Note: Descriptions are shown in the official language in which they were submitted.
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BENZO[H][1 ,61NAPHTHYRIDIN-2(1 H)-ONES AS BMX INHIBITORS, FOR USE AGAINST
CANCER
Related Application
The present case claims the benefit of, and priority to, GB01908171.0 filed on
7 June 2019
(07.06.2019), the contents of which are hereby incorporated by reference in
their entirety.
Field of the Invention
The present invention provides compounds having a tricyclic core of a
quinoline fused to a
pyridinone, and pharmaceutical compositions comprising such compounds. The
compounds
and compositions find use in methods of treatment, such as methods of treating
cancer.
Also provided are complexes comprising a compound of the invention covalently
bound to a
polypeptide, such as covalently bound to BMX, and polymorphs of these
complexes.
Background
Over the last couple of years, the development of irreversible kinase
inhibitors has gained
more traction both in academia and the pharmaceutical industry (Chaikuad et
al.; Singh et at
2018). Historically, irreversible inhibitors were considered problematic due
to their lack of
selectivity and safety issues related to the undesired engagement of off-
targets. However,
these potential liabilities can be overcome, and the development of covalent
small molecule
kinase inhibitors has recently seen renewed interest (Singh et at; Bail etal.;
Bourne etal.;
Lagoutte et at; Gilbert et at). Supporting the value and "renaissance" of
covalent inhibitors,
four small molecule entities have recently been approved by the FDA for
clinical use: Afatinib
(EGFR and HER2 inhibitor), lbrutinib and Acalabrutinib (BTK inhibitor),
Ositnertinib (EGFR
inhibitor) and Neratinib (EGFR and HER2 inhibitor) (Byrd et al.; Honigberg et
al.; Rabindran
et al.; Soria et at; Miller et at). However, not all kinases are accessible
for covalent binding,
as it depends on the target amino acid positioning (Zhang et at; Liu et at
Chem. Blot 2013;
Zhao et at; Lanning et at). One of such kinases of interest is the epithelial
and endothelial
tyrosine kinase (ETK), commonly known as bone marrow tyrosine kinase in
chromosome X
(BMX).
BMX is a major member of the TEC family of non-receptor tyrosine kinases,
together with
ITK, TEC, BTK and TXK (reviewed by Smith et aL and Norwood et at). TEC kinases
are
activated by many cell surface receptor-associated signalling complexes and
are recruited to
the plasma membrane or specific micro-environments by a variety of lipids and
proteins.
Through this mechanism, they are involved in signal transduction in response
to a myriad of
extracellular stimuli, including those mediated by growth factor receptors,
cytokine receptors,
G-protein coupled receptors, antigen-receptors, integrins and death receptors.
Moreover,
TEC kinases regulate many of the major signalling pathways, such as those for
PI3K, PKC,
PLCy, AKT, STAT3 and p-activated kinase 1 (PAK1) (see Jarboe et at and Qiu et
at) while
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being responsible for a variety of cell processes, including regulation of
gene expression,
calcium mobilization, actin reorganization/mofility and survivallapoptosis
(Smith et at and
Horwood et at).
BMX is widely expressed in granulocytes, monocytes, cells of epithelial and
endothelial
lineages, as well as brain, prostate, lung and heart and it is specifically
involved in
tumorigenicity, adhesion, motility, angiogenesis, proliferation and
differentiation (see
Wen et at; Guryanova et at; Kaukonen et at; Mano et at). Moreover, it has been
found to
be overexpressed in numerous cancer types including breast (Bagheri-Yarmand et
al.;
Chen et at; Cohen et at), prostate (Dai et at Cancer Res. 2006; Dai et at
Cancer Res.
2010), colon (Potter et at Neoplasia 2014) and cervical carcinoma (Li et at),
suggesting that
elevated levels of BMX increase cancer cell survival. In addition, BMX is also
required for
stem cell maintenance and survival (see Kaukonen et at) and its up-regulation
gives a
survival benefit to primary tumours and the cancer stem cells which are highly
resistant to
apoptosis and many chemotherapeutic agents. Homozygous BMX knockout mice have
a
normal life span without any obvious altered phenotype, suggesting that
therapies based on
BMX inhibition might have few side effects (Rajantie et at).
Therefore, taking into consideration the existence of multiple downstream
target proteins, the
integration in multiple and diverse signalling pathways, together with the
fact that it regulates
proliferation, migration and antiapoptotic effect, BMX emerges as a potential
target for
multiple aspects of cancer therapy. Recent studies also highlighted that
modulation of BMX
activity sensitizes cells to therapeutic agents improving response to
chemotherapy DNA
damaging agents or radiation. These studies show strong evidence that both
direct inhibition
of BMX or through modulation of related pathways result in an increased
therapeutic efficacy
(Potter et at Neoplasia 2014; Fox et al.; Potter et at Mot Cancer Ther. 2016).
Several Endothelial Growth Factor Receptor (EGFR) inhibitors in clinical
development were
shown to irreversibly alkylate BMX at a unique cysteine residue in the
aforementioned
fashion (Hur et at). These molecules are derived from reversible EGFR
receptors gefitinib
(Iressae) and erlotinib (Tarceva0), by adding a Michael acceptor moiety that
reacts with the
cysteine residue (Cys496) in the ATP binding site. This cysteine residue is a
unique
occurrence found in the ATP binding pocket and is present in all five members
of the
TEC-family kinase members. Therefore, by virtue of structural homology these
compounds
could also be covalent inhibitors of the other kinases in the TEC family.
BMX-IN-1 is one of the most potent BMX inhibitors (IC50: 8.0 nM) reported in
the literature
and also binds to BTK with very high affinity (IC50: 10.4 nM) (Liu etal. ACS
(hem. Blot
2013).
WO 2014/063054 describes compounds for use as inhibitors of Bone Marrow
Tyrosine
Kinase on Chromosome on X (BMX). This includes compounds having a tricyclic
core of a
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quinoline fused to a pyridinone at positions 3 and 4 of the quinoline. The
core is substituted
at the pyridinone ring nitrogen atom, and is further substituted at the 6-
position of the
quinoline ring. The 6-substituent contains a phenyl group, which may be
connected directly
to the 6-position of the quinoline ring, or may be connected via a C1_6
hydrocarbon linker,
such as an ethylene linker (-CHCH-).
Certain compounds exemplified in WO 2014/063054 are said to bind other
kinases, such as
BTK, rnTOR, BLK, TEC, TAK1, CLK1/2 and JAK3.
WO 2014/063054 notes that the disclosed compounds have antiproliferative
activity, and are
therefore suitable for use in treating cancerous cells, such as VVM and
lymphoma cell lines.
Liu et a/. discloses a compound for use as an inhibitor of BMX. The compound
has a
tricyclic core of a quinoline fused to a pyridinone at positions 3 and 4 of
the quinoline. The
core is substituted at the pyridinone ring nitrogen atom, and is further
substituted at the
6-position of the quinoline ring. The 6-substituent is a phenyl group which is
substituted with
sulfonamide.
The disclosed compound is shown to have antiproliferative activity against
panel of prostate
cancer cell lines
Wu et at (Scientific Reports) discloses a compound for use as an inhibitor of
Bruton's
tyrosine kinase (BTK). The compound has a tricyclic core of a quinoline fused
to a
pyridinone, similar to the tricyclic cores disclosed in WO 2014/063054 and Liu
et at The
6-substituent to the quinoline ring is a pyrazolyl group.
The disclosed compound is shown to suppress the inflammatory response in a
rheumatoid
arthritis model.
In further work, Wu et at (ACS Chem. Biol.) also show that the same compound
is an
inhibitor for B-Cell lymphoma.
Wang et at discloses compounds for use as inhibitors of BTK. The compound has
a tricyclic
core of a quinoline fused to a pyridinone, similar to the tricyclic cores
disclosed in
WO 2014/063054 and Liu et al. The 6-substituent to the quinoline ring is an
aromatic group,
such as phenyl, with the exception of one example compound where a phenyl
group is
attached to the 6-position via an ethylene linker.
Certain compounds are shown to have antiproliferative activity against a panel
of cancer cell
lines, including lung cancer, prostate cancer and colorectal carcinoma cell
lines.
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Jarboe et at and Liang et at are reviews of the known inhibitors of BMX and
BTX
respectively. The inhibitors disclosed in these reviews differ from those
described in the
literature cited above.
There is a need for alternative compounds for use in treating cancer, for
example where
such compounds covalently bind kinases such as BMX, BTK and TEC.
Summary of the Invention
In a general aspect the present invention provides compounds having a
quinoline ring fused
to a pyridinone, and more specifically a 2-pyridinone, at positions 3 and 4 of
the quinoline.
The core is substituted at the pyridinone ring nitrogen atom with a cyclic
group, and is further
substituted at the 7-position of the quinoline ring.
Also provided are related compounds where the 6-position of the quinoline ring
is
substituted, rather than the 7-position. The 6-substituent does not contain an
aromatic group
connected to the quinoline ring, or the 6-substituent does not contain an
aromatic group
connected to the quinoline ring via a C1_6 hydrocarbon linker.
The compounds of the invention may have improved binding to BMX and other
kinases
compared with compounds known in the art. The compounds of the invention may
also
have unexpected binding to BMX and other kinases, taking into account the
teaching in the
art, particularly with regards to the binding modes predicted by the prior
art. The compounds
of the invention may have an altered, such as improved, selectively to
kinases, such as
BMX, or an optimized physicochemical profile.
The compounds of the invention are suitable for forming complexes with kinases
such as
BMX, and such complexes are crystallisable. The crystal structures provide an
insight into
the mode of binding and may be used for the future development of inhibitors
with improved
efficacy and selectivity.
Accordingly, in a first aspect of the invention there is provided a compound
of formula (I):
A
7 401 1
and salts, solvates and protected forms thereof,
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wherein:
-A- is an optionally substituted cyclic group selected from arylene,
cycloalkylene and
heterocycylene, which cyclic group may be fused to a further ring;
-L- is a covalent bond or C1-6 alkylene;
-D is an acceptor group, such as a Michael acceptor group; and
-R7 is -L7A-L76-R7t1, where
-LM- is a covalent bond, or is selected from *-0-, *-S-, *-NH-, *-N(RN)-,
*-C(0)-, *-C(0)NH-, *-C(0)N(RN)-, *-NHC(0)-, *-N(RN)C(0)-, *-S(0)2NH-, *-
S(0)2N(RN)-,
*-NHS(0)2- and *-N(RN)S(0)2-, where -R" is Cse alkyl and the asterisk
indicates the point of
attachment to the quinoline;
-L7B- is selected from a covalent bond or selected from C-I-6 alkylene,
C2-6 alkenylene, C2-6 alkynylene and C2-6 heteroalkylene; and
-R7A is selected from optionally substituted cycloalkyl, heterocyclyl, and
aryl,
and when -L7B- is a covalent bond, -R74' is further selected from optionally
substituted alkyl,
alkenyl, alkynyl and heteroalkyl.
In a second aspect of the invention there is provided a compound of formula
(II):
DL 0
1
A
'N R6
I
SI
N
and salts, solvates and protected forms thereof,
wherein:
-A- is an optionally substituted cyclic group selected from arylene,
cycloalkylene and
heterocyclylene, which cyclic group may be fused to a further ring;
-L- is a covalent bond or CI-6 alkylene;
-D is an acceptor group, such as a Michael acceptor group; and
-R6 is -L6A-L6B-R6A, where
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-L6A- is a covalent bond or is selected from *-0-, *-S-, *-NH-, *-N(RN)-, *-
C(0)-,
*-C(0)NH-, *-C(0)N(RN)-, *-NHC(0)-, *-N(RN)C(0)-, *-S(0)2NH-, *-S(0)2N(RN)-, *-
NHS(0)2-
and *-N(RN)S(0)2-, where -RN is Cm alkyl and the asterisk indicates the point
of attachment
to the quinoline;
-L6B- is a covalent bond or is selected from Cia alkylene, C2_6 alkenylene,
C2_6 alkynylene and C2-6 heteroalkylene; and
-R6A is selected from optionally substituted cycloalkyl and heterocyclyl, and
when -L6B- is a covalent bond, -R6A is further selected from optionally
substituted alkyl,
alkenyl, alkynyl and heteroalkyl.
Optionally, the compounds of formula (II) are not either of the following
compounds:
0
0
H N H N
A-e
(110
1110 a
0 "Th 111110 N 1
N'Th N 1
I........õN
l.,,,N
10 I
0 I
N
N .
In a third aspect of the invention, there is provided a pharmaceutical
composition comprising
a compound of formula (I) according to the first aspect of the invention, or a
compound of
formula (II) according to the second aspect of the invention, together with a
pharmaceutically
acceptable carrier.
In a further aspect the present invention provides the use of a compound of
formula (I)
according to the first aspect of the invention, the use of a compound of
formula (II) according
to the second aspect of the invention, or the use of pharmaceutical
composition according to
the third aspect of the invention, in a method of treatment of the human or
animal body.
In yet a further aspect the present invention provides the use of a compound
of formula (I)
according to the first aspect of the invention, the use of a compound of
formula (II) according
to the second aspect of the invention, or the use of pharmaceutical
composition according to
the third aspect of the invention, in a method of treating a proliferative
disease, such as
cancer.
In a further aspect the present invention provides the use of a compound of
formula (I)
according to the first aspect of the invention, the use of a compound of
formula (II) according
to the second aspect of the invention, or the use of pharmaceutical
composition according to
the third aspect of the invention, in a method of treating an autoimmune
disease, such as
rheumatoid arthritis or lupus.
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The present invention also provides methods of treatment of a human or animal
body. Such
methods make use of the compounds and the compositions of the invention as
described
above, which may be administered in a therapeutically effective amount to a
subject.
The invention also provides a method of treating a cell, the method comprising
the step of
contacting a cell with a compound of formula (I) according to the first aspect
of the invention
or a compound of formula (II) according to the second aspect of the invention.
The cell may
be a proliferative cell, such a cancer cell. The method may be performed in
vitro or in viva
In one aspect the present invention provides a complex of a polypeptide
covalently bound to
a compound of formula (I) according to the first aspect of the invention, or a
compound of
formula (II) according to the second aspect of the invention.
In a further aspect there is provided a method of forming a complex, the
method comprising
the step of reacting a compound of formula (I) according to the first aspect
of the invention,
or a compound of formula (II) according to the second aspect of the invention,
with a
polypeptide.
In each of these aspects the polypeptide may be a protein, such as a kinase,
such as BMX
or BTK.
These and other aspects and embodiments of the invention are described in
further detail
herein.
Summary of the Figures
Figure 1 shows the change in BMX IC50 values for compounds 9-29 compared with
BMX-IN-1. Human recombinant BMX was incubated with the compounds and
phosphorylation of a biotinylated peptide measured by HTRF. Values are
expressed in
potency gain or loss (fold) against control BMX-IN-1 used in each set of
experiments. The
full library was tested in 4 different experiments, where BMX-IN-1 was always
used as
control. Compound 10 had a potency loss of 58-fold, while compounds 11-13 had
a potency
loss of more than 275-fold.
Figure 2 shows the mass spectrometric analysis of BMX together with a drug
conjugated
BMX. (A) Native MS analysis of hBMX. The measured molecular weight is
indicated. (B)
Denaturing MS analysis of drug conjugated hBMX. The measured molecular weight
is
indicated. (C) Tandem MS analysis of the drug conjugated trypfic peptide of
hBMX labeled
on the sequence (top) and MS/MS mass spectrum (bottom). The red asterisk
indicates the
drug conjugated Cys.
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Figure 3 shows the co-crystal structure exhibiting the binding mode of 24 to
hBMX kinase
catalytic domain. A) Covalent bond between the acrylamide of 24 and Cys496. B)
Non-
bonding interactions between 24 and Lys445 and 11e492. C) DFC-motif adopting
the out-like
conformation (D) Positioning of the sulfonamide aromatic ring pointing out of
the ATP
pocket.
Figure 4 show the flow cytometry results for the use of BMX-IN-1 and compounds
24-27 in
an apoptosis study in LNCaP prostate cancer cells.
Figure 5 shows the anti-proliferative activity in LNCaP cells of compounds 24-
26 in
combination with AKT1/2 (Ala inhibitor), Flutamide (androgen receptor
antagonist) and
LY293002 (P13K inhibitor). A) Cells co-treated with 24(3 pM), 25 (5 pM) and 26
(6 pM) with
AKT1/2 (1 pM); B) 24(3 pM), 25 (5 pM) and 26 (6 pM) with Flutamide (50 pM); C)
24
(3 pM), 25 (5 pM) and 26 (6 pM) with LY294002 (3 pM). Values are reported in %
cell
viability normalized to DMSO controls and are the mean of three individual
experiments
performed in triplicate. Determined P-values are illustrated as ns (P>0.05), *
(P s 0.05), **
(P s 0.01), *** (Ps 0.001) and ***-* (P 5 0.0001).
Figure 6 shows the induced targeted cell cytotoxicity on the B-cancer cell in
primary DLBCL
samples. A) Relative cell fraction (RCF) of the viable target cells for
increasing
concentrations of 25 in DMSO; relative cell fraction is the percentage of the
target cell
population. B) Normalized to the fraction of target cell population at
increasing concentration
in DMSO. C) 11 primary patient samples ranked by the drug response score (DRS)
of
compound 25 calculated as 1-mean of the RCF.
Detailed Description of the Invention
The compounds of the present invention are suitable for forming covalent bonds
to kinases
such as BMX.
The compounds of the invention possess a tricyclic core of a quinoline fused
to a pyridinone,
and more specifically a 2-pyridinone, at positions 3 and 4 of the quinoline.
The core is
substituted at the pyridinone ring nitrogen atom, and is further substituted
at the 6- or
7-positions of the quinoline ring.
The compounds previously described in the art possess a tricyclic core of a
quinoline fused
to a pyridinone at positions 3 and 4 of the quinoline, and the core is further
substituted at the
6-position of the quinoline ring. The substituent that is present at the 6-
position of the known
compounds is an aromatic group connected to the quinoline ring, either
directly or via a
C143 hydrocarbon linker.
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The compounds of formula (I) differ from those compounds known in the prior
art in that they
are substituted at the 7-position of the quinoline ring rather than the 6-
position.
The compounds of formula (II) differ from those compounds known in the prior
art in that
they are substituted at the 6-position with a substituent that does not
contain an aromatic
group connected to the quinoline ring, either directly or via a C1-6
hydrocarbon linker.
The compounds of formula (I) and (II) are described in further detail below.
Compounds of Formula (I)
The invention provides a compound of formula (I):
D
--y
0
A
''N 1
I
, 10 1 '
R
N
and salts, solvates and protected forms thereof,
wherein:
-A- is an optionally substituted cyclic group selected from thylene,
cycloalkylene and
heterocycylene, which cyclic group may be fused to a further ring;
-L- is a covalent bond or Ci-e alkylene;
-D is an acceptor group, such as a Michael acceptor group; and
-R7 is -L7A-L76-R7A, where
-L7A- is a covalent bond, or is selected from *-0-, *-S-, *-NH-, *-N(R")-,
*-C(0)-, *_C(0)N H-, *-C(0)N(R")-, *-NHC(0)-, *-N(R")C(0)-, *-S(0)2NH-, *S(0)N
(RN)_,
*-NHS(0)2- and *-N(RN)S(0)2-, where -R" is Ci_e alkyl and the asterisk
indicates the point of
attachment to the quinoline;
-L7B- is a covalent bond or selected from C1-6 alkylene, C2-6 alkenylene,
C2-6 alkynylene and C243 heteroalkylene; and
-R7A is selected from optionally substituted cycloalkyl, heterocyclyl, and
aryl,
and when -L7B- is a covalent bond, -R7A is further selected from optionally
substituted alkyl,
alkenyl, alkynyl and heteroalkyl.
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The substituent groups and the optional substituents groups for -R7A are
described in further
detail below.
Compounds of Formula (I1)
The invention also provides a compound of formula (I):
D
'Ir 0
A
'N 0
R6 I1
N
and salts, solvates and protected forms thereof,
wherein:
-A- is an optionally substituted cyclic group selected from arylene,
cycloalkylene and
heterocyclylene, which cyclic group may be fused to a further ring;
-L- is a covalent bond or Ci_6 alkylene;
-D is an acceptor group, such as a Michael acceptor group; and
-R6 is -L-L66-R, where
-L6A- is a covalent bond or is selected from *-0-, *-S-, *-NH-, *-N(RN)-, *-
C(0)-,
*-C(0)NH-, *-C(0)N(RN)-, *-NHC(0)-, *-N(RN)C(0)-, *-S(0)2NH-, *-S(0)2N(RN)-, *-
NHS(0)2-
and *-N(RN)S(0)2-, where -RN is C1.6 alkyl and the asterisk indicates the
point of attachment
to the quinoline;
-L6B- is a covalent bond or is selected from C1-6 alkylene, C2-6 alkenylene,
C2-6 alkynylene and C2-6 heteroalkylene; and
-R6A is selected from optionally substituted cycloalkyl and heterocyclyl, and
when -L6B- is a covalent bond, -RBA is further selected from optionally
substituted alkyl,
alkenyl, alkynyl and heteroalkyl.
The substituent groups and the optional substituents groups for -R6A are
described in further
detail below.
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In one embodiment, the compound of formula (II) may not be:
o
HWILCI-
o
0-Th s N 1
140 I
N
.
In one embodiment, the compound of formula (II) may not be:
o
HNiLd----
II N "Th 50
N
1
I
N
5
.
The compounds identified above are disclosed in Liang et at as compounds 7 and
8
(Liang et at 2017). Compounds from this document are also discussed in
W02013154778.
These compounds are tested for their antiviral activity against the dengue
virus in a viral
10 focus-forming assay and a viral protein accumulation assay. They are not
disclosed as
suitable for use in the treatment of any disease associated with altered
kinase activity, such
as proliferative diseases, and they are not said to bind TEC, or any other
kinase.
This exclusion applies only to those aspects of the present invention that
relate to
compounds of formula (II), compositions containing compounds of formula (II)
and the uses
of such compounds and compositions in methods of medical treatment,
particularly insofar
as they relate to the use in antiviral treatments, such as treatment of a
dengue virus
infection.
-A-
The group -A- is a cyclic group which is a substituent at the nitrogen ring
atom of the
pyridi none ring within the tricyclic core.
The worked examples in the present exemplify the use of phenylene as the
cyclic group -A-_
It is known from the art that other cyclic groups may be used at this
position. For example,
WO 2014/063054 describes compounds having a range of cyclic and bicyclic
groups,
including phenylene, pyridinene, tetrahydroquinolinylene and
tetrahydroisoquinolinylene
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amongst others (these are the groups -C- and -F- in this prior art
disclosure). Thus, the
compounds of the present case are not limited to the use of a phenylene group
at -A-.
The cyclic group is substituted with the group -L-D, and it is optionally
further substituted, for
example with one or more groups -RA. Each cyclic group may be monocyclic or
may be a
series of fused rings, such as a bicyclic ring.
The group -A- may be a cyclic group selected from arylene, cycloalkylene and
heterocyclylene which cyclic group may be fused to a further ring. Each cyclic
group is
optionally substituted with one or more substituents -RA. Preferably the
cyclic group is
substituted with one further substituent, -RA.
The group -A- is preferably a cyclic group having 6, 9 or 10 ring atoms only.
Each of the ring
atoms may be a carbon ring atom, and optionally one of the ring atoms may be a
nitrogen
ring atom.
Where -A- comprises two or more fused rings, it is preferred that the ring
attached to the
nitrogen ring atom of the pyridone group is a 6-membered ring. The ring that
is fused to the
6-membered ring is preferably a 5- or a 6-membered ring.
Where -A- is arylene, this may be carboarylene or heteroarylene. The arylene
may be
monocyclic, or may comprise a plurality of fused rings. Where a plurality of
rings is present,
the ring connected to the pyridone group is aromatic. The other rings are
optionally
aromatic. The other rings may be fully unsaturated or partially unsaturated.
The other rings
may be independently selected from aromatic, cycloalkyl and heterocyclyl
rings.
A carboarylene group may be selected from phenylene (C6 carboarylene),
naphthylene and
tetralinylene (Cio arylene).
A heteroarylene group may be C5_10 heteroarylene, such as Cs.6 heteroarylene.
The heteroarylene may be selected from pyridinylene (C6); indolylene,
isoindolylene,
benzoimidazolylene, indolinylene and isoindolinylene (C9); and
tetrahydroquinolinylene and
tetrahydroisoquinolinylene (C10)-
Where a number of atoms is given, this refers to the total number of ring
atoms, including
carbon and hetero (nitrogen) ring atoms as appropriate.
Where -A- is cycloalkylene this may be Cs_io cycloalkylene, such as C5_10
alkylene. The
cycloalkylene may be monocyclic, or may comprise a plurality of fused rings. A
cycloalkylene group may be partially unsaturated (but not aromatic).
Where a plurality of rings is present, the ring connected to the pyridone
group is non-
aromatic, and is preferably a fully saturated ring. The other rings are
optionally aromatic.
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The other rings may be fully unsaturated, partially unsaturated or saturated.
The other rings
may be independently selected from aromatic, cycloalkyl and heterocyclyl
rings.
A cycloalkylene group may be selected from cyclopentylene (CS), cyclohexylene
(C6);
tetralinylene and decalinylene (Ci0), such as cyclohexylene.
Where -A- is heterocyclylene this may be C3-10 heterocyclylene, such as
C5,-10 heterocyclylene. A heterocyclylene has one or two ring heteroatoms,
with each ring
heteroatonn selected from 0, S and N(H). The ring heteroatonn is not connected
to the
nitrogen ring atom of the pyridone. A heterocyclylene group may be partially
unsaturated
(but not aromatic).
The heterocyclylene may be monocyclic, or may comprise a plurality of fused
rings. Where
a plurality of rings is present, the ring connected to the pyridone group is
non-aromatic, and
is preferably a fully saturated ring. The other rings are optionally aromatic.
The other rings
may be fully unsaturated, partially unsaturated or saturated. The other rings
may be
independently selected from aromatic, cycloalkyl and heterocyclyl rings.
A heterocyclylene may be selected from pyrrolidinylene, tetrahydorfuranylene,
tetrahydrothiophenylene, pyrrolinylene (Cs); piperidinylene, piperazinylene,
tetrahydropyranylene, dioxanylene, thianylene, dithianylene, morpholinylene
and
thionnorpholinylene (C6); indolinylene, decahydroisoquinolinylene,
decahydroquinolinylene
and tetrahydroquinoline, and tetrahydroisoquinoline (Cio).
Where the cyclic group has 6 ring atoms only, the group -L-D is preferably
provided at the
3-position (where the 1-postion is the point of attachment to the nitrogen
ring atom of the
pyridone group).
Where the cyclic group has 6 ring atoms only, the group -L-D is preferably
provided at the
3-position and any further substituents -RA may be provided at one or more of
the 2-, 4-, 5-
and 6-positions (again, where the 1-postion is the point of attachment to the
nitrogen ring
atom of the pyridone group). Preferably the cyclic group is not substituted at
the 2- or
6-positions. Typically, the substituent is provided at the 4-position. It is
most preferred that
a group -RA is provided at the 4-position and the group -L-D is provided at
the 3-position.
Preferably, the group -A- is an optionally substituted group selected from
phenylene,
pyridinylene, indolylene, isoindolylene, benzoimidazolylene, indolinylene,
isoindolinylene,
tetrahydroquinolinylene and tetrahydroisoquinolinylene.
More preferably, the group -A- is an optionally substituted group selected
from phenylene,
pyridinylene, indolylene, 1,2,3,4-tetrahydroquinolinylene and indolinylene.
The cyclic group is connected to both the pyridone of the tricyclic core and -
L-. The cyclic
group may be optionally further substituted, such as substituted with one,
two, three or four
further substituents -RA.
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Where -A- is phenylene, this may be pheny1-1,3-ene. Here, the 1-position is
the carbon ring
atom attached to the pyridone nitrogen.
Where the phenylene is substituted with -RA these may be provided at one or
more of the 2-,
4-, 5- and 6-positions, and preferably at one or more of the 4- and 5-
positions, as noted
above. Preferably, the phenylene is monosubstituted, and is substituted at the
4-position.
When -A- is pyridinylene, this may be a pyridinylene selected from the group
consisting of
pyridiny1-2,3-ene, pyridiny1-2,4-ene, pyridiny1-2,5-ene, pyridiny1-2,6-ene,
pyridiny1-3,4-ene,
and pyridiny1-3,5-ene. Here, the 1-position is the nitrogen ring atom.
Preferably, the pyridinylene is unsubstituted or monosubstituted with -RA. The
pyridinylene
may be substituted at a carbon ring atom that is at the 2-, 3-, 4-, 5- or 6-
position, where that
position is available for substitution.
When -A- is indolylene this may be indolyI-1,6-ene, where the 1-position is
nitrogen ring
atom. The indolylene may be connected to -L- via the nitrogen ring atom. Here,
the
indolylene may be connected to pyridine via the 6-position.
The indolylene is unsubstituted or monosubstituted with -RA. The indolylene is
preferably
substituted on the benzene ring with -RA.
Where -A- is isoindolylene this may be isoindolyI-2,5-ene, where the 2-
position is nitrogen
ring atom. The isoindolylene may be connected to -L- via the nitrogen ring
atom. Here, the
isoindolylene may be connected to pyridine via the 5-position.
The isoindolylene is unsubstituted or monosubstituted with -RA. The
isoindolylene is
preferably substituted on the benzene ring with -RA.
Where -A- is benzoimidazolylene this may be benzoimidazolyI-1,6-ene, where the
1-position
is nitrogen ring atom. The benzoimidazolylene may be connected to -L- via the
nitrogen ring
atom. Here, the benzoimidazolylene may be connected to pyridine via the 6-
position.
The benzoimidazolylene is unsubstituted or monosubstituted with -RA. The
benzoimidazolylene is preferably substituted on the benzene ring with -RA.
When -A- is indolinylene this may be indoliny1-1,6-ene, where the 1-position
is nitrogen ring
atom. The indolinylene may be connected to -L- via the nitrogen ring atom.
Here, the
indolinylene may be connected to pyridine via the 6-position.
The indolinylene is unsubstituted or monosubstituted with -RA. The
indolinylene is preferably
substituted on the benzene ring with -RA.
Where -A- is isoindolinylene this may be isoindoliny1-2,5-ene, where the 2-
position is
nitrogen ring atom. The isoindolinylene may be connected to -L- via the
nitrogen ring atom.
Here, the isoindolinylene may be connected to pyridine via the 5-position.
The isoindolinylene is unsubstituted or monosubstituted with -RA. The
isoindolinylene is
preferably substituted on the benzene ring with -RA.
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Where -A- is tetrahydroquinolinylene (or 1,2,3,4-tetrahydroquinolinylene) this
may be
tetrahydroquinoliny1-1,7-ene, where the 1-position is nitrogen ring atom. The
tetrahydroquinolinylene may be connected to -L- via the nitrogen ring atom.
Here, the
tetrahydroquinolinylene may be connected to pyridine via the 7-position. The
tetrahydroquinolinylene is unsubstituted or monosubstituted with -RA. The
tetrahydroquinolinylene is preferably substituted on the benzene ring with -
RA.
Where -A- is tetrahydroisoquinolinylene (or 1,2,3,4-
tetrahydroisoquinolinylene) this may be
tetrahydroisoquinoliny1-2,6-ene, where the 1-position is nitrogen ring atom.
The
tetrahydroisoquinolinylene may be connected to -L- via the nitrogen ring atom.
Here, the
tetrahydroisoquinolinylene may be connected to pyridine via the 6-position.
The
tetrahydroisoquinolinylene is unsubstituted or monosubstituted with -RA. The
tetrahydroisoquinolinylene is preferably substituted on the benzene ring with -
RA.
Preferably, the group -A- is optionally substituted phenylene, such as
phenylene substituted
with one further substituent -RA. Most preferably the phenylene is pheny1-1,3-
ene, optionally
substituted at the 4-position. Here, the 1-position is the carbon ring atom
attached to the
pyridone nitrogen.
The group -A- may be optionally substituted indolinylene, such as indoliny1-
1,6-ene, where
the 1-position is nitrogen ring atom. The indolinylene is preferably
unsubstituted_
-RA
The group -RA is a substituent to the cyclic group -A-. The cyclic group -A-
may have one or
more substituents, each of which is -RA. In one embodiment, the cyclic group -
A- is not
substituted with -RA, it is monosubstituted with -RA or it is disubstituted
with -RA. Preferably,
however, the cyclic group is not substituted with -RA, or it is
monosubstituted with -RA.
In the worked examples of the present case, the group -A- is not further
substituted or is
further substituted with methyl. It is known from the art that other
substituent groups may be
provided to the group -A-, whilst maintaining biological activity. For
example,
WO 2014/063054 describes a large range of possible substituent groups (these
are the
groups -Re and -RF in this prior art). Thus, the compounds of the present case
are not
limited to those where -A- is not further substituted or is further
substituted with methyl.
Where -RA is present, it is typically a substituent to a ring carbon atom.
Each group -RA is independently selected from -LAA-RAA, halo, hydroxy (-OH),
amino (-NH2),
thiol (-SH), cyano, nitro, and carboxy (-COON), where:
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-LAA- is a covalent bond or is selected from *-C(0)-, *-S(0)-, *-S(0)2- *-
N(H)C(0)-,
*-N(RN)C(0)-, *-N(H)S(0)-, *-N(RN)S(0)-, *-N(H)S(0)2-, *-N(RN)S(0)2-, *-N(H)-,
and _N(RN)_,
where -R14 is C1-6 alkyl, and the asterisk indicates the point of attachment
to the cyclic group;
and
-RAA is selected from optionally substituted alkyl, alkenyl, alkynyl,
cycloalkyl,
heterocyclyl and aryl.
