Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR TREATING
ANDROGEN RECEPTOR POSITIVE FORMS OF CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No.
62/819,487, filed
March 15, 2019, U.S. Provisional Application No. 62/819,482, filed March 15,
2019, U.S.
Provisional Application No. 62/819,472, filed March 15, 2019, U.S. Provisional
Application No.
62/819,490, filed March 15, 2019, U.S. Provisional Application No. 62/819,476,
filed March 15,
2019, U.S. Provisional Application No. 62/821,660, filed March 21, 2019, and
International
Application No. PCT/U52019/039936, filed June 28, 2019, each of which is
incorporated by
reference herein in its entirety.
TECHNICAL FIELD
[0002] This disclosure relates to compositions and methods for inhibiting CREB-
binding protein
(CBP). Compositions for inhibiting CBP are useful, for example, in
pharmaceutical compositions
for the treatment of certain Androgen Receptor dependent forms of cancer.
BACKGROUND
[0003] Growth and proliferation of hormone sensitive tumors are dependent on
oncogenic
signaling programs driven by corresponding nuclear hormone receptors. The
androgen receptor
(AR), a key driver in prostate cancer and subsets of breast cancers, controls
the expression of about
100 androgen-responsive target genes. Expression of these AR target genes is
important for normal
tissue development and cellular activities but can have pathological effects
that underlie tumor
initiation and progression. Direct targeting of androgen biosynthesis and
androgen interaction with
the AR can provide clinical utility. However, acquired resistance to these
therapies can circumvent
ligand-driven AR function while retaining continued dependence on AR-driven
transcriptional
programs.
[0004] Nuclear receptors are part of multiprotein complexes involving co-
activators and
co-repressors which control the impact of the nuclear receptor on its
downstream target genes.
Within the AR-associated multiprotein complex, CBP/P300 are critical co-
activators of AR,
modifying the chromatin environment surrounding the nuclear receptor to
increase its intrinsic
transcriptional activity and recruiting additional co-factors. Given the co-
regulatory relationship
between the AR and CBP/P300, inhibition of CBP/P300 activity offers a rational
approach to
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suppress AR-dependent oncogenic programs in AR-dependent tumors such as breast
and prostate
cancers.
[0005] There are no approved therapeutics specifically aimed at metastatic
castrate-resistant
(mCRPC) patients for whom androgen antagonists and taxane therapies have
proven ineffective.
These include patients with tumors harboring structurally altered androgen
receptors, including
the AR-v7 splice form, which continue to promote the AR transcriptional
program in a ligand-
independent manner, unaffected by androgen antagonists. This population
represents an unmet
clinical need.
SUMMARY
[0006] The present invention provides methods of and compositions for treating
AR+ cancers,
including patients diagnosed with certain forms of AR+ cancer that are
resistant to other
treatments, such as patients resistant or refractory to apalutamide,
darolutamide or enzalutamide
(e.g., patients with disease progression or with disease refractory to
treatment with enzalutamide),
by the administration of a CBP Inhibitor compound (e.g., compounds of formula
(I)) to a patient
in need thereof. The CBP Inhibitor compositions are preferably used in a
therapeutically effective
amount to inhibit CBP and antagonize androgen receptor signaling, leading to
clinical benefit in
AR+ Triple Negative Breast Cancer (TNBC), and mCRPC expressing the AR-v7
splice form. AR
is an oncogenic driver in prostate cancer and progression of the disease
towards castration- and
drug-resistance is associated with aberrations of AR such as amplification of
AR, mutations in the
LBD and increase in a splice variant of AR lacking the LBD (AR-v7). AR is also
expressed in a
subset of TNBC in which it substitutes for ER and drives ER signaling via
binding to ER response
elements. Finally, ER is the main oncogenic driver in ER+ breast cancer.
Resistance to hormone
deprivation in this subtype of breast cancer can lead to mutations in ER LBD
leading to ligand-
independent growth. In one aspect, the CBP Inhibitor is useful to treat
hormone-receptor positive
subjects with relapsed or refractory-to-hormone-therapy cancers (e.g., by
antagonizing ER activity
in these subjects).
[0007] The present disclosure is based in part on the discovery that compounds
of formula (I):
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(R2)11
-1-
\ /
R1
N ¨
N.----9..
N OH
0
0 0 (I)
and pharmaceutically acceptable salts thereof, wherein:
R1 is H or ¨OH;
each R2 is independently selected from C1-C6 alkyl (e.g., methyl), halogen,
¨CN, and
¨0R3, (e.g., methoxy) wherein the alkyl is optionally substituted with one or
more halogen;
each R3 is independently H or Ci-C6 alkyl (e.g., methyl), wherein the alkyl is
optionally
substituted with one or more halogen; and
n is an integer selected from 0, 1, 2, 3, 4 or 5, wherein n is preferably 0,
1, 2, or 3, provide
an active moiety useful for the treatment of AR-positive cancer, such as
certain AR-positive forms
of breast cancer (e.g., TNBC) and prostate cancer (e.g., CRPC).
[0008] The disclosure includes the use of compounds of formula (I), and
pharmaceutically
acceptable salts thereof, for the treatment of diseases or disorders
associated with the inhibition of
CBP, including certain AR-positive forms of cancer, including the AR-v7 splice
form of AR. As
an essential cofactor in AR-driven transcription, including that of androgen-
independent AR
variants, CBP/P300 is an attractive target for development of novel therapy to
meet these patients'
needs.
[0009] In some embodiments, the compound of formula (I) is Compound 1:
F . 0/
OH
N-
Nh.9....
N
0 OH
0 0
I (1)
or a stereoisomer and/or pharmaceutically acceptable salt thereof.
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[0010] In some embodiments, Compound 1 is the first eluting isomer when eluted
by preparative
HPLC under the conditions defined in Example 1.2.
[0011] In some embodiments, the compound of formula (I) is Compound 2:
OH
N-
0 OH
0 0
(2)
or a stereoisomer and/or pharmaceutically acceptable salt thereof.
[0012] In some embodiments, Compound 2 is the first eluting isomer when eluted
by preparative
HPLC under the conditions defined in Example 1.3.
[0013] In some embodiments, the compound of formula (I) is Compound 3:
F 4.0 0>-F
OH
N-
0 OH
0 o
1
(3)
or a stereoisomer and/or pharmaceutically acceptable salt thereof.
[0014] In some embodiments, Compound 3 is the second eluting isomer when
eluted by
preparative HPLC under the conditions defined in Example 1.4.
[0015] In some embodiments, the compound of formula (I) is Compound 4:
0
.,,OH
0 0
(4)
or a stereoisomer and/or pharmaceutically acceptable salt thereof.
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[0016] Compounds of formula (I) are active in enzalutamide-resistant
preclinical models, and thus
represent a potential novel therapy for these patients with refractory or
resistant disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 is a table of compounds in accordance with various embodiments
of the disclosure.
[0018] Figure 2 is a series of reaction schemes for chemical syntheses of
compounds of Formula
(I) and useful intermediates in the preparation of compounds of Formula (I).
[0019] Figure 3 is an immunoblot showing H3K27Ac, total H3, and 13-actin
protein expression
levels from a breast cancer cell line exposed to increasing concentrations of
compound 1 for 24h.
