 >40
Compounds 57 (6.5), 52 (6.11), 54 (6.16), 28 (6.18), 31 (6.21), 75 (6.31), 118
(6.33), 120 (6.35),
123 (6.38) and 150 (6.58) exhibit good enzyme inhibition and below 10uM IC50
of cytotoxicity in
SW620 cells. Any one of compounds 57, 52, 54, 28, 31, 75,118, 120,123 and 150
is particularly
useful in the methods of treatment, combination therapies, pharmaceutical
compositions and
pharmaceutical combinations of this invention.
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