Where -RAA is optionally substituted, each optional substituent may be
selected from halo,
hydroxy (-OH), amino (-NH2), thiol (-SH), cyano (-CN), nitro, carboxy (-COON),
and phenyl,
and where -RAA is cycloalkyl, heterocyclyl or aryl, the optional substituent
is further selected
from alkyl, such as C1-6 alkyl, such as methyl and ethyl.
The group -LAA- is preferably a covalent bond. Thus, here the group -RAA is
directly
connected to the cyclic group -A-. This is preferred.
The group -RAA may be alkyl, such as C1-6 alkyl, such as methyl or ethyl.
The group -RAA may be alkenyl, such as C2-6 alkenyl, such as ethenyl or
propenyl.
The group -RAA may be alkynyl, such as C24alkynyl, such as ethynyl or
propynyl.
An alkyl, alkenyl or alkynyl group may be linear or branched.
The group -RAA may be cycloalkyl, such as C3-14 cycloalkyl, such as
cyclopentyl and
cyclohexyl. The cycloalkyl group may be monocyclic, or may contain two or more
fused
rings, where each ring is a cycloalkyl ring. A cycloalkyl group is
nonarornatic. A cycloalkyl
ring may be unsaturated or partially saturated or fully saturated (but not
aromatic).
The group -RAA may be heterocyclyl, such as C3_14 heterocydyl, such as
pyrrolinyl,
piperidinyl, tetrahydrofuranyl and tetrahydropyranyl. The heterocyclyl group
may be
monocyclic, or may contain two or more fused rings, where one ring is a
heterocyclyl ring
and the other rings may be cycloalkyl or heterocyclyl rings. The heterocyclyl
group is
nonaromatic. Each ring in the heterocyclyl group may be unsaturated or
partially saturated
or fully saturated (but not aromatic).
The group -RAA may be aryl, such as carboaryl and heteroaryl.
The carboaryl may be C6_14 carboaryl, such as phenyl and naphthyl.
The heteroaryl may be C6-14 heteroaryl, such as C6-10 heteroaryl, such as
pyridinyl, pyrrolyl,
furanyl and thiophenyl.
A halo group may be selected from fluoro, chloro, bromo and iodo, such as
fluoro.
Preferably, the group -RA is -Rm1/4 or halo, such as -RA is alkyl or halo,
such as methyl or
fluoro.
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Most preferably, -RA is methyl or ethyl, such as most preferably methyl.
-L-
The group -L- is a link between the cyclic group -A- and the acceptor group -
D. The group
-L- may be a covalent bond or alkylene.
In the worked examples of the present case the group -L- is a covalent bond.
It is known from the art that other linkers may be used to connect the
acceptor group to the
cyclic group. For example, WO 2014/063054 describes compounds where the group -
A-
may be connected to a Michael acceptor group via a linker that is a
hydrocarbon chain
(these are the groups -L- and -V- in this prior art). Thus, the compounds of
the present case
are not limited to those where the cyclic group -A- connects directly to -D.
Where -L- is alkylene this may be C1-6 alkylene, such as C14 alkylene, such as
methylene or
ethylene. An alkylene group is a saturated aliphatic group. The alkylene group
may be
linear or branched.
Preferably -L- is a covalent bond. Here, the cyclic group -A- connects
directly to -D.
-D
The group -D is an acceptor group, which is suitable for reaction with a
nucleophilic group
present within a polypeptide, such as a protein. The acceptor group is
preferably reactive
with thiol functionality, which may be present within the side chain of a
cysteine amino acid
residue of a polypeptide.
In the present case, the compounds of the invention are preferably reacted
with a kinase,
such as those described herein, including BMX, to form a complex of the
compound with the
kinase. Here, the compound is covalently linked to the kinase via the acceptor
group and a
site on the kinase.
In the worked examples of the present case, the group -D is an amide
connecting to an
ethenyl group. It is known from the art that other acceptor group may be used
at this
position. For example, WO 2014/063054 describes compounds having a range of
acceptor
groups, (these are the groups -R and -IR in this prior art). Thus, the
compounds of the
present case are not limited to the use of an amide connecting to an ethenyl
group at -D.
The acceptor group may contain an 0,13-unsaturated carbonyl group or 0,13-
unsaturated
thiocarbonyl group.
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The acceptor group may be -X-M, where -X- is a covalent bond or -Lm-, and -M
is selected
from alkenyl, alkynyl, heterocyclyl, alkyl substituted with cyano, and cyano.
The group -M may contain an unsaturated bound, such as a carbon-carbon double
bond, for
example where -M is alkenyl, or heterocyclyl. Preferably this group is
provided a,13 to the
group -X-. This is particularly preferred where -X- contains a carbonyl group
(-C(0)-). The
unsaturated bond may be a carbon-carbon triple bond, for example where -M is
alkynyl. The
unsaturated bond may be a carbon-nitrogen triple bond, for example where a
cyano group is
present.
The group -IY- may be selected from *-C(0)-, *-S(0)-, *-S(0)2- *-N(H)C(0)-, *-
N(RN)C(0)-,
*-N(H)S(0)-, *-N(RN)S(0)-, *-N(H)S(0)2-, *-N(RN)S(0)2-, *-N(H)-, and -N(RN)-,
where -RN is
C1-6 alkyl, and the asterisk indicates the point of attachment to -L- (and
where -L- is a
covalent bond, this is the point of attachment to -A-).
When the group -A- is connected to -Li& via a nitrogen ring atom (for example,
when -L- is a
covalent bond), -X- may be a covalent bond group, or -X- may be -IY- where -
124- is selected
from *-C(0)-, *-S(0)-, and *-S(0)2-.
The group -124- is typically *-C(0)-, *-S(0)-, *-S(0)2-, *-N(H)C(0)- or *-
N(RN)C(0)-, such as
*-N(H)C(0)- or *-N(RN)C(0)-, such as *-N(H)C(0)-.
The group -M comprises the reactive functionality for forming a covalent bond
to a
polypeptide, such as a kinase. Typically, the reactive functionality is
electrophilic, and more
specifically, together with group -X-, it is a Michael acceptor group.
Where -M is alkenyl this may be C24 alkenyl. The alkenyl group may contain one
carbon-
carbon double bond. Preferably the double bond is conjugated with a carbonyl
group
present as -X- (thus, providing a 048-unsaturated carbonyl group or a,8-
unsaturated
thiocarbonyl group).
Where -M is alkynyl this may be C2-6 alkynyl. The alkynyl group may contain
one carbon-
carbon triple bond. Preferably the triple bond is conjugated with a carbonyl
group present
as -X-.
Where -M is heterocyclyl this may be a saturated or partially unsaturated
heterocyclyl. The
heterocyclyl may be a C3-7 heterocyclyl, such as a C3 or a C5 heterocyclyl.
A heterocyclyl group may be optionally substituted with carbonyl (-C(0)-) at
an available ring
carbon atom. Preferably, where the carbonyl is present, it may be a
substituent to a ring
carbon atom that is a to a ring heteroatom, such as a ring nitrogen atom, for
example to from
an internal (endo) amide or imide.
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Where the heterocyclyl is a C3 heterocyclyl it is preferably unsaturated.
A C3 heterocyclyl may be selected from aziridinyl, oxiranyl and thiiranyl.
Where the heterocyclyl is a C5 heterocyclyl it is preferably partially
saturated, and preferably
contains a single carbon-carbon double bond.
In one embodiment -M is maleimidyl, which is connected to -X- through the ring
nitrogen
atom. A maleirnidyl group is a C5 nitrogen heterocyclyl group where each of
the carbon ring
atoms a to the ring nitrogen atom are substituted with carbonyl.
The group -M may be alkyl substituted with cyano. The alkyl group may be Ci_io
alkyl, such
as Cim alkyl, substituted with cyano. The alkyl may be methyl or ethyl, such
as methyl,
substituted with cyano.
In one embodiment, -M is selected from optionally substituted alkenyl,
optionally substituted
alkynyl, cyano, and alkyl substituted with cyano.
It is preferred that -M is optionally substituted alkenyl, more preferably
optionally substituted
ethenyl, and most preferably ethenyl (-CH=CH2).
The group -D, such as -L-D together, may be selected from -N(H)C(0)CHCH2,
-N(H)C(0)CH2CN, -N(H)C(0)CCH, -N(H)C(0)CN, -N(H)C(0)CHCHMe, and
-N(H)C(0)CCMe.
The group -D, such as -L-D together, is preferably -N(H)C(0)CHCH2.
For the avoidance of doubt, the group -CF3 is not an acceptor group. Thus, -D
cannot be
-CIF3.
-R7
The group -R7 is a substituent at the 7-position of the quinoline ring. In
contrast, the
compounds known in the art are substituted only at the 6-position. The present
inventors
have shown that a range of different groups are tolerated at this position.
The inventors understand that those substituents previously used at the 6-
position may be
provided alternatively at the 7-position. Thus, WO 2014/063054 describes
compounds
where the tricyclic core is substituted at the 6-position of the quinoline
ring of that core. The
substituent that is present at the 6-position is typically an aromatic group
connected to the
quinoline ring, either directly or via a C143 hydrocarbon linker. Such a
substituent may be
provided at the 7-position within the compounds of the invention. Thus, -R7
may contain
aryl. Here, the presence of a 7-susbituent group may be associated with an
improved
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biological activity compared with a related compounds having the same
substituent at the
7-position.
Moreover, alternative groups may be used at the 7-position, where such groups
have never
been described for use at the 6-position. As noted above, WO 2014/063054
describes
substituent to the tricyclic core that contain an aryl group. The inventors
have shown that
alternative groups may be used as substituents to the tricyclic core, for
example including
substituted cycloalkyl and heterocyclyl, amongst others. The presence of such
groups may
be associated with a comparable or improved biological activity compared with
those
compounds substituted at the 6-position, or those compounds having an aryl
group within
the substituent to the tricyclic core.
The group -R7 is -L7A-L7B-R7A, where
-L7A- is a covalent bond, or is selected from *0 *NH *-NH-, *-N(RN)-,
*-C(0)-, *_C(0)N H-, *-C(0)N(RN)-, *-NHC(0)-, *-N(RN)C(0)-, *-S(0)2NH-, *-
S(0)2N(RN)-,
*-NHS(0)2- and *-N(RN)S(0)2-, where -RN is C1.6 alkyl and the asterisk
indicates the point of
attachment to the quinoline;
-L7B- is a covalent bond or selected from C14 alkylene, C2-6 alkenylene,
C2-6 alkynylene and C2-6 heteroalkylene; and
-R7A is selected from optionally substituted cycloalkyl, heterocyclyl, and
aryl,
and when -L7B- is a covalent bond, -R7A is further selected from optionally
substituted alkyl,
alkenyl, alkynyl and heteroalkyl.
A group -R7A may be optionally substituted with one or more groups -RS. Where
two or more
groups -RS are present, each -RS may be the same or different. These optional
substituents
are defined in detail below.
Preferably, the group -R7 contains a nitrogen atom. Such may be provided where
-R74 is
heterocyclyl or aryl (for example, heteroaryl), or where the group -Rs contain
a nitrogen
atom, for example where -R8 includes a sulfonamide group.
The groups -L7A- and -L76- are linkers that connect the quinoline ring of the
core to the group
-Rm. Alternatively, the group -R7A may be connected directly to the quinoline
ring. In this
case, each of -L7A- and -L7B- is a covalent bond.
Preferably, each of -L7A- and -L76- is a covalent bond. Here -R7 is -R7A.
Thus, -WA is
connected directly to the tricyclic ring.
In another embodiment, -R7 is -L7B-R7A, and preferably -L78- is C2_6
alkenylene.
In another embodiment, -R7 is -L7A-R7A, and preferably -L7A- is -NH- or -N(RN)-
.
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When -R7A is aryl it may be carboaryl or heteroaryl.
A carboaryl group may be C6.14 carboaryl, such as phenyl or naphthyl, and
preferably phenyl.
A heteroaryl group may be C5-14 heteroaryl, such as C5_10 heteroaryl, such as
C5_0 heteroaryl.
An aryl group may be monocyclic, or may comprise a plurality of fused rings.
Where a
plurality of rings is present, the ring connected to -L7B- is aromatic. The
other rings are
optionally aromatic. The other rings may be fully unsaturated or partially
unsaturated. The
other rings may be independently selected from aromatic, cycloalkyl and
heterocyclyl rings.
In one embodiment, -Wit is optionally substituted aryl, such as optionally
substituted phenyl,
pyridinyl, pyrrolyl, oxazolyl, thiophenyl, innidazolyl, pyrazolyl, oxazolyl,
thiazolyl, quinolinyl
and isoquinolinyl.
Preferably, -IVA is optionally substituted phenyl or pyridinyl, such as
phenyl. The phenyl or
the pyridinyl is optionally substituted, such as optionally nnonosubstituted.
When -R7A is heterocyclyl it may be C3-14 heterocyclyl.
A heterocyclyl has one or two ring heteroatoms, with each ring heteroatom
selected from 0,
S and N(H). The heterocyclyl may connect to -1213- via a ring carbon atom or a
ring nitrogen
atom, where present.
A heterocyclyl may be partially unsaturated (but not aromatic).
The heterocyclyl may be nnonocyclic, or may comprise a plurality of fused
rings. Where a
plurality of rings is present, the ring connected to -1213- is non-aromatic,
and is preferably a
fully saturated ring. The other rings are optionally aromatic. The other rings
may be fully
unsaturated, partially unsaturated or saturated. The other rings may be
independently
selected from aromatic, cycloalkyl and heterocyclyl rings.
The heterocyclyl group may be selected from piperidinyl, piperazinyl,
morpholinyl, and
thiomorpholinyl, such as selected from piperidinyl and piperazinyl.
When -R7A is cydoalkyl it may be C3_10 cycloalkyl, such as C5_10 cycloalkyl.
The cycloalkyl
may be nnonocyclic, or may comprise a plurality of fused rings. A cycloalkyl
group may be
partially unsaturated (but not aromatic).
Where a plurality of rings is present, the ring connected to -L713- is non-
aromatic, and is
preferably a fully saturated ring_ The other rings are optionally aromatic.
The other rings
may be fully unsaturated, partially unsaturated or saturated. The other rings
may be
independently selected from aromatic, cycloalkyl and heterocyclyl rings.
A cycloalkyl group may be selected from cydopentylene (Cs), cyclohexylene
(C6);
tetralinylene and decalinylene (C10), such as cyclohexylene.
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When -R7A is alkyl it may be C14 alkyl, such as C14 alkyl, such methyl or
ethyl. The alkyl
group may be linear or branched. The alkyl may be optionally substituted.
When -R7A is alkenyl it may be C24 alkenyl, such as C24 alkenyl, such ethenyl.
The alkenyl
group may be linear or branched. The alkenyl may be optionally substituted.
When -R7A is alkynyl it may be C24 alkynyl, such as 024 alkynyl, such ethynyl.
The alkynyl
group may be linear or branched. The alkynyl may be optionally substituted.
When -R7A is heteroalkyl it may be 024 heteroalkyl, such as 034 heteroalkyl.
The alkynyl
group may be linear or branched. A heteroalkyl group is an alkyl group where
one or two
carbon atoms is replaced with a heteroatonn selected from 0, S and N(H). The
heteroatonn
does not replace a carbon atom at the terminal of the alkyl group. The
heteroalkyl group
may be connected via a heteroatom, or alternatively it may be connected via a
carbon atom.
The group -R7A is preferably selected from optionally substituted aryl and
heterocyclyl.
-R6
The group -R6 is a substituent at the 6-position of the quinoline ring. The
substituent does
not contain an aromatic group connected to the quinoline ring, or the 6-
substituent does not
contain an aromatic group connected to the quinoline ring via a C1-6
hydrocarbon linker
The compounds known in the art are substituted at the 6-position with an
aromatic group,
which is directly linked to the quinoline ring, or is connected via a C143
hydrocarbon linker.
See, for example, the compounds described in WO 2014/063054.
As noted above, two compounds having a non-aromatic group bound directly to
the
quinoline at 6-postion are known from Liang et at (Liang et aL 2017). These
compounds
may be excluded from the definition for the compounds of formula (II).
The compounds are not for use in the treatment of proliferative diseases, such
as cancer,
and they are not disclosed for use as binders to any kinase. Rather, the
compounds are
used for their antiviral activity.
The group -R6 is -L6A-L6B-R6A, where
-L6A- is a covalent bond or is selected from *-0-, *-5-, *-NH-, *-N(RN)-, *-
C(0)-,
*-C(0)NH-, *-C(0)N(RN)-, Ik-NHC(0)-, '*-N(RN)C(0)-, *-S(0)2NH-, *-S(0)2N(RN)-,
*-NHS(0)2-
and *-N(RN)S(0)2-, where -RN is C1.6 alkyl and the asterisk indicates the
point of attachment
to the quinoline;
-L6B- is selected from a covalent bond or selected from C1-8 alkylene,
C24 alkenylene, C24 alkynylene and C24 heteroalkylene; and
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-R6A is selected from optionally substituted cycloalkyl and heterocyclyl, and
when -L6B- is a covalent bond, -R6A is further selected from optionally
substituted alkyl,
alkenyl, alkynyl and heteroalkyl.
The group -R6 may not be morpholinyl, such as morpholin-4-y1 (that is, a
morpholinyl group
connected to the quinolone ring via the morpholine ring nitrogen). Such a
limitation may be
limited to unsubstituted nnorpholinyl groups only.
The group -R6 may not be optionally substituted piperazinyl, such as
optionally substituted
piperazin-1-yl. More specifically, -R6 may not be 4-phenylpiperazin-1-yl.
A group -R6A may be optionally substituted with one or more groups -Rs. Where
two or more
groups -RS are present, each -Rs may be the same or different These optional
substituents
are defined in detail below.
Preferably, the group -R6 contains a nitrogen atom. Such may be provided where
-R6A is
heterocyclyl, or where the group -Rs contain a nitrogen atom, for example
where -Rs
includes a sulfonamide group.
The groups -La- and -L6B- are linkers that connect the quinoline ring to the
group -R6A.
Alternatively, the group -R" may be connected directly to the quinoline ring.
In this case,
each of -L6A- and -L6B- is a covalent bond.
Preferably, each of -L6A- and -L66- is a covalent bond. Here -R6 is -R6A.
Thus, -R6A is
connected directly to the tricyclic ring.
In another embodiment, _Re is _Les_ReA, and preferably -Les- is C2_43
alkenylene.
In another embodiment, -R6 is -L6A-R6A, and preferably -L6A- is -NH- or -N(RN)-
.
When -R6A is heterocyclyl it may be C3-14 heterocyclyl, such as C5-7
heterocyclyl, such as
Cs 6 heterocyclyl.
A heterocyclyl has one or two ring heteroatonns, with each ring heteroatonn
selected from 0,
S and N(H). The heterocyclyl may connect to -L7B- via a ring carbon atom or a
ring nitrogen
atom, where present.
A heterocydyl may be partially unsaturated (but not aromatic).
The heterocyclyl may be nnonocyclic, or may comprise a plurality of fused
rings. Where a
plurality of rings is present, the ring connected to -L68- is non-aromatic,
and is preferably a
fully saturated ring. The other rings are optionally aromatic. The other rings
may be fully
unsaturated, partially unsaturated or saturated. The other rings may be
independently
selected from aromatic, cycloalkyl and heterocyclyl rings.
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The heterocydyl group may be selected from piperidinyl, piperazinyl,
morpholinyl, and
thiomorpholinyl, such as piperidinyl, piperazinyl, and thiomorpholinyl, such
as selected from
piperidinyl and piperazinyl or selected from piperidinyl and thiomorpholinyl.
The heterocydyl group may not be nriorpholinyl, for example where each of -L6A-
and -L66- is
a covalent bond.
The heterocydyl group may not be piperazinyl, for example where each of -OA-
and -Let is
a covalent bond.
When -R6A is cydoalkyl it may C3_140 cycloalkyl, such as C5_10 cycloalkyl. The
cycloalkyl may
be monocyclic, or may comprise a plurality of fused rings. A cycloalkyl group
may be
partially unsaturated (but not aromatic).
Where a plurality of rings is present, the ring connected to -L66- is non-
aromatic, and is
preferably a fully saturated ring. The other rings are optionally aromatic.
The other rings
may be fully unsaturated, partially unsaturated or saturated. The other rings
may be
independently selected from aromatic, cycloalkyl and heterocyclyl rings.
A cycloalkyl group may be selected from cyclopentylene (C5), cyclohexylene
(C6);
tetralinylene and decalinylene (Cio), such as cyclohexylene.
When -R6A is alkyl it may be C14 alkyl, such as C14 alkyl, such methyl or
ethyl. The alkyl
group may be linear or branched. The alkyl may be optionally substituted.
When -IR" is alkenyl it may be C24 alkenyl, such as C24 alkenyl, such ethenyl.
The alkenyl
group may be linear or branched. The alkenyl may be optionally substituted.
When -R6A is alkynyl it may be C24 alkynyl, such as C24 alkynyl, such ethynyl.
The alkynyl
group may be linear or branched. The alkynyl may be optionally substituted.
When -R6A is heteroalkyl it may be 024 heteroalkyl, such as C34 heteroalkyl.
The heteroalkyl
group may be linear or branched. A heteroalkyl group is an alkyl group where
one or two
carbon atoms is replaced with a heteroatom selected from 0, S and N(H). The
heteroatom
does not replace a carbon atom at the terminal of the alkyl group. The
heteroalkyl group
may be connected via a heteroatom, or alternatively it may be connected via a
carbon atom.
The group -R6A is preferably optionally substituted heterocydyl.
In one embodiment, the group -R6 contains no aromatic functional group, for
example, the
group -R6 does not contain a phenyl group.
-Rs
The group -Rs may be provided as a substituent to the group -R7A or the group -
R6A. This
substituent is optionally present. Typically each of -R7A and -R6A is
unsubstituted, or is
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monosubstituted with -Rs. In other embodiments each of -Rm and -R6A is
provided with two
or more substituents -Rs.
The group -Rs may be a substituent to a carbon atom within the group -R" or -
Rm. Here,
the group -Rs is -Rsc.
The group -R8 may be a substituent to a nitrogen atom within the group -1R6A
or -Rm. Here,
the group -RS is -Rs".
In one embodiment, each -Rsc is independently selected from -L-R, halo,
hydroxy (-OH),
amino (-NH2), thiol (-SH), cyano, nitro, and carboxy (-00011), where:
-Lse- is a covalent bond or is selected from *-C(0)-, *-S(0)-, *-S(0)2- *-
N(H)C(0)-,
*-N(RN)C(0)-, *-N(H)S(0)-, *-N(RN)S(0)-, *-N(H)S(0)2-, *-N(RN)S(0)2-, *-N(H)-,
and _N(RN)_,
where -RN is Ci-e alkyl, and the asterisk indicates the point of attachment to
RCA or -Rm; and
-Rss is selected from optionally substituted alkyl, alkenyl, alkynyl,
cycloalkyl,
heterocyclyl and aryl.
In one embodiment, -Lsc- is a covalent bond or is selected from *-N(H)S(0)-, *-
N(RN)S(0)-,
*-N(H)S(0)2-, *-N(RN)S(0)2-, such as -Lsc- is a covalent bond or *-N(R")S(0)2--
In one embodiment, each -Rs" is independently selected from -L-R, where:
-Ls"- is a covalent bond or is selected from *-C(0)-, *-S(0)-, *-S(0)2- , and
the
asterisk indicates the point of attachment to R6A or -Rm; and
-Rss is selected from optionally substituted alkyl, alkenyl, alkynyl,
cycloalkyl,
heterocyclyl and aryl.
In one embodiment, -Lse- is a covalent bond or is selected from *-S(0)-, *-
S(0)2-, such as
-Lse- is a covalent bond or *-S(0)2-.
Where -Rss is alkyl it may be Ci_g alkyl, such as C14 alkyl, such methyl or
ethyl. The alkyl
group may be linear or branched. The alkyl may be optionally substituted.
When -Rss is alkenyl it may be C2-6 alkenyl, such as C24 alkenyl, such
ethenyl. The alkenyl
group may be linear or branched. The alkenyl may be optionally substituted.
When -Rss is alkynyl it may be C2-6 alkynyl, such as C2-4 alkynyl, such
ethynyl. The alkynyl
group may be linear or branched. The alkynyl may be optionally substituted.
When -Rss is cycloalkyl it may Cs_io cycloalkyl, such as C5_10 cycloalkyl. The
cycloalkyl may
be monocyclic, or may comprise a plurality of fused rings. A cycloalkyl group
may be
partially unsaturated (but not aromatic).
Where a plurality of rings is present, the ring connected to -Ls"- or -LsQ is
non-aromatic, and
is preferably a fully saturated ring. The other rings are optionally aromatic.
The other rings
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may be fully unsaturated, partially unsaturated or saturated. The other rings
may be
independently selected from aromatic, cydoalkyl and heterocyclyl rings.
A cycloalkyl group may be selected from cyclopentylene (Cs), cyclohexylene
(C6);
tetralinylene and decalinylene (Ci0), such as cyclohexylene.
When -Rss is heterocyclyl it may be C3-14 heterocyclyl.
A heterocyclyl has one or two ring heteroatoms, with each ring heteroatom
selected from 0,
S and N(H). The heterocyclyl may connect to
or -Lsc- via a ring carbon atom or a ring
nitrogen atom, where present.
A heterocyclyl may be partially unsaturated (but not aromatic).
The heterocyclyl may be monocyclic, or may comprise a plurality of fused
rings. Where a
plurality of rings is present, the ring connected to -Ls"- or -Lsc- is non-
aromatic, and is
preferably a fully saturated ring. The other rings are optionally aromatic.
The other rings
may be fully unsaturated, partially unsaturated or saturated. The other rings
may be
independently selected from aromatic, cycloalkyl and heterocyclyl rings.
The heterocyclyl group may be selected from piperidinyl, piperazinyl,
morpholinyl, and
thiomorpholinyl.
When -Rss is aryl it may be carboaryl or heteroaryl.
A carboaryl group may be C6-14 carboaryl, such as phenyl or naphthyl, and
preferably phenyl.
A heteroaryl group may be 06-14 heteroaryl, such as Cs_10 heteroaryl, such as
C6.6 heteroaryl.
An aryl group may be monocyclic, or may comprise a plurality of fused rings.
Where a
plurality of rings is present, the ring connected to -Ls"- or -Lsc- is
aromatic. The other rings
are optionally aromatic. The other rings may be fully unsaturated or partially
unsaturated.
The other rings may be independently selected from aromatic, cycloalkyl and
heterocyclyl
rings.
In one embodiment, ass is optionally substituted aryl, such as optionally
substituted phenyl,
pyridinyl, pyrrolyl, oxazolyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl,
thiazolyl, quinolinyl
and isoquinolinyl.
Preferably -Rss is alkyl.
Where -Rss is optionally substituted, each optional substituent may be
selected from the
group consisting of halo (such as -F, -Cl, -Br, and -I), hydroxy (-OH), amino
(-NH2), thiol
(-SH), cyano (-CN), nitro, carboxy (-000H), and phenyl, and where -R88 is
cycloalkyl,
heterocyclyl or aryl, the optional substituent is further selected from alkyl,
such as C1-6 alkyl,
such as methyl and ethyl.
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Exemplary Compounds of Formula (I)
The compounds of formula (I) may be a compound as set out below:
HN
RA 10
N
7 001 I
R N
and salts, solvates and protected forms thereof, wherein -RA, -R7 and -M have
the
same meanings as given above. Here, the phenylene group attached to the
pyridone
nitrogen is monosubstituted with -RA. This group may be provided at any one of
the 2-, 4-,
5- or 6-positions, and preferably at the 4-position.
The compounds of formula (I) may be a compound as set out below:
0
HN.-1-LM
0
N
7 141 I 4_,
R N
and salts, solvates and protected forms thereof, wherein -R7 and -M have the
same
meanings as given above.
The compounds of formula (I) may be a compound as set out below:
0
N
7 140 I
R N
and salts, solvates and protected forms thereof, wherein -R7 and -M have the
same
meanings as given above.
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Exemplary Compounds of Formula (1)
The compounds of formula (II) may be a compound as set out below:
HNAM
RA
0
N
R6
and salts, solvates and protected forms thereof, wherein -RA, -R6 and -M have
the
same meanings as given above. Here, the phenylene group attached to the
pyridone
nitrogen is monosubstituted with -RA. This group may be provided at any one of
the 2-, 4-,
5- or 6-positions, and preferably at the 4-position.
The compounds of formula (II) may be a compound as set out below:
0
HN
N 0
R6
401
and salts, solvates and protected forms thereof, wherein -R6 and -M have the
same
meanings as given above.
The compounds of formula (II) may be a compound as set out below:
0
00 o
N
Re
and salts, solvates and protected forms thereof, wherein -R8 and -M have the
same
meanings as given above.
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Complex
In one aspect, the present invention also provides a compound of formula (I)
or a compound
of formula (II) covalently bound to a polypeptide. This combination may be
referred to as a
complex of the compound with the polypeptide.
The polypeptide typically contains a threonine amino acid residue, and the
compound of
formula (I) or (II) is bound to the polypeptide through the side-chain
functionality of this
threonine residue.
A complex may be formed by contacting a compound of formula (I) or (II) with a
polypeptide.
The compounds of formula (I) and (II) are provided with an acceptor group,
such as a
Michael acceptor group, that is suitable for reaction with a side chain
functionality of an
amino residue of the polypeptide, such as a thiol functionality of a cysteine
residue.
In one embodiment, the polypeptide is a kinase.
The kinase may be selected from a kinase family selected from TEC, EGFR. JAK,
Src, FAK,
PI3K, mTOR, Liver Kinase B1, Pkb, PAK1, TAM, Abl and PDPK1.
A TEC kinase family member may be selected from the group consisting of BMX,
BTK, ITK,
TEC and TXK.
An EGFR kinase family member may be selected from the group consisting of
EGFR,
ERBB2, ERBB3 and ERB134.
A JAK kinase family member may be selected from the group consisting of JAK1,
JAK2,
JAK3 and TYK2.
A Src kinase family member may be selected from the group consisting of FYN,
SRC, YES1,
BLK, FGR, LCK, HCK, and LYN.
A FAK kinase family member may be PTK2.
A PI3k kinase family member may be selected from the group consisting of
PIK3CA,
PIK3C13, PIK3C7 and PIK3C45.
A mTOR kinase family member may be mTOR.
A Liver Kinase B1 kinase family member may be Liver Kinase B1.
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A Pkb kinase family member may be selected from the group consisting of ATK1,
ATIC and
ATK3.
A PAK1 kinase family member may be PAK1.
A TAM kinase family member may be selected from AXL and MERTK.
A Abl kinase family member may be Abl 1.
A PDPK1 kinase family member may be PDPK1.
Preferably the kinase is a TEC kinase family, and most preferably the kinase
is BMK or BTK,
such as BMX.
The kinase may be a human kinase.
The polypeptide is an enzyme with kinase activity, where the polypeptide has
an amino acid
sequence as set out in SEQ ID Nos.: 1 to 6, or a variant thereof.
In one embodiment, the polypeptide may comprise a polypeptide having at least
35%, 45%,
55%, 65%, 75%, 85%, 95%, 98%, 99% or 100% identity to any one of SEQ ID Not: 1
to 6,
such as SEQ ID No.: 1.
The kinase may comprise a TH domain, and typically comprise a cysteine residue
in the
pocket of the active site.
The kinase may be BMX, such as a BMX comprising a polypeptide having the amino
acid
sequence as set out in SEQ ID No.: 1. A compound of formula (I) or (II) may be
bound to
the side chain of the Cys 496 residue of BMX.
Where the kinase is not a BMX kinase, the compound may be bound to a cysteine
residue
that corresponds to the Cys 496 residue of BMX.
Kinase¨ General Information
Amino acid sequence identity and similarity may be measured using standard
bioinformatics
software tools, such as the freely available EMBOSS, or BLAST, software tools.
Default
parameters are generally used. For example EMBOSS Needle pairwise sequence
alignment can be used to determine amino add sequence identity. EMBOSS Needle
pairwise sequence alignment, which uses the Needleman-Wunsch algorithm (J.
Mol. Biol.
(48): 444-453 (1970)), can be used to determine amino acid sequence
similarity, for example
using default parameters and using a BLOSUM scoring matrix such as the
BLOSUM62
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scoring matrix. Default parameters may be used with a gap creation penalty =
12 and gap
extension penalty =4. Use of GAP may be preferred but other algorithms may be
used, e.g.
BLAST or TBUkSTN (which use the method of Altschul et al. (1990) J. Mol. Biol.
215: 405-
410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85:
2444-
2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol.
147: 195-
197), generally employing default parameters.
Percent (%) amino acid sequence identity with respect to a reference sequence
is defined as
the percentage of amino add residues in a candidate sequence that are
identical with the
amino acid residues in the reference sequence, after aligning the sequences
and introducing
gaps, if necessary, to achieve the maximum percent sequence identity, and not
considering
any conservative substitutions as part of the sequence identity. Percent
identity values may
be determined by VVU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-
480
(1996)). VVU-BLAST-2 uses several search parameters, most of which are set to
the default
values. The adjustable parameters are set with the following values: overlap
span = 1,
overlap fraction = 0.125, word threshold (T) = 11. A % amino acid sequence
identity value is
determined by the number of matching identical residues as determined by VVU-
BLAST-2,
divided by the total number of residues of the reference sequence (gaps
introduced by WU-
BLAST-2 into the reference sequence to maximize the alignment score being
ignored),
multiplied by 100.
Percent (%) amino acid sequence alignment coverage with respect to a reference
sequence
is defined as the percentage of amino acid residues in the candidate sequence
in
comparison to the number of amino acid residues in the reference sequence,
after aligning
the sequences.
A variant polypeptide may be a truncated polypeptide. Any truncation may be
used as long
as the truncated polypeptide still has kinase activity. Truncations may remove
one or more
residues from the N- and/or C-terminus of the polypeptide, which residues are
non-essential
for kinase activity. Appropriate truncations may be routinely identified by
systematic
truncation of sequences of varying length from the N- and/or C-terminus.