[0020] Figure 4 is a graph showing the in vivo activity of Compound 1 in a
cell line derived
xenograft model of AR+ triple negative breast cancer.
[0021] Figure 5 is an immunoblot showing AR and AR-v7 expression levels in an
AR-v7+ prostate
cancer cell line after 24h exposure to increasing concentrations of Compound
3.
[0022] Figure 6 is a graph showing the in vivo activity of Compound 4 in a
patient-derived
xenograft model of prostate cancer resistant to enzalutamide.
DETAILED DESCRIPTION
[0023] The present disclosure encompasses the recognition that compounds of
formula (I) are
CBP Inhibitor Compounds, defined herein as compounds having one or more of the
following
characteristics when tested according to the HTRF biochemical Assay Protocol
below in Example
2: (1) a CBP IC50 value of less than 1 (1M; and (2) a CBP IC50 value of
between 0.001 and 1 (1M.
CBP Inhibitor Compounds can be of formula (I):
(12_ )n
\ /
Ri
N¨
N----9._
N OH
0
00 (I)
or a pharmaceutically acceptable salt thereof, wherein R1, R2, and n are as
described above.
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[0024] Unless otherwise indicated herein, all isomeric forms of specified
chemical compounds are
provided by the present disclosure, including mixtures thereof (e.g., S, R and
racemic orientations
at each chiral center). If the compound contains a double bond, the
substituent may be in the E or
Z configuration. If the compound contains a disubstituted cycloalkyl, the
cycloalkyl substituent
may have a cis- or trans- configuration. All tautomeric forms are also
intended to be included.
[0025] Compounds of formula (I) and Group A, unless otherwise indicated, may
exist in their
tautomeric form. All such tautomeric forms are contemplated herein as part of
the present
disclosure.
[0026] The compounds of formula (I) and Group A, unless otherwise indicated,
may contain one
or more stereocenters, and, therefore, exist in different stereoisomeric
forms. It is intended that
unless otherwise indicated all stereoisomeric forms of the compounds of
formula (I) and Group A,
as well as mixtures thereof, including racemic mixtures, form part of the
present disclosure. In
addition, the present disclosure embraces all geometric and positional
isomers. For example, if a
compound of formula (I) or Group A incorporates a double bond or a fused ring,
both the cis- and
trans-forms, as well as mixtures, are embraced within the scope of the
disclosure. Each compound
herein disclosed includes all the enantiomers that conform to the general
structure of the
compound. The compounds may be in a racemic or enantiomerically pure form, or
any other form
in terms of stereochemistry. The assay results may reflect the data collected
for the racemic form,
the enantiomerically pure form, or any other form in terms of stereochemistry.
[0027] Diastereomeric mixtures can be separated into their individual
diastereomers based on their
physical chemical differences by methods well known to those skilled in the
art, such as, for
example, by chromatography and/or fractional crystallization. Enantiomers can
be separated by
converting the enantiomeric mixture into a diastereomeric mixture by reaction
with an appropriate
optically active compound (e.g., chiral auxiliary such as a chiral alcohol or
Mosher's acid chloride),
separating the diastereomers and converting (e.g., hydrolyzing) the individual
diastereomers to the
corresponding pure enantiomers. Also, some of the compounds of formula (I) or
Group A may be
atropisomers (e.g., substituted biaryls) and are considered as part of this
disclosure. Enantiomers
can also be separated by use of a chiral HPLC column.
[0028] The compounds of formula (I) or Group A may form acid addition salts,
which may be
pharmaceutically acceptable salts. The disclosure also includes pharmaceutical
compositions
comprising one or more compounds as described herein, or a pharmaceutically
acceptable salt
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thereof, and a pharmaceutically acceptable carrier. In some embodiments,
pharmaceutical
compositions reported herein can be provided in a unit dosage form (e.g.,
capsule, tablet or the
like). In some embodiments, pharmaceutical compositions reported herein can be
provided in an
oral dosage form. In some embodiments, an oral dosage form of a compound of
formula (I) or
Group A can be a capsule. In some embodiments, an oral dosage form of a
compound of formula
(I) or Group A is a tablet. In some embodiments, an oral dosage form comprises
one or more
fillers, disintigrants, lubricants, glidants, anti-adherents and/or anti-
statics. In some embodiments,
an oral dosage form is prepared via dry blending. In some embodiments, an oral
dosage form is a
tablet and is prepared via dry granulation.
[0029] A CBP Inhibitor compound of the present disclosure can be dosed at a
therapeutically
effective level. A Selective CBP Inhibitor compound of the present disclosure
can be dosed at a
therapeutically effective level.
Compounds of the Disclosure
[0030] In one aspect, the disclosure relates to compounds of formula (I):
(12_ )n
\ /
Ri
N-
N----9._
N OH
0
00 (I)
or a pharmaceutically acceptable salt thereof, wherein:
R1 is H or ¨OH;
each R2 is independently selected from C1-C6 alkyl, halogen, ¨CN, and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen;
each R3 is independently H or Ci-C6 alkyl, wherein the alkyl is optionally
substituted with one or more halogen; and
n is an integer selected from 0-5, wherein n is preferably 0, 1, 2, or, 3.
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[0031] In some embodiments, R1 is H or ¨OH; each R2 is independently selected
from ¨F, ¨Cl,
¨CH3, ¨CHF2, ¨CN, and ¨0R3; each R3 is independently selected from ¨CH3,
¨CHF2, and
¨CH(CH3)2, and n is selected from 0, 1, 2, and 3.
[0032] In some embodiments, compounds of Formula (I) are provided wherein R2
is ¨Cl, ¨CH3,
¨CHF2, ¨CN, ¨OCH3, ¨OCHF2, ¨OCH(CH3)2. In some embodiments, R2 is ¨F, ¨CH3,
¨CHF2,
¨CN, or ¨0R3. In some embodiments, R2 is ¨F, ¨Cl, ¨CHF2, ¨CN, or ¨0R3. In some
embodiments,
R2 is ¨F, ¨Cl, ¨CH3, ¨CN, or ¨0R3. In some embodiments, R2 is ¨F, ¨Cl, ¨CH3,
¨CHF2, or ¨0R3.
In some embodiments, R2 is ¨F, ¨Cl, ¨CH3, ¨CHF2, or ¨CN.
[0033] In one embodiment, the disclosure provides compounds of formula (lb):
R2c R2b
R2d R2a
R
R2e 1
N¨
N
OH
0
0 0 (Ib)
or a pharmaceutically acceptable salt thereof, wherein:
R1 is H or ¨OH;
R2a is selected from H, C1-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
CHF2);
R2b is selected from H, C1-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
CHF2);
R2' is selected from H, C1-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen;
R2d is selected from H or halogen (e.g., F or Cl);
R2e is selected from H, C1-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
¨CN, and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
methyl); and
R3 is H or C1-C6 alkyl (e.g., methyl), wherein the alkyl is optionally
substituted with one
or more halogen (e.g., CHF2).