A variant polypeptide comprise one or more additional amino acids. A variant
polypeptide
may comprise an affinity tag for purifying the variant polypeptide, such as a
poly-histidine
tag, a T7 tag or a GST tag. An affinity tag may be located at the N- or C-
terminus.
Alternatively or additionally, the variant polypeptide may further comprise a
leader sequence
at the N-terminus. The leader sequence may be useful for directing secretion
and/or
intracellular targeting of the polypeptide in a recombinant expression system.
Leader
sequences are also known as signal peptides and are well known in the art.
Alternatively or
additionally, the polypeptide may further comprise a label such as a
fluorescent label_
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Amino add substitutions may be conservative amino acid substitutions, in which
an amino
add of a given sequence is substituted by an amino acid having similar
characteristics. For
example, where a hydrophobic amino add (e.g. Leu) is substituted by another
hydrophobic
amino acid (e.g. Ile). Amino acids and conservative substitutions are shown in
the table
below. A conservative substitution may be defined as a substitution within an
amino acid
class and/or a substitution that scores positive in the BLOSUM62 matrix.
Conservative
Conservative
Amino acid Amino acid
substitution
substitution
Ala Ser Leu
Ile; Val
Arg Lys Lys
Arg; Gin; Glu
Asn Gin; His Met
Leu; Ile
Asp Glu Phe
Met; Leu; Tyr
Cys Ser Ser
Thr
Gin Asn Thr
Ser
Glu Asp Trp
Tyr
Gly Pro Tyr
Trp; Phe
His Asn; Gin Val
Ile; Leu
Ile Leu; Val
Salts, Solvates and Other Forms
Examples of salts of compound of formula (I) and (II) include all
pharmaceutically acceptable
salts, such as, without limitation, add addition salts of strong mineral acids
such as HCI and
HBr salts and addition salts of strong organic acids such as a methanesulfonic
acid salt.
Further examples of salts include sulphates and acetates such as
trifluoroacetate or
trichloroacetate.
A compound of formula (I) or (II) can also be formulated as prodrug. Prodrugs
can include a
compound herein described in which one or more amino groups are protected with
a group
which can be cleaved in vivo, to liberate the biologically active compound.
In one embodiment a compound of formula (I) or (II) is provided as a prodrug.
A reference to a compound of formula (I) or (II), or any other compound
described herein, is
also a reference to a solvate of that compound. Examples of solvates include
hydrates.
A compound of formula (I) or (II), or any other compound described herein,
includes a
compound where an atom is replaced by a naturally occurring or non-naturally
occurring
isotope. In one embodiment the isotope is a stable isotope. Thus a compound
described
here includes, for example deuterium containing compounds and the like. For
example, H
may be in any isotopic form, including 1H, 2H (0), and 3H (T); C may be in any
isotopic form,
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including 12C, "C, and 14C; 0 may be in any isotopic form, including 160 and
180; and the
like.
Certain compounds of formula (I) or (II), or any other compound described
herein, may exist
in one or more particular geometric, optical, enantiomeric, diasteriomeric,
epimeric, atropic,
stereoisomeric, tautomeric, conformational, or anomeric forms, including but
not limited to,
cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-
forms; R-, S-, and
rneso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-,
and enolate-
forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and 8-forms;
axial and
equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and
combinations
thereof, hereinafter collectively referred to as "isomers" (or "isomeric
forms").
Note that, except as discussed below for tautomeric forms, specifically
excluded from the
term "isomers," as used herein, are structural (or constitutional) isomers
(i.e., isomers which
differ in the connections between atoms rather than merely by the position of
atoms in
space). For example, a reference to a nnethoxy group, -OCH3, is not to be
construed as a
reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly,
a reference to
ortho-chlorophenyl is not to be construed as a reference to its structural
isomer, meta-
chlorophenyl. However, a reference to a class of structures may well include
structurally
isomeric forms falling within that class (e.g., Ci4a1ky1 includes n-propyl and
iso-propyl; butyl
includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-,
and para-
methoxypheny1).
Unless otherwise specified, a reference to a particular compound includes all
such isomeric
forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the
preparation
(e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation
and
chromatographic means) of such isomeric forms are either known in the art or
are readily
obtained by adapting the methods taught herein, or known methods, in a known
manner.
One aspect of the present invention pertains to compounds in substantially
purified form
and/or in a form substantially free from contaminants.
In one embodiment, the substantially purified form is at least 50% by weight,
e.g., at least
60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight,
e.g., at least 90%
by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g.,
at least 98% by
weight, e.g., at least 99% by weight.
Unless specified, the substantially purified form refers to the compound in
any
stereoisomeric or enantiomeric form. For example, in one embodiment, the
substantially
purified form refers to a mixture of stereoisomers, i.e., purified with
respect to other
compounds. In one embodiment, the substantially purified form refers to one
stereoisomer,
e.g., optically pure stereoisomer. In one embodiment, the substantially
purified form refers
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to a mixture of enantiomers. In one embodiment, the substantially purified
form refers to an
equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one
embodiment,
the substantially purified form refers to one enantiomer, e.g., optically pure
enantiomer.
In one embodiment, the contaminants represent no more than 50% by weight,
e.g., no more
than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20%
by weight,
e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no
more than 3%
by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight
Unless specified, the contaminants refer to other compounds, that is, other
than
stereoisomers or enantiomers. In one embodiment, the contaminants refer to
other
compounds and other stereoisomers. In one embodiment, the contaminants refer
to other
compounds and the other enantiomer.
In one embodiment, the substantially purified form is at least 60% optically
pure (i.e., 60% of
the compound, on a molar basis, is the desired stereoisonner or enantiomer,
and 40% is the
undesired stereoisomer or enantiomer), e.g., at least 70% optically pure,
e.g., at least 80%
optically pure, e.g., at least 90% optically pure, e.g., at least 95%
optically pure, e.g., at least
97% optically pure, e.g., at least 98% optically pure, e.g., at least 99%
optically pure.
Methods of Treatment
The compounds of formula (I) or (II), or pharmaceutical formulations
containing these
compounds, are suitable for use in methods of treatment and prophylaxis. The
compounds
may be administered to a subject in need thereof.
The compounds of formula (I) or (II) are for use in a method of treatment of
the human or
animal body by therapy. In some aspects of the invention, a compound of
formula (I) or (II)
may be administered to a mammalian subject, such as a human, in order to treat
a
proliferative disease, such as cancer.
Another aspect of the present invention pertains to use of a compound of
formula (I) or (II) in
the manufacture of a medicament for use in treatment. In one embodiment, the
medicament
comprises a compound of formula (I) or (II).
The compounds of the present case may be useful for the treatment of a
proliferative
disease, such as cancer_
The cancer may be selected from breast, prostate, colon and cervical cancers,
leukaemia,
myeloma and non-Hodgkin's lymphoma.
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The compounds of the invention may be used to treat an autoimmune disease. The
autoimmune disease may be for example rheumatoid arthritis or lupus (see, for
example.
Honignerg et aL, 2010, Xia et aL, 2010, Chalmers et at, 2015 and Rankin et aL,
2013).
The compounds of the invention may be used to treat a disease associated with
kinase
activity, such as elevated kinase activity.
Treatment
The term "treatment," as used herein in the context of treating a condition,
pertains generally
to treatment and therapy, whether of a human or an animal (e.g., in veterinary
applications),
in which some desired therapeutic effect is achieved, for example, the
inhibition of the
progress of the condition, and includes a reduction in the rate of progress, a
halt in the rate
of progress, alleviation of symptoms of the condition, amelioration of the
condition, and cure
of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is
also included.
For example, use with patients who have not yet developed the condition, but
who are at risk
of developing the condition, is encompassed by the term "treatment"
The term "therapeutically-effective amount," as used herein, pertains to that
amount of a
compound, or a material, composition or dosage form comprising a compound,
which is
effective for producing some desired therapeutic effect, commensurate with a
reasonable
benefit/risk ratio, when administered in accordance with a desired treatment
regimen_
The term "treatment" includes combination treatments and therapies, in which
two or more
treatments or therapies are combined, for example, sequentially or
simultaneously.
Formulations
In one aspect, the present invention provides a pharmaceutical composition
comprising a
compound of formula (I) or (II) together with a pharmaceutically acceptable
carrier.
While it is possible for the compound of formula (I) or (II) to be
administered alone or
together with the second agent, it is preferable to present it as a
pharmaceutical formulation
(e.g., composition, preparation, medicament) comprising at least one compound
of formula
(I) or (II), as described herein, together with one or more other
pharmaceutically acceptable
ingredients well known to those skilled in the art, including, but not limited
to,
pharmaceutically acceptable carriers, diluents, excipients, adjuvants,
fillers, buffers,
preservatives, anti-oxidants, lubricants, stabilisers, solubilisers,
surfactants (e.g., wetting
agents), masking agents, colouring agents, flavouring agents, and sweetening
agents. The
formulation may further comprise other active agents, for example, other
therapeutic or
prophylactic agents.
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Thus, the present invention further provides pharmaceutical compositions, as
defined above,
and methods of making a pharmaceutical composition comprising admixing at
least one
compound of formula (I) or (II), as described herein, together with one or
more other
pharmaceutically acceptable ingredients well known to those skilled in the
art, e.g., carriers,
diluents, excipients, etc. If formulated as discrete units (e.g., tablets,
etc.), each unit
contains a predetermined amount (dosage) of the compound. The composition
optionally
further comprises the second active agent in a predetermined amount.
The term "pharmaceutically acceptable," as used herein, pertains to compounds,
ingredients, materials, compositions, dosage forms, etc., which are, within
the scope of
sound medical judgment, suitable for use in contact with the tissues of the
subject in
question (e.g., human) without excessive toxicity, irritation, allergic
response, or other
problem or complication, commensurate with a reasonable benefit/risk ratio.
Each carrier,
diluent, excipient, etc. must also be "acceptable" in the sense of being
compatible with the
other ingredients of the formulation.
Suitable carriers, diluents, excipients, etc. can be found in standard
pharmaceutical texts, for
example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing
Company,
Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition,
2005.
The formulations may be prepared by any methods well known in the art of
pharmacy. Such
methods include the step of bringing into association the compound of formula
(I) or (II) with
a carrier which constitutes one or more accessory ingredients. In general, the
formulations
are prepared by uniformly and intimately bringing into association the
compound with
carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then
shaping the product, if
necessary.
Formulations may suitably be in the form of liquids, solutions (e.g., aqueous,
non-aqueous),
suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-
in-oil), elixirs,
syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated
tablets), granules,
powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin
capsules),
cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels,
pastes,
ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
Formulations may suitably be provided as a patch, adhesive plaster, bandage,
dressing, or
the like which is impregnated with one or more compounds and optionally one or
more other
pharmaceutically acceptable ingredients, induding, for example, penetration,
permeation,
and absorption enhancers. Formulations may also suitably be provided in the
form of a
depot or reservoir.
The compound may be dissolved in, suspended in, or admixed with one or more
other
pharmaceutically acceptable ingredients.
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Formulations suitable for oral administration (e.g., by ingestion) include
liquids, solutions
(e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous),
emulsions
(e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets,
granules, powders,
capsules, cachets, pills, ampoules, boluses.
Formulations suitable for buccal administration include mouthwashes, losenges,
pastilles, as
well as patches, adhesive plasters, depots, and reservoirs. Losenges typically
comprise the
compound in a flavoured basis, usually sucrose and acacia or tragacanth.
Pastilles typically
comprise the compound in an inert matrix, such as gelatin and glycerin, or
sucrose and
acacia. Mouthwashes typically comprise the compound in a suitable liquid
carrier.
Formulations suitable for sublingual administration include tablets, losenges,
pastilles,
capsules, and pills.
Formulations suitable for oral transnnucosal administration include liquids,
solutions (e.g.,
aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions
(e.g., oil-in-
water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches,
adhesive plasters,
depots, and reservoirs.
Formulations suitable for non-oral transmucosal administration include
liquids, solutions
(e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous),
emulsions
(e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes,
ointments, creams,
lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
Formulations suitable for transdermal administration include gels, pastes,
ointments,
creams, lotions, and oils, as well as patches, adhesive plasters, bandages,
dressings,
depots, and reservoirs.
Tablets may be made by conventional means, e.g., compression or moulding,
optionally with
one or more accessory ingredients.
Ointments are typically prepared from the compound and a paraffinic or a water-
miscible
ointment base.
Emulsions are typically prepared from the compound and an oily phase, which
may
optionally comprise merely an emulsifier (otherwise known as an ennulgent), or
it may
comprises a mixture of at least one emulsifier with a fat or an oil or with
both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a lipophilic
emulsifier which acts
as a stabiliser. It is also preferred to include both an oil and a fat.
Together, the
emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying
wax, and the wax
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together with the oil and/or fat make up the so-called emulsifying ointment
base which forms
the oily dispersed phase of the cream formulations.
Formulations suitable for intranasal administration, where the carrier is a
liquid, include, for
example, nasal spray, nasal drops, or by aerosol administration by nebuliser,
include
aqueous or oily solutions of the compound. As an alternative method of
administration, a dry
powder delivery may be used as an alternative to nebulised aerosols.
Formulations suitable for intranasal administration, where the carrier is a
solid, include, for
example, those presented as a coarse powder having a particle size, for
example, in the
range of about 20 to about 500 microns which is administered in the manner in
which snuff is
taken, i.e., by rapid inhalation through the nasal passage from a container of
the powder
held close up to the nose.
Formulations suitable for pulmonary administration (e.g., by inhalation or
insufflation therapy)
include those presented as an aerosol spray from a pressurised pack, with the
use of a
suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane,
dichoro-
tetrafluoroethane, carbon dioxide, or other suitable gases. Additionally or
alternatively, a
formulaton for pulmonary administration may be formulated for administration
from a
nebuliser or a dry powder inhaler. For example, the formulation may be
provided with
carriers or liposomes to provide a suitable particle size to reach the
appropriate parts of the
lung, to aid delivery of an appropriate does to enhance retention in the lung
tissue.
Formulations suitable for ocular administration include eye drops wherein the
compound is
dissolved or suspended in a suitable carrier, especially an aqueous solvent
for the
compound.
Formulations suitable for rectal administration may be presented as a
suppository with a
suitable base comprising, for example, natural or hardened oils, waxes, fats,
semi-liquid or
liquid polyols, for example, cocoa butter or a salicylate; or as a solution or
suspension for
treatment by enema.
Formulations suitable for vaginal administration may be presented as
pessaries, tampons,
creams, gels, pastes, foams or spray formulations containing in addition to
the compound,
such carriers as are known in the art to be appropriate.
Formulations suitable for parenteral administration (e.g., by injection),
include aqueous or
non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions,
suspensions), in which
the compound is dissolved, suspended, or otherwise provided (e.g., in a
liposome or other
microparticulate). Such liquids may additional contain other pharmaceutically
acceptable
ingredients, such as anti-oxidants, buffers, preservatives, stabilisers,
bacteriostats,
suspending agents, thickening agents, and solutes which render the formulation
isotonic with
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the blood (or other relevant bodily fluid) of the intended recipient. Examples
of excipients
include, for example, water, alcohols, polyols, glycerol, vegetable oils, and
the like.
Examples of suitable isotonic carriers for use in such formulations include
Sodium Chloride
Injection, Ringers Solution, or Lactated Ringers Injection. Typically, the
concentration of the
compound in the liquid is from about 1 ng/mL to about 100 pg/mL, for example
from about
ng/mL to about 10 pg/mL, for example from about 10 ng/mL to about 1 pg/mL. The
formulations may be presented in unit-dose or multi-dose sealed containers,
for example,
ampoules and vials, and may be stored in a freeze-dried (lyophilised)
condition requiring
only the addition of the sterile liquid carrier, for example water for
injections, immediately
10 prior to use. Extemporaneous injection solutions and suspensions may be
prepared from
sterile powders, granules, and tablets.
Kits
One aspect of the invention pertains to a kit comprising (a) a compound of
formula (I) or (II),
or a composition comprising a compound as defined in any one of formula (I) or
(II), e.g.,
preferably provided in a suitable container and/or with suitable packaging;
and
(b) instructions for use, e.g., written instructions on how to administer the
compound or
composition.
The written instructions may also include a list of indications for which the
compound of
formula (I) or (II) is a suitable treatment.
In one embodiment, the kit further comprises (c) a second active agent, or a
composition
comprising the second active agent. Here, the written instructions may also
include a list of
indications for which the second active agent, together with the compound of
formula (I) or
(II), is suitable for treatment.
Routes of Administration
A compound of formula (I) or (II), a second agent, or a pharmaceutical
composition
comprising the compound of formula (I) or (II), may be administered to a
subject by any
convenient route of administration, whether systemically/peripherally or
topically (i.e., at the
site of desired action).
Routes of administration include, but are not limited to, oral (e.g., by
ingestion); buccal;
sublingual; transdernnal (including, e.g., by a patch, plaster, etc.);
transnnucosal (including,
e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular
(e.g., by eyedrops);
pulmonary (e.g., by inhalation or insuffiation therapy using, e.g., via an
aerosol, e.g., through
the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by
pessary);
parenteral, for example, by injection, including subcutaneous, intradermal,
intramuscular,
intravenous, intraarterial, intracardiac, intrathecal, intraspinal,
intracapsular, subcapsular,
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intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular,
subarachnoid, and
intrasternal; by implant of a depot or reservoir, for example, subcutaneously,
intracranially or
intramuscularly.
The Subject/Patient
The subject/patient may be a chordate, a vertebrate, a mammal, a placental
mammal, a
marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a
rat, a mouse),
murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird),
canine (e.g., a dog),
feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine
(e.g., a sheep), bovine
(e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,
marmoset, baboon),
an ape (e.g., gorilla, chimpanzee, orang-utan, gibbon), or a human.
Furthermore, the
subject/patient may be any of its forms of development, for example, a foetus.
In one preferred embodiment, the subject/patient is a human.
It is also envisaged that the invention may be practised on a non-human animal
having a
microbial infection. A non-human mammal may be a rodent. Rodents include rats,
mice,
guinea pigs, chinchillas and other similarly-sized small rodents used in
laboratory research.
Cell Treatment
The present invention provides a method of treating a cell or a population of
cells with a
compound of formula (I) or a compound of formula (II), the method comprising
the step of
contacting a cell or cell population with a compound of formula (I) or a
compound of
formula (II).
The method may be performed in vitro or in vivo.
A cell or cell population may be obtained from a subject, such as a subject
described herein.
A cell may be treated to limit or prevent its proliferation.
The cell may be a proliferative cell, such as cancer cell. The cancer cell may
be selected
from breast, prostate, colon and cervical cancer cells.
Examples of cancer cells for treatment include prostate cancer cells, such as
the LCNCaP,
PC-3 and DU-145 and 22RV-1 cell lines exemplified herein, and further selected
from
chronic lymphocytic leukemia cell lines, JVM-3, MEC-2, M01043 and WaC3CD5 cell
lines.
The method of treating a cell or a cell population may include the step of
determining the
proliferation of a cell or a cell population.
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The methods may be used to determine the suitability of a compound for use in
methods of
treatment.
Combination Therapy and Co-Treatment
A compound of formula (I) may be administered in conjunction with a second
agent.
Administration may be simultaneous, separate or sequential.
The appropriate route of administration and how the compound of formula (I)
and the second
agent are administered will depend on the pharmacokinetics of the compound of
formula (I)
and the second agent
By "simultaneous" administration, it is meant that a compound of formula (I)
and a second
agent are administered to a subject in a single dose by the same route of
administration.
By "separate" administration, it is meant that a compound of formula (I) and a
second agent
are administered to a subject by two different routes of administration which
occur at the
same time. This may occur for example where one agent is administered by
infusion and
the other is given orally during the course of the infusion.
By "sequential" it is meant that the two agents are administered at different
points in time,
provided that the activity of the first administered agent is present and
ongoing in the subject
at the time the second agent is administered.
Generally, a sequential dose will occur such that the second agent is
administered within
48 hours of administration of the compound of formula (I), preferably within
24 hours, such
as within 12, 6, 4, 2 or 1 hour(s) of the first agent. Alternatively, the
active agent may be
administered first, followed by the compound of formula (I).
Ultimately, the order and timing of the administration of the compound and
second agent in
the combination treatment will depend upon the pharmacokinetic properties of
each.
The amount of the compound of formula (I) to be administered to a subject will
ultimately
depend upon the nature of the subject and the disease to be treated. Likewise,
the amount
of the active agent to be administered to a subject will ultimately depend
upon the nature of
the subject and the disease to be treated.
The second agent may be a substance which is a kinase inhibitor, for example a
PI3K or
AKT inhibitor. For example the second agent may be LY294002 (CAS number:
154447-36-
6) or AKT1/2 kinase inhibitor. The second agent may be an androgen receptor
inhibitor, for
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example Flutamide (also known under trade name Eulexin). The second agent may
also act
as an anti-cancer agent, such as one used to treat prostate cancer.
Other Preferences
Each and every compatible combination of the embodiments described above is
explicitly
disclosed herein, as if each and every combination was individually and
explicitly recited.
Various further aspects and embodiments of the present invention will be
apparent to those
skilled in the art in view of the present disclosure.
"and/or where used herein is to be taken as specific disclosure of each of the
two specified
features or components with or without the other. For example "A and/or B" is
to be taken as
specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if
each is set out individually
herein.
Unless context dictates otherwise, the descriptions and definitions of the
features set out
above are not limited to any particular aspect or embodiment of the invention
and apply
equally to all aspects and embodiments which are described.
Certain aspects and embodiments of the invention will now be illustrated by
way of example
and with reference to the figures described above.
Experimental and Results
Genera/ Methods and Materials
Starting materials were obtained from commercial suppliers and used without
further
purification unless otherwise stated. BMX-IN-1 was acquired from Calbiochem.
Column
chromatography was carried out with Merck Si60 (60-200 pm) silica gel columns
as the
stationary phase and analytical grade solvents as the eluent unless otherwise
stated. All
reactions using anhydrous conditions were performed under an argon atmosphere
in oven-
dried glassware.
Reactions were followed by thin layer chromatography (TLC) using coated silica
gel plates
(Merck, aluminium sheets, silica gel 60 coated with fluorescent indicator
F254) and
visualized by UV light and ninhydrin staining (if required). Proton magnetic
resonance (1H
NMR) spectra were recorded at 300 MHz on a Bruker Fourier 300 spectrometer and
are
reported as follows: chemical shifts 6 (ppm) (multiplicity, coupling constant
in J (Hz), number
of protons). Multiplicities are labelled s, singlet; d, doublet; t, triplet;
m, multiplet; br, broad;
or a combination of these.
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Total ion current traces were obtained for electrospray positive and negative
ionization
(ES+/ES-) on a Waters Acquity QDa detector. Analytical chromatographic
conditions used
for the LC/MS analysis were as follows. The column was a Cortecs C18 2.7 pM
(4.6 mm x
50 mm). Solvent A was an aqueous solvent consisting of Mink) water with 0.01%
Formic
Add and solvent B was acetonitrile with 0.01% Formic Acid. Additional
chromatographic
parameters were as follows: flow rate, 0.5 mi./min; injection volume, 5 pL;
column
temperature, 40 C; and UV wavelength range, 210-400 nm. The purity of all
tested
compounds was a95% using the analytical method described above unless
otherwise
stated.
High resolution mass spectrometry (HRMS) analysis were performed at the Unidad
de
Espectrometria de Masas e Proteonnica of the Universidad de Santiago de
Connpostela.
Samples were recorded on a Bruker Da!tonics microTOF ESI-TOF mass
spectrometer.
Calculated and exact m/z values are indicated in DaItons.
Synthesis
Compounds
The compounds prepared in this study, including compounds of the invention and
comparator compounds are set out in Table 1 below.
A compound of the invention includes compounds 24-27, where a group -R7 is
present.
These are compounds of formula (I).
A compound of the invention includes compounds 20-23, where a group -R6 is
present.
These are compounds of formula (II).
Compounds 9A-9E and 10-19 are provided as comparative examples for the useful
understanding of the invention. These compounds are of a type described in
WO 2014/063054.
Compound 28 is provided as a further reference example.
Compound 29 is an intermediate compound useful for the preparation of the
compounds of
formula (II).
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Table 1 - Compounds
HN yim.40 0
Hpator
HNI-s#
011 et 1 4
0 411
-4140,100it -1- a '
6 b Nip
NI 0
-
0
41 ---
- N
9A (BMX-IN-1) ge
9C 90
Li. A, Hr-
e-"CN
MI
5
- NH
to' 0
Paso siti 0 ,,tu
el
9 u 4.11 , 40
410
41 1-4) a II
1
I it isial 11 I 0 up . I
-
9E 10
11 12
Hri---r
MAL. 3`%-oss NIEL
U 0
` 4 00. 0
. =
6 I
/ 0
_AI
_it 40 4 I Qtril s 41t .
0. w a
. a II
41 -
Is _
-
.
13 14
15 16
4451-0'. #451-4 -
leiXe 14/E0
F3c,iiiicocr,10
0
NCO& ritc,
L----4:1-,
.......mcc9
17 13
19 20
ia
gn Hwyli.....#, u wity
iim
o
o-Thni
a
o
tr-A1141 0
wi m I
* -:--
1111:::SH: I
-6-t1
-
21 22
23 24
-1-, HNLe P1NLe
0
Itt
.a...õN 9 .
4.
01
it
Ai I
I -- IIC 0 --
r4 0
4 If
ja.
"11
- = .
ii pig
*KW
25 26
27 23
0
HNIA-00.
=
' II .
Er a __
W la
29
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Synthetic Procedure
The synthesis of the compounds of the invention was devised using the
synthetic pathway
developed for the known inhibitor BMX-IN-1 (see Liu et at ACS Chem. Biol.
2013).
However, some modifications were introduced, either to improve the yield of
intermediate
steps or because the reported procedure failed in our hands (see Scheme 1).
Scheme 1 - Synthetic route for the preparation of compounds 9a-e, 10-13 and 14-
23.
Br OH
CI 0
*
b)
EtO2C
El0 Br 41 CO2Et Br ist -.. =
NH 0
* + 1 --._ CO2E1
N N _ --IN.- c)
Himip._ Br
4 I41-12
N
1
2
3a-e
NO2 NO2
NO2
. 4i
)
N
I NH 0
õcut_e 4111
NH
'0-1 (I)
411
Br . ..,... _gag I.) Br
. ." H
Br
'
is
"-..._
OH
N N
F N
6a-e
5a-e
4a-e
NO2
N1-12
R2
4 alla II 0
* 0
HNe
N 1
I It) 11.141 N 6
R2 a 1
N I
¨Ow- R2 a
¨OW- R2 0 .......
g*IIP N.- ....
1111 N
N
la-e 8a-e
71-0 81-0 9a-e
10-13
14-23
24-27
a) Reflux, 24 h; Ph20, 240 C, 5h, 40%; b) SOCl2, reflux, 5h, 100%; c) aniline,
dioxane, 90 C, 6 h,
90%; d) NaBH4, Et0H, r.t., overnight, 100%; e) Dess-Martin Reriodinane, DCM,
r.t., 3 h, 75%; t)
Triethylphosphonoacetate, K2CO3, Et0H, 100 C, overnight, 60%; g) boronic
acid/ester, PdC12(PPh3)2,
Na2CO3, dioxane, 90 C, ovemight, 50% or amine, Pd(OAc)2, R-BINAP, Cs2CO3,
dioxane, 85 C1
overnight, 80%; h) Fe, NH4CI, Et0H/H20, 80 C, 2 h, 100%; i) acyl chloride,
DIPEA, THF, -10 C to rt.
5 h, 25% or alkyl halide, K2CO3, DMF, rt., overnight, 35%.
Briefly, the formation of the bromohydroxyl quinoline 1 was achieved through a
two-step
reaction. First, the reaction between 4-bromoaniline and the malonate is
carried out at ca.
140-150 C overnight and the cyclization is carried out in diphenyl ether at
225 C overnight
Despite several reports in the literature using a wide range of temperature
values
(Price et at; Ramsey et at; Lin et at; Reis et at), it was found that when
employing gram
amounts of starting materials, a strict control of the temperature between 230
C and 240 C
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was crucial to obtain the product in high yields (Rivilli et at). From 1,
intermediate 2 was
achieved with 100% yield by refluxing in SOCl2.
Preparation of anilines 3 was accomplished via nucleophilic aromatic
substitution with the
required aniline. While the reduction of the ester 3 to alcohol 4 was achieved
with NaBF14,
the oxidation to aldehyde 5 could not be performed with Mn02 as originally
described (Liu et
at. ACS Chem. Biol. 2013). The oxidation could be achieved with Pyridinium
chlorochromate
(PCC) with Na0Ac, however, using Dess-Martin Periodinane (DMP) the oxidation
was
smoother with less side product& Therefore, the alcohols 4 were oxidized with
DMP
originating the aldehydes 5. The Homer-Wadsworth-Emmons (HVVE) cydization with
triethylphosphonoacetate afforded intermediates 6.
In the next step, intermediates 7 were obtained via Suzuki cross-coupling with
the
corresponding boronic esters or acids or Buchwald-Hartwig aminations. The
amination
procedure was not straightforward, and attempts were made with Pd2(dba)3 as
palladium
source, while screening K2CO3 or Cs2CO3 as bases, together with BINAP, XPhos
or
tBuXPhos as phosphines. Finally, successful reactions were obtained employing
Pd(OAc)2,
Cs2CO3 and RBINAP in dioxane at 90 C overnight. Reduction of the nitro group
into amine
was not performed following the literature procedure (SnCl2) (Liu et al. ACS
Chem. Biol.
2013). Instead, a more environmentally-friendly reagent was employed (Fe/NH4CI
in boiling
Et0H) affording intermediates 8 in nearly quantitative yields. Final compounds
9-23 were
obtained by acylation of intermediate 8 with the corresponding acyl chloride
or by
nucleophilic substitution with 4-bromobut-2-enetrile and methyl 4-
bromocrotonate to afford
12 and 13, respectively. Analogues 24-27 were prepared in the same way, using
3-bromoaniline as starting material (Scheme 2).
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Scheme 2- Synthetic route for the preparation of compounds 24-27
NO2
R2 a
OH
CI 0
00 a
gill Br EU
NH 0 ....... CO2Et I))
0 1 t cco
to
-NI.
N-.. = -e--"=-=
Et02i 21Et 13r 11411
NH,
Br tilla N
1'
2'
3%.13,t1
NOT, NO2
NO2 NO2
R2 0
4R2 lali 0 R-,
-
'11-1P R2 N I 9) litillij N sir a I) NH 0
e) W NH -4--k)
%-.. a. . H to --... =H
-== .--
R4 N Br N Br
N Br N
Ta,b.d Wad
roAd 4:13,13,d
0
..A...4.0%
NH2 HN
R, s R2 a 0
S
Lc
I
1.1 .1 N R4 N Re, N
Ife,b,d
24-27
a) Reflux 24h; Ph20, 240 C, 5h, 40%; b) SOCl2, reflux, 5h, 100%; c) aniline,
dioxane, 90 C, overnight,
80%; d) N8BH4, Et0H, r.t., ovn, 90%; e) Dess-Martin Periodinane, DCM, r.t.,
3h, 80%; t)
Triethylphosphonoacetate, K2CO3, Et0H, 100 C, overnight, 70%; g) boronic
acid/ester, PdC12(PPh3)2,
Na2CO3, dioxane, 90 C, ovn, 20% or amine, Pd(OAc)2, R-BINAP, Cs2CO3, dioxane,
85 C, overnight,
90%; h) Fe, NH4CI, Et0H/H20, 80 C, 2h, 70%; i) acryloyl chloride, DIPEA, THF, -
10 C to r.t. 5h, 15%.
In order to prepare compound 28, first a Suzuki coupling between intermediate
3a and the
boronic ester was performed under the described conditions (see conditions g)
in
Scheme 1), followed by reduction of the nitro group with Fe/NH4C1 and
acylation with
acryloyl chloride (Scheme 3). Finally, compound 29 was prepared from
intermediate 6a
which was reduced with SnCl2, followed by acylation with acryloyl chloride
(Scheme 4).
Scheme 3- Synthetic route for the preparation of compound 28
yii..
11D2 NO2
NI-12 MN
.. 6,to!n 11 4113 Nfri Et onell a,
er,thotia 2_.' j) .....1- 101-n-Ir 1)--iiii..- 6 W
NV
.5- Clair2"14
3a 11411
INT2 Mt
a) 4-(methanesulfonylamino) phenyl boronic acid pinacol ester, PdC12(PPh3)2,
Na2CO3, dioxane, 90 C,
overnight, 73%; b) Fe, NH4C1, Et0H/H20, 80 C, 2h, 88%; c) acryloyl chloride,
DIPEA, THF, -10 C to
RT, 5h, 10%.
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Scheme 4- Synthetic route for the preparation of compound 29
0
NO2 NH2
FIN)L-1-
.1 Br I_3110,..
a
Br a
111111j N
N
ea !NTS
29
a) SnC12, Et0Ac, 85 C, 2 h, 68%; b) acyl chloride, DIPEA, THF, -10 C to RT,
5h, 95%.
6-Bromo-4-hydroxyquinoline-3-carboxylate (1)
Diethyl 2-(ethoxymethylene) malonate (11.7 mL; 58.13 mmol) and 4-bromoaniline
(10 g;
58.13 mmol) were heated to 145 C. After 23 h, the solvent was evaporated
affording an off-
white solid. Ph20 (25 mL) was added and the reaction heated to 245 C. After
6h, no more
intermediate was detected by TLC (Et0Ac:Hexane 20:80). Upon cooling to r.t., a
precipitate
was formed, and hexane was added to induce more precipitation. The precipitate
was
filtered, washed with Et0Ac and dried in vacuum to afford the title compound
as an off-white
solid (6.9 g; 40% yield).