[0034] In one embodiment, the disclosure provides compounds of formula (lb):
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R2c R2b
R2d R2a
R
R2e 1
N¨
N
OH
0
0 0 (Ib)
or a pharmaceutically acceptable salt thereof, wherein:
R1 is ¨OH;
R2a is selected from H, Ci-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
CHF2);
R2b is selected from H, Ci-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
CHF2);
R2' is selected from H, Ci-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen;
R2d is selected from H or halogen (e.g., F or Cl);
R2e is selected from H, C1-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
¨CN, and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
methyl); and
R3 is H or C1-C6 alkyl (e.g., methyl), wherein the alkyl is optionally
substituted with one
or more halogen (e.g., CHF2).
[0035] In one embodiment, the disclosure provides compounds of formula (lb):
R2c R2b
R2d R2a
R
R2e 1
N¨
N
OH
0
0 0 (Ib)
or a pharmaceutically acceptable salt thereof, wherein:
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R1 is H;
R2a is selected from H, Ci-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
CHF2);
R2b is selected from H, Ci-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
CHF2);
R2' is selected from H, Ci-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen;
R2d is selected from H or halogen (e.g., F or Cl);
R2e is selected from H, C1-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
¨CN, and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
methyl); and
R3 is H or C1-C6 alkyl (e.g., methyl), wherein the alkyl is optionally
substituted with one
or more halogen (e.g., CHF2).
[0036] In one embodiment, the disclosure provides compounds of formula (lb):
R2c R2b
R2d R2a
R
R2e 1
N¨
N
OH
0
0 0
(Ib)
or a pharmaceutically acceptable salt thereof, wherein:
R1 is H or -OH;
R2a is selected from H, C1-C6 alkyl (e.g., methyl), halogen (e.g., F or Cl),
and ¨0R3,
wherein the alkyl is optionally substituted with one or more halogen (e.g.,
CHF2);
R2b is H;
R2' is H;
R2d is independently selected from H or halogen (e.g., F or Cl);
R2e is H; and
R3 is independently H or C1-C6 alkyl (e.g., methyl), wherein the alkyl is
optionally
substituted with one or more halogen (e.g., CHF2).
[0037] In one embodiment, the disclosure provides compounds of formula (lb):
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R2c R2b
R2d R2a
R
R2e 1
N¨
N
OH
0
0 0 (Ib)
or a pharmaceutically acceptable salt thereof, wherein:
R1 is H or -OH;
R2a is selected from H, and ¨0R3;
R2b is H;
R2' is H;
R2d is independently selected from H or halogen (e.g., F or Cl);
R2e is H; and
R3 is independently H or Ci-C6 alkyl (e.g., methyl), wherein the alkyl is
optionally
substituted with one or more halogen (e.g., CHF2).
[0038] In some embodiments, Selective CBP Inhibitor Compounds of formula (I)
are provided.
In some embodiments, Selective CBP Inhibitor Compounds of Formula (lb) are
provided. The
present disclosure encompasses the recognition that compounds of formula (I)
are CBP Inhibitor
Compounds, defined herein as compounds having one or more of the following
characteristics
when tested according to the HTRF biochemical Assay Protocol below in Example
2: (1) a CBP
IC50 value of less than 1 i.tM; and (2) a CBP IC50 value of between 0.001 and
1 i.i.M.
[0039] In some embodiments, the disclosure relates to compounds of formula (I)
that are of a
formula selected from Group A:
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(R2), (R2)õ
(1.._....... 1
e \
\ 3.
OH OH
N-
Ni....(
0
?... Ni,,Q
:.
0 i.
N N )--OH OH
O 0 0 0
I (Al), I (A2),
(R2) (R2)
,, ,,
.,,0,_, ='10H
N- N-
0 i/.
N
0
i....9.. Ni...0
N N -OH OH
O 0 0 0
1 (A3), I (A4),
(r-2 (R2),,
OH '10H
,.9., Nh
N-
Ni .9.,
N N
OH OH
0 0
O 0 0 0
1 (A5), I (A6),
(R2),, (R2L
\ 31
(7____..1
e \
OH .'10H
N-
N...0
0 0
:.
N N
//-0H OH
O 0 0 0
1 (A7), I (A8),
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(R2) (R2),,
N-
0
OH 0//--OH
0 0 0 0
(A9), I (A10),
(R2) (R2),,
N¨ N-
0 OH
0OH
0 0 0 0
(All), and I (Al2),
and pharmaceutically acceptable salts thereof.
[0040] In some embodiments, the disclosure relates to a compound of formula
(I) selected from
Figure 1. In Figure 1, "Eluted Isomer" refers to the order in which the
compound eluted by
preparative HPLC.
[0041] In some embodiments, R1 is H or ¨OH. In some embodiments, R1 is H. In
some
embodiments, R1 is ¨OH.
[0042] In some embodiments, each R2 is independently selected from C1-C6
alkyl, halogen,
¨CN, and ¨0R3, wherein the alkyl is optionally substituted with one or more
halogen. In some
embodiments, R2 is Ci-C6 alkyl, wherein the alkyl is optionally substituted
with one or more
halogen. In some embodiments, R2 is Ci-C6 alkyl, wherein the alkyl is
substituted with one
halogen. In some embodiments, R2 is Ci-C6 alkyl, wherein the alkyl is
substituted with two
halogens. In some embodiments, R2 is selected from ¨CH3 and ¨CHF2. In some
embodiments, R2
is ¨CH3. In some embodiments, R2 is ¨CHF2. In some embodiments R2 is halogen.
In some
embodiments, R2 is selected from ¨F and ¨Cl. In some embodiments, R2 is ¨F. In
some
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embodiments, R2 is ¨Cl. In some embodiments, R2 is ¨CN. In some embodiments,
R2 is ¨0R3,
wherein R3 is as described herein.
[0043] In some embodiments, each R3 is independently C1-C6 alkyl, wherein the
alkyl is optionally
substituted with one or more halogen. In some embodiments, R3 is Ci-C6 alkyl,
wherein the alkyl
is substituted with one halogen. In some embodiments, R3 is Ci-C6 alkyl,
wherein the alkyl is
substituted with two halogens. In some embodiments, R3 is selected from
¨CH3, ¨CHF2, and propyl. In some embodiments, R3 is ¨CH3. In some embodiments,
R3 is
¨CHF2. In some embodiments, R3 is propyl.
[0044] In some embodiments, n is selected from 0, 1, 2, and 3. In some
embodiments, n is 0. In
some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n
is 3.
[0045] In some embodiments, n is 2 and R2 is CH3 in one instance and F in the
second instance.
In some embodiments, n is 2 and R2 is ¨OCH3 in one instance and Cl in the
second instance. In
some embodiments, n is 2 and R2 is ¨OCH3 in one instance and ¨OCH3 in the
second instance. In
some embodiments, n is 2 and R2 is ¨CH3 in one instance and F in the second
instance. In some
embodiments, n is 2 and R2 is F in one instance and F in the second instance.
In some embodiments,
n is 2 and R2 is ¨CHF2 in one instance and F in the second instance. In some
embodiments, n is 2
and R2 is ¨OCH(CH3)2 in one instance and F in the second instance. In some
embodiments, n is 2
and R2 is ¨OCHF2 in one instance and F in the second instance. In some
embodiments, n is 3 and
R2 is ¨OCH3 in one instance, F in the second instance, and F in the third
instance. In some
embodiments, n is 3 and R2 is ¨OCH3 in one instance, ¨OCH3 in the second
instance, and F in the
third instance.