1H NMR (300 MHz, De-DMS0): 58.60 (s, 1H), 8.22 (d, J = 2.4 Hz, 1H), 7.82 (dd,
J = 8.7,
2.4 Hz, 1H), 7.58 (d, J = 8.7 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 1.27 (t, J =
7.1 Hz, 3H).
Ethyl 6-bronno-4-chloro-3-quinolinecarboxylate (2)
6-bromo-4-hydroxy-3-quinolinecarboxylate (13.4 g; 45.25 mmol) was suspended in
SOCl2
(130 mL; 1.792 mol) and the mixture heated to 80 C. After 5 h, a clear yellow
solution was
obtained. The solvent was evaporated and the solid co-evaporated with DCM (5
x) to
remove residual HCI. It was dried in vacuum to afford the title compound as a
light-yellow
solid (14.4 g; 100% yield).
1H NMR (300 MHz, CDC13): 6 9.37 (s, 1H), 8.71 (d, J = 2.0 Hz, 1H), 8.56 (d, J
= 9.0 Hz, 1H),
8.12 (dd, J = 9.0, 2.0 Hz, 1H), 4.53 (q, J = 7.1 Hz, 2H), 1.47 (t, J = 7.1 Hz,
3H).
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General Procedure A: Nucleophilic Aromatic Substitution
Ethyl 6-bromo-4-((4-methy1-3-nitrophenyl)amino)quinoline-3-carboxylate (3a)
Ethyl 6-bromo-4-chloro-3-quinolinecarboxylate 2 (800 mg; 2.543 mmol) and 4-
methy1-5-
nitroaniline (387 mg; 2.543 mmol) were mixed in dioxane (15 mL) and heated to
90 C. After
7 h, TLC analysis (50% Et0Ac/Hexane) no longer detected starting materials.
The yellow
suspension was cooled to r.t., diluted with H20 and NaOH (1 M) added until pH
= 8 was
reached. Et0Ac was added and the phases were separated. The aqueous phase was
further extracted with Et0Ac (2 x) and the combined organics were washed with
brine and
dried over MgSO4. After filtration, the solvent was evaporated to afford the
title compound
as a bright-yellow solid (980 mg; 90% yield).
1H NMR (300 MHz, CDC13): 6 10.38 (s, 1H), 9.29 (s, 1H), 7.90 (d, J = 9.4 Hz,
1H), 7.74 (dq, J
= 4.3, 2.2 Hz, 2H), 7.65(d, J =2.5 Hz, 1H), 7.25 (d, J = 8.3 Hz, 1H), 7.08
(dd, J = 8.3,
2.5 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 2.58 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H).
Ethyl 6-bromo-4-((3-methy1-5-nitrophenyl)annino)quinoline-3-carboxylate (3b)
Prepared using General Procedure A, reacting intermediate 2 with 3-methyl-5-
nitroaniline.
Compound 3b was isolated as a yellow solid (730 mg; 89% yield).
1H NMR (300 MHz, CDC1a): 6 10.35 (s, 1H), 9.30 (s, 1H), 7.92 (d, J = 8.8 Hz,
1H), 7.79-7.73
(m, 3H), 7.61 (s, 1H), 7.11 (s, 1H), 4.45 (q, J = 7.1 Hz, 2H), 2.39 (s, 3H),
1.45 (t, J = 7.1 Hz,
3H). 13C NMR (75.5 MHz, CDC1a): 6 167.9, 151.5, 150.2, 149.6, 149.0, 143.5,
141.2, 135.1,
132.1, 128.4, 127.5, 120.8, 119.7, 119.2, 113.2, 109.1, 61.9, 14.4. HRMS
(ES1): m/z [M +
H]+ calc. for C19H-17BrN304: 430.0397; found: 430.0401.
Ethyl 6-bromo-4-((2-methy1-5-nitrophenyl)amino)quinoline-3-carboxylate (3c)
Prepared using General Procedure A, reacting intermediate 2 with 2-methyl-5-
nitroaniline.
The reaction required heating at 90 C during 23 h followed by 4 h at 110 C.
Compound 3c
was isolated as a yellow solid (730 mg, 89% yield).
1H NMR (300 MHz, De-DMS0): 59.29 (s, 1H), 8.87 (s, 1H), 8.52 ¨8.47 (m, 1H),
7.98 ¨ 7.90
(m, 3H), 7.66 (d, J = 2.4 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 3.85 (q, J = 7.1
Hz, 2H), 2.44 (s,
3H), 1.04(t, J = 7.1 Hz, 3H). HRMS (ES1): nn/z [M + H]+ calc. for
C19H17BrN304: 430.0397;
found: 430.0400. 13C NMR could not be acquired because the compound is not
sufficiently
soluble in D6-DMSO, D6-acetone, Da-acetonitrile or D4-methanol.
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Ethyl 6-bromo-4-((3-nitrophenyl)amino)quinoline-3-carboxylate (3d)
Prepared using General Procedure A, reacting intermediate 2 with 3-
nitroaniline. The
reaction time was 8 h. Compound 3d was isolated as an orange solid (640 mg:
81% yield).
1H NMR (300 MHz, CDCI3): 510.41 (s, 1H), 9.32 (s, 1H), 8.01 -7.91 (m, 2H),
7.86 (t, J =2.2
Hz, 1H), 7.79 - 7.70 (m, 2H), 7.51 - 7.42 (m, 1H), 7.28 - 7.23 (m, 1H), 4.47
(q, J = 7.1 Hz,
2H), 1.47 (t, J = 7.2 Hz, 3H). 13C NMR (75.5 MHz, CDCI3): 6 167.9, 151.5,
150.1, 149.5,
149.0, 143.7, 135.1, 132.1, 130.0, 128.3, 126.6, 120.8, 119.4, 119.0, 115.9,
109.4, 62.0,
14.3. HRMS (ESI): in& [M + H]+ calc. for Ci8H1513rN304: 416.0240; found:
416.0245.
Ethyl 6-bromo-4-((4-methoxy-3-nitrophenyl)amino)quinoline-3-carboxylate (3e)
Prepared using the General Procedure A, reacting intermediate 2 with 4-
nnethoxy-3-
nitroaniline. Compound 3e was isolated as an orange solid (780 mg; 92% yield).
1H NMR (300 MHz, CDCI3): 6 10.43 (s, 1H), 9.26 (s, 1H), 7.88 (d, J = 9.3 Hz,
1H), 7.73 -
7.70 (m, 2H), 7.59 (d, J = 2.7 Hz, 1H), 7.22 - 7.18 (m, 1H), 7.04 (d, J = 9.0
Hz, 1H), 4.45 (q,
J = 7.1 Hz, 2H), 3.98 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H). 13C NMR (75.5 MHz,
CDCI3): 5168.2,
151.7, 151.1, 150.4, 149.7, 139.7, 135.2, 134.9, 132.1, 128.4, 128.1, 120.4,
119.7, 118.9,
114.6, 107.8, 61.8, 57.1, 14.4. HRMS (ESI): ink [M + F1]+ calc. for C19H-
1713rN305: 446.0346;
found: 446.0347.
Ethyl 7-bromo-4-(4-methyl-3-nitrophenylamino)quinoline-3-carboxylate (3S)
Prepared using General Procedure A, reacting intermediate 2' with 4-methyl-5-
nitroaniline.
The reaction was completed after 3 h. Compound 31 was isolated as a yellow
solid (1.85 g;
85% yield).
1H NMR (300 MHz, CDCI3): 6 10.54 (s, 1H), 9.31 (s, 1H), 8.26 (d, J = 2.0 Hz,
1H), 7.69
(d, J = 2.4 Hz, 1H), 7.48 (d, J= 9.1 Hz, 1H), 7.34 (dd, J= 9.1, 2.0 Hz, 1H),
7.28 (m, 1H),
7.13 (dd, J= 8.3, 2.4 Hz, 1H), 4.48 (q, J = 7.1 Hz, 2H), 2.60 (s, 3H), 1.49
(t, J= 7.1 Hz, 3H).
13C NMR (75.5 MHz, CDCI3): 6 168.0, 152.1, 151.8, 151.3, 149.6, 141.5, 133.7,
132.4,
129.6, 128.8, 127.3, 126.4, 126.2, 117.9, 117.7, 108.0, 61.9,20.1, 14.4. HRMS
(ESI): rink
[M + H]+ calc. for C19F117BrN304: 430.0397; found: 430.0393.
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Ethyl 7-bromo-4-(3-methyl-5-nitrophenylarnino)quinoline-3-carboxylate (313')
Prepared using General Procedure A, reacting intermediate 2' with 3-methyl-5-
nitroaniline.
Reaction time was 17 h. The crude was washed with cold Et0Ac to remove
unreacted
aniline. Compound 3b' was isolated as an orange solid (740 mg: 58% yield).
1H NMR (300 MHz, CDCI3) 610.41 (s, 1H), 9.31 (s, 1H), 8.35 - 8.08 (m, 1H),
7.79(s, 1H),
7.63(s, 1H), 7.46 (d, J = 9.1 Hz, 1H), 7.37 - 7.28 (m, 1H), 7.10 (s, 1H), 4.46
(q, J = 7.0 Hz,
2H), 2.39(s, 3H), 1.46 (t, J = 7.1 Hz, 3H). HRMS (ESI): m/z [M + H]+ calc. for
C1eH17BrN304:
430.0397; found: 430.0390.
Ethyl 7-bromo-4-(3-nitrophenylamino)quinoline-3-carboxylate (3d')
Prepared using General Procedure A, reacting intermediate 2' with 3-
nitroaniline.
Reaction time was 17 h. Compound 3d' was isolated as an orange solid (2.529:
95% yield).
1H NMR (300 MHz, CDCI3): 6 10.41 (s, 1H), 9.31 (s, 1H), 8.24 (brs, 1H), 7.79
(s, 1H), 7.63
(s, 1H), 7.46 (d, J = 9.1 Hz, 1H), 7.34 -7.28 (m, 1H), 7.10 (s, 1H), 4.46 (q,
J = 7.1 Hz, 2H),
2.39 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H). it NMR (75.5 MHz, CDCI3): 6 168.0,
152.3, 151.7,
151.2, 149.1, 144.0, 132.9, 130.3, 129.0, 127.2, 126.9, 126.3, 119.0, 118.2,
116.0, 108.8,
62.0, 14.4. HRMS (ES!): m/z [M + H]+ calc. for C181-115BrN304: 416.0240;
found: 416.0244.
General Procedure a Ester to Alcohol Reduction
(6-Bromo-44(4-methyl-3-nitrophenyl)annino)quinolin-3-yOnnethanol (4a)
Sodium borohydride (5.44 g; 143.99 nnmol; 15 equiv.) was added portionwise to
a stirred
solution of 3a (4.13 g; 9.599 mmol) in Et0H (35 mL) at 0 C. After 15 h, TLC
analysis (50%
Et0Ac/Hexane) showed disappearance of starting material. The orange solution
was cooled
in an ice-bath and quenched with NH4C1aq. The mixture was partitioned between
H20 and
Et0Ac. The phases were separated and the aqueous phase further extracted with
Et0Ac
(2 x). The combined organics were washed with brine, dried over MgSO4 and
evaporated to
dryness to afford the title compound as an orange solid (3.73 g; 100% yield).
(6-Bromo-4-((3-methyl-5-nitrophenyl)amino)quinolin-3-yl)methanol (4b)
Prepared using the procedure described for 4a. Compound 4b was obtained as an
orange
solid (630 mg; 100%).
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1H NMR (300 MHz, 0e-DMS0): 69.07 (s, 1H), 9.00 (s, 1H), 8.24 (s, 1H), 8.00 (d,
J = 8.9 Hz,
1H), 7.87 (d, J = 8_9 Hz, 1H), 7.48 (s, 1H), 7.28 (s, 1H), 6.85 (s, 1H), 5.47
(t, J = 5.4 Hz, 1H),
4.48, (d, J = 5.4 Hz, 2H), 2.31 (s, 3H). 13C NMR (75.5 MHz, d6-DMS0): 6 152.2,
148.6,
146.9, 146.1, 140.6, 139.9, 132.3, 131.7, 128.7, 125.5, 125.3, 121.3, 119.6,
114.3, 106.5,
58.2, 20.9. HRMS (ESI): ink [M + H]+ calc. for Ci7HisBrN303: 388.0291; found:
388.0293.
(6-Bromo-44(2-methy1-5-nitrophenyl)annino)quinolin-3-yOnnethanol (4c)
Prepared using the procedure described for 4a. Compound 4c was obtained as an
orange
solid (250 mg; 68% yield).
1H NMR (300 MHz, De-DMS0): 59.03 (s, 1H), 8.20 (d, J = 2.1 Hz, 1H), 8.09 (s,
1H), 8.00 (d,
J = 8.9 Hz, 1H), 7.87 (dd, J = 8.9, 2.2 Hz, 1H), 7.68 (dd, J = 8.3, 2.3 Hz,
1H), 7.50 (dd, J =
8.3, 0.9 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H), 5.47 (t, J = 5.5 Hz, 1H), 4.39,
(d, J = 5.5 Hz, 2H),
2.50 (s, 3H). 13C NMR (75.5 MHz, d6-DMS0): 6 152.0, 147.0, 146.5, 144.4,
141.5, 134.5,
132.4, 131.7, 131.5, 128.2, 125.7, 125.2,119.5, 115.0, 109.2, 58.3, 18.4. HRMS
(ES!): mtz
[M + H]+ calc. for C17Hi5BrN303: 388.0291; found: 388.0293.
(6-Bromo-4-((3-nitrophenyl)amino)quinolin-3-yl)methanol (4d)
Prepared using the procedure described for 4a. Purification was carried out by
column
chromatography over silica-gel (eluent: MeOH:DCM 0:100 to 5:95) to give
compound 4d as
a yellow solid (250 mg; 42% yield).
1H NMR (300 MHz, De-DMS0): 59.06 (d, J = 5.4 Hz, 2H), 8.21 (d, J = 2.0 Hz,
1H), 7.99 (d, J
= 8.9 Hz, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.64(d, J = 8.0 Hz, 1H), 7.49- 7.42
(m, 2H), 7.00(d,
J = 8.0 Hz, 1H), 5.47 (t, J = 5.1 Hz, 1H), 4.48 (d, J = 5.1 Hz, 2H). 13C NMR
(75.5 MHz, De-
DMS0): 6 152.3, 148.6, 147.0, 146.3, 139.9, 132.4, 131.7, 130.5, 128.8, 125.5,
125.3,
120.9, 119.7, 113.7, 109.1, 58.2. HRMS (ESI): rniz [M + H]+ calc. for
C1eH13BrN303:
374.0135; found: 374.0134.
(6-Bromo-4-((4-nnethoxy-3-nitrophenyl)amino)quinolin-3-yl)methanol (4e)
Prepared using the procedure described for 4a. Compound 4e was obtained as an
orange
solid (600 mg; 93% yield)
1H NMR (300 MHz, 0e-DMS0): 68.97 (s, 1H), 8.67 (s, 1H), 8.26 (d, J = 2.1 Hz,
1H), 7.94 (d,
J = 8.9 Hz, 1H), 7.83 (dd, J = 8.9, 2.1 Hz, 1H), 7.32 - 7.18 (m, 2H), 7.00
(dd, J = 9.0, 2.8 Hz,
1H), 5.40 (t, J = 5.4 Hz, 1H), 4.42 (d, J = 5.4 Hz, 2H), 3.85 (s, 3H). 13C NMR
(75.5 MHz, d6-
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DMS0): 6 152.2, 147.0, 146.0, 141.1, 139.3, 138.2, 132.1, 131.7, 131.7, 126.5,
125.5,
124.6, 122.3, 119.2, 115.5, 112.3, 58.5, 56.9. HRMS (ESI): rrilz [M + H]+
calc. for
C17H-15BrN304: 404.0240; found: 404.0244.
(7-Bromo-4-(4-methyl-3-nitrophenylannino)quinolin-3-yOmethanol (4a')
Prepared using the procedure described for 4a. Compound 4a2 was isolated as an
orange
solid (770 mg; 100% yield).
1H NMR (300 MHz, De-DMS0): 69.00 (s, 1H), 8.83 (s, 1H), 8.23 (d, J= 2.0 Hz,
1H), 7.88 (d,
J= 9.0 Hz, 1H), 7.68 (dd, J= 9.0, 2.1 Hz, 1H), 7.32 - 7.21 (m, 2H), 6.86 (dd,
J= 8.4, 2.5 Hz,
1H), 5.43 (t, 1= 5.4 Hz, 1H), 4.51 (d, J= 5.4 Hz, 2H), 2.39 (s, 3H). 13C NMR
(75.5 MHz,
De-DMS0): 6152.9, 149.2, 149.1, 144.2, 141.7, 133.4, 131.3, 129.2, 127.8,
125.8, 123.0,
122.5, 122.4, 120.2, 110.6, 58.3, 18.9. HRMS (ES!): tniz [M + H]+ calc. for
C17F115BrN303:
388.0291; found: 388.0293.
(7-Bromo-4-(3-methyl-5-nitrophenylamino)quinolin-3-yOmethanol (4b')
Prepared using the procedure described for 4a. Compound 4b' was isolated as a
yellow
solid (620 mg; 96% yield).
1H NMR (300 MHz, De-DMS0): 69.03 (s, 1H), 8.94 (s, 1H), 8.24 (d, J= 2.0 Hz,
1H), 7.88 (d,
J= 9.0 Hz, 1H), 7.68 (dd, J= 9.0, 2.0 Hz, 1H), 7.48 - 7.45 (m, 1H), 7.25 (m,
1H), 6.84 (brs,
1H), 5.47 (t, J= 5.4 Hz, 1H), 4.52 (d, J= 5.4 Hz, 2H), 2.29 (s, 3H). HRMS
(ESI): raiz
[M + H]+ cab. for C17H1eBrN303: 388.0291; found: 388.0294.
(7-Bromo-4-(3-nitrophenylamino)quinolin-3-yl)methanol (4d')
Prepared using the procedure described for 4a. Compound 4d' was isolated as an
orange
solid (810 mg; 91% yield).
1H NMR (300 MHz, De-DMS0): 6 9.03(s, 2H), 8.25 (d, J = 2.0 Hz, 1H), 7.87 (d, J
= 9.0 Hz,
1H), 7.69 (dd, J = 9.0, 2.1 Hz, 1H), 7.62 (ddd, J = 8.1, 2.2, 0.9 Hz, 1H),
7.48- 7.44 (m, 1H),
7.41 (d, J= 8.1 Hz, 1H), 5.82 (s, 1H), 5.51 (t, J= 5.5 Hz, 1H), 4.52 (d, J=
5.5 Hz, 2H).
HRMS (ESI): ralz [M + Hp- calc. for CieHi3BrN303: 374.0135; found: 374.0134.
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General Procedure C: Alcohol Oxidation to Aldehyde
6-Bromo-4-((4-methyl-3-nitrophenyl)amino)quinoline-3-carbaldehyde (5a)
The alcohol 4a (2.34 g; 6.029 mmol) was suspended in DCM (150 mL) and the
mixture
cooled to 0 C. DMP (3.83 g; 9.041 mmol; 1.5 equiv.) was added portionwise and
the
reaction warmed to r.t. After 2 h, TLC analysis (5% Me0H in DCM) showed that
the reaction
was completed. The solution was cooled to 0 C, and NaOH (1 M) was slowly
added. The
mixture was stirred for 15 min at rt.. H20 was added and the phases were
separated. The
aqueous phase was further extracted with DCM (3 x). The combined organics were
washed
with brine, dried over MgSat and taken to dryness to afford the title compound
as a yellow
solid (1.759; 75% yield).
6-Bromo-4-((3-methyl-5-nitrophenyl)amino)quinoline-3-carbaldehyde (5b)
Prepared using the procedure described for 5a. Compound 5b was obtained as an
orange
solid (420 mg; 72% yield).
1H NMR (300 MHz, CDCI3): 6 11.26 (s, 1H), 10.10(s, 1H), 8.90 (s, 1H), 7.97-
7.88 (m, 2H),
7.83-7.74 (m, 2H), 7.66 (d, J= 2.1 Hz, 1H), 7.34-7.27(m, 1H), 2.46(s, 3H). 13C
NMR (75.5
MHz, CDCI3): 6 193.4, 155.0, 150.2, 149.7, 149.0, 141.6 (2), 136.0, 132.2,
129.5, 128.7,
121.4, 119.2, 118.9, 115.3, 113.7,21.5. HRMS (ESI): mit [M + H]+ calc. for
C17F113BrN303: 386.0135; found: 386.0142.
6-Bromo-4-((2-methyl-5-nitrophenyl)amino)quinoline-3-carbaldehyde (Sc)
Prepared using the procedure described for 5a. Compound Sc was obtained as a
yellow
solid (165 mg; 72% yield).
1H NMR (300 MHz, De-DMS0): 6 10.18 (s, 1H), 9.94 (s, 1H), 8.97 (s, 1H), 8.07-
8.02 (m, 2H),
7.93-7.88 (m, 3H), 7.67 (m, 1H), 2.42 (s, 3H). 1H NMR (300 MHz, CDCI3): 6
11.28 (s, 1H),
10.11 (s, 1H), 8.90 (s, 1H), 8.11 (dd, J = 8.4,2.1 Hz, 1H), 7.91 (d, J = 8.9
Hz, 2H), 7.75 (dd,
J = 9.0, 2.1 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 1.9 Hz, 1H), 2.48
(s, 3H). HRMS
(ES!): trt/z [M + H]+ calc. for C17H13BrN303: 386.0135; found: 386.0136.
6-Bromo-4-((3-nitrophenyl)amino)quinoline-3-carbaldehyde (5d)
Prepared using the procedure described for 5a. Compound 5d was obtained as an
orange
solid (200 mg; 65% yield).
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1H NMR (300 MHz, CDCI3): 511.27 (s, 1H), 10.12 (s, 1H), 8.93 (s, 1H), 8.14
(dd, J = 8.1,2.2
Hz, 1H), 8.03 (t, J =2.2 Hz, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.78 (dd, J =
9.0,2.1 Hz, 1H), 7.64
(d, J = 2.1 Hz, 1H), 7.60-7.53 (m, 1H), 7.45 (d, J = 8.0 Hz, 1H). 1313 NMR
(75.5 MHz,
CDCI3): 6 193.4, 154.9 150.1, 149.7, 149.1, 142.0, 136.0, 132.2, 130.6, 128.8,
128.6, 120.8,
119.2, 119.1, 118.0, 113.9. HRMS (ESI): /rift [M + H]+ calc. for C1eHliBrN303:
371.9978;
found: 371.9986.
6-Bromo-44(4-methoxy-3-nitrophenyparnino)quinoline-3-carbaldehyde (5e)
Prepared using the procedure described for 5a. Compound 5e was obtained as an
orange
solid (370 mg; 70% yield). 1H NMR (300 MHz, CDCI3): 611.33 (s, 1H), 10.07 (s,
1H), 8.84
(s, 1H), 7.88 (dd, J = 8.9, 2.1 Hz, 1H), 7.77-7.70 (m, 2H), 7.63 (d, J = 2.1
Hz, 1H), 7.38 (dd, J
= 8.8, 2.7 Hz, 1H), 7.14 (d, J = 8.9 Hz, 1H), 4.02 (s, 3H). 13C NMR (75.5 MHz,
CDCI3): 6
193.3, 155.2, 151.6, 151.0, 149.7, 139.7, 135.8, 133.0, 132.1, 129.9, 128.7,
121.5, 119.0,
118.8, 114.8, 112.9, 57.1. HRMS (ESI): m/z [M + H]+ calc. for C17H1313rN304:
402.0084;
found: 402.0088.
7-Bromo-4-(4-methy1-3-nitrophenylarnino)quinoline-3-carbaldehyde (5a')
Prepared using the procedure described for 5a. Compound 5a' was obtained as a
brown
solid (220 mg; 85% yield).
1H NMR (300 MHz, CDCI3): 6 11.29 (s, 1H), 10.06(s, 1H), 8.85 (s, 1H), 8.18 (d,
J = 2.0 Hz,
1H), 7.80 (d, J= 2_4 Hz, 1H), 7.40 -7.32 (m, 2H), 7.30¨ 7.24 (m, 2H), 2.62 (s,
3H).
13C NMR (75.5 MHz, CDCI3): 6 193.3, 155.9, 151.8, 151.6, 149.7, 139.8, 134.1,
132.9,
131.4, 128.8, 127.9, 127.5, 127.4, 119.5, 116.6, 113.3, 20.2. HRMS (ESI): miz
[M + H]+
calc. for Ci7H1313rN303: 386.0135; found: 386.0135.
7-Brorno-4-(3-methyl-5-nitrophenylarnino)quinoline-3-carbaldehyde (5b')
Prepared using the procedure described for 5a. Compound 5b' was obtained as an
orange
solid (480 mg; 82% yield).
1H NMR (300 MHz, De-DMS0): 610.37 (s, 1H), 10.11 (s, 1H), 9.03 (s, 1H), 8.22
(d, J= 2.1
Hz, 1H), 7.92 (d, J= 9.1 Hz, 1H), 7.78 ¨ 7.75 (m, 2H), 7.67 (dd, J= 9.0,2.1
Hz, 1H), 7.38 (s,
1H), 2.34 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for C17H1313rN303: 386.0135;
found:
386.0127.
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Prepared using the procedure described for 5a. Compound 5dP was obtained as a
brown
solid (790 mg; 99% yield).
1H NMR (300 MHz, CDCI3): 511.32 (s, 1H), 10.10(s, 1H), 8.92 (s, 1H), 8.23 (d,
J= 2.0 Hz,
1H), 8.17 ¨ 8.05 (m, 1H), 8.03 ¨ 8.01 (m, 1H), 7.55(t, J= 8.1 Hz, 1H), 7.50 ¨
7.41 (m, 1H),
7.38 (d, J= 9.1 Hz, 1H), 7.29 (dd, J = 9.1, 2.0 Hz, 1H). HRMS (ESI): mit [M +
H]+ calc. for
C16H11BrN303: 371.9978; found: 371.9975.
General Procedure a Homer- Wadsworth-Emmons (HWE) cyclization
9-Bromo-1-(4-methyl-3-nitrophenyl)benzo[h][1,6]naphthyridin-2(1H)-one (6a)
The aldehyde 5a (1.74 g; 4.505 mmol), triethylphosphonoacetate (894 pL; 4.505
mmol) and
K2CO3 (1.87 g; 13.516 mmol; 3 equiv.) were mixed in dry Et0H (30 mL) in a
sealed tube
under Argon. The mixture was heated to 100 C overnight After 16 h, the
reaction was
cooled to r.t. and the solvent evaporated. The crude was partitioned between
H20 and
Et0Ac. The aqueous phase was further extracted with Et0Ac (3 x) and the
combined
organics washed with brine, dried over MgSO4 and taken to dryness to afford
the title
compound as a dark brown solid (1.62 g; 88% yield).
9-Bromo-1-(3-methyl-5-nitrophenyl)benzo[h][1,6]naphthyridin-2(1H)-one (6b)
Prepared using the procedure described for fia. Compound 6b was obtained as a
brown
solid (320 mg; 74% yield).
1H NMR (300 MHz, CDCI3): 58.97 (s, 1H), 8.33 (s, 1H), 8.09 ¨ 7.88 (m, 3H),
7.67 (dd, J =
8.9, 2.1 Hz, 1H), 7.53 (s, 1H), 6.95 (d, J = 9.5 Hz, 1H), 6.80 (d, J = 2.1 Hz,
1H), 2.58 (s, 3H)_
13C NMR (75.5 MHz, CDCI3): 6 162.9, 150.8, 149.4, 148.2, 142.7, 141.2, 140.6,
139.7,
135.6, 133.5, 132.6, 127.5, 125.1, 122.9, 121.4, 120.3, 118.44, 113.8, 21.5.
HRMS (ESI):
tri/z [M + H]+ calc. for C19H1313rN303: 410.0135; found: 410.0132.
9-Bromo-1-(2-methyl-5-nitrophenyl)benzo[h][1,6]naphthyridin-2(1H)-one (6c)
Prepared using the procedure described for 6a. Compound fic was obtained as a
dark
brown solid (165 mg; 68% yield).
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1H NMR (300 MHz, CDC13): 6 9.02 (s, 1H), 8.45 (dd, J= 8.5, 2.4 Hz, 1H), 8.11
¨7.97 (m,
3H), 7.74 ¨ 7.68 (m, 2H), 7.00(d, J= 9.5 Hz, 1H), 6.80(d, J = 2.0 Hz, 1H),
2.21 (s, 3H).
HRMS (ES!): m/z [M + H]+ calc. for C19F113BrN303: 410.0135; found: 410.0133.
9-Bromo-1-(3-nitrophenyl)benzo[h][1,6]naphthyridin-2(1H)-one (6d)
Prepared using the procedure described for 6a. Compound 6d was obtained as a
dark
brown solid (150 mg; 71% yield).
1H NMR (300 MHz, CDC13): 6 9.01 (s, 1H), 8.53 (dd, J= 8.3, 2.1 Hz, 1H), 8.21
(t, J= 2.1 Hz,
1H), 8.00 (dd, J= 10_4, 9_2 Hz, 2H), 7.87 (t, J= 8.1 Hz, 1H), 7.80 ¨ 7.64 (m,
2H), 6.97 (d, J=
9.5 Hz, 1H), 6.80 (d, J= 2.1 Hz, 1H). HRMS (ES!): m/z [M + H]+ calc. for
C1eHliBrN303:
395.9978; found: 395.9976.
9-Bromo-1-(4-methoxy-3-nitrophenyl)benzo[h][1,6]naphthyridin-2(1H)-one (6e)
Prepared using the procedure described for 6a. Compound 6e was obtained as a
dark
brown solid (255 mg; 65% yield)
1H NMR (300 MHz, De-DMS0): 6 9.17 (s, 1H), 8.32(d, J= 9.5 Hz, 1H), 8.17(d, J =
2.5 Hz,
1H), 7.98(d, i= 8_9 Hz, 1H), 7.82 (m, 2H), 7.69(d, J = 9.0 Hz, 1H), 6.96(d, J=
9.4 Hz, 1H),
6.83 (d, J = 2.0 Hz, 1H), 4.07 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for
C19H13BrN304:
426.0084; found: 426.0090.
8-Bromo-1-(4-methyl-3-nitrophenyObenzo[h][1,6]naphthyridin-2(1H)-one (6a')
Prepared using the procedure described for 6a. Compound 6a7 was obtained as a
brown
solid (220 mg; 96% yield).
1H NMR (300 MHz, De-DMS0): 6 9.17 (s, 1H), 8.31 (d, J = 9.5 Hz, 1H), 8.23(m,
2H), 7.81 ¨
7.68 (m, 2H), 7.39 (dd, J= 9.4, 2_3 Hz, 1H), 6.94 (d, J= 9.5 Hz, 1H), 6.73 (d,
J= 9.4 Hz,
1H), 2.68 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for C19H13BrN303: 410.0135;
found:
410.0134.
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(6b')
Prepared using the procedure described for 6a. Compound 6b' was obtained as a
brown
solid (345 mg; 81% yield).
1H NMR (300 MHz, De-DMS0): 59.18 (s, 1H), 8.36 - 8.27 (m, 3H), 8.24 (d, J= 2.3
Hz, 1H),
7.76 (brs, 1H), 7.36 (dd, J = 9.5, 2.3 Hz, 1H), 6.95 (d, J = 9.4 Hz, 1H), 6.69
(d, J = 9.4 Hz,
1H), 2.48 (s, 3H). HRMS (ESI): rn/z. [M + H]+ calc. for C1eH13BrN303:
410.0135; found:
410.0132.
8-Bronno-1-(3-nitrophenyl)benzo[h][1,6]naphthyr1d1n-2(1H)-one (6d')
Prepared using the procedure described for 6a. Compound 64:12 was obtained as
a brown
solid (500 mg; 60% yield).
1H NMR (300 MHz, De-DMS0): 59.18 (s, 1H), 8.49 (m, 2H), 8.32 (d, Jr 9.5 Hz,
1H), 8.24
(d, J = 2.3 Hz, 1H), 7.91 -7.89 (m, 2H), 7.34 (dd, J = 9.4, 2.3 Hz, 1H), 6.95
(d, J = 9.5 Hz,
1H), 6.65 (d, 1= 9.4 Hz, 1H). HRMS (ESI): rniz [M + H]+ calc. for
CieHliBrN303: 395.9978;
found: 395.9975.
General Procedure E: Suzuki Coupling
N-(4-(1-(4-Methyl-3-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yOphenyl)methanesulfonamide (7a)
The bromo-quinoline 6a (280 mg; 0.683 mmol), 4-(methanesulfonylamino) phenyl
boronic
add pinacol ester (243 mg; 0.819 mmol; 1.2 equiv.), PdC12(PPh3)2 (48 mg; 0.068
mmol;
0.1 equiv.) and Na2CO3 (1.025 ml; 2 M, 2.049 mmol; 3 equiv.) were mixed in
dioxane (3 mL)
under Argon. The mixture was heated to 90 C overnight After 16 h, TLC analysis
(MeOH:DCM 5:95) showed that the reaction was completed. The mixture was cooled
to RT
and filtered through a Celite pad. The pad was further washed with Et0H and
Me0H/DCM
(10%) until no more product was detected by TLC. The solvent was evaporated
and the
crude applied in a silica column with a gradient up to 2% Me0H in DCM. The
desired
fractions were collected and evaporated to dryness to afford the title
compound as a yellow
solid (455 mg; 69%).
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N-(4-(1-(3-Methy1-5-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)methanesulfonamide (7b)
Prepared using the procedure described for 7a. Compound 7b was obtained as a
dark
yellow solid (160 mg: 64% yield).