[0046] In some embodiments, R1 is H and n is 0.
[0047] In some embodiments, R1 is ¨OH and n is 0.
[0048] In some embodiments, R1 is ¨OH, n is 2, and R2 is ¨F in one instance
and ¨0R3 in the
second instance, wherein R3 is ¨CH3.
[0049] In some embodiments, R1 is ¨OH, n is 2, and R2 is ¨F in one instance
and ¨0R3 in the
second instance, wherein R3 is ¨CHF2.
[0050] In some embodiments, the compound of formula (I) is Compound 1:
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F ii 01
OH
N¨
No.9..._
N
0 OH
0 0
I (1)
or a pharmaceutically acceptable salt thereof.
[0051] In some embodiments, the compound of formula (I) is (1R,3R)-3-[(7S)-2-
[(R)-(5-fluoro-
2-methoxyphenyl)(hydroxy)methyl]-6-(methoxycarbony1)-7-methyl-3H,6H,7H,8H,9H-
imidazo[4,5-fiquinolin-3-yl]cyclohexane-l-carboxylic acid.
[0052] In some embodiments, the compound of formula (I) is (1R,3R)-3-((S)-2-
((R)-(5-fluoro-2-
methoxyphenyl)(hydroxy)methyl)-6-(methoxycarbonyl)-7-methyl-6,7,8,9-tetrahydro-
3H-
imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic acid.
[0053] In some embodiments, the compound of formula (I) is Compound 2:
*
OH
N¨
Ni,.9..
N
L 0 OH
0 0
I (2)
or a pharmaceutically acceptable salt thereof.
[0054] In some embodiments, the compound of formula (I) is 3-((7S)-2-
(hydroxy(phenyl)methyl)-
6-(methoxycarbony1)-7-methy1-6,7,8,9-tetrahydro-3H-imidazo[4,5-fiquinolin-3-
y1)cyclohexane-
1-carboxylic acid.
[0055] In some embodiments, the compound of formula (I) is the first eluting
isomer of 3-((7S)-
2-(hydroxy(phenyl)methyl)-6-(methoxycarbony1)-7-methyl-6,7,8,9-tetrahydro-3H-
imidazo[4,5-
fiquinolin-3-y1)cyclohexane-1-carboxylic acid when eluted from a preparative
HPLC using the
conditions defined in Example 1.3.
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[0056] In some embodiments, the compound of formula (I) is (1R,3R)-3-((S)-2-
((R)-
hydroxy(phenyl)methyl)-6-(methoxycarbony1)-7-methyl-6,7,8,9-tetrahydro-3H-
imidazo[4,5-
fiquinolin-3-y1)cyclohexane-1-carboxylic acid.
[0057] In some embodiments, the compound of formula (I) is Compound 3:
F 4.0 0>-F
OH
N-
0 OH
01 0
(3)
or a pharmaceutically acceptable salt thereof.
[0058] In some embodiments, the compound of formula (I) is 3-((7S)-2-((2-
(difluoromethoxy)-5-
fluorophenyl)(hydroxy)methyl)-6-(methoxycarbonyl)-7-methyl-6,7,8,9-tetrahydro-
3H-
imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic acid.
[0059] In some embodiments, the compound of formula (I) is the first eluting
isomer of 3-((7S)-
2-((2-(difluoromethoxy)-5-fluorophenyl)(hydroxy)methyl)-6-(methoxycarbonyl)-7-
methyl-
6,7,8,9-tetrahydro-3H-imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic
acid when eluted
from a preparative HPLC using the conditions defined in Example 1.5.
[0060] In some embodiments, the compound of formula (I) is (1R,3R)-3-((S)-2-
((R)-(2-
(difluoromethoxy)-5-fluorophenyl)(hydroxy)methyl)-6-(methoxycarbonyl)-7-methyl-
6,7,8,9-
tetrahydro-3H-imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic acid.
[0061] In some embodiments, the compound of formula (I) is Compound 4:
0
OH
0 0
(4)
or a pharmaceutically acceptable salt thereof.
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[0062] In some embodiments, the compound of formula (I) is 3-((S)-2-benzy1-6-
(methoxyc arbony1)-7-methy1-6,7 ,8,9-tetrahydro-3H-imidazo [4,5-f] quinolin-3 -
yl)c yclohex ane-1-
carboxylic acid.
[0063] In some embodiments, the compound of formula (I) is (1R,3R)-3-((S)-2-
benzy1-6-
(methoxyc arbony1)-7-methy1-6,7 ,8,9-tetrahydro-3H-imidazo [4,5-f] quinolin-3 -
yl)c yclohex ane-1-
carboxylic acid.
[0064] The disclosure is also based in part on the recognition that compounds
of formula (I) are
Selective CBP Inhibitor Compounds, defined herein as CBP Inhibitors having a
BRD4 IC50 value
greater than that of their CBP IC50 value, preferably wherein its BRD4 IC50
value is greater than 1
i.tM (e.g., 1 micromolar to 10 micomolar, or greater), wherein the IC50 values
are determined as in
the procedures set forth in the assay described in Example 2. In some
embodiments, compounds
of formula (I) can be Selective CBP Inhibitor Compounds, wherein the BRD4 IC50
value is greater
than 500 nM (e.g., 500 nanomolar to 10 micomolar, or greater), wherein the
IC50 values are
determined as in the procedures set forth in the assay described in Example 2.
The disclosure is
also based in part on the recognition that Compound 1 is a Selective CBP
Inhibitor Compound,
defined herein as a CBP Inhibitor having a BRD4 IC50 value greater than that
of its CBP IC50
value, preferably wherein its BRD4 IC50 value greater than 1 i.tM (e.g., 1
micromolar to 10
micomolar, or greater), wherein the IC50 values are determined as in the
procedures set forth in the
assay described in Example 2.
[0065] The discovery includes the use of one or more compounds of formula (I),
and
pharmaceutically acceptable salts thereof, in pharmaceutical preparations for
the treatment of
patients diagnosed with a disease or disorder associated with the inhibition
of CBP (e.g., certain
forms of cancer). The compositions comprising one or more compounds of formula
(I), and
pharmaceutically acceptable salts thereof, can be obtained by certain
processes also provided
herein. In some embodiments, a Selective CBP Inhibitor Compound of formula (I)
is used to treat
breast cancer (e.g., TNBC) or prostate cancer. In some embodiments, a
Selective CBP Inhibitor
Compound of Formula (Ib) is used to treat an AR+ form of cancer, including AR+
breast cancer
or prostate cancer. The use of a Selective CBP Inhibitor Compound of formula
(I) is provided for
treatment of a patient diagnosed with a AR+ form of cancer, such as AR+ breast
cancer (e.g., AR+
TNBC) or AR+ prostate cancer (e.g., a AR-v7+ form of prostate cancer).
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[0066] In some embodiments, the discovery includes the use of (1R,3R)-3-[(7S)-
2-[(R)-(5-fluoro-
2-methoxyphenyl)(hydroxy)methyll-6-(methoxycarbony1)7-methyl-3H,6H,7H,8H,9H-
imidazo [4,5-f] quinolin-3-yl] cyclohexane- 1-carboxylic acid (Compound 1),
and pharmaceutically
acceptable salts thereof, in pharmaceutical preparations for the treatment of
patients diagnosed
with a disease or disorder associated with the inhibition of CBP (e.g.,
certain forms of cancer).