1H NMR (300 MHz, De-DMS0): 69.92 (brs, 1H), 9.15 (s, 1H), 8.45¨ 8.28 (m, 3H),
8.10 (d, J
= 8.6 Hz, 1H), 7.98 (dd, J= 8.7, 1.9 Hz, 1H), 7.89 (s, 1H), 7.20 (d, J = 8.6
Hz, 2H), 7.15 ¨
7.02 (m, 3H), 6.95 (d, J = 9.5 Hz, 1H), 3.03 (s, 3H), 2.5 (s, 3H). 1H NMR (300
MHz, CDCI3):
69.01 (s, 1H), 8.29 (s, 1H), 8.19(d, J= 8.7 Hz, 1H), 8.09 (m, 1H), 8.03 (d, J=
9.5 Hz, 1H),
7.86 (dd, J= 8.7, 1.9 Hz, 1H), 7.59 (s, 1H), 7.19(d, J= 8.6 Hz, 2H), 7.12 (d,
J= 1.9 Hz, 1H),
7.04 (d, J= 8.6 Hz, 2H), 6.96 (d, J= 9.4 Hz, 1H), 6.57 (s, 1H), 3.07 (s, 4H),
2.56 (s, 3H).
HRMS (ES!): raiz [M + H]+ calc. for C201-121N405S: 501.1227; found: 501.1224.
N-(4-(1-(2-Methy1-5-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)methanesulfonamide (7c)
Prepared using the procedure described for 7a. Compound 7c was obtained as an
orange
solid (145 mg; 77% yield).
1H NMR (300 MHz, De-acetone): 6 9.14 (s, 1H), 8.74 (brs, 1H), 8.53 ¨ 8.44 (m,
2H), 8.34 (d,
J= 9.5 Hz, 1H), 8.15(d, J= 8.6 Hz, 1H), 8.03 ¨ 7.91 (m, 2H), 7.38 ¨ 7.31 (m,
2H), 7.23 ¨
7.13 (m, 3H), 6.95 (d, 1= 9.5 Hz, 1H), 3.04 (s, 3H), 2.23 (s, 3H). HRMS (ESI):
tn/z [M + H]+
calc. for C2eH2iN405S: 501.1227; found: 501.1223.
N-(4-(1-(3-NitrophenyI)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)methanesulfonamide (7d)
Prepared using the procedure described for la. Compound 7d was obtained as a
yellow
solid (85 mg; 51% yield).
1H NMR (300 MHz, De-DMS0): 6 9.93(s, 1H), 9.15 (s, 1H), 8.62 (s, 1H), 8.58 ¨
8.47 (m,
1H), 8.34 (d, J= 9.5 Hz, 1H), 8.10 (d, J= 8.7 Hz, 1H), 7.97 (m, 3H), 7.18 (d,
J= 8.2 Hz, 2H),
7.10 ¨ 6.99 (m, 3H), 6.96(d, J= 9.4 Hz, 1H), 3.03 (s, 3H). HRMS (ESI): mit [M
+ H]+ calc.
for C2sHieN405S: 487.1071; found: 487.1071.
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N-(4-(1-(4-Methoxy-3-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyOmethanesulfonamide (7e)
Prepared using the procedure described for 7a. Compound 7e was obtained as a
dark
yellow solid (120 mg; 42% yield).
1H NMR (300 MHz, De-acetone): 69.06 (s, 1H), 8.83- 8.74 (brs, 1H), 8.24 (d, J=
9.5 Hz,
1H), 8.15- 8.09 (nn, 2H), 7.99 (dd, J = 8.7, 1.9 Hz, 1H), 7.82 (dd, J= 8.9,
2.6 Hz, 1H), 7.64
(d, J = 9.0 Hz, 1H), 7.40 (d, J= 8.6 Hz, 2H), 7.30(d, J= 2.2 Hz, 1H), 7.27 (d,
J= 2.1 Hz,
1H), 7.21 (dd, J = 1.9, 0.6 Hz, 1H), 6.88 (d, J= 9.5 Hz, 1H), 4.14 (s, 3H),
3.06 (d, J = 0.9 Hz,
3H). HRMS (ESI): m/z [M + H]+ calc. for C26H2iN40eS: 517.1176; found:
517.1176.
N-(4-(1-(4-Methyl-3-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8-
yl)phenyl)methanesulfonamide (7a')
Prepared using the procedure described for la. Compound 7a' was obtained as an
orange
solid (76 mg; 21%).
1H NMR (300 MHz, De-DMS0): 69.97 (s, 1H), 9.16 (s, 1H), 8.33 - 8.30 (m, 2H),
8.26 (s,
1H), 7.84- 7.77 (m, 4H), 7.57 (dd, J = 9.3, 2.2 Hz, 1H), 7.30 (d, J = 8.7 Hz,
2H), 6.92 - 6.86
(m, 2H), 3.04 (s, 3H), 2.69 (s, 3H). HRMS (ESI): raiz [M + H]+ calc. for
C26H2111405S:
501.1227; found: 501.1227.
N-(4-(1-(3-Methyl-5-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8-
yOphenyOmethanesulfonannide (7b')
Prepared using the procedure described for 7a. Compound 7b' was obtained as an
orange
solid (400 mg; 95%).
HRMS (ESI): rniz [M + H]+ calc. for C26F121 N405S: 501.1227; found: 501.1232.
N-(4-(1-(3-Nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8-
yOphenyl)methanesulfonamide (7d')
Prepared using the procedure described for 7a. Compound 7c1' was obtained as
an orange
solid (90 mg; 31%).
1H NMR (300 MHz, De-DMS0): 69.92 (brs, 1H), 9.16 (s, 1H), 8.52 - 8.49 (m, 2H),
8.34 -
8.29 (m, 2H), 7.95 - 7.93 (m, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.52 (dd, J =
9.3, 2.2 Hz, 1H),
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7.29 (d, 1= 8.8 Hz, 2H), 6.91 (d, J= 9.4 Hz, 1H), 6.76 (d, J= 9.3 Hz, 1H),
3.03 (s, 3H).
HRMS (ESI): miz [M + F1]+ calc. for C2sHi9N405S: 487.1071; found: 487.1069.
Ethyl 4-(4-methy1-3-nitrophenylamino)-6-(4-(methylsulfonamido)phenyl)quinoline-
3-
carboxylate (Intermediate 1 - INT1)
Prepared using the procedure described for 7a, from intermediate 3a. Compound
INT1 was
obtained as a yellow solid (265 mg; 73%).
1H NMR (300 MHz, CDC13): 6 10.52 (s, 1H), 9.29 (s, 1H), 8.10 (d, 1= 8.7 Hz,
1H), 7.87 (dd, J
= 8.7, 2.0 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.67 (d, J = 2.5 Hz, 1H), 7.59 ¨
7.41 (m, 2H),
7.20- 7.16(m, 5H), 4.46 (q, J= 7.1 Hz, 2H), 3.03 (s, 3H), 2.58 (s, 3H), 1.47
(t, J= 7.1 Hz,
3H). HRMS (ESI): ink [M + H]+ calc. for C26H25N406S: 521.1489; found:
521.1486.
N-(4-(1-(4-Methy1-3-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)butane-1-sulfonamide (7f ¨ precursor of 14)
Prepared using General Procedure E, reacting intermediate 6a with
4-(butylsulfonamido)phenylboronic acid. Compound if was isolated as a yellow
solid
(200 mg; 53% yield)
1H NMR (300 MHz, De-DMS0): 69.98 (s, 1H), 9.13 (s, 1H), 8.44 ¨ 8.26 (m, 2H),
8.09 (d, J = 8.7 Hz, 1H), 7.98 (d, J= 8.6 Hz, 1H), 7.82-7.74 (m, 2H), 7.24 (d,
J= 8.2 Hz, 2H),
7.09 (d, J = 8.2 Hz, 2H), 6.95 (d, J= 8.2 Hz, 2H), 3.12 (t, J = 7.9 Hz, 3H),
2.65 (s, 3H), 1.65
(m, 2H), 1.36 (m, 2H), 0.84 (t, J = 72 Hz, 3H). HRMS (ES!): Ink [M + H]+ calc.
for
C29H271\14055: 543.1697; found: 543.1694.
N-(3-(1-(4-Methy1-3-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)methanesulfonamide (7g ¨ precursor of 15)
Prepared using General Procedure E, reacting intermediate 6a with
3-(methanesulfonylamino)phenylboronic add pinacol ester. Compound 7g was
isolated as
an orange solid (235 mg; 67% yield).
1H NMR (300 MHz, De-DMS0): 69.81 (s, 1H), 9.16 (s, 1H), 8.34 (d, J= 9.5 Hz,
1H), 8.29
(m, 1H), 8.15(d, J = 8.6 Hz, 1H), 7.91 (dd, J= 8.7, 1.9 Hz, 1H), 7.79 (d, J=
1.3 Hz, 2H),
7.35(t, J = 7.8 Hz, 1H), 7.27 ¨7.15 (m, 2H), 7.05(d, J= 1.8 Hz, 1H), 6.95(d,
J= 9.4 Hz,
1H), 6.58 (dt, J= 8.0, 1.2 Hz, 1H), 3.00 (s, 3H), 2.66 (s, 3H). HRMS (ESI):
miz [M + H]+ calc.
for C2e H21 N405S: 501.1227; found: 501.1228.
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Methyl 4-(1-(4-methy1-3-nitropheny1)-2-oxo-1,2-
dihydrobenzo[h][1,6]naphthyridin-9-
yOphenylcarbarnate (7h ¨ precursor of 16)
Prepared using General Procedure E, reacting intermediate 6a with
4-(methoxycarbonylamino)benzeneboronic acid. Compound 7h was isolated as an
orange
solid (166 mg; 50% yield).
1H NMR (300 MHz, De-DMS0): 59.82 (s, 1H), 9.13 (s, 1H), 8.33 (dd, J= 9.4, 1.5
Hz, 2H),
8.08 (dd, J= 8.6, 1.4 Hz, 1H), 8.04 ¨ 7.93 (m, 1H), 7.86 ¨7.72 (m, 2H), 7.56 ¨
7.46 (m, 2H),
7.06 (dd, J= 8.7, 1.5 Hz, 2H), 7.03 ¨ 6.91 (m, 2H), 3.69 (s, 3H), 2.68 (s,
3H). HRMS (ES1):
rn/z [M + H]+ calc. for C27H2iN405: 481.1506; found: 481.1507.
1-(4-Methyl-3-nitropheny1)-9-(pyridin-4-y1)benzo[h][1,6]naphthyridin-2(1H)-one
(71 ¨precursor
of 17)
Prepared using General Procedure E, reacting intermediate 6a with pyridine-4-
boronic acid
hydrate. Compound 7i was isolated as an orange solid (90 mg; 45% yield).
1H NMR (300 MHz, De-DMS0): 69.21 (s, 1H), 8.64 ¨ 8.56 (m, 2H), 8.40 ¨ 8.31 (m,
2H), 8.20
¨8.14 (m, 1H), 8.11 (dd, J = 8_7, 1.9 Hz, 1H), 7.87 ¨ 7.75 (m, 3H), 7.15 ¨
7.12 (m, 2H), 6.98
(d, J = 9.5 Hz, 1H), 2+66(s, 3H). HRMS (ES1): mfr [M + H]+ calc. for
C24H1711403: 409.1295;
found: 409.1293.
5-(1-(4-Methyl-3-nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yOpicolinonitrile
(7j ¨ precursor of 18)
Prepared using General Procedure E, reacting intermediate 6a with 6-
(cyanopyridin-3-
yl)boronic acid. Compound 7j was isolated as an orange solid (92 mg; 44%
yield).
1H NMR (300 MHz, De-DMS0): 59.22 (s, 1H), 8.50 (m, 1H), 8.36 (d, J= 9.5 Hz,
1H), 8.29
(s, 1H), 8.20 (d, J= 8.6 Hz, 1H), 8.16- 8.14 (m, 1H), 7.80 (m, 2H), 7.65 ¨
7.52 (m, 3H), 6.98
(d, J= 9.5 Hz, 1H), 2.66 (s, 3H). HRMS (ES1): m/z [M + H]+ calc. for C251-
116Ne0s: 434.1248;
found: 434.1245.
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yObenzo[h][1,6]naphthyridin-2(1H)-
one (7k ¨ precursor of 19)
Prepared using General Procedure E, reacting intermediate 6a with 2-
trifluoronnethyl(pyridin-
5-yl)boronic acid. Compound 7k was isolated as an orange solid (110 mg; 47%
yield).
1H NMR (300 MHz, 06-DMS0): 5 9.22 (s, 1H), 8.53 (d, J= 2.1 Hz, 1H), 8.36 (d,
J= 9_5 Hz,
1H), 8.29 (s, 1H), 8.20 (d, J= 8.7 Hz, 1H), 8.13 (dd, J= 8.7, 1.9 Hz, 1H),
7.95 (d, J = 8.2 Hz,
1H), 7.83(m, 1H), 7.65 ¨ 7.54 (m, 3H), 6.97(s, 1H), 2.62(s, 3H). HRMS (ESI):
m/z1M + H]+
calc. for C2eH1eF3N403: 477.1169; found: 477.1168.
General Procedure F: Buchwald-Hartwig Coupling
1-(4-Methyl-3-nitropheny1)-9-(piperidin-1-yObenzo[h][1,6]naphthyridin-2(1H)-
one (71-
precursor of 20)
The bromo-quinoline 6a (250 mg; 0.609 mmol), piperidine (180 pL; 1.827 mmol; 3
equiv.),
Pd(OAc)2 (8.4 mg; 37.4 pmol; 0.06 equiv.), R-BINAP (46 mg: 73.1 pmol; 0.12
equiv.) and
Cs2CO3 (595 mg, 1.827 mmol; 3 equiv.) were mixed in dioxane (5 mL) under
Argon. The
mixture was heated to 90 C overnight. After 24 h, LCMS analysis showed that
the reaction
was completed. The mixture was cooled to r.t. and the solvent evaporated. The
crude was
partitioned between Et0Ac and sat NaHCO3. The phases were separated and the
aqueous
phase further extracted with Et0Ac (2 x). The combined organics were washed
with brine
and dried over MgSO4. After filtration, the solvent was evaporated to afford
the title
compound as a yellow solid. The compound was used without further purification
(180 mg;
71% yield).
1H NMR (300 MHz, De-DMS0): 68.87 (s, 1H), 8.26 ¨ 8.18 (m, 2H), 7.84 (d, J= 9.2
Hz, 1H),
7.76 (d, J = 8.2 Hz, 1H), 7.70 (dd, J = 8.2, 2.1 Hz, 1H), 7.50- 7.46 (m, 1H),
6.85 (d, J = 9_4
Hz, 1H), 6.23 (d, J = 2.6 Hz, 1H), 2.74 (m, 4H), 2.60 (s, 3H), 1.45 (m, 6H).
HRMS (ES!): mit
[M + H]+ calc. for C24H23N403: 415A765; found: 415.1763.
1-(4-Methy1-3-nitropheny1)-9-morpholinobenzo[h][1,6]naphthyridin-2(1H)-one (7m
¨precursor
of 21)
Prepared using General Procedure F, using morpholine as amine. Compound 7m was
isolated as an orange solid (290 mg; 95% yield).
1H NMR (300 MHz, 0e-DMS0): 58.91 (s, 1H), 8.35 ¨ 8.17 (m, 2H), 7.89 (d, J= 9.2
Hz, 1H),
7.81 ¨7.65 (m, 2H), 7.53 (dd, J = 9_2, 2.6 Hz, 1H), 6.87 (d, J = 9.4 Hz, 1H),
6.23 (d, J = 2.6
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Hz, 1H), 3.62 (m, 4H), 2.68 (m, 4H), 2.58 (s, 3H). HRMS (ESI): mai [M + H]+
calc. for
Cz3H2-1N404: 417.1557; found: 417.1557.
1-(4-Methy1-3-nitropheny1)-9-(4-(methylsulfonyl)piperazin-1-
yl)benzo[h][1,6]naphthyridin-
2(1H)-one (7n ¨ precursor of 22)
Prepared using General Procedure F, using 1-(rnethylsulfonyl)piperazine as
amine.
Compound 7n was isolated as a yellow solid (285 mg; 95% yield).
1H NMR (300 MHz, CDCI3): 6 8.83 (s, 1H), 8.10¨ 7.86 (m, 3H), 7.69¨ 7.52 (m,
2H), 7.35
(dd, J = 9.3, 2.6 Hz, 1H), 6.90 (d, J = 9.3 Hz, 1H), 6.36 (d, J = 2.6 Hz, 1H),
3.25 (t, J = 5.0
Hz, 4H), 2.95 ¨2.89 (m, 2H), 2.85 ¨ 2.82 (m, 2H), 2.82 (s, 3H), 2.71 (s,
3H).HRMS (ESI):
ink [M + H]+ calc. for C24h124N505S: 494.1493; found: 494.1492.
9-(4-(Dimethylamino)piperidin-1-y1)-1-(4-methyl-3-
nitrophenyObenzo[h][1,6]naphthyridin-
2(1H)-one (7o ¨ precursor of 23)
Prepared using General Procedure F, using N,N-dimethylpiperidin-4-amine as
amine.
Compound 70 was isolated as a yellow solid (215 mg; 77% yield).
1H NMR (300 MHz, 06-DMS0): 58.87 (s, 1H), 8.23 (m, 2H), 7.85 (d, i= 9.2 Hz,
1H), 7.77
(d, 1= 8.4 Hz, 1H), 7.70 (dd, J= 81,2.1 Hz, 1H), 7.51 (m, 1H), 6.85 (d, J= 9.4
Hz, 1H),
6.25 (d, J = 2.6 Hz, 1H), 3.23¨ 3.18 (m, 2H), 2.62 (s, 3H), 2.44 ¨2.37 (m,
2H), 2.15 (s, 3H),
2.15(m, 1H), 1.66¨ 1.62 (m, 2H), 1.28 ¨ 1.23 (m, 2H). HRMS (ESI): m/z [M + H]+
calc. for
C26H28N503: 458.2187; found: 458.2185.
8-(4-(Methylsulfonyl)piperazin-1-y1)-1-(3-
nitrophenyl)benzo[h][1,6]naphthyridin-2(1H)-one
(7d'n'- precursor of 27)
Prepared using the General Procedure F, reacting intermediate 6d with
1-(methylsulfonyl)piperazine. Compound 7d'n' was obtained as a brown solid
(355 mg;
98%).
HRMS (ES!): nilz [M + F1]+ calc. for C23H22N505S: 480.1336; found: 480.1330.
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General Procedure G: Nitro to amine reduction with Fe/NH4C1
N-(4-(1-(3-Amino-4-methylphenyI)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yOphenyOmethanesulfonannide (8a)
Intermediate 7a (335 mg; 0.669 mnnol) was suspended in Et0H (40 mL) and heated
to
reflux. Fe (224 mg; 4.016 mmol; 6 equiv.) and NRICI (215 mg; 4.016 mmol; 6
equiv.) in H20
(20 nnL) were added and the mixture heated to reflux. After 2 h, TLC analysis
(MeOH:DCM
1:9) showed that the reaction was completed. The hot mixture was filtered
through a Celite
pad and the pad further washed with Et0H and MeOH:DCM (2:8). The solvent was
evaporated and the crude partitioned between H20 and Et0Ac. The phases were
separated
and the aqueous phase was further extracted with Et0Ac (3 x). The combined
organics
were washed with brine, dried over MgSO4 and taken to dryness to afford the
title compound
as an off-white solid (330 mg; 100% yield).
N-(4-(1-(3-Amino-5-methylphenyI)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)methanesulfonamide (8b)
Prepared using the procedure described for 8a. Compound 8b was obtained as a
bright-
yellow solid (85 mg; 70% yield).
1H NMR (300 MHz, De-DMS0): 6 9.90 (s, 1H), 9.07 (s, 1H), 8.25 (d, J= 9.4 Hz,
1H), 8.03 (q,
J= 8.9 Hz, 2H), 7.70 (s, 1H), 7.35 ¨ 7.21 (m, 4H), 6.87 (d, J= 9.5 Hz, 1H),
6.66 (s, 1H), 6.48
(s, 1H), 6.29 (s, 1H), 5.40 (s, 2H), 3.04 (s, 3H), 2.25 (s, 3H). HRMS (ESI):
al& [M + Hp- calc.
for C26F123N403S: 471.1485; found: 471.1484.
N-(4-(1-(5-Amino-2-methylphenyI)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yOphenyl)methanesulfonannide (8c)
Prepared using the procedure described for 8a. Compound 8c was obtained as an
orange
solid (70 mg; 68% yield).
1H NMR (300 MHz, De-DMS0): 69.91 (s, 1H), 9.11 (s, 1H), 8.30(d, J= 9.5 Hz,
1H), 8.12 ¨
7.97 (m, 2H), 7.53 (d, J = 1.8 Hz, 1H), 7.32 (d, J = 8.7 Hz, 2H), 7.27 ¨ 7.16
(m, 3H), 6.92 (d,
J= 9.4 Hz, 1H), 6.79 (dd, J= 8.2, 2.3 Hz, 1H), 6.45 (d, J= 2.3 Hz, 1H), 5.29
(s, 2H), 3.05 (s,
3H), 1.80(s, 3H). HRMS (ESI): mit [M + H]+ calc. for C26H23N403S: 471.1485;
found
471.1485.
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N-(4-(1-(3-Nitropheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)methanesulfonamide (8d)
Prepared using the procedure described for 8a. Compound 8d was obtained as a
dark
yellow solid (60 mg; 92% yield).
1H NMR (300 MHz, d6-DMS0): 69.91 (s, 1H), 9.09 (s, 1H), 8.27(d, J= 9.5 Hz,
1H), 8.11 ¨
7.95 (m, 2H), 7.60 (d, J = 1.9 Hz, 1H), 7.36 ¨7.27 (m, 3H), 7.23 (d, J = 8.8
Hz, 2H), 6.92 ¨
6.79 (m, 2H), 6.57 (dd, J= 6.7, 1_2 Hz, 2H), 5.55 (brs, 2H), 3.04 (s, 3H).
HRMS (ES1): m/z
[M + H]+ calc. for C25F121N403S: 457.1329; found: 457.1328.
N-(4-(1-(3-Amino-4-methoxypheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)methanesulfonamide (8e)
Prepared using the procedure described for 8a. Compound 8e was obtained as an
orange
solid (85 mg; 78% yield).
1H NMR (300 MHz, De-DMS0): 6 9.93 (s, 1H), 9.07 (s, 1H), 8.26 (d, J = 9.5 Hz,
1H), 8.05 (d,
J= 8.7 Hz, 1H), 7.97 (dd, J= 8.7, 1.9 Hz, 1H), 7.37(d, J= 1.9 Hz, 1H), 7_32
¨7.18 (m, 4H),
7.05(d, Jr 8.3 Hz, 1H), 6.88 (d, Jr 9.4 Hz, 1H), 6.67 ¨ 6.53 (m, 2H), 5.11 (s,
2H), 3.91 (s,
3H), 3.04 (s, 3H). HRMS (ES1): rn/z [M + H]+ calc. for C261-123N404S:
487.1435; found:
487.1433.
N-(4-(1-(3-Amino-4-methylphenyI)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8-
yOphenyOmethanesulfonarnide (8a')
Prepared using the procedure described for 8a. Compound 8a' was obtained as a
bright-
yellow solid (80 mg; 71% yield).
1H NMR (300 MHz, 0e-DMS0): 6 9.96(s, 1H), 9.10 (s, 1H), 8.26 ¨ 8.23 (m, 2H),
7.84 (d, J=
8.7 Hz, 2H), 7.52 (dd, J= 9.4, 2.2 Hz, 1H), 7.30 (d, J= 8.7 Hz, 2H), 7.15 (m,
2H), 6.84 (d, J
= 9.4 Hz, 1H), 6.56 (d, J= 2.1 Hz, 1H), 6.48 (dd, J= 7.8, 2.1 Hz, 1H), 5_20
(s, 2H), 3.04 (s,
3H), 2.21 (s, 3H). HRMS (ES1): m/z [M + H]+ calc. for C26H23N403S: 471.1485;
found:
471.1483.
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N-(4-(1-(3-Amino-5-methylpheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8-
yl)phenyl)methanesulfonamide (8b')
Prepared using the procedure described for 8a. Compound 8b' was obtained as an
orange
solid (295 mg; 70% yield).
1H NMR (300 MHz, De-DMS0): 69.99 (s, 1H), 9.09 (s, 1H), 8.25 ¨ 8.22 (m, 2H),
7.86 - 7.81
(m, 4H), 7.54 (dd, J= 9A, 2.3 Hz, 1H), 7.30 (d, J= 8.5 Hz, 2H), 7.21 (d, J=
9.3 Hz, 1H),
6.84 (d, J = 9.4 Hz, 1H), 6.61 (s, 1H), 6.34 (s, 2H), 3.04 (s, 3H), 2.22 (s,
3H). HRMS (ESI):
m/z [M + H]+ calc. for C26H23N403S: 471.1485; found: 471.1483.
N-(4-(1-(3-Aminopheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8-
yl)phenyl)methanesulfonamide (8d')
Prepared using the procedure described for 8a. Compound 8d' was obtained as a
brown
solid (140 mg; 85% yield).
1H NMR (300 MHz, De-DMS0): 69.09 (s, 1H), 8.26 ¨ 8.22 (m, 2H), 7.79 ¨ 7.77 (m,
2H), 7.50
(dd, J= 9.3, 2.2 Hz, 1H), 7.29 ¨ 7.23 (m, 3H), 7.12 (d, J= 9.3 Hz, 1H), 6.85 ¨
6.78 (m, 2H),
6.53¨ 6.50 (m, 2H), 5.42 (s, 2H), 2.97 (s, 3H). HRMS (ESI): m/z [M + H]+ calc.
forC2.5H21N403S: 457.1329; found: 457.1329.
Ethyl 4-(3-amino-4-methylphenylamino)-6-(4-(methylsulfonamido)phenyl)quinoline-
3-
carboxylate (Intermediate 2¨ I NT2)
Prepared using the procedure described for 8a. Compound INT2 was obtained as a
yellow
solid (190 mg; 88%).
1H NMR (300 MHz, De-DMS0): 610.14 (s, 1H), 9.88 (s, 1H), 8.94 (s, 1H), 8.08
(d, J= 2.0
Hz, 1H), 8.04 ¨ 7.96 (m, 1H), 7.92(d, J= 8.7 Hz, 1H), 7.67 ¨ 7.50 (m, 2H),
7.39(d, J = 8.7
Hz, 2H), 7.23 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 7.9 Hz, 1H), 6.45 (d, J = 2.2
Hz, 1H), 6.33 (dd,
J= 7.9, 2.2 Hz, 1H), 4.19 (q, J= 7.1 Hz, 2H), 3.02 (s, 3H), 2.11 (s, 3H),
1.29(t, J= 7.1 Hz,
3H). HRMS (ESI): rn/z [M + H]+ calc. for C26H27N404S: 491.1753; found:
491.1748.
N-(4-(1-(3-Amino-4-methylpheny1)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenyl)butane-1-sulfonamide (8f ¨ precursor of 15)
Prepared using the procedure described for 8a. Compound 81 was obtained as a
bright-
yellow solid (163 mg; 91% yield).
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1H NMR (300 MHz, De-DMS0): 59.92 (s, 1H), 9.06 (s, 1H), 8.25 (d, J= 9.5 Hz,
1H), 8.04 (d,
J= 8.6 Hz, 1H), 7.96 (dd, J= 8.7, 1.9 Hz, 1H), 7.70- 7.48 (m, 2H), 7.39 (d, J
= 1.9 Hz, 1H),
7.30 - 7.13 (m, 5H), 6.87 (d, J = 9.4 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 6.51
(dd, J = 7.8, 2.1
Hz, 1H), 3.12 (t, J= 7.4 Hz, 2H), 2.25 (s, 3H), 1.76 - 1.58 (m, 2H), 1.47 -
1.29 (m, 2H), 0.85
(t, J = 7.3 Hz, 3H). HRMS (ESI): In& [M + H]+ calc. for C29H291\1403S:
513.1955; found:
513.1954.
N-(3-(1-(3-Amino-4-methylphenyI)-2-oxo-1,2-dihydrobenzo[h][1,6]naphthyridin-8-
yl)phenyl)methanesulfonamide (8g - precursor of 16)
Prepared using the procedure described for 8a. Compound 8g was obtained as a
bright-
yellow solid (75 mg; 94% yield).
HRMS (ES!): mitz [M + H]+ calc. for C26H23N4035: 471.1484; found: 471.1485.
Methyl 4-(1-(3-amino-4-methylphenyI)-2-oxo-1,2-
dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenylcarbannate (8h - precursor of 17)
Prepared using the procedure described for 8a. Compound 8h was obtained as a
bright-
yellow solid (147 mg; 100% yield).
1H NMR (300 MHz, De-DMS0): 59.77 (s, 1H), 9.06 (s, 1H), 8.25 (d, J= 9.4 Hz,
1H), 8.08 -
7.91 (m, 2H), 7.51 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 1.8 Hz, 1H), 7.22 - 7.18
(m, 3H), 6.87 (d,
J = 9.4 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 6.51 (dd, J = 7.8, 2.1 Hz, 1H),
5.25 (brs, 2H), 3.70
(s, 3H), 2.27 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for C2+123N403:
451.1765; found:
451.1763.
1-(3-Amino-4-methylphenyI)-9-(pyridin-4-yl)benzo[h][1,6]naphthyr1din-2(1H)-one
(8i -
precursor of 18)
Prepared using the procedure described for 8a. During phase separation, the
aqueous
phase (pH-5) was adjusted to pH 7 with NaHCO3. Compound 8i was obtained as an
orange
solid (75 mg; 100% yield).
1H NMR (300 MHz, De-DMS0): 69.14 (s, 1H), 8.64 - 8.55 (m, 2H), 8.28 (d, 1= 9.5
Hz, 1H),
8.11 (s, 2H), 7.51 (s, 1H), 7.31 - 7.23 (m, 2H), 7.20(d, J= 8.2 Hz, 1H),
6.90(d, J= 9.4 Hz,
1H), 6.66 (d, J = 2.1 Hz, 1H), 6.49 (dd, 1= 7.8, 2.1 Hz, 1H), 5.33 (s, 2H),
2.25 (s, 3H).
HRMS (ES!): m/z [M + H]+ calc. for C24E119N40: 379.1553; found: 379.1552.
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9-
yppicolinonitrile (8j ¨ precursor of 19)
Prepared using the procedure described for 8a. Compound 8j was obtained as an
orange
solid (79 mg; 97% yield).
HRMS (ES1): m/z [M + H]+ calc. for C2.51-118N50: 404.1506; found: 4041507.
1-(3-Amino-4-rnethylpheny1)-9-(6-(trifluororriethyppyridin-3-
yl)benzo[h][1,6]naphthyridin-
2(1H)-one (8k ¨ precursor of 20)
Prepared using the procedure described for 8a. Compound 8k was obtained as an
orange
solid (99 mg; 100% yield).
1H NMR (300 MHz, De-DMS0): 6 9.15(s, 1H), 8.67 (d, J = 2.1 Hz, 1H), 8.29 (d, J
= 9.5 Hz,
1H), 8.13 (brs, 2H), 7.95 (m, 1H), 7.88 (m, 1H), 7.33 (s, 1H), 7.19 (d, J= 7.8
Hz, 1H), 6.92
(d, J = 9.4 Hz, 1H), 6.64 (d, J= 2.1 Hz, 1H), 6.51 (dd, J= 7.7, 2.1 Hz, 1H),
5.33 (s, 2H), 2.21
(s, 3H). HRMS (ES!): m/z [M + H]+ calc. for C25H18F3N40: 447.1427; found:
447.1426.
1-(3-Amino-4-methylpheny1)-9-(piperidin-1-yObenzo[h][1,6]naphthyridin-2(1H)-
one (81-
precursor of 21)
Prepared using the procedure described for 8a. Compound 81 was obtained as a
yellow solid
(134 mg; 85% yield).
HRMS (ES1): m/z [M + H]+ calc. for C24E1251\140: 385.2023; found: 385.2025.
1-(3-Amino-4-rnethylpheny1)-9-rnorpholinobenzo[h][1,6]naphthyridin-2(1H)-one
(8m ¨
precursor of 22)
Prepared using the procedure described for 8a. Compound 8m was obtained as an
orange
solid (141 mg; 95% yield).
HRMS (ES1): nv'z [M + H]+ calc. for C23H23N402: 387.1816; found: 387.1817.
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1-(3-Amino-4-methylpheny1)-9-(4-(methylsulfonyl)piperazin-1-
yl)benzo[h][1,6]naphthyridin-
2(1H)-one (8n)
Prepared using the procedure described for 8a. Compound 8n was obtained as a
yellow
solid (285 mg; 88% yield).
1H NMR (300 MHz, C0CI3): 68.79 (s, 1H), 7.94 (dd, J= 16.9, 9.3 Hz, 2H), 7.33
(dd, J= 9_2,
2.6 Hz, 1H), 7.27 (m, 1H), 6.90 (d, J= 9.4 Hz, 1H), 6.78 (d, J= 2.6 Hz, 1H),
6.68(d, J= 6.4
Hz, 2H), 3.83 (s, 2H), 3.26- 3.22 (m, 4H), 2.99 -2.96 (m, 4H), 2.82 (s, 3H),
2.26 (s, 3H).
HRMS (ES!): m/z [M + H]+ calc. for C24H2eNe03S: 4641751; found: 464.1748.
1-(3-amino-4-methylphenyI)-9-(4-(dimethylamino)piperidin-1-
yl)benzo[h][1,6]naphthyridin-
2(1H)-one (8o - precursor of 23)
Prepared using the procedure described for 8a. The reaction required 12 eq. of
Fe and
NH4CI and 5 h reaction time at 80 C. During phase separation, the aqueous
phase (pH-5)
was adjusted to pH 8 with NaHCO3_ Compound 8o was obtained as a yellow solid
(85 mg;
46% yield).
1H NMR (300 MHz, De-DMS0): 68.81 (s, 1H), 8.16 (d, Jr 9.4 Hz, 1H), 7.79 (d, Jr
9.1 Hz,
1H), 7.46 (dd, J= 9.4,2.4 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 6.79 (d, J= 9.4,
1H), 6.71 (brs,
1H), 6.57 (d, J = 1_8 Hz, 1H), 6.49 - 6.37 (m, 1H), 5.22 (s, 2H), 3.34 -3.30
(m, 2H), 2_50 (s,
3H), 2.42 - 2.36 (m, 2H), 2.16 (s, 6H), 2.16(m, 1H), 1.70- 1.66 (m, 2H), 1.30 -
1.22 (m,
2H). HRMS (ESI): rn/z [M + H]+ calc. for C261-130N50: 428.2445; found:
428.2442.
1-(3-aminopheny1)-8-(4-(methylsulfonyl)piperazin-1-yObenzo[h][1,6]naphthyridin-
2(1H)-one
(8d'n' - precursor of 27)
Prepared using the procedure described for 8a. Compound 8d'n' was obtained as
an
orange solid (225 mg; 71% yield).
1H NMR (300 MHz, CDCI3): 6 8.92 (s, 1H), 8.14(d, J = 9.4 Hz, 1H), 7.28 - 7.21
(m, 2H),
7.03 - 6.96 (m, 1H), 6.86 (d, J = 9.8 Hz, 1H), 6.77 -6.68 (m, 2H), 6.46 (brs,
2H), 5.43 (s,
2H), 3.43 (m, 4H,) 3.20 (m, 4H), 2.89 (s, 3H). HRMS (ESI): m/z [M + H]+ calc.
for
C23H2411503S: 450.1594; found: 450.1600
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General Procedure H: Nitro to Amine reduction with SnCl2
1-(3-Amino-4-methylphenyI)-9-bromobenzo[h][1,6]naphthyridin-2(1H)-one
(Intermediate 3 ¨
I NT3)
Intermediate 6a (1.03 g; 2.511 mmol) was suspended in Et0Ac (50 mL) and SnCl2
(2.86 g;
15.065 mmol; 6 equiv.) was added. The mixture was heated to 85 C and after 2
h, the
reaction cooled to r.t and saturated NaHCO3 aq. was added. The phases were
separated
and the aqueous phase further extracted with ethyl acetate (2 x). The combined
organics
were washed with brine, dried over Mg304, taken to dryness to afford INT3 as a
brownish
solid (650 mg; 68% yield).
1H MIR (300 MHz, De-DMS0): 69.11 (s, 1H), 8.25 (d, J = 9.4 Hz, 1H), 7.91 (d,
J= 8.9 Hz,
1H), 7.78 (dd, J = 8.9,2.1 Hz, 1H), 7.18 (d, J= 7.8 Hz, 1H), 6.99 (d, J = 2.1
Hz, 1H), 6.90 (d,
J= 9.4 Hz, 1H), 6.53 (d, J = 2.1 Hz, 1H), 6.45 (dd, J= 7.7, 2.1 Hz, 1H), 5.24
(s, 2H), 2.22 (s,
3H). HRMS (ESI): mfr [M + H]+ calc. for C1eH1eBrN30: 380.0393; found:
380.0390.
General Procedure I: Acylation
N-(2-Methyl-5-(9-(4-(methylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yl)phenyl)acrylamide (9a; BMX-IN-1)
A stirred solution of 8a (90 mg; 0.191 mmol) in dry THE (20 rrt) was cooled in
an ice-bath to
-10 C for 20 min. DIPEA (133 uL; 0.765 mmol; 4 equiv.) was added and the
mixture stirred
for 10 min at T < 4 C. After 10 min, acryloyl chloride was added and the
mixture further
stirred at -10 C for 10 min. and then 1 h at r.t. THF was then evaporated and
the crude
redissolved in Et0Ac and washed three times with NaHCO3 (4%). The organics
were dried
over MgSO4 and taken to dryness. The crude was applied in a silica column and
eluted with
a gradient from 100:0 to 96:4 in DCM:Me0H. The desired fractions were
collected and
taken to dryness to afford the title compound as a white solid. (20 mg; 20%
yield).
1H NMR (300 MHz, De-DMS0): 69.85 (s, 1H), 9.78 (s, 1H), 9.11 (s, 1H), 8.30 (d,
J= 9.4 Hz,
1H), 8.08(d, J= 8.7 Hz, 1H), 7.98 (dd, J= 8.7, 1.9 Hz, 1H), 7.69(s, 1H),
7.52(d, J= 8.1 Hz,
1H), 7.23-7.20 (m, 6H), 6.91 (d, J= 9.4 Hz, 1H), 6.57 (dd, J= 17.2, 10.2 Hz,
1H), 6.19 (d, J
= 17.2 Hz, 1H), 5.74 (d, J= 10.2 Hz, 1H), 3.01 (s, 3H), 2.42 (s, 3H). HRMS
(ESI): m/z [M +
H]+ calc. for C291-12eN404S: 525.1591; found: 525.1586. HPLC Purity: 98.9%.
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N-(3-Methy1-5-(9-(4-(nnethylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yl)phenyl)acrylamide (9b)
Prepared using the procedure described for 9a. Compound 9b was obtained as a
light-
yellow solid (8 mg; 10% yield).
1H NMR (300 MHz, De-DMS0): 610.42 (s, 1H), 9.92 (s, 1H), 9.12 (s, 1H), 8.31
(d, J= 9.5
Hz, 1H), 8.08 (d, J= 8.8 Hz, 1H), 8.00 (dd, J= 8.8, 1.9 Hz, 1H), 7.78(s, 1H),
7.57 (brs, 1H),
7.44 (d, J= 1.9 Hz, 1H), 7.20 (brs, 4H), 7.09 (s, 1H), 6.92 (d, J= 9.4 Hz,
1H), 6.39 (dd, J=
17.0, 10.0 Hz, 1H), 6.22 (dd, 1= 17.0, 2.2 Hz, 1H), 5.73 (dd, J= 9.9,2.1 Hz,
1H), 3.03 (s,
3H), 2.36 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for C291-125N4048: 525.1591;
found:
525.1576. HPLC Purity: 93.1%.
N-(4-Methy1-3-(9-(4-(methylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yl)phenyl)acrylamide (9c)
Prepared using the procedure described for 9a. Compound 9c was obtained as a
light-
yellow solid (40 mg; 55% yield).
1H NMR (300 MHz, De-DMS0): 6 10.40 (s, 1H), 9.91 (brs, 1H), 9.14 (s, 1H), 8.35
(d, J= 9.5
Hz, 1H), 8.10 (d, J= 8.7 Hz, 1H), 8.01 (dd, J= 8.7, 1.9 Hz, 1H), 7.83 (dd, J=
8.4, 2.2 Hz,
1H), 7.72 (d, i= 2.1 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.32 (d, J= 1.8 Hz,
1H), 7.19 (m, 4H),
6.96 (d, J = 9.4 Hz, 1H), 6.39 (dd, 1= 17.0, 10.0 Hz, 1H), 6.22 (dd, J= 17.0,
2.2 Hz, 1H),
5.74 (dd, J= 10.0, 2.2 Hz, 1H), 3.03 (s, 3H), 1.91 (s, 3H). HRMS (ESI): m/z [M
+ H]+ calc.
for C29H25N404S: 525.1591; found: 525.1587. HPLC Purity: 99.5%.
N-(3-(9-(4-(Methylsulfonamido)phenyI)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-
yl)phenyl)acrylamide (9d)
Prepared using the procedure described for 9a. Compound 9d was obtained as a
light-
yellow solid (27 mg; 50% yield).
1H NMR (300 MHz, De-DMS0): 610.50 (s, 1H), 9.91 (s, 1H), 9.12 (s, 1H), 8.31
(d, J= 9.4
Hz, 1H), 8.08 (d, J= 8.7 Hz, 1H), 8.03 ¨ 7.91 (m, 2H), 7.83 (brs, 1H), 7.63
(t, J= 8.1 Hz,
1H), 7.34 (brs, 1H), 7.18 (m, 5H), 6.92 (d, J= 9.4 Hz, 1H), 6.42 (dd, J= 16.9,
10.0 Hz, 1H),
6.26 (dd, J= 16.9,2.2 Hz, 1H), 5.77 (dd, J = 9.7, 1.9 Hz, 1H), 3.03 (s, 3H).
HRMS (ESI): infr
[M + H]+ calc. for C28H23N4045: 511.1435; found: 511.1433. HPLC Purity: 98.0%.
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N-(2-Methoxy-5-(9-(4-(methylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yl)phenyl)acrylamide (9e)
Prepared using the procedure described for 9a. Compound 9e was obtained as a
light-
yellow solid (38 mg; 43% yield).
1H NMR (300 MHz, De-DMS0): 6973 (s, 1H), 9.10 (s, 1H), 8.29 (d, J= 9.5 Hz,
1H), 8.19 ¨
8.10 (m, 1H), 8.07(d, J= 8.6 Hz, 1H), 7.97 (d, J = 8.7 Hz, 1H), 7.36 ¨ 7.20
(m, 8H), 6.91 (d,
J= 9.4 Hz, 1H), 6.74 (dd, J= 17.0, 10.2 Hz, 1H), 6.13 (dd, J= 17.0, 2.1 Hz,
1H), 5.68 (dd, J
= 10.3, 2.1 Hz, 1H), 4.00 (s, 3H), 3.01 (s, 3H). HRMS (ES1): ink [M + H]+
calc. for
C29H25N40eS: 541.1540; found 541.1542. HPLC Purity: 97.0%.
N-(2-Methy1-5-(9-(4-(methylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yl)phenyl)but-2-enamide (10)
Prepared using the procedure described for 9a. Compound 10 was obtained as a
pale-
yellow solid (30 mg; 41%).
1H NMR (300 MHz, De-DMS0): 69.86 (s, 1H), 9.55 (s, 1H), 9.10 (s, 1H), 8.29 (d,
J= 9.5 Hz,
1H), 8.02 (q, J= 8.6 Hz 2H), 7.69 (m, 1H), 7.49 (d, 1= 8.1 Hz, 1H), 7.21 (brs,
6H), 6.91 (d, J
= 9.4 Hz, 1H), 6.73 (dd, J= 15.1, 7.2 Hz, 1H), 6.27 (d, J= 15.3 Hz, 1H), 3.02
(s, 3H), 2.41
(s, 3H), 1.83 (s, 3H). HRMS (ES1): ink [M + H]+ calc. for C3oHnN404S:
539.1748; found:
539.1746. HPLC Purity: 99.3%.
3-Methyl-N-(2-methy1-5-(9-(4-(methylsulfonannido)pheny1)-2-
oxobenzo[h][1,6]naphthyridin-
1(2H)-yl)phenyl)but-2-enamide (11)
Prepared using the procedure described for 9a. Compound 11 was obtained as a
pale-
yellow solid (20 mg; 32%).
1H NMR (300 MHz, d6-DMS0): 6 9.39 (s, 1H), 9.10 (s, 1H), 8.29 (d, J = 9.5 Hz,
1H), 8.08 ¨
7.96 (m, 2H), 7.72 (brs, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.16 (m, 6H), 6.91 (d,
J = 9.4 Hz, 1H),
6.03 (s, 1H), 2.98 (s, 3H), 2.40 (s, 3H), 2.04 (s, 3H), 1.84 (s, 3H). HRMS
(ES1): ink [M + H]+
calc. for C3iHnN.404S: 553.1904; found: 553.1909. HPLC Purity: 99.3%.
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N-(5-(9-(4-(Butylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-y1)-
2-
nnethylphenypacrylamide (14)
Prepared using the procedure described for 9a. Compound 14 was obtained as a
pale-
yellow solid (33 mg; 20%).
1H NMR (300 MHz, De-DMS0): 69.92 (s, 1H), 9.81 (s, 1H), 9.11 (s, 1H), 8_30 (d,
J= 9.4 Hz,
1H), 8.07(d, J= 8_6 Hz, 1H), 7.98 (d, J= 9.0 Hz, 1H), 7.69 (s, 111), 7.50 (d,
J= 8.1 Hz, 1H),
7.19 (brs, 6H), 6.91 (d, J= 9.5 Hz, 1H), 6.57 (dd, J= 17.1, 9.4 Hz, 1H), 6.19
(d, J= 17.0 Hz,
1H), 5.74 (d, J= 10.1 Hz, 1H), 3.09 (t, J= 7.8 Hz, 2H), 2.40 (s, 3H), 1.74 ¨
1.54 (m, 2H),
1.35 (q, J = 7.4 Hz, 2H), 0.83 (t, i = 7.3 Hz, 3H). HRMS (ESI): rniz [M + H]+
calc. for
C32H3iN404S: 567.2061; found 567.2060. HPLC Purity: 95.2%.
N-(2-Methy1-5-(9-(3-(methylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yl)phenyl)acrylamide (15)
Prepared using the procedure described for 9a. Compound 15 was purified by
semi-prep
HPLC with a gradient 25:75 until 50:50 with a mixture (95:5 ACN:NaHCO3 10 mM)
:
(NaHCO3 10 nnM). The title compound was obtained as a white solid (8 mg; 4%).
1H NMR (300 MHz, CDC13): 6 8.98 (s, 1H), 8.68 (s, 1H), 8.17 (d, J= 8.7 Hz,
1H), 8.07¨ 7.94
(m, 2H), 7.88 (dd, J = 8_7, 1.9 Hz, 1H), 7.54 ¨ 7.32 (m, 5H), 7.25 (m, 1H),
7.09 (dd, J = 8.1,
2.2 Hz, 1H), 6.98 ¨ 6.84 (m, 2H), 6.56 (dd, J= 16.9, 1.3 Hz, 1H), 6.36 (dd, 1=
16.9, 10.2 Hz,
1H), 5.85 (dd, J= 10_2, 13 Hz, 1H), 2.98 (s, 3H), 2.34 (s, 3H). HRMS (ES!):
Ink [M + H]+
calc. for C29H2eN404S: 525.1591; found 525.1594. HPLC Purity: 94.3%.
Methyl 4-(1-(3-acrylamido-4-methylphenyI)-2-oxo-1,2-
dihydrobenzo[h][1,6]naphthyridin-9-
yl)phenylcarbamate (16)
Prepared using the procedure described for 9a. Compound 16 was obtained as a
pale-
yellow solid (55 mg; 36%).
1H NMR (300 MHz, De-DMS0): 69.75 (s, 2H), 9.09 (s, 1H), 8.29 (d, J= 9.5 Hz,
1H), 8.15 ¨
7.90 (m, 2H), 7.69 (d, J = 2.0 Hz, 1H), 7.53 - 7.46 (m, 3H), 7.32 ¨ 7.08 (m,
4H), 6.90 (d, J =
9.4 Hz, 1H), 6.58 (dd, J = 17.0, 10.1 Hz, 1H), 6.19 (dd, J = 17.0, 2.0 Hz,
1H), 5.73 (d, J =
10.4 Hz, 1H), 3.68 (s, 3H), 2.44 (s, 3H). HRMS (ESI): mit [M + H]+ calc. for
C301-125N404:
505.1870; found 505.1869. HPLC Purity: 97.8%.
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N-(2-Methyl-5-(2-oxo-9-(pyridin-4-yObenzo[h][1,6]naphthyridin-1(2H)-
yl)phenyl)aarylarnide
(17)
Prepared using the procedure described for 9a. Compound 17 was obtained as a
yellow
solid (29 mg; 20%).
1H NMR (300 MHz, De-DMS0): 6976 (s, 1H), 9.17 (s, 1H), 8.56 (s, 2H), 832 (d,
J= 9.5 Hz,
1H), 8.16-8.12 (m, 2H), 7.73 (s, 1H), 7.52 (d, J= 8.1 Hz, 1H), 7.38 (s, 1H),
7.26-7.20 (m,
3H), 6.94 (d, J= 9_6 Hz, 1H), 6.57 (dd, J= 16.5, 9.6 Hz, 1H), 6.20 (d, J= 16.5
Hz, 1H), 5.74
(d, J= 9.3 Hz, 1H), 2.43 (s, 3H). HRMS (ESI): rn/z [M + H]+ calc. for
C2+124\402: 433.1659;
found: 433.1656. HPLC Purity: 95.8%.
N-(5-(9-(6-Cyanopyridin-3-y1)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-y1)-2-
methylphenypacrylamide (18)
Prepared using the procedure described for 9a. Compound 18 was obtained as a
light
yellow solid (14 mg; 17%).
1H NMR (300 MHz, De-DMS0): 69.75 (s, 1H), 9.19 (s, 1H), 8.61 (dd, J= 2.3, 0.8
Hz, 1H),
8.32 (d, J = 9.5 Hz, 1H), 8.16- 8.13 (m, 2H), 8.06 (dd, J = 8.2, 0.8 Hz, 1H),
7.85 (dd, J = 8.2,
2.3 Hz, 1H), 7.67 (d, J= 2.2 Hz, 1H), 7.51 (d, J= 8.1 Hz, 1H), 7.25 ¨ 7.22 (m,
2H), 6.94 (d, J
= 9.4 Hz, 1H), 6.55 (dd, J= 17.0, 10_1 Hz, 1H), 6.17 (dd, J = 17.0, 2.0 Hz,
1H), 5.73 (dd, J =
10.1, 2.0 Hz, 1H), 2.42 (s, 3H). HRMS (ESI): rniz [M + H]+ calc. for C281-
120N502: 458.1612;
found: 458.1610. HPLC Purity: 95.3%.
N-(2-Methyl-5-(2-oxo-9-(6-(trifluoromethyl)pyridin-3-
yObenzo[h][1,6]naphthyridin-1(2 Hy
Aphenyl)aaylamide (19)
Prepared using the procedure described for 9a. Compound 19 was obtained as a
yellow
solid (20 mg; 18%).
1H NMR (300 MHz, De-DMS0): 69.77 (s, 1H), 9.18 (s, 1H), 8.66 (s, 1H), 832 (d,
J= 9.4 Hz,
1H), 8.17-8.14 (m, 2H), 7.87 (s, 2H), 7.67 (d, J= 2.2 Hz, 1H), 7.51 (d, 1= 8.0
Hz, 1H), 7.25-
7.22 (m, 2H), 6.94 (d, J= 9.4 Hz, 1H), 6.55 (dd, J= 17.1, 10.2 Hz, 1H), 6.17
(dd, J= 17.1,
2.2 Hz, 1H), 5.73 (d, J = 10.2 Hz, 1H), 2.39 (s, 3H). HRMS (ES!): miz [M + H]+
calc. for
C28H20F3N402: 501.1533; found: 501.1534_ H PLC Purity: 98.3%.
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N-(2-Methyl-5-(2-oxo-9-(piperidin-1-yObenzo[h][1,6]naphthyridin-1(2H)-
yl)phenyl)acrylamide
(20)
Prepared using the procedure described for 9a. Compound 20 was further
purified by
preparative TLC eluting with DCM:Me0H (96:4) to afford the title compound as a
yellow solid
(70 mg; 34%).
1H NMR (300 MHz, CDCI3): 58.76 (s, 1H), 8.26(s, 1H), 8.03 ¨ 7.79 (m, 3H), 7.38
¨ 7.27 (m,
2H), 6.97 - 6.90 (m, 2H), 6.62 (d, J = 2.6 Hz, 1H), 6.33¨ 6.26 (m, 2H), 5.67
(t, J = 5.9 Hz,
1H), 2.74 (m, 4H), 2.29 (s, 3H), 1.48 (m, 6H). HRMS (ESI): mfr [M + H]+ calc.
for
CnHnN402: 439.2129; found: 439.2127. HPLC Purity: 98.2%.
N-(2-Methyl-5-(9-morpholino-2-oxobenzo[h][1,6]naphthyridin-1(2H)-
yl)phenyl)acrylamide
(21)
Prepared using the procedure described for 9a. Compound 21 was further
purified by
preparative TLC eluting with DCM:Me0H (95:5) to afford the title compound as a
light-yellow
solid (36 mg; 29%).
1H NMR (300 MHz, CDCI3): 58.80 (s, 1H), 8.17 (s, 1H), 7.97 (t, J= 9.4 Hz, 2H),
7.87 (s, 1H),
7.38¨ 7.28 (m, 2H), 6.99 - 6.92 (m, 2H), 6.64 (d, J = 2.6 Hz, 1H), 6.41 ¨6.26
(m, 2H), 5.70
(m, 1H), 3.68(t, J= 4.8 Hz, 4H), 2.88 ¨ 2.62 (m, 4H), 2.28 (s, 3H). HRMS
(ESI): infr [M +
H]+ calc. for C261-125N403: 441.1921; found: 441.1920. HPLC Purity: 97.0%.
N-(2-Methyl-5-(9-(4-(rnethylsulfonyl)piperazin-1-y1)-2-
oxobenzo[h][1,6]naphthyridin-1(2H)-
yl)phenyl)acrylamide (22)
Prepared using the procedure described for 9a. Compound 22 was obtained as a
yellow
solid (45 mg; 25%).
1H NMR (300 MHz, CDCI3): 58.83 (s, 1H), 8.11 ¨7.95 (m, 4H), 7.35¨ 7.30 (m,
2H), 6.96 (d,
J= 9.3 Hz, 2H), 6.68(d, J = 2.6 Hz, 1H), 6.34-6.30 (m, 2H), 5.71 (m, 1H), 3.29
¨ 3.02 (m,
4H), 2.97 ¨ 2.93 (m, 4H), 2.76 (s, 3H), 2.29 (s, 3H). HRMS (ESI): rn/z [M +
H]+ calc. for
C27F1281\1504S: 518.1857; found: 518.1859_ HPLC Purity: 96.7%.
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N-(5-(9-(4-(Dimethylamino)piperidin-1-y1)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-
y1)-2-
nnethylphenyl)acrylamide (23)
Prepared using the procedure described for 9a. Compound 23 was obtained as a
light-
yellow solid (9 mg; 10%).
1H NMR (300 MHz, CDC13): 58.77 (s, 1H), 8.25 (s, 1H), 8.09 ¨ 7.84 (m, 3H),
7.42 ¨ 7.27 (m,
2H), 6.96 ¨ 6.91 (m, 2H), 6.64 (d, J= 2.6 Hz, 1H), 6.40 ¨ 6.23 (m, 2H), 5.75¨
5.58 (m, 1H),
3.30 (t, J = 12.1 Hz, 2H), 2.43 ¨ 2.29 (m, 3H), 2.29 (s, 3H), 2.26 (s, 6H),
1.70- 1.66 (m, 2H),
1.44¨ 1.38 (m, 2H). HRMS (ESI): mivz [M + H]+ calc. for C29H32N502: 482.2551;
found:
482.2551. HPLC Purity: 99.5%.
N-(2-Methy1-5-(8-(4-(methylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yl)phenyl)acrylamide (24)
Prepared using the procedure described for 9a. Compound 24 was obtained as a
yellow
solid (35 mg; 43%).
1H NMR (300 MHz, De-DMS0): 6 9.98(s, 1H), 9.68 (s, 1H), 9.13(s, 1H), 8.37 ¨
8.23 (m,
2H), 7.83 (d, J= 8.6 Hz, 2H), 7.67 (s, 1H), 7.58 ¨7.41 (m, 2H), 7.29 (d, J=
8.7 Hz, 2H), 7.19
(dd, J= 8.0, 2.2 Hz, 1H), 6.98 (d, J= 9.3 Hz, 1H), 6.88 (d, J= 9.4 Hz, 1H),
6.56 (dd, J=
17.1, 10.1 Hz, 1H), 6.19 (dd, J = 17.0, 2.1 Hz, 1H), 5.74 (d, J = 10.1 Hz,
1H), 3.03 (s, 3H),
2.40 (s, 3H). HRMS (ESI): tniz [M + H]+ calc. for C29H2eN404S: 525.1591; found
525.1591.
HPLC Purity: 95.1%.
N-(3-Methy1-5-(8-(4-(methylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yOphenyl)acrylamide (25)
Prepared using the procedure described for 9a. Compound 25 was further
purified by
preparative TLC eluting with DCM:Me0H (97:3) to afford the title compound as a
yellow solid
(10 mg; 9%).
1H NMR (300 MHz, De-DMS0): 6 10.39 (s, 1H), 9.98 (brs, 1H), 9.14 (s, 1H), 8.31
¨8.28 (m,
2H), 7.84 (d, J = 8A Hz, 2H), 7.74 (s, 1H), 7.59 ¨ 7.54 (m, 2H), 7.28 (d, J =
8.3 Hz, 2H), 7.01
¨ 6.96 (m, 2H), 6.89(d, J= 9.4 Hz, 1H), 6.43 (dd, J= 17.0, 10.0 Hz, 1H), 6.23
(dd, J= 17.0,
2.1 Hz, 1H), 5.76 (dd, 1= 9.9, 2.1 Hz, 1H), 3.03 (s, 3H), 2.37 (s, 3H). HRMS
(ESI): raiz
[M + H]+ calc. for C29H251\1404S: 525.1591; found 525.1595. HPLC Purity:
97.1%.
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N-(3-(8-(4-(Methylsulfonamido)pheny1)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-
yl)phenyl)acrylamide (26)
Prepared using the procedure described for 9a. Compound 26 was obtained as a
light-
yellow solid (20 mg; 18%).
1H NMR (300 MHz, De-DMS0): 6 10.42 (s, 1H), 9.94 (brs, 1H), 9.15 (s, 1H), 8.32
¨ 8.28 (m,
2H), 7.89(d, J= 8.2 Hz, 1H), 7.85 ¨ 7.76 (m, 3H), 7.61 (t, J = 8.1 Hz, 1H),
7.52 (dd, J= 9.3,
2.2 Hz, 1H), 7.29 (d, J= 8.7 Hz, 2H), 7.17 (dd, 3= 7.9, 1.1 Hz, 1H), 6.91 (dd,
J= 9.4, 8.5 Hz,
2H), 6.43 (dd, J = 17.0, 10.0 Hz, 1H), 6.24 (dd, J = 17.0, 2.1 Hz, 1H), 5.77
(dd, J = 9.9,
2.0 Hz, 1H), 3.03 (s, 3H). HRMS (ESI): raiz [AA + H]+ calc. for C28H23N40.45:
511.1435; found:
511.1439. HPLC Purity: 98.0%.
N-(3-(8-(4-(methylsulfonyl)piperazin-1-yI)-2-oxobenzo[h][1,6]naphthyridin-
1(2H)-
yl)phenyl)acrylamide (27)
Prepared using the procedure described for 9a. Compound 27 was obtained as a
light-
yellow solid (10 mg; 6%).
1H NMR (300 MHz, De-DMS0): 6 10.44 (s, 1H), 8.97 (s, 1H), 8.19 (d, J= 9.4 Hz,
1H), 7.86
(m, 1H), 7.74 (t, J= 2.0 Hz, 1H), 7.59 (t, J= 7.9 Hz, 1H), 7.30 (d, J= 2.8 Hz,
1H), 7.12 (ddd,
J = 7.8,2.0, 1.0 Hz, 1H), 6.99 (dd, J = 9.9, 2.9 Hz, 1H), 6.74 (d, J = 9.4 Hz,
1H), 6.64 (d, J =
9.8 Hz, 1H), 6.43 (dd, 1= 16.9, 10.0 Hz, 1H), 6.24 (dd, 1= 17.0,2.1 Hz, 1H),
5.78 (dd, J=
10.0 Hz, 2.0 Hz, 1H), 3.45 (t, J= 5.1 Hz, 4H), 3.19 (t, J= 5.1 Hz, 4H), 2.89
(s, 3H). HRMS
(ES1): rn(z [M + H]+ calc. for C26H2eN5048: 504.1700; found: 504.1703. HPLC
Purity: 96.8%.
Ethyl 4-(3-acrylamido-4-methylphenylamino)-6-(4-
(rnethylsulfonamido)phenyOquinoline-3-
carboxylate (28)
Prepared using the procedure described for 9a. Compound 28 was obtained as a
yellow
solid (10 mg; 10%).
1H NMR (300 MHz, CDC13): 610.59 (s, 1H), 9.20 (s, 1H), 8.05 ¨ 7.93 (m, 1H),
7.85 - 7.75
(m, 3H), 7.46 (s, 1H), 7.23¨ 7.06 (m, 5H), 6.81 (d, J = 7.8 Hz, 1H), 6.47 ¨
6.18 (m, 2H), 5.73
(d, J= 9.6 Hz, 1H), 4.44 (q, J= 7.1 Hz, 2H), 3.01 (s, 3H), 2.33 (s, 3H), 1.45
(t, J= 7.1 Hz,
3H). HRMS (ES1): ink [M + H]+ calc. for C29H29N40e5: 545.1853; found 545.1849.
HPLC
Purity: 95.5%.
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N-(5-(9-Bromo-2-oxobenzo[h][1,6]naphthyridin-1(2H)-y1)-2-
methylphenyl)acrylamide (29)
Prepared using the procedure described for 9a. Compound 29 was obtained as an
off-white
solid (40 mg; 85%).
1H NMR (300 MHz, De-DMS0): 59.68 (s, 1H), 9.15 (s, 1H), 8.29 (d, J= 9.5 Hz,
1H), 7.94 (d,
J= 8.8 Hz, 1H), 7.79 (dd, J= 8.8, 2.1 Hz, 1H), 7.66 (s, 1H), 7.53 (d, J= 8.0
Hz, 1H), 7.19
(dd, J = 8.0, 2.2 Hz, 1H), 6.94(d, J= 9.4 Hz, 1H), 6.84 (d, J = 2.1 Hz, 1H),
6.59 (dd, J =
17.0, 10.1 Hz, 1H), 6.20 (dd, 1= 17.0, 2.1 Hz, 1H), 5.74 (dd, J= 10.0,2.1 Hz,
1H), 2.42 (s,
3H). HRMS (ESI): ire& [M + H]+ calc. for C22HirBrN302: 434.0499; found:
434.0497. HPLC
Purity: 92.2%.
General Procedure J: Alkylation
Methyl 4-(2-methyl-5-(9-(4-(methylsulfonannido)pheny1)-2-
oxobenzo[h][1,6]naphthyridin-
1(2H)-yl)phenylamino)but-2-enoate (13)
To a stirred solution of 8a (73 mg; 0.155 mmol) and K2CO3 (28 mg; 0.202 mmol;
1.3 equiv.)
in DMF (2 nnL) at 0 C, methyl-4-brornocrotonate (28 mg; 0.155 mmol; 1 equiv.)
in DMF
(2 mL) was slowly added over 1 h, and the mixture stirred at 0 C. The reaction
was allowed
to warm to r.t. overnight and after 20 h TLC analysis (5% Me0H in DCM) showed
complete
consumption of starting material. The solvent was evaporated and the crude
partitioned
between Et0Ac and saturated NaHCO3 aq. The phases were separated and the
aqueous
phase further extracted with Et0Ac (2 x). The combined organics were dried
over MgS0.4
and taken to dryness. The crude applied in a silica column and eluted with a
gradient from
100:0 to 97:3 (DCM:Me0H). The desired fractions were collected and taken to
dryness to
afford the title compound as a light-yellow solid (30 mg; 34% yield).
1H NMR (300 MHz, De-DMS0): 69.09 (s, 1H), 8.26 (d, J = 9.5 Hz, 1H), 8.08- 8.00
(m, 2H),
7.48- 7.39 (m, 4H), 7.33 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 6.89
(d, J = 9.4 Hz,
1H), 6.79 - 6.72 (m, 1H), 6.66 (d, J = 2.0 Hz, 1H), 6.48 (dd, J = 7.7, 2.0 Hz,
1H), 6_05 (d, J =
15.7 Hz, 1H), 5.38 (m, 0.5H), 5.28 (brs, 2H), 4.57 (d, J = 4.5 Hz, 1H), 4.42
(dt, J = 14.3, 7.3
Hz, 0.5H), 3.65 (s, 3H), 3.10 (s, 3H), 2.21 (s, 3H). HRMS (ESI): rniz [M + H]+
calc. for
C311-129N405S: 569.1853; found: 569.1853_ HPLC Purity: 99.5% (2 isomers with
1:5.3 ratio).
N-(4-(1-(3-(3-Cyanoallylamino)-4-methylpheny1)-2-oxo-1,2-
dihydrobenzo[h][1,6]naphthyridin-
9-yl)phenyl)methanesulfonamide (12)
Prepared using the procedure described for 13. Compound 12 was obtained as a
pale-
yellow solid (25 mg; 39%).
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1H NMR (300 MHz, De-DMS0): 59.10 (s, 1H), 8.27 (d, J = 9.4 Hz, 1H), 8.08 ¨8.03
(m, 2H),
7.47¨ 7.35 (m, 4H), 7.33 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 7.8 Hz, 2H), 6.89
(dd, J = 9.4, 2.3
Hz, 2H), 6.67 (m, 1H), 6.49 (dd, J= 7.8, 2.3 Hz, 1H), 5.87 (dd, J= 31.2, 13.6
Hz, 0.5H), 5.30
(brs, 2H), 4.64 (d, J= 6.6 Hz, 0.5H), 4.54 (d, 1= 5.0 Hz, 0.5H), 4.26 (dl, J=
13.5, 6.5 Hz,
0.5H), 3.11 (s, 3H), 2.20 (s, 3H). HRMS (ESI): m/z [M + H]+ calc. for
C31H25N503S:
536.1751; found: 536.1751. H PLC Purity: 99.0% (2 isomers with 1:1.65 ratio).
Solubility, Lipophilicity, and PAMPA Permeability
The modifications introduced in the scaffold also sought to improve the
physicochemical
profile of the new analogues. BMX-IN-1 is a lipophilic molecule (cLogP = 3.94)
with limited
water solubility (LogS = -5.98) (see Table 1 below). The removal of the
sulfonamide
aromatic ring rendered compounds with less lipophilic character and also
increased water
solubility. More specifically, analogues 20-23 showed that the introduction of
secondary
cyclic amines is well tolerated and able to reduce cLogP up to 0.7 units and
increase LogS
by 1.6 units, when introduced in the 7-position of the quinolone ring. Most
interestingly,
installation of 1-(methylsulfonyl)piperazine at the 7-position gave compound
27, the
analogue with the best in silk lipophilicity and water solubility profile:
cLogP = 2.32 and
LogS = - 4.36.