The compositions comprising Compound 1 and pharmaceutically acceptable salts
thereof can be
obtained by certain processes also provided herein.
[0067] In some embodiments, the discovery includes the use of Compound 2, and
pharmaceutically acceptable salts thereof, in pharmaceutical preparations for
the treatment of
patients diagnosed with a disease or disorder associated with the inhibition
of CBP (e.g., certain
forms of cancer). The compositions comprising Compound 2 and pharmaceutically
acceptable
salts thereof can be obtained by certain processes also provided herein.
[0068] In some embodiments, the discovery includes the use of Compound 3, and
pharmaceutically acceptable salts thereof, in pharmaceutical preparations for
the treatment of
patients diagnosed with a disease or disorder associated with the inhibition
of CBP (e.g., certain
forms of cancer). The compositions comprising Compound 3 and pharmaceutically
acceptable
salts thereof can be obtained by certain processes also provided herein.
[0069] In some embodiments, the discovery includes the use of Compound 4, and
pharmaceutically acceptable salts thereof, in pharmaceutical preparations for
the treatment of
patients diagnosed with a disease or disorder associated with the inhibition
of CBP (e.g., certain
forms of cancer). The compositions comprising Compound 4 and pharmaceutically
acceptable
salts thereof can be obtained by certain processes also provided herein.
Methods of Synthesizing the Compounds
[0070] The compounds of the present disclosure may be made by a variety of
methods, including
standard chemistry. Suitable synthetic routes are depicted in the examples
given below.
[0071] The compounds of the present disclosure, i.e., compounds of Formula
(I), (II), or Group A
or a pharmaceutically acceptable salt thereof, may be prepared by methods
known in the art of
organic synthesis as set forth in part by the synthetic schemes depicted in
the examples. In the
schemes described below, it is well understood that protecting groups for
sensitive or reactive
groups are employed where necessary in accordance with general principles or
chemistry.
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Protecting groups are manipulated according to standard methods of organic
synthesis (T. W.
Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis", Third
edition, Wiley, New
York 1999). These groups are removed at a convenient stage of the compound
synthesis using
methods that are readily apparent to those skilled in the art. The selection
processes, as well as the
reaction conditions and order of their execution, shall be consistent with the
preparation of
compounds of Formula (I), (II), or Group A.
[0072] Those skilled in the art will recognize stereocenters exist in the
compounds of Formula (I),
(II), or Group A. Accordingly, the present disclosure includes both possible
stereoisomers (unless
otherwise indicated and/or specified in the synthesis) and includes not only
racemic compounds
but the individual enantiomers and/or diastereomers as well. Unless otherwise
indicated, when a
compound is desired as a single enantiomer or diastereomer, it may be obtained
by stereospecific
synthesis or by resolution of the final product or any convenient
intermediate. Resolution of the
final product, an intermediate, or a starting material may be affected by any
suitable method known
in the art. See, for example, "Stereochemistry of Organic Compounds" by E. L.
Eliel, S. H. Wilen,
and L. N. Mander (Wiley-lnterscience, 1994).
Methods of Using the Compounds
[0073] In some embodiments, compounds of formula (I) are tool compounds useful
for studying
the effects of CBP/p300 inhibition in vitro or in an in vivo model. In vitro,
the tool compounds of
formula (I) may be useful for studying the effects of CBP/p300 inhibition on
purified proteins,
cellular extracts, in intact cells and cell line models, and the like. In
vivo, the tool compounds of
formula (I) may be useful for studying the effects of CBP/p300 inhibition in
cell line derived
xenografts, in patient derived xenografts, in knock-in mouse model, in knock-
out mouse models,
and the like.
[0074] Preferably, the disclosure provides pharmaceutical preparations for the
treatment of
patients diagnosed with AR+ cancer. In particular, compounds provided herein
can be formulated
as an active pharmaceutical composition comprising one or more compounds of
formula (I) (or a
pharmaceutically acceptable salt and/or enantiomer thereof) useful for
treatment of prostate
cancer, including metastatic castrate resistant prostate cancer (CRPC), and/or
AR+ breast cancers
including locally advanced or metastatic AR+ breast cancer. For example, the
inhibition of
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CBP/P300 can target AR transcriptional activity through H3K27Ac, reduction of
AR target gene
expression, or reduction of AR expression with, ultimately, a reduction in
proliferation. In
addition, CBP/P300 BRD inhibitors present the possibility of suppressing ER-
driven signaling in
hormone-receptor positive breast cancers. In some embodiments, the
pharmaceutical composition
comprises Compound 1. In some embodiments, the pharmaceutical composition
comprises
Compound 2. In some embodiments, the pharmaceutical composition comprises
Compound 3. In
some embodiments, the pharmaceutical composition comprises Compound 4.
[0075] Compounds and compositions described herein are inhibitors of CBP
having a lower
inhibition concentration compared to its inhibition concentration with respect
to BRD4.
[0076] Methods of treatment (e.g., by inhibiting CBP) can comprise
administering to a subject in
need thereof a therapeutically effective amount of (1) a compound of formula
(I) or a
pharmaceutically acceptable salt thereof; (2) a pharmaceutical composition
comprising one or
more compounds of formula (I) or a pharmaceutically acceptable salt thereof
and a
pharmaceutically acceptable carrier.
[0077] Methods of treatment (e.g., by inhibiting CBP) can comprise
administering to a subject in
need thereof a therapeutically effective amount of (1) Compound 1 or a
pharmaceutically
acceptable salt thereof; (2) a pharmaceutical composition comprising Compound
1 or a
pharmaceutically acceptable salt thereof and a pharmaceutically acceptable
carrier.
[0078] Methods of treatment (e.g., by inhibiting CBP) can comprise
administering to a subject in
need thereof a therapeutically effective amount of (1) Compound 2 or a
pharmaceutically
acceptable salt thereof; (2) a pharmaceutical composition comprising Compound
2 or a
pharmaceutically acceptable salt thereof and a pharmaceutically acceptable
carrier.
[0079] Methods of treatment (e.g., by inhibiting CBP) can comprise
administering to a subject in
need thereof a therapeutically effective amount of (1) Compound 3 or a
pharmaceutically
acceptable salt thereof; (2) a pharmaceutical composition comprising Compound
3 or a
pharmaceutically acceptable salt thereof and a pharmaceutically acceptable
carrier.
[0080] Methods of treatment (e.g., by inhibiting CBP) can comprise
administering to a subject in
need thereof a therapeutically effective amount of (1) Compound 4 or a
pharmaceutically
acceptable salt thereof; (2) a pharmaceutical composition comprising Compound
4 or a
pharmaceutically acceptable salt thereof and a pharmaceutically acceptable
carrier.
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[0081] The pharmaceutical compositions may be orally administered in any
orally acceptable
dosage form. Accordingly, a patient and/or subject can be selected for
treatment using a compound
described herein by first evaluating the patient and/or subject to determine
whether the subject is
in need of inhibition of CBP, and if the subject is determined to be in need
of inhibition of CBP,
then administering to the subject a composition described herein.