Considering that BMX-IN-1 does not have an optimal physicochemical profile, it
was
anticipated that the analogues could have limited membrane permeability. Cell
membrane
permeability is of utmost importance for any drug molecule, even more if a
molecule is
targeting cytoplasmic proteins. For assessment of drug permeability, we relied
on parallel
artificial membrane permeability assay (PAMPA) performed at Pion Inc. The
PAMPA
EvolutionTm instrument was used to determine permeability and we observed that
the vast
majority of the analogues have a high permeability (Table 1).
Once again, the solubilizing motifs introduced in the sulfonamide region led
to an increase in
effective permeability. Based on the values of cLogP calculated for molecules
9-29, it is
believed that the increased permeability may be mostly due to conformational
aspects, such
as intramolecular hydrogen bonding more than cLogP that does not consider
three-
dimensional conformation.
From the data given in Table 2, it can be seen that the analogues with higher
cLogP (14, 16,
19, 28) are the compounds with more limited solubility (LogS), which was also
observed in
the PAMPA assay. In addition, very lipophilic compounds such as 11 and 13
display the
highest permeability rates but also the least lipophilic compounds (21, 22, 26
and 27) display
good permeability rates, therefore reinforcing the hypothesis
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In summary, the modifications introduced did improve the overall profile of
the analogues in
most cases, including compounds 26 and 27.
Colloidal Aggregation
Colloidal aggregation is the major source of false positive readouts in
screening assays. To
rule out unspecific binding of compounds 9-29 to BMX particle sizes were
measured using
dynamic light scattering (DLS). The data shows that, albeit with limited
solubility, the
synthesized compounds do not form aggregates at the relevant inhibitory
concentrations
(see Table 1).
Dynamic light scattering (Zetasizer Nano S. Malvern, UK) was used to determine
compound
colloidal aggregation. The particle sizes were measured at 25 C. A 10 mM stock
solution of
test compound was prepared in DMSO, following dilution with deionized and
filtered water to
obtain an analyte solution of 10 pM (0.1% DMSO). Colloidal aggregation was
measured
through sequential dilutions at 10 pM, 1 pM and 0.1 pM.
Table 1: In silico cLogP and LogS calculation and in vitro artificial membrane
permeability
(PAMPA) and colloidal aggregation (DLS) determination.
PAMPA/ PAMPA/
Compound eLogP LogS
DLS
Pe
%R
9A 3_94
-5.98 Fe: ianEtrol 55+8 = 10
':tAibittiy1130-11
Stairs.caateatIrd:
911 3.88 _5.98 0,1Ftik
sizi 35 7
NAMMiti
9C 3.87 -5.98
11+1 51+6
9D 359 -5.64 ; YU i
48+3
9E 3_60 -5.76
'Ago 55+8 rr,
= k
10 4_19 -6.21 ,
- 33+4
11 4_51 -6.77
s 31+4 54+9
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12 4.06 -6.27 ' 45t3 39 2
13 4.22 -6.43 45*9 , 30 16
14 4_92 -7.04
15 192 _5.98 lozag 17 24 pM
16 4.43 -6./3
3
10 iatel
17 3.98 -5.33 24 13. 10 3 iOiM-
18 3_83 -5.69 ,µ 24j2 18 5
19 4.97 -6.25
20 4.00 -5.52 r 301/42 18 1 = 110.pg
21 3_22 -4.61 , 22,th1, 19 3 1004
22 256 -4.71 1.8th,1µ 19 6 10,144'
'
23 3.72 -5A3 39 5 1OjLM
24 3.96 _5,98 trwri,t7::litip 13th1 1 phil
tinlif 410.
000gbisi
25 4.02 -5.98 -*INA49,6.nr0144.M 10R18 , 0-1 pM
liar6 PtIA
26 3.59 -5.64 ,9 13 8 14 1OLM
27 2.32 -4.36 12,th2 42 14 10 pM
28 4.33 -7.01
29 4.03 -5.32 ,32*4 1 1 10pM'
Artificial Membrane Permeability (PAMPA)
The PAMPA EvolutionTm instrument was used to determine permeability, at Pion
Inc. In
PAMPA, a sandwich is formed such that each composite well is divided into two
chambers,
separated by a 125 pm thick microfilter disc (0.45 pm pores), coated with Pion
GIT-0
phospholipid mixture. The effective permeability, Pe (X 10-6 cm/sec), of each
compound was
measured at pH 6.8 in the donor compartment using low-binding, low UV Prisma
buffer. The
drug-free acceptor compartment was filled with acceptor sink buffer containing
a scavenger
at the start of the test. The proprietary scavenger mimics serum proteins and
blood
circulation, thus creating sink conditions. Aqueous solutions of studied
compounds are
prepared by diluting and thoroughly mixing 3 pL of DMSO stock in 600 pL of
Prisma HT
buffer. Final concentration of organic solvent (DMSO) in aqueous buffer is
0.5% (v/v).
The reference solution is identical to the donor at time zero, so that any
surface adsorption
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effects from the plastic ware is compensated. The PAMPA sandwich was assembled
and
allowed to incubate for about 15 hours. The solutions in the donor compartment
were un-
stirred within duration of the experiment. Thus, the thickness of the aqueous
boundary layer
expected to be about 1,000 pm. The sandwich was then separated, and both the
donor and
receiver compartments were assayed for the amount of drug present by
comparison with the
UV spectrum obtained from reference standards. Mass balance was used to
determine the
amount of material remaining in the membrane filter and on the plastic (%R).
All values are
reported as the average of quadruplicates.
In silico cLog and LogS
cLogP and LogS were calculated using SwissADME software (Daina et at). cLog P
is a
consensus value obtained as the arithmetic mean of five freely available
predictive models
(XLOGP3 (Cheng eta!), VVLOGP (Wildman eta!), MLOGP (Moriguchi et at; Moriguchi
et at), SILICOS-IT and iLOGP (Daina et at) and LogS is the arithmetic mean of
two
topological methods (ESOL model: Delaney et at.; Ali eta!)
PAMPA: Pe is effective permeability (x 10-6 cm/sec) measured directly from
assay at pH 6.8
and %R is membrane retention. All values are reported as the average of
quadruplicates.
Und label refers to compounds with extremely low solubility for which UV
limits were below
the detection limits therefore considered undetected. Compounds were labelled
as high
permeability (green), medium permeability (orange) or low permeability (red).
DLS is
measured at 10 pM, 1 pM and 100 nM. The maximum soluble concentration ¨ at
which no
aggregates are observed - is indicated and the colour code indicates if the
compound forms
aggregates at the ICso concentration (green ¨ no aggregation at ICso
concentration; red -
aggregation at ICso concentration.
Structure-Activity Relationship
The structure-activity relationship (SAR) scoping was directed at establishing
the limitations
of the tool chemotype, elucidating what kind of substituents were tolerated in
each position
and establishing the optimal vector and positioning of different
functionalities.
In order to explore the SAR, the library of compounds was tested in an ICso
enzymatic assay
against recombinant BMX, performed by Eurofins-CEREP (France). The results are
shown
in Figure 1.
We initially focused our modifications in two main regions corresponding to -D
and -R6 in the
compounds of formula (II), and also introduced minor changes region
corresponding to -A- in
the compounds of the invention, for systematic modulation.
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Covalent inhibition of BMX is thought to occur via alkylabon of Cys496, which
is located at
the lip of the ATP binding site. In a stepwise approach, the importance and
tolerability of
different substituents in the regions corresponding to -D and -A- were
investigated, which
regulates the electrophilic attack by the cysteine and other potential
nucleophiles.
The substituents to the cyclic group -A- play an unexpected relevant role for
the activity,
affording different reactivity patterns arising from non-covalent
interactions. Introducing a
strong electron donating group like methoxy (0Me) (9E) as a substituent to a
phenylene
cyclic group decreases potency by 4-fold while the weak electron donating
group methyl has
different effects depending on its positioning about the phenylene ring.
Moving the methyl substituent to the phenylene 6-position abolishes target
inhibition (9C)
while a methyl positioning in the 5-position slightly improved it by 2-fold
(96). Even more
striking is the effect of no substituent in the ring (9D) increasing
inhibition by 6-fold. Since
the electronic influence of the methyl substituents in the different positions
is not expected to
account for these differences, it is believed that a conformational effect may
play an
important role. A 6-substituent may increase the constraints for fitting into
the pocket, while
the removal of the methyl groups affords less spatial restriction.
The binding process is partially regulated by the nucleophilic attack at the
acceptor group,
-M. As such, the modification of the acceptor group was envisioned to
determining if
different electrophiles would influence binding.
The results clearly show that modifications of the acceptor group can lead to
abrogation of
binding. Introducing one (10) or two (11) terminal methyl groups to an alkene
acceptor
decreases inhibition. Also, inverting the acceptor moiety (13), or introducing
a conjugated
alkene and nitrile (12), can limit binding. Therefore, the acceptor group was
not modified in
subsequent scaffolds.
The focus of the research then moved on to the nature of the groups at the -R6
position.
Docking studies with BMX-IN-1 had previously suggested that a major
interaction occurs at
the distal sulfonamide which regulates the interaction with Lys445 (Liu et a/.
ACS Chem.
Blot). To evaluate the importance of this region in the activity of the
molecule, we replaced
the phenyl-containing group at -R6 of BMX-IN-1 with a variety of alternative
aromatic and
non-aromatic substituents.
Compound 14 was prepared in order to understand if this position could be a
good option for
placing a long chain substituent. Clearly, the results showed that a long
substituent is not
tolerated most probably because fitting into the pocket is impaired. Based on
this
information, we explored new substituents that could afford different
interactions.
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Moving the sulfonamide to the meta position of the phenyl ring at the 6-
position (15) only
afforded a 2-fold gain in potency.
To gather further insight into the relevance of the interaction with Lys445
new modalities
were provided at the quinolone 6-position. The carbamate functionality (16) is
related to
amide-ester hybrids as it participates in hydrogen bonding through the
carboxyl group and
the backbone NH. Its ability to modulate inter- and intramolecular
interactions prompted us
to use this functionality, also reinforced by the chemical and proteolytic
stability, and ability to
permeate cell membranes (Ghosh et at).
Following the same principles, the use of 4-pyridine (17) and substituted 3-
pyridines (18
and 19) sought to lower lipophilicity by replacing the aryl ring with a
heterocycle.
Unfortunately, neither the carbamate (16) nor 4-pyridine (17) substituents
provided large
increase in activity (only a 1-fold increase) and the substituted pyridines
(18 and 19)
decreased affinity by 3- and 9-fold.
One of the research goals was to improve the drug likeness of the compounds
for use. With
an eye towards optimization of physicochemical properties it was an aim of the
project to
lower the lipophilic profile of BMX-IN-1 and to improve its limited
solubility. Here,
consideration was given to disruption of possible rr-Tr stacking interactions
due to the high
number of aromatic rings and introduced reduced forms of parent heterocycles,
such as
piperidines and piperazines, aiming to improve the drug profile.
It was observed that the analogues bearing piperidine (20), dimethylamino-
piperidine (23)
and morpholine (21) afforded similar inhibition while the sulfonamide-
piperazine (22)
performed slightly better improving activity by 3-fold. These data suggested
that a major
interaction was not occurring at this protein site and in order to confirm
these observations
compound 29 was prepared.
By virtually removing any group capable of interacting with the Lys445
residue, the binding
was not affected. This observation prompted us to a more thorough assessment
of
alternative regions of the parent molecule. More specifically, the relevance
of the tricyclic
core was assessed by disrupting the central unit. The acceptor group and the
sulfonamide
regions were not modified but the fused pyridinone ring was cleaved leaving a
free ester
functionality (28). The results clearly show that the tricyclic central core
is required since this
analogue lost 7-fold in potency. With this information in hand, we sought to
prepare
compound 24. Since the initial analogues with substituents at the quinoline 6-
position only
improved binding slightly, compounds having substituents at the 7-position
were considered
as alternatives.
A striking improvement of 14-fold in potency clearly showed that the 7-
position is the best
positioning for substituents to the quinolone-containing core. Analogues 25,
26 and 27 were
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also prepared, in which the structural groups from the 6-substituted analogues
were used, to
afford compounds having a preferred overall profile. From the earlier work on
the
6-substituted analogues compounds 9B and 9B were chosen as appropriate
comparators.
These are the compounds with the phenylene cyclic group -A- is unsubstituted
(9D) or is
substituted with methyl at the 4-position (9B). The core has a phenyl
substituent at the
6-position, and that phenyl is itself substituted with a sulfonamide, which
drastically reduces
cLogP while increasing solubility and permeability.
All the new analogues displayed improved potency in comparison to BMX-IN-1 (14-
, 6- and
4-fold, respectively for compounds 25, 26 and 27). Importantly, in order to
obtain a direct
comparison between leads 24-27, all the compounds were tested in the same
assay, once
again using BMX-IN-1 as a control in the experiment and re-testing compound
24. These
results demonstrate that positioning substituents at the quinoline 7-position
opens new
possibilities regarding effective binding in the pocket and potential
conjugation without
affecting activity.
Ligand efficiency (LE) and lipophilic efficiency (LipE) are two important
metrics of
¶druglikeness" which are associated with improved prospects for good drug
properties such
as e.g. bioavailability. These depend on the molecule's activity and
physicochemical
properties and are used as criteria for progression of the most promising
candidates across
drug discovery pipelines (Bembenek et at; Perola; Hann et at).
LE is used to compare binding efficacy of inhibitorslligands relative to their
size while LipE is
used as comparative binding efficacy taking into consideration the
lipophilicity of the
molecules. Given the structural similarity between BMX-IN-1 and analogues 24-
26, only 27
reflects a major improvement in LipE, empowered by the drastic reduction in
cLogP due to
the introduction of an aliphatic amine. On the other hand, the LE improvement
is driven by
the increased potency of all analogues rather than a decrease in the size of
the molecule.
To date, all the reported BMX inhibitors also display the ability to inhibit
Bruton's tyrosine
kinase (BTK). In order to determine if our leads were selective binders of
BMX, we also
evaluated binding against BTK (Eurofins). For the BTK IC50 assay analogues
with higher
BMX inhibitory capacity - 24 and 25- were selected, as well as analogue 27,
which presents
the best LE and LipE improvement (and also offers the possibility of
derivatization).
The results showed that all the compounds are potent BTK inhibitors, in the
low nanomolar
range (Table 2 below). The same inhibitory trend is observed with an increase
of 62-, 33-
and 15-fold potency with 25, 24 and 27, respectively, in comparison to BMX-IN-
1.
Interestingly, BMX-IN-1 displays 7-fold higher ICso against BTK when compared
to BMX.
Consequently, the new analogues offer a greater improvement of LE and LipE
metrics
regarding BTK binding, in comparison to BMX-IN-1.
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Table 2: Biochemical IC60, LE and LipE for compounds BMX-IN-1 and 24-27
against BMX
and BTK.
BMX BTK
BMX BTK
Compound
IC50 Or) IC50 (tW)
(LE/LipE) (LE/LipE)
BMX-IN-1 50.2 = 362 = 0.26/3.36
0.23/2.50
24 7.5 11.1
0.29/4.17 0.29/3.99
25 3.5 5.8
0.30/4.43 0.30/4.22
26 9.1 Nd
0.30/4.45 Nd
27 13.7 24.3
0.30/5.54 0.29/5.29
LE ¨ Ligand efficiency; LipE ¨ Lipophihc efficiency; Nd ¨ not determined
Biochemical Kinase Assay
BMX kinase activity (IC60) was performed at CEREP-France. Briefly, the
inhibition of the
human recombinant Bmx kinase is quantified by measuring the phosphorylation of
the
substrate biotiny1-8A8A13AEEEPQYEEIPIYLELLP using a human recombinant enzyme
expressed in insect cells and the HTRF detection method. The compounds for
testing were
incubated for 60 min at room temperature and the results expressed as a
percent of control
specific activity. BTK kinase activity (IC60) was performed at Discover-X
through a
radiometric assay.
The results are shown in Figure 1_
Differential Scanning Fluorimetrit
DSF, a fluorescence-based thermal shift assay, was used to study His6-BMX
thermal
stabilization upon ligand binding (Niesen et at; Fedorov et at), thus
providing an indication
of the directed interactions between the target-protein and the reported
inhibitors.
The purified recombinant human His6-BMX protein was subjected to thermal
scanning in the
absence and presence of the experimental compounds described herein, and the
protein
melting temperature (Tm) was calculated from the melting curve.
As shown in Table 3, BMX-IN-1 increases the Tm value by 8.04 C. Compounds 11,
12 and
13 showed virtually no changes in the protein melting temperature, suggesting
that a low
affinity or no interaction at all may be occurring, corroborating the results
obtained in the
enzymatic assays.
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Compounds 24 and 27 stabilized the protein increasing Tm by 11.34 C and
10.81 C, once
more suggesting the direct binding of 24 and 27 to BMX with higher affinity.
Table 3: Melting temperature (Tin) shift calculated with a DSF assay
in .1.1õ4.4tett... v.". 4
, yõ,t4 111 ++. t Vt." r = ID rttatt
p 1 a 44 L-R = r ,
p r , 7 `t.-1 4,. i= 4 r frip tictiffSttffr r, kt, p.<
' ,C= 6iev "SlEr 34-71L-c,.. 4 -45riµi 0.4 174-r
crt, triCo JariffeChira "..40t-S. 414 *144. tiENNO
ifteitagnaptiSinfitt, gan 1:11,74.etirACIN Alidt
F71011 'j'nfirtiltirfartAtiki,tifirk4 ..-µ154L
, trt tiro.
t4, k1/4-41"0.i4 41-1,,tv *v. 4:01, p 4,4A IA <==',g
a a len
BMX-IN-1 6017 032 52
13+0 11 8.04 032
,
9C 5412+0_06 51 92+0 31
220 0.06
J X ^
9E 5797+0_01 51 92+0 31
.&05 0.O1
11 52.26+0.11 51.92+0.31
034+0.11
an. aiii "
13 52.44 0.06 51 .92 0 31
0.52 0.06
Pik:4 A ag..11.1 AtIkhe.1.
tjtek.o....7 0,40)44:1.1,1*
*qs:i6wptoRcalerAit
0144,:/ VP; tr
Al 1 11:4,,,...)it-P
15 60 .72 02 8
51 .93 0 22 8.79 0.28
. .
. . .
41 ;et' 4
tc.
1õ-mn= tt 'err
ArArVibt"-f Y-1/ Orthr
}if*"/c* iiNtriMZIketigrev4
VI
tgo. its % .1"V' .. Ar
,
17 59_55 01131
51.931-022 7 62 0 01
s,141/ !4,4c4"4151r;i " v vii4f " ' pp4s:r6tri=
ti-z.tact 700 (4 (AM - .1E-01
14 4.t.1..4,*...-tbetv.,.
'43u1414,Cattittatie4.;441. 011414,aarrN4C->N4Z.Azi,A.
Iric..44ak44.10.,Appet:NAokoLs.:Kre
silit chs", to,
...ne fit ecni r4kra 0,041vo si-tySori". ..1.904srvi ;Fr:
19 58 .64 0 .13
51 .82 0 .19 6.82 0.13
, :4"5(.1 = 6 ir d, tch
A = rt
, n
r 1 ran 40.14424:1141:14
evis",õ
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¨89¨
.21 60.50+0.06 51.93+022
8.57+01)6
' = s = twee eliktforir)'irty',.,e- psi
int; r I is
= ;iezilet
IlrY h,
PACIOV; nolo, vki,
, e ''^fy ii4Sa a
23 60.5910_11 51.931-022
8.661LL11
25 61.43+0.48 52.13+0.11
9.3010.48
,
õ
27 62_941-0_06 52.13i-0_11
10_81+01)6
DSF was performed in MicroAmpTm EnduraPlateT" Optical 96-Well Clear Reaction
Plates
with Barcode (Applied Biosystems, Life Technologies, California, USA) using a
QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Pre-incubation
of the
protein with the compound for 2 hours at 4 C was required prior to DSF
experiments. The
final reaction mixture (20 pL of total volume) contained 4 pg of His6-BMX, 4-
fold of Protein
Thermal Shift"' Dye (Applied Biosystems) diluted in protein buffer solution,
and 100 pM of
compound. The temperature was increased from 25 C to 90 C with an increment
rate of
0.016 C/s. Excitation and emissions filters were applied for Protein Thermal
Shift Tm Dye
(470 nm and 520 nm, respectively) and for ROX reference dye (580 nm and 623
nm,
respectively). The melting temperatures were obtained by taking the midpoint
of each
transition.
Surface Plasmon Resonance
The putative interaction of the synthetic compounds with BMX in real time was
analysed by
using Surface Plasmon Resonance (SPR). SPR is a sensitive spectroscopic method
that
can be used as a primary tool to screen interacting molecules or as a
validation tool for
interactions previously identified by other methods () [55].
To validate the HTRF and DSF results reported here, compounds that showed a
significant
potency gain or loss (fold) against control BMX-IN-1 were injected at
different concentrations
on BMX immobilized surfaces (sensorgrams not shown). As expected, BMX-IN-1 was
shown to interact with BMX with high affinity (Ku = 69 nM) as observed in the
biochemical
assay. The results show that compounds 9C, 10 and 12 interact transiently with
the target,
with fast association and dissociation kinetics, making it possible to
calculate only the affinity
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at steady state (Kass). This observation is in accordance with the lack of
inhibitory capacity
observed in the enzymatic assay and it reinforces the hypothesis that steric
hindrance is
hampering the protein-compound association
The enzymatic assay results are also supported by the kinetic evaluation of 9D
and 9E
interaction with BMX. One compound, 90, shows a K0 value in the 1-digit
nanonnolar range
whilst compound 9E has lower affinity than the control BMX-IN-1 (69 nM).
Higher affinity interactions were observed, as expected, for compounds 24 to
27, showing
comparable association rates (Ken from 5.4 x 104 to 1. 4 x
105 M-1s-') but, most
importantly, very slow dissociation rates (Kon, < 1 x 10-4 s1), in agreement
with the covalent
nature of the interaction.
Table 4: Kinetic Constants Calculates from Surface Plasmon Resonance
_____________________________________________ Erceefflarti
mait WatiF:Ztrir
1Z4- , ' :414.01fr:õ.
a.tifietI
3 1-9010.11-440froo
=
Mem,' eAsittiAltetsb47/1- 14Pg&VRWIR7k ,detiprecepON
BMX-IN-1 Nd 74x 101 5_10 x 10-4
6.9 x 10-n
= 4s4 NV*
90 Nd 1.4 x 104 < 1 x 104
< 1 x 108
r itta .µ /ref "Rag EA 114.: 3 AY' .44 44+45kt -JAN:
1, atv 0,110,0
goipht istitiiipaspt ghtip-ropto-ws
4 itteaki4444.- ACIrerVitV-
11,401te erti40 41P el- Fi?"30611.4 41,P 4
µ. I" '44m, -5 rt s ?hi
10 3.9 x 10-b Nd Nd Nd*
, A
24 Nd 1.4 x 10i < 1 x 104 Nd*
26 Nd 7.2 x 104 < 1 x 10-4 Nd*
z4,`-g ry =
ISPArir ilkk'
. = tr'..gsv
riZ4 4
Nd ¨ not determined; *Immeasurable KD due to very prolonged off-rates (outside
instrument
specification).
SPR Experiments were carried out in a Biacore 4000 instrument (Biacore AB, GE
Healthcare Life Sciences, Uppsala, Sweden) at 25 C. His6-BMX protein was
diluted to
10 pg/nriL in sodium acetate pH 5.5, in the presence of 5 pM staurosporine and
immobilized
onto CM5 (Series S) sensor chips, using the standard amine coupling procedure.
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Prior to immobilization, the carboxymethylated surface of the chip was
activated with
400 mM 1-ethyl-3-(3- dimethylaminopropy1)-carbodiimide and 100 mM N-
hydroxysuccinirnide
for 10 min. FIBS-N (10 nriM HEPES pH 7.4, 150 nriM NaCI) was used as the
background
buffer. Protein was coupled to the surface after 2 to 10 min injection times,
at a flow rate of
pL/min, in order to reach 1,500 to 3,500 response units (RU). The remaining
activated
carboxymethylated groups were blocked with a 7 min injection of 1 M
ethanolamine pH 8.5.
Compounds were pre-diluted in DMSO to 50 times the desired highest tested
concentration
10 and diluted afterwards in running buffer (20 rriM HEPES pH 7.4, 150 mM
NaC1, 1 mM DTT,
0.1 mM EGTA, 0.05 % (v/v) TWEEN-20, 5 mM MgCl2) in order to reach 2% of DMSO
concentration. A DMSO solvent correction (1%-3%) was performed to account for
variations
in bulk signal and to achieve high-quality data.
Each compound was injected over immobilized HiserBMX for 220 $ (30 pL min-1;
association
phase) followed by 600 to 2,000 s of buffer flow (dissociation phase) at a
maximum
concentration of 0.5 pM or 10 pM for high and low affinity binders,
respectively, and diluted
five times in 2-fold dilution series. All sensorgrams were processed by first
subtracting the
binding response recorded from the control surface (reference spot), followed
by subtracting
the buffer blank injection from the reaction spot. All datasets were fit to a
simple 1:1
Langmuir interaction model with the provided Biacore 4000 Evaluation software,
to
determine kinetic rate constants (k, kofF) or steady-state affinity (Koss).
Native Mass Spectrometric Analysis
In order to further confirm the covalent binding mode between the protein and
the ligand(s),
a mass spectrometry study was performed using compound 24 as probe. The
truncated
human BMX was analysed by native MS and the protein mass found was 30899 Da
(see
Figure 2(A)).
The protein was then treated with 24 and directly analysed by denaturating MS
(as detailed
further below). The mass found upon incubation with 24 is 31,424 Da which is
525 Da larger
than the apo-form of hBMX (Figure 2(B)). This result suggests covalent
conjugation of a
single molecule of 24 to hBMX. Furthermore, proteomics analysis of drug
conjugated hBMX
indicates the drug covalently interacts with the cysteine residue at position
496 (Figure 2(C)).
For native MS analysis, a protein sample was buffer exchanged to 200 mM
ammonium
acetate, pH7.6 and analyzed on a modified Q-exactive hybrid quadrupole-
Orbitrap mass
spectrometer (Thermo Fisher Scientific) [69] using gold-coated glass needles
(Hernandez et
at). Typical native MS settings are a source fragmentation voltage of 50 V and
capillary
temperature of 30 C. Denaturing MS analysis of drug conjugated protein was
performed by
liquid chromatography-MS (LC-MS) using a Dionex UltiMate 3000 RSLC Nano system
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coupled with a LTC) Orbitrap XL hybrid ion trap-Orbih-ap spectrometer (Thermo
Fisher
Scientific). The protein sample was directly loaded onto a C18 trap cartridge
(Acclaim
PepMap100, C18, 1 mm x 5 mm Thermo Scientific), desalted with 100% buffer A
(100%
H20 and 0.1% formic acid) at a flow rate of 10 pUmin for 10 min, eluted and
separated onto
a C18 column (Acclaim PepMap100, C18, 75 pm x 15 cm, Thermo Scientific) with a
linear
gradient from 0% to 100% buffer B (50% isopropanol, 45% acetonitrile, 5% H20
and
0.1% formic acid) at a flow rate of 300 nUmin in 50 min.
Typical MS conditions were a spray voltage of 1.8 kV and capillary temperature
of 300 C.
The LTQ-Orbitrap XL was set up in positive ion mode with ion trap scanning
(m/z 335-2000).
The proteomics analysis of drug conjugated protein was performed on the same
LC-MS
system with minor changes. Tryptic digested peptides were loaded to a C18 trap
cartridge,
desalted with 100% buffer A (100% H20 and 0.1% formic acid) at 20 pl/min for 5
min and
separated on a C18 analytical column with a linear gradient from 0% to 60%
buffer B (80%
acetonitrile, 20% H20 and 0.1% formic acid) at flow rate of 300 nUmin.
LTQ-XL was operated in data-dependent acquisition mode with one full MS scan
followed by
5 MS/MS scans with collision-induced dissociation. For full MS scan, the mass
range was
set to 335 to 2,000 m/z at a resolution of 60,000. For tandem MS scan, the CID
normalized
energy was 35%.
Crystallization and Structure Determination of BMX in Complex with Inhibitor
To further characterize the inhibition mechanism and binding mode of the
compounds
described herein, a variety of commercial crystallization screens were tested
in order to
obtain a protein crystal suitable for X-ray diffraction.
Crystals were grown through co-crystallization of BMX protein with the
inhibitor 24 (details
given below). The X-ray crystal structure of BMX in complex with inhibitor 24
was
determined to 2.0 A resolution, with a well-defined electron density map
around the BMX
ATP binding pocket where the inhibitor is bound. The values of the equivalent
isotropic
atomic displacement parameters for the ligand atoms within the pocket are
comparable to
those of the protein atoms they are interacting with, an indication of full
ligand occupancy of
the binding site. Not surprisingly, an increase is observed in the sulfonamide
aromatic ring,
since this group is more exposed to the solvent and hence more mobile.
The crystal structure shows the expected covalent binding between the
acrylamide warhead
and Cys496 see (see Figure 3(A)). Other major interactions of the inhibitor
with the enzyme
active site are mediated through polar non-bonding interactions between the
nitrogen in the
quinoline ring and 11e492 and quite unexpectedly between Lys445 and the oxygen
located in
the fused pyridinone ring (see Figure 3(B)).
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Contrary to what was proposed by the docking studies with BMX-IN-1, the
interaction with
Lys445 is not observed at the sulfonamide group provided on the phenyl
substituent to the
core, but at the tricyclic quinoline core itself.
The polar interaction between 24 and Lys445 is actually one of the key points
to regulate
BMX activity. The conserved In Lys interacts with aC-helix Glu residue in
order to form a
salt bridge required for ATP catalysis. The binding of 24 to Lys445 alters
this interaction
between the in Lys and the aC-helix Glu and consequently inactivates BMX.
Other
hydrophobic interactions occur between the aromatic rings of 24 and the side
chains of
Tyr491, Ala443, Va1431, and Leu543 (data not shown). Compound 24 is further
stabilized
by a hydrogen bond between a water molecule and the carbonyl oxygen of the
acrylamide
group. A second water molecule stabilizes the first via a hydrogen bond, and
forms
hydrogen bonds with the peptide nitrogen of Cys496 and the terminal amine
group of
Asn499.
The crystal structure also shows that the DFG-motif adopts an out-like
conformation
(Figure 3(C)), where the Asp554 side chain is positioned in the back cleft,
away from the
ATP binding pocket, and the Phe555 aromatic ring points up into the gatekeeper
region
blocking the /33 Lys445-aC Glu460 ion pair formation. Both the activation loop
and the DFG-
out-like conformation are similar to what is observed in the only reported BMX
crystal
structure with the non-covalent inhibitors Dasatinib and PP2 (Muckelbauer et
at). The
positioning of the BMX DFG-motif is reminiscent of an inactive conformation or
DFG-out,
typically found in BTK and other kinases inactive structures (Sultan at at),
and it is also
commonly observed in type II inhibitor complexes (Zhao et at) [58] (data not
shown).
The positioning of the sulfonamide-substituted phenyl ring is also of outmost
importance.
Contrary to the docking results with BMX-IN-1, this group is not interacting
with any
important residue and it is in fact pointing out of the ATP pocket (see Figure
3(D)). This
observation is of critical importance because it allows for the introduction
of a linker or
chemical handle in this region of the molecule. Since it is not sterically
hindered by other
residues, one can expect the linker or handle to remain outside the pocket,
and therefore not
to significantly influence the inhibitor binding capacity.
The guidelines for plasmid construction and vector cloning are described by
Muckelbauer et a/. The expression of BMX protein using Sf-9 cells, as well as
the
purification process, were optimized to improve sample quality at the end of
purification, in
order to increase the likelihood of protein crystallization (manuscript in
preparation). The
purified BMX tyrosine kinase was concentrated to a final concentration of 10
mg/mL
(according to Muckelbauer et at) and pre-incubated for 2 hours at 20 C with a
2-fold
concentration of the inhibitor 24.
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The trials were carded out using the sitting-drop vapor diffusion method on
the Mosquito
LCP crystallization robot (TP Labtech Ltd, Hertfordshire, UK). The drops
consisted of
0.150 pL of the reservoir solution mixed with an equal volume of the protein
sample,
equilibrated against a 45 pL reservoir. The crystals appeared after 2 days in
a lead
condition consisting of 0.2 M imidazole-malate buffer, pH 5.5, with 42% v/v
PEG 600_
The better-shaped crystals were analyzed at the European Synchrotron Radiation
Facility
(ESRF) in Grenoble, France. An X-ray diffraction data set to 2.0 A was
collected at ESRF
beamline ID30A-3 with a Dectris EIGER X 4M detector from a cry0000led crystal
at 100 K.
The diffraction data were processed with AutoPROC and XDS (RCiegg et at). Two
diffraction datasets were obtained: in the first, a spherical region of
reciprocal space to 2.2 A
resolution was defined, and in the second a triaxial ellipsoidal region to a
maximal resolution
of 1.95 A was selected with the STARANISO module of AutoPROC (Riiegg et at).
The
structure of hBMX in complex with ligand 24 was determined by molecular
replacement with
PHASER (Vonrhein et at)as implemented in the CCP4 program suite (McCoy et at;
Potterton et at) using the PDB entry 3SXS (Muckelbauer et at) as a search
model, without
including ligands and water molecules.
Two independent copies of the search model were located in the crystal
structure, and
model rebuilding was carded out with BUCANEER (Pofterton et at) and COOT
(Cowtan).
Initial structure refinement was undertaken with REFMAC (Emsley et at). The
stereochemical restraint dictionary for ligand 24 was created with JUGAND
(Murshudov
et at) and the ligand was manually fitted into the electron density using
COOT. Refinement
was continued with PHENIX (Lebedev et at), alternating with manual model
editing in COOT
between refinements against sA-weighted 21Fol-IFcl and IFol-IFcl electron
density maps. In
the final refinement cycles, hydrogen atoms were added and refined in
calculated positions,
Translation-Libration-Screw rigid-body anisotropic atomic displacement
parameters were
refined, water molecules added automatically and the relative weights between
the
crystallographic and stereochemical energy terms optimized.
Each BMX molecule was divided into 4 rigid-body segments, estimated from the
TLSMD
server (Adams et at) using the isotropic atomic displacement parameters from a
previous
refinement run. The final refinement was carried out to 2.0 A against the
STARANISO
dataset. Figures were prepared with PYMOL (Painter et at).
Kinase Selectivity
As mentioned previously, most of the BMX inhibitors reported to date offer
poor selectivity
since they are both BMX and BTK inhibitors. Their cellular effect is often
attributed to off-
target activity either upstream or downstream of BMX signalling pathways. In
order to
investigate in which targets our new analogues could have an effect, we tested
the most
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potent analogue (25) against a panel of 36 BMX-related kinases in the Eurofins
DiscoveRx's
KINOMEscan platform at the concentration of 1 pM.
From the wide number of accessible cysteines residues distributed across the
whole kinonne
not all are available for covalent modification [11-13]. BMX belongs to a
restricted group
including other 10 kinases that share an equivalently placed cysteine in the
ATP binding
pocket. This group comprises members from the TEC family (BTK, ITK, TXK and
TEC), the
EGFR family (EGFR, Her2, Her4), JAK3, BLK and dual specificity nnitogen-
activated protein
kinase kinase 7 (MAP2K7)_ Therefore, the TEC, EGFR and JAK families were
included in
the screening, as well as the Src family and Lkb1, which also have a cysteine
within the
same sequence alignment Also included were kinases involved in upstream (Sic,
FAK,
PI3K, nnTOR, PDK1) and downstream (Akt, PAK1, TAM) regulation of BMX
signalling
pathway and nonreceptor tyrosine protein kinase Abl. The KinomeScan platform
is a binding
assay and the screening showed that compound 25 shows strong binding affinity
against all
the members of TEC family that share an equivalently placed cysteine and
within these,
higher affinity is observed towards BMX, BTK and TEC (see Table 5 below).
Table 5: Kinase Selectivity of Compound 25 using KinomeScan Technology.
rirratalgr.414-,;11 v. Nµfir474-40- rahtfa4 I
,
t
*
fairna;tixar,14101-0-the = taam, , A cy.,,citple410/4
BMX
1.3
,
.
TEC
ITK 4.7
DIrafax:404iiica,
_______________________________________________________________________________
___________ ' T :
all Net
TXK
3.4
;maw q : :0 1 Alt tri
r
ale? MOPS 1 s
1
ergagOOP10400=ERBB2
89
i 404440.Mitaro r=S
1,1k* ;,.. :etc Vs tronklyti
fraffeaStd k r ,
e latypo, .1 fr*
Nagferotaft
r vz of,topirig
noir 4nAltw.....
altartAA , & . s - ii
tianteauts
ERBIM 66
tssimainiajitonrAN-
kly,tat/P*40.
t4,.. ,,,= ;4.,
r :4-iii-ro': ,aa ',' -''' I' kik 41,,,Q,
Paw"
1,,,,,VS:tAtchiAlitig al '444"..4434avdielttkektqre ir,,w,N.,04-4:4 bue...,,k.
rtN, =
3/44.4imaitniai1/441;w514Yet ."1- v>, QiitfAcTevtitry..e,44404.-.4 44
ei,,,,,,..wievil Atli
ti-Apeapayee.>449404 t i ' - - a -., ,- - - i .. , ,, -
µ4. = s , . , J.
Pii9a1,73071,06ay JAK2(JH1dornain-catalytic) 81
talettirOWRWW4
IT, .7';','"e41/2640401e
Allikeneiratilaft, -
4140.54-pleAvb int417 ,
livtrAti& \I:ft;
Wi. toitv.40.!
ifitiliatal, ., 1
, once law,"
10 -, rotor 241inis TYK2WH1domain-
catalytic) 100
, oti-,..)4efio 0%A.-1/44 9.1
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i
pri-Prz.74-474,7A74..Fensys)
ittersle, r =:., õ '<, .fr ;Sir - ,
jiaing, ,P0Ae4...%*phingvie SRC 92
q=-i014,940-clifreftekiti
:Ã,A wovivii0/14.4441.-74 '1
;1fentv01:kokr=41*-ei
vitt,1,õ ,,,,,iii , -õ,4 ..õ i- =03,1V4 =
gls- [1:-VadVitiAti k -4.µ`.
pie46/9111-getreer"."%rd.0
Ipastotv amp
r ,440,1000N4100,4 -., BLK 16
it-'474,04***49trivi ,
4.6)fr..4, . Irr.s. Tweiiti j'it- i 'n241 ,
i ir st.rn=ll - Vat itneph-04eamj 4 .
''',.1,ARtrattailepterMatiN
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I i74. 41141411;d4444.0W1 :
c itike.13tkiarYN'n tpetKVIT c .., -1 4,1 th h
ViciyitAt it :SAWaVintip LCK 80
irim At3b-,Skipmattc3/434õ
fAtArc iisay-Mitry riles
,datreirutvivrammilermaNi
, Ras" ,.Ã1, Awe isa= 441c1,--#. ,
Pnktsttleseq44:3 vzifii- 0mi:it .
iit.orp,estd 1,41,
_NAr Vir= 1
'''itN141-4. VONA tiht, +5,40µ 3 11 .4' 7
N.. ' . . ' ' - 7; = ..at t LYN 100
44 "1E iwctiil- hffikkee
W1.44,0,14 a lithfl. e{
,N IP. V ti:,ri=== ;21, {,( r 11/41f e 7Mli
gl
0
PIK3CA 79
,
is
PI3K
PIK3CG
64
,
49,100.r.tuto04,.eattaVienrate kt)'
mTOR
MTOR 100
-
, r,
AKT1
100
, Pith
r
,., ________________________________________________________ =
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.,
, 1 Abl
ABLI-phosphorylated 100
r
, =
,
The results for primary screen binding interactions at 1 pM concentration are
reported as %
DMSO control.
As stated above, the TEC family has high sequence similarity and in particular
residues in
the ATP binding kinase domain share 40-65% identity and 60-80% similarity. The
ATP
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binding sites are also highly conserved between the TEC and Src families with
14 identical
residues out of 18 that comprise the ATP binding pocket. More specifically,
BMX shares a
57% similarity to Src and most importantly, one of the key determinants of
kinase selectivity -
the gatekeeper residue - is a Thr in both the Src family and the TEC family
members except
ITK [56]. It is therefore not surprising that 25 also binds Blk (and JAK3)
while no affinity was
observed with other targets. These results show that compound 25 is a good
probe molecule
for TEC kinases and suggest that any cellular activity mediated by 25 is
probably due to
inhibition of any of the TEC kinases rather than any off-target inhibition of
upstream and
downstream BMX regulator&
Cellular Activity Assay
LNCaP and PC-3 Cell Growth Assay
The role of BMX in different pathologies is not yet fully validated.
Notwithstanding, it has
been implicated in many regulatory mechanisms and despite the absence of a BMX
dependent disease model, prostate cancer cell lines have been used to evaluate
inhibitors
anti-proliferative effects in a cellular context The activation of BMX in
response to PI3K
signalling is just one of the mechanisms through which the levels of BMX
became increased
in prostate cancer (Chau etal.; Guo et (V.).
To determine the effect of the most potent analogues in prostate cancer cells,
the ability of
compounds 24-27 to inhibit the proliferation of LNCaP and PC-3 prostate cancer
cell
Lines was tested, using CellTiter-Glow .
The androgen-receptor negative PC-3 cells are resistant to the treatment, with
no significant
anti-proliferative effect at the maximum concentration tested (10 pM). In
LNCaP - androgen-
receptor positive cells a different profile was observed. BMX-IN-1 and 24
showed a GI50 of
1.4 pM and 2.8 pM, respectively. Compound 27 was the least active (G150 10 pM)
while 25
and 26 showed a Glso around 5 pM, as shown in Table 6.
Table 6: Antiproliferative activity of compounds BMX-IN-1 and 24-27 against
LNCaP and
PC-3 prostate cancer cell lines.
Cell Line BMX-IN-1 24
25 26 27
LNCaP 1.7 0.7 1.5 2.3
6.6 2.3 7.7 1.4 9.3 0.4
PC-3 11.5 2.2 11.4 4.1
12.1 0.7 10.1 1.3 Nd
Proliferation in LNCaP and PC-3 was measured following 96 h incubation with
the drugs. GI50
values are reported in pM and are the mean of three individual experiments
performed in triplicate.
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Cells were seeded in white, opaque-bottom 96-well plates at 5,000 cells/well
(LNCaP) or
2000 cells/well (PC-3) in a total volume of 100 pL of culture media. Serial
diluted
compounds (2-fold) in 100 pL media were added to the cells 24 hours later.
After 96 hours
incubation cellular viability was assessed by CellTiter-Glo (Promega)
according to the
manufacturers instructions. The values were normalized to vehicle and IC50 was
calculated
using GraphPad Prism software.
Propidiunn Iodide assay
In order to determine whether the growth inhibition was due to apoptosis, we
carried out
Fluorescence assisted cell sorting (FACS) analysis, using Propidium Iodide
staining (PI).
LNCaP cells were incubated with BMX-IN-1 and 24-27 for 64h, at 10 pM and
results showed
that no marked differences were observed in the percentage of necrotic events
when
compared to the vehicle control, showing that in these conditions, these
analogues do not
cause increased cell death (see Figure 4).
It is not surprising that all the compounds show a low proliferation
inhibitory potential in
prostate cancer cell lines and it remains questionable whether modulation of
BMX alone is
relevant or not towards anti-proliferative effects (Price et al.). In fact, a
large body of
evidence is present in the literature showing that selective or dual BMXJBTK
inhibitors have
poor anti-proliferative effects in BMX-dependent models, most probably from
dynamic
compensation of signalling mechanisms. Focus has been made on the modulation
of BMX
activity to sensitize cells to others therapeutic agents since anti-
proliferative effects are only
observed in combination with inhibitors of related pathways (Fox etal.; Fox
etal.).
BMX-IN-1 against RV-1 cells could only be potentiated with the Akt inhibitor
MK2206 (Liu et
al.); another inhibitor, ABT-737 only induces apoptosis upon co-treatment with
PI3K
inhibitors (Li et al.); the dual BMX/BTK inhibitor CTNO6 requires a co-
treatment with the
autophagy inhibitor chloroquine (CC) or docetaxel to inhibit PC-3 cells growth
(Guo et at)
and a similar profile is observed with the dual BMXJSrc inhibitor CTA095
synergizing with
CQ and paditaxel (Guo et at). Available data shows that BMX is a key
regulatory protein but
not an effector and the combinatorial approach is believed to be the most
effective.
Consequently, this collection of compounds can become useful molecules for
combinatorial
treatments.
LNCaP cells were seeded in 24 well-plates at 8,000 cells/well in in a total
volume of 500 pL
of culture media and incubated for 24 hours to allow for attachment. After
this time, 5 pM of
each compound diluted in culture medium was added to the cells. After 64 hours
of
treatment, cells were harvested after trypsinization (TrypLE Express,
LifeTechnologies,
USA) into round-bottom FACs tubes, and washed with 10 % FBS in PBS. Cells were
then
re-suspended in 5 pWmL of propidium iodide diluted in wash buffer and analyzed
directly
after 15 minutes using an LSR Fortessa X-20 flow cytometer equipped with a 488
nm laser
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and a 670LP and 695/40BP combination of filters. The results for compounds 24-
27 and
BMX-IN-1 are shown in Figure 4 as percentages of controls (i.e. vehicles) and
represent
average SD (of triplicates).
Additional Data
irreversible binding efficacy relative to BMX-IN-1
The inactivation of BMX occurs in a two-step process that is governed by two
parameters:
the affinity of the initial non-covalent binding, KI, and the rate of the
subsequent covalent
bond-forming reaction with the thiol of the cysteine residue, kinact. The rate
of inactivation
(kinact/KI) is second-order, which describes the efficiency of covalent bond
formation.
Therefore, we evaluated the irreversible binding efficiency of our rationally
designed
compounds. The kinetic analysis is presented in Table 7, reveals that compound
25 exhibits
the best binding fit with the target, with a binding affinity of 323 pM. This
represents an
increase in excess of 10-fold relative to BMX-IN-1 (KI: 4.07 nM). The other
leads, display a
similar binding affinity among themselves (1.93-2.52 nM), lower than 25 and
approximately
2-fold higher than BMX-IN-1. However, the rate of covalent bond formation of
the bound
inhibitor (determined by kKinact) shows that compounds 24, 25, and 26 showed
slightly
improved efficiency (0.335, 0.378 and 0.443 min-1, respectively) in comparison
to BMX-IN-1
(0.217 min-1) and 27 (0.166 min-1). Consequently, the irreversible binding
efficiency of 25
(19.4 pM-15-1) is the highest of the series, whereas BMX-IN-1 shows the lowest
result
(0.89 pM-15-1) relative to the remaining inhibitors. Overall, these results
provide quantitative
evidence that the improved activity is mostly driven by changes in the binding
complementarity between the compound and target rather than faster rate of
covalent
binding. Thus, taking into account that all the analogues have the same
Michael acceptor
moiety, the enhanced activity must be a result of the structural modifications
introduced in
the scaffold.
Table 7. Determination of the kinetic parameters K1, kinact, kinactilca
Compound Ki [nM]
kinact (min-l] kinacal [pM-1s-1]
2513 0.32 0.05
0.378 0.034 19.4 1.55
26b 1.93 0.18
0.443 0.003 3.86 0.34
24 2.52 0.01
0.335 t 0.001 2.22 0.01
27 2.15 0.13
0.166 0.003 1.29 0.10
BMX-IN-1 4.07 0.06
0.217 0.005 0.89 0.20c
aResults tested in duplicate, showing mean S.D..
bResults obtained from two independent studies, showing mean S.D..
Value with a 0.06 uM-'s-' deviation from published results. (Wang at at).
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lntracellular BMX inhibition
To validate target affinity and identification for 25, we performed an
intracellular target
engagement kinase assay with HEK293 cells expressing NanoLuc -BMX fusion
vector with
Promega's NanoBRET1u TE Intracellular Kinase Assay. The cell proliferation
depends on
BMX kinase activity that was used to monitor the cellular activity of the
compounds (IC50). As
shown in Table 8, the IC50 determination showed the inhibitory capacity of 25
(IC50: 44.8 nM)
is 10 times greater than BMX-IN-1 (IC50: 495 nM), which aligns with the
previous
observations of an increased biochemical potency with similar activity
difference.
Assay performed at Reaction Biology Corporation (USA), with concentrations
tested in
duplicate, showing mean S.D. Cells were treated for 1h and IC50 values were
calculated
and plotted by using GraphPad Prism 8 based on a sigmoidal dose response
curve.
Table 8: Intracellular target engagement in HEK293 cells transiently
transfected with BMX
expressing NanoLuca-BMX.
Compound
IC5c, (nM)
BMX-IN-1
495 35.7
44.8 5.4
In-cell target engagement was performed at the Reaction Biology Corporation
(USA) using
the NanoBRETTm technology. Very briefly, HEK296 cells, purchased from ATCC,
were
transfected with BMX and treated in duplicate with test compounds. BMX-IN-1 or
JS25, and
with the reference compound Dasatinib, for 1 hour of incubation. Compounds
were diluted
10 times with 3-fold dilution, starting at 1 pM. Curve fits were performed
only when %
NanoBret signal at the highest concentration of compounds was less than 55%.
The IC50
values were determined using the GraphPad Prism 8 (USA).
Cancer cell growth inhibition by BMX inhibitors.
The role of BMX in different pathologies is not yet fully validated.
Nevertheless, it has been
implicated in many regulatory mechanisms and despite the absence of a BMX
dependent
disease model, prostate cancer cell lines have been used to evaluate anti-
proliferative
effects of the inhibitors in a cellular context. In a previous experiment
(unpublished results)
we screened several inhibitors in a collection of cell lines representing
prostate, brain, blood,
breast, ovary, lung, bone marrow and lymphoid tumour tissues. Compounds were
incubated
with cells for 72 h in a 386 well-plate format to monitor dose-dependent
impact on viable cell
growth by using the CellTiter-Glo luminescent assay, which quantifies ATP and
the
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presence of metabolically active cells. The study included 24, BMX-IN-1 and
the structurally
similar compounds 10 and 11 which do not bind to BMX (Figure 1).
The results presented in Table 9 show that 10 and 11 (non-binders) have little
or no effect on
viable cell growth of the majority of the tested cell lines. BMX-IN-1
demonstrated more
potent inhibitory effects relative to 24 in the four prostate cancer cell
lines that were included
in the panel, 22RV1, PC3, LNCaP and 0U145, particularly in those dependent on
androgen
receptor signaling (LNCaP and 22RV1). In contrast androgen receptor negative
cells
(DU145 and PC3) were overall more resistant to treatment In addition, 24
showed potent
inhibitory effects against LNCaP and 22RV1 but also against PC3, which are
androgen
receptor negative cells. Furthermore, both compounds were also potent
inhibitors of viable
cell growth for R84 (11) (lymphoblast) and DAUDI (T-Iymphoblast) cells, in
which BTK is
highly overexpressed. Altogether, these results demonstrate BMX inhibition
impacts viable
cell growth of prostate cancer cells and prompted us to further investigate
the importance of
the androgen receptor and related BMX pathways in these cell lines.
Table 9: Viable cell growth inhibition of compounds BMX-IN-1, 10, 11 and 24 in
a panel of
prostate, brain, blood, breast, ovary, lung and lymphoid cancer cells.
Tissue Cell line BMX-IN-1
JS24 JS10 JS11
LNCaP 1.81 0.05 4.4
NC 9.7 NC 10.41 NC
22RV1 2.07 0.06
6.66 0.09 4.86 0.11 7.3 NC
Prostate
PC3 10.98 1.13
4.8 NC ND 20.12 NC
DU145 17.7 NC
ND ND ND
U-87M6 533 019 5.04 0.01
ND ND
Brain
SK-N-MC 2.36 NC
8.53 0.44 11.19 NC 8.24 NC
Jurkat 5.99 NC 5.48
ND 9.71 1.48 6.36 0.17
Blood
Kasumi 3.13 0.06
5.12 0.12 4.37 0.04 10.14 0.07
MDA-MB- 23.61 0.48
Breast ND ND ND
231
CA0V3 7.68 0.13 8.56
NC 17.30 NC 19.31 NC
Ovary
OVCAR3 ND
ND ND ND
Bone
H1299 ND
7.28 0.37 19.42 NC ND
Marrow
Lung RS4(11) 1.176 0.06
2.09 NC 5.06 NC 6.66 NC
Lymphoid DAUDI 1.68 0.07
1.27 0.05 2.57 0.09 4.57 0.12
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Compound activity was profiled against 14 human cell lines from different
tissues in a 384-
well format, opaque white assay plates at 500-1000 cells per well using a semi-
automated
system. Cells were incubated at 37 C and 5% CO2. Compound stocks were plated
in a 384-
well format in 11-point and 2-fold concentration ranges. Compounds were pin-
transferred
into duplicate assay plates and incubated for72h. ATP levels were assessed by
CellTiter-
Glo (Promega) according to the manufacturers instructions. The values were
normalized to
vehicle and G150 was calculated using GraphPad Prism 8.When ambiguous fit was
observed
curves were top (100%) and bottom (0%) constrained and G150 was determined
with 4-P
least squares fit. In these cases SD is not calculated by GraphPad Prism 8.
Co-treatment of LNCaP cells with 24-26 and androgen receptor antagonist, PI3K
and AKT
inhibitors.
As shown above, BMX inhibition alone induces limited cell death in BMX-
expressed cell lines
owing to the existence of compensatory mechanisms in signalling pathways. To
evaluate the
use of BMX inhibitors in combination treatment regimens, the synergistic anti-
proliferative
effects of BMX inhibitors when combined with other therapeutic agents, which
pre-sensitize
prostate cancer cells was examined. For this purpose, LNCaP cells were co-
treated in a
combinatorial fashion with compounds 24-26, AKT1/2 (AKT inhibitor), Flutamide
(androgen
receptor antagonist) and LY294002 (PI3K inhibitor). Cell viability was
evaluated after 5 days
with CellTiter-Glo and compared with the overall anti-proliferative effects
of the compounds
alone. An optimization study was performed by screening several concentrations
to
determine the ideal conditions to obtain initial viability above 80% with the
individual
inhibitors alone. Based on these results, we tested 24 (at 3 pM), 25 (5 pM)
and 26 (6 pM)
with AKT112 (1 pM), Flutamide (50 pM) and LY294002 (3 pM).
LNCaP cells were seeded in 96 well-plates at 5000 cells/well in in a total
volume of 100 pL
of culture media and incubated for 24 hours to allow for attachment. After
incubation, cells
were treated in triplicate, in a combinatorial-fashion with 24 (2 pM and 3
pM), 25 (5 pM and 6
pM), 26 (6 pM), AKT1/2 (1 pM and 2 pM), Flutamide (25 pM and 50 pM), and PI3K
inhibitor
(3 pM and 3.5 pM). The results are shown in Figure 5.
Although the control concentrations of 24-26 and the inhibitors did not have
an effect on
reducing cell viability upon co-treatment, a marked viability decrease was
observed in all
tested conditions. With AKT1/2 a decrease in cell viability ranging from 48%
(with 24) to
63% (with 26) was observed, relative to control AKT1/2. With Flutamide, the
most effective
combination was with compound 25 (63% cell viability reduction) and the least
effective with
24(44% reduction). Finally, co-treatment with LY294002 decreased cell
viability by 35%
(with 24 and 26) and 59% (with 25). Overall, these results demonstrate a
synergistic effect
between 24-26 and AKT1/2, Flutamide and LY294002 in cancer cell proliferation
capable of
overcoming the compensatory mechanisms of BMX inhibition, and open the
possibility of
becoming useful molecules for drug combination approaches.
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Targeted cell cytotoxicity in patient samples
Compound 25 was tested through 11 Diffuse Large B-cell lymphoma (DLBCL)
samples from
hospital patients to quantify its ability to induce targeted cell cytotoxicity
on the B-cancer cell
fraction versus non-transformed cells. CD2O+CD79a+ markers (double and single
positive
cells) were used to determine the target cancer fraction. Results are shown in
Figure 6.
Figure 6A shows the relative cell fraction (RCF) of the viable target cells
for increasing
concentrations of 25 in DMSO. Relative cell fraction of <1.0 (hashed line)
indicates on-target
cytotoxic response, >1.0 indicates general cytotoxicity or off-target
cytotoxic response.
Figure 6B shows this normalized to the fraction of target cell population at
increasing DMSO
concentrations. Figure 6C shows 11 primary patient samples ranked by the drug
response
score (DRS) of compound 25 calculated as 1-mean of the RCF.
Data is 11 biological repeats, each concentration point for each sample was
performed in
4 replicates, at a single 72 h hour incubation time point The drug response
score (DRS) has
been previously shown to correlate to clinical response for late stage
hematological cancer
patients (Snijder et at). Viable target cells are defined as cytotoxicity-
marker negative and
diagnostic marker (C019, CD20, and/or CD79a) positive B-cells. As can be seen
in Figure 6,
compound 25 has an "on target" effect in 7 out of 11 patient samples.
Sequence Listings
SEQ ID No. 1: BMX Amino Acid Sequence
MDTKS I LEEL LLKRSQQKKKMSENNYKERLFVLTKTNL SYYEYDKMKRGSRKGS I E IKKI
RCVEKVNLEEQTPVERQYPFQIVYKDGL LYVYASNEE SRSQWLKALQKE I RGNPHLLVKY
HSGFFVDGKFLCCQQSCKAAPGCTLWEAYANLHTAVNEEKHRVP TFPDRVLKIPRAVPVL
KMDAPS SSTT LAQYDNESKKNYGSQPPS SSTS LAQYDSNSKK I YGSQPNFNMQY I P REDF
P DWWQVRK LK SS S S SEDVAS SNQKERNVNHT T SK I SWEFP ES S SSEEE ENLDDYDWFAGN
ISRS Q S EQLLRQKGKE GAFMVRN S SQVGMYTVSLFSKAVNDKKGTVKHYHVHTNAENKLY
LAENYCFDS IPKLI HYHQHNSAGMI TRLRHPVSTKANKVPDSVSLGNGIWELKREE ITLL
KELGSGQFGVVQLGKWKGQYDVAVKMIKEGSMSEDEFFQEAQTMMKLSHPKLVKFYGVCS
KEYP I Y IVTEY I SNGCLLNYLRSHGKGLEP SQLLEMCYDVCEGMAFLE SHQF IHRDLAAR
NCLVDRDLCVKVSDFGMTRYVLDDQYVS SVGTKFPVKWSAPEVFHYFKYS SKSDVWAFGI
LMWEVFS LGKQP YD LYON SQVVLKVSQGHRLYRP HLASDT I YQ IMY SCWH E LP EKRPTFQ
QLLSSIEPLREKDKH
>spIP51813IBMX HUMAN Cytoplasmic tyrosine-protein kinase BMX OS=Homo sapiens
OX=9606 GN=BMX PE=1 SV=1
Reference: Uniprot (https://www.uniprotorciluniprot/P51813)
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SEQ ID No. 2: BTK sequence - Isofonn BTK-A: canonical sequence
MAAV I LE S IF LKRSQQKKKT SP LNFKKRLF LLTVHKLSYY EY DFERGRRGSKKG S I DVEK
I TCVETVVP EKNPP PERQ IP RRGEESSEME QI SI IERFPYPFQVVYDE GP LYVF SP TEE L
RKRWI HQLKNVI RYNSDLVQKYHP CFWIDGQYLC CS QTAKNAMGCQ I LENRNGS LKPGS S
HRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDE
YF I LEESNLPWWRARDKNGQEGYI P SNYVTEAED S I EMYEWYSKHMTRSQAEQLLKQEGK
EGGF IVRD SSKAGKYTVSVFAKSTGDPQGV IRHYVVCSTPQSQYY LAEKH LF ST I P EL IN
YHQHNSAG L I SRLKYPVSQQNKNAPSTAGLGYGSWE IDPKDLTFLKELGTGQFGVVKYGK
WRGQYDVAIKMIKEGSMSEDEF IEEAKVMMNLSHEKLVQLYGVCTKQRP IF I I TEYMANG
CLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDF
GL SRYVLDDEYT S SVG SKFPVRWSPPEVLMYSKF SSKSD I WAFGVLMWE I YSLGEMPYER
FTNSETAEHIAQGLRLYRPHLASEKVYT IMYSCWHEKADERPTFKILLSNILDVMDEES
>splQ06187IBTK HUMAN Tyrosine-protein kinase BTK OS=Homo sapiens OX=9606
GN=BTK PE=1 SV=3
Reference: Uniprot (https://wvvw.uniprot.orciluniprot/Q06187#sequences)
SEQ ID No. 3: BTK sequence - Isoform BTK-C
MASWS I QQMVI GCP LCGRHC SGGEHTGE LQKEEAMAAVI LES IFLKRSQQKKKT SP LNFK
KRLF LLTVHKLSYYEYDFERGRRGSKKGS I DVEKI TCVETVVPEKNPPPERQ IPRRGEE S
SEMEQ I S I IERFPYPFQVVYDEGP LYVF SP TEELRKRW IHQLKNVI RYNS D LVQKY HP CF
WI DGQYLCCSQTAKNAMGCQ I LENRNGS LKP G SS HRKTKKP LPP TPEE DQ I LKKPLPP EP
AAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYF ILEE SNLPWWRARDKNGQEGY I P S
NYVTEAEDS IEMYEWYSKHMIRSQAEQLLKQEGKEGGF IVRDSSKAGKYTVSVFAKSTGD
PQGVIRHYVVCSTPQSQYYLAEKHLFST IP EL INYHQHNSAGL I S RLK YPVS QQNKNAP S
TAGLGYGSWE I DPKDL TF LKELGTGQFGVVKYGKWRGQYDVAIKMI KE GSMSEDEF IEEA
KVMMNLSHEKLVQLYGVCTKQRP IF I I TEYMANG CLLNYLREMRHRFQ TQQLLEMC KDVC
EAMEYLE SKQFLHRDLAARNCLVNDQGVVKVSDF GLSRYVLDDEYT SSVGSKFPVRWSPP
EVLMYSKF SSKSD IWAFGVLMWE I YSLGKMPYERFTNSETAEH IAQGLRLYRPHLASEKV
YT IMYSCWHEKADERP TF K I LLSN I LDVMDEES
>splQ06187-2IBTK HUMAN Isoform BTK-C of Tyrosine-protein kinase BTK OS=Homo
sapiens OX=9606 GN=BTK
Reference: Uniprot (https://www.un1protorciluniproti006187#seauences)
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SEQ ID No. 4: TEC Kinase
MNFNT I LEE I L I KRSQQKKK T S P LNYKE RLFVLT KSMLT Y YE GRAEKK YRKGF I DVSK
IK
CVE IVKNDDGVIPCQNKYPFQVVHDANTLY I FAP SP QSRD LWVKKLKE E I KNNNN IMIKY
HPKFWTDGSYQCCRQTEKLAPGCEKYNLFESS I RKALPPAPE TKKRRPPP P I P LEEEDNS
EE IVVAMYDFQAAEGHDLRLERGQEYL I LE KNDVHWWRARDKYGNEGY IF SNYVTGKKSN
NLDQYEWYCRNMNRSKAEQLLRSEDKEGGFMVRDSSQPGLYTVSLYTKFGGEGSSGFRHY
HI KE TTT SPKKYYLAEKHAF GS IPE I I EYHKHNAAGLVTRLRYP VSVKGKNAP T TAGF SY
EKWE I NP SEL TFMRE L GS G L F GVVRLGKWRAQYKVA I KA I RE GAMCEE DF I EEAKVMMKL
THPKLVQLYGVCTQQKP I Y IVTEFMERGCLLNFL RQRQGH FS RDVL LSMCQDVCEGMEY L
ERNSF I HRDLAARNCLVSEAGVVKVSDF GMARYVLDDQYT SSSGAKETVKWCPPEVFNYS
RF S SKS DVWSFGVLMWEVFTEGRMPFEKYTNYEVVTMVTRGHRLYQPKLASNYVYEVMLR
CWQEKPEGRP SFEDLLRT IDELVECEETFGR
>spIP42680ITEC_HUMAN Tyrosine-protein kinase Tec OS=Homo sapiens OX=9606
GN=TEC PE=1 SV=2
Reference: Uniprot (https://vvww.uniprot.orciluniprot/P426804sequences)
SEQ ID No. 5: ITK Kinase
MNNF I LLEEQL I KK SQQKRRT S P SNFKVRFFVLTKASLAYFEDRHGKKRT LEGS IELS RI
KCVE IVKSD I SIP CHYKYPFQVVHDNYL LYVFAPDRE SRQRWVLALKEETRNNNSLVP KY
HPNFWMDGKWRCCSQLEKLATGCAQYDP TKNASKKPLPP TPEDNRRPLWEPEETVVIALY
DYQTNDPQELALRRNEEYCL LD S SE I HWWRVQDRNGHEGYVP S SYLVEKSPNNLET YEWY
NKS I SRDKAE K LLLDT GKEGAFMVRDSR TAGTYTVSVFTKAVVSENNP C I KHYH I KETND
NP KRYYVAEK YVFD SI PLLI NYHQHNGGGLVTRL RYPVCF GRQKAPVTAGLRYGKWVI DP
SELTFVQE I G SGQFGLVH LGYWLNKDKVAI KT I REGAMSEEDF I EEAEVMMKLS HPKLVQ
LYGVCLEQAP I CLVFEFMEH GCL SDYLR TQRGLFAAETLL GMCLDVCE GMAYLEEACVI H
RDLAARNCLVGENQVI KVSDF GMTRFVLDDQYTS ST GTKFPVKWASPEVF SF SRYS SK SD
VWSFGVLMWEVFSEGKIPYENRSNSEVVED I S TGFRLYKPRLAS THVYQ IMNHCWKERPE
DRPAFSRLLRQLAE IAESGL
>splQ0888111TK_HUMAN Tyrosine-protein kinase ITKTISK OS=Homo sapiens OX=9606
GN=ITK PE=1 SV=1
Reference: Uniprot (https://www.uniprot.orchiuniprot/008881#sequences)
SEQ ID No. 6: TXK Kinase
MI LS SYNT I Q SVF CCC CC C SVQKRQMRTQ I S L STDEELPEKYTQRRRPWLSQLSNKKQSN
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T GRVQP SKRKPLPP LPP SEVAEEKI QVKALYDFLPREP CNLALRRAEEYL I LEKYNPHWW
KARDRLGNEGL I P SNYVTENKI TNLE I YEWYHRNI TRNQAEHLLRQESKE GAFI VRDSRH
LG SY T I SVFMGARRSTEAAI KHYQ I KKNDSGQWYVAERHAFQS IPEL IWY HQHNAAGLMT
RLRYPVGLMGSCLPATAGFSYEKWE I DP SE LAF I KE IGSGQFGVVHLGEWRSH I QVAIKA
INEGSMSEEDF I EEAKVMMKLS HSKLVQLYGVCI QRKP LY IVTEFMENGCLLNYLRENKG
KLRKEMLLSVCQD I CEGMEYLERNGY I HRDLAARNCLVS S TCIVKI SDFGMTRYVLDDEY
VS SF GAKFP IKWSPPEVFLFNKYSSKSDVWSFGVLMWEVF TEGKMPFENKSNLQVVEAIS
EGFRLYRPHLAPMS IYEVMYSCWHEKP E GRP TFAELLRAVTE IAETW
>spIP426811TXK_HUMAN Tyrosine-protein kinase TXK OS=Honno sapiens OX=9606
GN=TXK PE=1 SV=3
Reference: Uniprot (https://www.uniprot.oro/uniprot/P42681#seauences)
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