[0082] The CBP Inhibitor compounds and compositions are useful, for example,
in suppressing
AR-driven transcriptional programs through inhibition of the CBP/P300 BRD,
including AR-v7
variants. The growth inhibitory effect of Compounds 1, 2 and 4 and
enzalutamide was determined
across a panel of prostate cancer cell lines including androgen-dependent and
independent models
as well as AR-negative cell lines. Compound 1 induced a concentration-
dependent reduction of
H3K27Ac, a mark specific to CBP/P300, in an AR positive breast cancer cell
line (Figure 3).
Compounds 1, 2, and 4 reduced the mRNA expression of TMPRSS2 and XBP1 in an AR
positive
breast cancer cell line. Compound 1, 2 and 4 inhibit proliferation of breast
cancer cell lines after
days continuous exposure to the drug, and the cell lines with high expression
of AR mRNA are
more sensitive than those with low expression. Compound 1 treatment produced a
tumor growth
inhibition in an AR positive breast cancer cell line derived xenograft model
(Figure 4). In some
embodiments, an AR positive breast cancer cell line can be MDA-MB-453.
Treatment of prostate
cancer cells with Compound 2 led to the reduction of both full length and
variant forms of the AR
including AR-v7 (Figure 5). Compounds 1, 2, and 4 reduced AR target genes
TMPRSS2 and
KLK3, as well as MYC in a concentration-dependent manner in an AR-v7+ prostate
cancer cell
line. In some embodiments, an AR-v7+ prostate cancer cell line can be 22Rv1.
Compounds 1, 2,
and 4 had a potent and concentration-dependent growth inhibitory effect in all
AR+ cell lines,
including AR-v7 harboring cell lines. Treatment with Compound 4 at 40
mg/kg/dose daily Monday
- Thursday repeated weekly or 80 mg/kg/dose Monday and Thursday (twice weekly)
repeated
weekly resulted in a strong antitumor response in a patient-derived xenograft
model resistant to
enzalutamide (Figure 6).
[0083] Enzalutamide is an androgen receptor inhibitor indicated for the
treatment of patients with
castration resistant prostate cancer or metastatic castration-sensitive
prostate cancer. Patients
receiving enzalutamide can also receive a gonadotropin-releasing hormone
(GnRH) analog
concurrently or can have had bilateral orchiectomy. Enzalutamide is an
androgen receptor inhibitor
that acts on different steps in the androgen receptor signaling pathway.
Enzalutamide has been
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shown to competitively inhibit androgen binding to androgen receptors; and
consequently, inhibits
nuclear translocation of androgen receptors and their interaction with DNA. A
major metabolite,
N-desmethyl enzalutamide, exhibited similar in vitro activity to enzalutamide.
Enzalutamide
decreased proliferation and induced cell death of prostate cancer cells in
vitro, and decreased tumor
volume in a mouse prostate cancer xenograft model.
[0084] In some embodiments, compounds of formula (I), or pharmaceutically
acceptable salts
thereof, are indicated for the treatment of patients with castration resistant
prostate cancer or
metastatic castration-sensitive prostate cancer. Patients receiving a compound
of formula (I), or a
pharmaceutically acceptable salt thereof, can also receive gonadotropin-
releasing hormone (GnRH)
analog concurrently or can have had a bilateral orchiectomy.
[0085] Enzalutamide was active against androgen-dependent cell lines such as
VCaP but was
inactive in AR- negative cell lines as well as AR-v7+ cells. Conversely,
Compound 1 had a potent
and concentration-dependent growth inhibitory effect in all AR+ cell lines
including an AR-v7+
cell line (IC50 =0.6 i.t1\4). Compound 1 was also inactive against AR- cell
lines. This is consistent
with the proposed mechanism of action of Compound 1.
[0086] Without being bound to theory, it is believed that CBP/P300 can
interact with AR directly
via the NR1D domain in CBP/P300. However, contrary to other nuclear receptors,
this interaction
is not believed to be dependent on ligand binding. Also, CBP/P300 can interact
with both the LBD
of AR and its N-terminal domain. Both these factors are of relevance in
castrate resistant prostate
cancer and it is expected that CBP/P300 interacts with AR-v7 very similarly as
with AR.
CBP/P300 also interact with AR indirectly via the co-factor TIP60/SRC-1 which
itself interacts
with AR. This approach can differ from direct receptor antagonists and can
have the advantage of
being unaffected by structural variations in the AR ligand binding domain
(LBD). Such CBP/P300
BRD inhibitors can have activity in a number of cancers dependent upon nuclear
hormone-receptor
transcriptional programs, such as metastatic CRPC and locally advanced or
metastatic AR+ breast
cancers. The therapeutic benefits of AR inhibition have been demonstrated
clinically in these
cancers, but with limitations that highlight the need for alternative
approaches with long term
benefit and mechanisms of resistance that are non-overlapping with anti-
androgen therapy. For
example, the inhibition of CBP/P300 can target AR transcriptional activity
through reduction of
H3K27 acetylation, reduction of AR target gene expression, and/or reduction of
AR expression,
ultimately leading to a reduction in proliferation. CBP/P300 inhibitor-
mediated inhibition of AR
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activities is expected to be insensitive to AR LBD structural variations, and
thus insensitive to
LBD-related mechanisms of resistance to androgen antagonists. Finally,
CBP/P300 BRD
inhibitors can be useful for suppressing ER-driven signaling in hormone-
receptor positive breast
cancers.
[0087] AR aberrations in the LBD (splice variant v7, AR mutations) can render
the receptor
ligand-independent and insensitive to AR antagonists. Approximately twenty AR
mRNA splice
variants have been identified, with a subset that are constitutively active.
Notably, all biologically
active forms of the AR retain the NTD; drugs that target the NTD have the
potential to impact all
AR forms, including those that may drive resistance to AR-LBD-targeting
therapies. Of the AR
variants, only AR-v7 and ARv567es have been detected at the protein level and
AR-v7 has been
the most studied. Notably, in cases of mCRPC where men were initially treated
with an AR
antagonist, those with AR-v7-positive circulating tumor cells (CTC) showed
poorer PSA response,
and shorter PFS and OS, compared to those negative for AR-v7 CTCs.
Furthermore, in blood
samples from mCRPC patients, the frequency of AR-v7 protein detection in CTC
nuclei increased
from 3% of samples from patients following first line of therapy to 31% of
samples for the third
or more lines of therapy. Findings such as these point to the potential for
using AR-v7 as a patient
selection biomarker, and its likely utility for determining which men with
mCRPC may benefit
from AR antagonist treatment versus chemotherapy.
[0088] Various methods for measuring and defining AR positivity can be
employed to select
patients to receive a pharmaceutical composition comprising one or more
compounds of formula
(I). In the case of prostate cancer, AR expression is largely maintained in
the target population of
relapse patients. Furthermore, AR and AR-v7 protein or mRNA can be detected in
CTC while AR
mutations and AR amplification can be detected from circulating tumor DNA
(ctDNA). Current
IHC methods for measuring AR in breast cancer vary on multiple factors
including the antibody
used, the IHC methodology, and the cut-off criterion for positivity (Safarpour
et al., Am. J. Cancer
Res., 2014, 4:353-368). Most of the studies use the AR441 antibody clone from
Dako (Agilent)
and 1% or 10% positive nuclei as the threshold for positivity. Overall, the
frequency of AR+ TNBC
has been reported to be 20% to 30%.
[0089] Prostate cancers resistant to androgen deprivation or androgen
antagonists remain an unmet
need. In some examples, the inhibition of CBP/P300 BRD, with its
differentiated mechanism of
antagonizing the AR-driven transcriptional program, is used as a treatment for
these patients, with
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potential utility as an earlier line of therapy. Pharmaceutical compositions
comprising one or more
compounds of formula (I) can be used to treat certain forms of prostate
cancer. Preferably,
compositions comprising one or more compounds of formula (I) are useful for
the inhibition of
CBP/P300 BRD, with a differentiated mechanism of antagonizing the AR-driven
transcriptional
program (e.g., as an early line of therapy). Pharmaceutical compositions
comprising one or more
compounds of formula (I) can be used to treat mCRPC, or other cancers
dependent on AR-driven
transcription.
[0090] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising one or more compounds of formula (I) to
patients
diagnosed with mCRPC with progressive castration-resistant disease who have
failed or been
intolerant to at least two prior systemic therapies, including at least one
androgen antagonist-based
therapy with evaluable disease (anti-androgen+LHRH analog, enzalutamide, or
abiraterone) and
either a rising PSA with or without detectable metastatic disease per standard
definitions or
neuroendocrine features. Compounds of formula (I) are useful for treatment of
patients diagnosed
with an AR-v-7 variant form of AR. In some methods, patients diagnosed with
mCRPC with AR-
v7 positive circulating tumor cells (CTC) can be treated with the
pharmaceutical composition
comprising one or more compounds of formula (I). In some methods, patients
diagnosed with
disease progression after treatment with enzalutamide can be treated with the
pharmaceutical
composition comprising one or more compounds of formula (I).
[0091] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising Compound 1 to patients diagnosed with
mCRPC with
progressive castration-resistant disease who have failed or been intolerant to
at least two prior
systemic therapies, including at least one androgen antagonist-based therapy
with evaluable
disease (anti-androgen+LHRH analog, enzalutamide, or abiraterone) and either a
rising PSA with
or without detectable metastatic disease per standard definitions or
neuroendocrine features.
Compound 1 is also useful for treatment of patients diagnosed with an AR-v-7
variant form of AR.
In some methods, patients diagnosed with mCRPC with AR-v7 positive circulating
tumor cells
(CTC) can be treated with the pharmaceutical composition comprising Compound
1. In some
methods, patients diagnosed with disease progression after treatment with
enzalutamide can be
treated with the pharmaceutical composition comprising Compound 1.
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[0092] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising
(1R,3R)-3-((S )-2-((R)-(5-fluoro-2-
methoxyphenyl)(hydroxy)methyl)-6-(methoxycarbonyl)-7-methyl-6,7,8,9-tetrahydro-
3H-
imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic acid to patients
diagnosed with mCRPC
with progressive castration-resistant disease who have failed or been
intolerant to at least two prior
systemic therapies, including at least one androgen antagonist-based therapy
with evaluable
disease (anti-androgen+LHRH analog, enzalutamide, or abiraterone) and either a
rising PSA with
or without detectable metastatic disease per standard definitions or
neuroendocrine features.
(1R,3R)-3-((S)-2-((R)-(5-fluoro-2-methoxyphenyl)(hydroxy)methyl)-6-
(methoxycarbonyl)-7-
methyl-6,7,8,9-tetrahydro-3H-imidazo [4,5-f] quinolin-3 -yl)c yclohexane- 1-
carboxylic acid is also
useful for treatment of patients diagnosed with an AR-v-7 variant form of AR.
In some methods,
patients diagnosed with mCRPC with AR-v7 positive circulating tumor cells
(CTC) can be treated
with the pharmaceutical composition comprising (1R,3R)-3-((S)-2-((R)-(5-fluoro-
2-
methoxyphenyl)(hydroxy)methyl)-6-(methoxycarbonyl)-7-methyl-6,7,8,9-tetrahydro-
3H-
imidazo [4,5-f] quinolin-3-yl)c yclohex ane-1 -c arbo xylic acid. In some
methods, patients diagnosed
with disease progression after treatment with enzalutamide can be treated with
the pharmaceutical
composition comprising (1R,3R)-3-((S)-2-((R)-(5-fluoro-2-
methoxyphenyl)(hydroxy)methyl)-6-
(methoxycarbonyl)-7-methyl-6,7,8,9-tetrahydro-3H-imidazo [4,5-f] quinolin-3 -
yl)c yclohex ane-1-
carboxylic acid.
[0093] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising Compound 2 to patients diagnosed with
mCRPC with
progressive castration-resistant disease who have failed or been intolerant to
at least two prior
systemic therapies, including at least one androgen antagonist-based therapy
with evaluable
disease (anti-androgen+LHRH analog, enzalutamide, or abiraterone) and either a
rising PSA with
or without detectable metastatic disease per standard definitions or
neuroendocrine features.
Compound 2 is also useful for treatment of patients diagnosed with an AR-v-7
variant form of AR.
In some methods, patients diagnosed with mCRPC with AR-v7 positive circulating
tumor cells
(CTC) can be treated with the pharmaceutical composition comprising Compound
2. In some
methods, patients diagnosed with disease progression after treatment with
enzalutamide can be
treated with the pharmaceutical composition comprising Compound 2.
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[0094] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising Compound 3 to patients diagnosed with
mCRPC with
progressive castration-resistant disease who have failed or been intolerant to
at least two prior
systemic therapies, including at least one androgen antagonist-based therapy
with evaluable
disease (anti-androgen+LHRH analog, enzalutamide, or abiraterone) and either a
rising PSA with
or without detectable metastatic disease per standard definitions or
neuroendocrine features.
Compound 3 is also useful for treatment of patients diagnosed with an AR-v-7
variant form of AR.
In some methods, patients diagnosed with mCRPC with AR-v7 positive circulating
tumor cells
(CTC) can be treated with the pharmaceutical composition comprising Compound
3. In some
methods, patients diagnosed with disease progression after treatment with
enzalutamide can be
treated with the pharmaceutical composition comprising Compound 3.
[0095] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising Compound 4 to patients diagnosed with
mCRPC with
progressive castration-resistant disease who have failed or been intolerant to
at least two prior
systemic therapies, including at least one androgen antagonist-based therapy
with evaluable
disease (anti-androgen+LHRH analog, enzalutamide, or abiraterone) and either a
rising PSA with
or without detectable metastatic disease per standard definitions or
neuroendocrine features.
Compound 4 is also useful for treatment of patients diagnosed with an AR-v-7
variant form of AR.
In some methods, patients diagnosed with mCRPC with AR-v7 positive circulating
tumor cells
(CTC) can be treated with the pharmaceutical composition comprising Compound
4. In some
methods, patients diagnosed with disease progression after treatment with
enzalutamide can be
treated with the pharmaceutical composition comprising Compound 4.
[0096] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising (1R,3R)-3-((S )-2-benzy1-6-
(methoxycarbony1)-7-
methy1-6,7 ,8,9-tetrahydro-3H-imidazo [4,5-f] quinolin-3 -yl)c yclohexane- 1-
carboxylic acid to
patients diagnosed with mCRPC with progressive castration-resistant disease
who have failed or
been intolerant to at least two prior systemic therapies, including at least
one androgen antagonist-
based therapy with evaluable disease (anti-androgen+LHRH analog, enzalutamide,
or abiraterone)
and either a rising PSA with or without detectable metastatic disease per
standard definitions or
neuroendocrine features. (1R,3R)-3-((S)-2-benzy1-6-(methoxycarbony1)-7-
methyl-6,7,8,9-
tetrahydro-3H-imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic acid is
also useful for
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treatment of patients diagnosed with an AR-v-7 variant form of AR. In some
methods, patients
diagnosed with mCRPC with AR-v7 positive circulating tumor cells (CTC) can be
treated with the
pharmaceutical composition comprising (1R,3R)-3-((S )-2-benzy1-6-
(methoxycarbony1)-7-
methy1-6,7 ,8,9-tetrahydro-3H-imidazo [4,5-f] quinolin-3 -yl)c yclohexane- 1-
carboxylic acid. In
some methods, patients diagnosed with disease progression after treatment with
enzalutamide can
be treated with the pharmaceutical composition comprising (1R,3R)-3-((S)-2-
benzy1-6-
(methoxyc arbony1)-7-methy1-6,7 ,8,9-tetrahydro-3 H-imidazo [4,5-f] quinolin-3
-yl)c yclohex ane-1-
carboxylic acid.
[0097] Pharmaceutical compositions comprising one or more compounds of formula
(I) are useful
for the treatment of certain forms of AR+ breast cancer. For example, the
suppression of AR
and/or ER transcriptional activity with a compound of formula (I) (or a
pharmaceutical
composition comprising one or more compounds of formula (I)) can be useful in
treating antitumor
effects in AR+ breast cancers, including TNBC and metastatic ER+ tumors, via
its inhibition of
CBP/P300 BRD. In some embodiments, the suppression of AR and/or ER
transcriptional activity
with a compound of formula (I) (or a pharmaceutical composition comprising one
or more
compounds of formula (I)) can be useful in treating antitumor effects in AR+
breast cancers,
including TNBC and ER+ tumors, via its inhibition of CBP/P300 BRD. In some
embodiments, the
suppression of AR and/or ER transcriptional activity with Compound 1 (or a
pharmaceutical
composition comprising Compound 1) can be useful in treating antitumor effects
in AR+ breast
cancers, including TNBC and metastatic ER+ tumors, via its inhibition of
CBP/P300 BRD. In
some embodiments, the suppression of AR and/or ER transcriptional activity
with Compound 1
(or a pharmaceutical composition comprising Compound 1) can be useful in
treating antitumor
effects in AR+ breast cancers, including TNBC and ER+ tumors, via its
inhibition of CBP/P300
BRD. In some embodiments, the suppression of AR and/or ER transcriptional
activity with
Compound 2 (or a pharmaceutical composition comprising Compound 2) can be
useful in treating
antitumor effects in AR+ breast cancers, including TNBC and ER+ tumors, via
its inhibition of
CBP/P300 BRD. In some embodiments, the suppression of AR and/or ER
transcriptional activity
with Compound 3 (or a pharmaceutical composition comprising Compound 3) can be
useful in
treating antitumor effects in AR+ breast cancers, including TNBC and ER+
tumors, via its
inhibition of CBP/P300 BRD. In some embodiments, the suppression of AR and/or
ER
transcriptional activity with Compound 4 (or a pharmaceutical composition
comprising Compound
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4) can be useful in treating antitumor effects in AR+ breast cancers,
including TNBC and ER+
tumors, via its inhibition of CBP/P300 BRD.
[0098] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising one or more compounds of formula (I) to
patients
diagnosed with invasive breast carcinoma with triple negative status (per
College of American
Pathologists [CAP] guidelines) and detectable AR expression in >1% of tumor
cells, with
progressive disease who has failed at least two prior systemic therapies with
evaluable disease or
invasive breast carcinoma AR positive >1% and ER, PR, or HER positive (per CAP
guidelines)
with progressive disease who has failed at least three prior systemic
therapies. Some methods
include administering a therapeutically effective amount of a pharmaceutical
composition
comprising one or more compounds of formula (I) to patients diagnosed with
Her2- breast cancer.
Some methods include administering a therapeutically effective amount of a
pharmaceutical
composition comprising one or more compounds of formula (I) to patients
diagnosed with ER-,
PR-, or ER-/PR- breast cancer.
[0099] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising Compound 1 to patients diagnosed with
invasive breast
carcinoma with triple negative status (per College of American Pathologists
[CAP] guidelines) and
detectable AR expression in >1% of tumor cells, with progressive disease who
has failed at least
two prior systemic therapies with evaluable disease or invasive breast
carcinoma AR positive >1%
and ER, PR, or HER positive (per CAP guidelines) with progressive disease who
has failed at least
three prior systemic therapies. Some methods include administering a
therapeutically effective
amount of a pharmaceutical composition comprising Compound 1 to patients
diagnosed with
Her2- breast cancer. Some methods include administering a therapeutically
effective amount of a
pharmaceutical composition comprising Compound 1 to patients diagnosed with ER-
, PR-, or ER-
/PR- breast cancer.
[0100] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising Compound 2 to patients diagnosed with
invasive breast
carcinoma with triple negative status (per College of American Pathologists
[CAP] guidelines) and
detectable AR expression in >1% of tumor cells, with progressive disease who
has failed at least
two prior systemic therapies with evaluable disease or invasive breast
carcinoma AR positive >1%
and ER, PR, or HER positive (per CAP guidelines) with progressive disease who
has failed at least
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three prior systemic therapies. Some methods include administering a
therapeutically effective
amount of a pharmaceutical composition comprising Compound 2 to patients
diagnosed with
Her2- breast cancer. Some methods include administering a therapeutically
effective amount of a
pharmaceutical composition comprising Compound 2 to patients diagnosed with ER-
, PR-, or ER-
/PR- breast cancer.
[0101] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising Compound 3 to patients diagnosed with
invasive breast
carcinoma with triple negative status (per College of American Pathologists
[CAP] guidelines) and
detectable AR expression in >1% of tumor cells, with progressive disease who
has failed at least
two prior systemic therapies with evaluable disease or invasive breast
carcinoma AR positive >1%
and ER, PR, or HER positive (per CAP guidelines) with progressive disease who
has failed at least
three prior systemic therapies. Some methods include administering a
therapeutically effective
amount of a pharmaceutical composition comprising Compound 3 to patients
diagnosed with
Her2- breast cancer. Some methods include administering a therapeutically
effective amount of a
pharmaceutical composition comprising Compound 3 to patients diagnosed with ER-
, PR-, or ER-
/PR- breast cancer.
[0102] Some methods include administering a therapeutically effective amount
of a
pharmaceutical composition comprising Compound 4 to patients diagnosed with
invasive breast
carcinoma with triple negative status (per College of American Pathologists
[CAP] guidelines) and
detectable AR expression in >1% of tumor cells, with progressive disease who
has failed at least
two prior systemic therapies with evaluable disease or invasive breast
carcinoma AR positive >1%
and ER, PR, or HER positive (per CAP guidelines) with progressive disease who
has failed at least
three prior systemic therapies. Some methods include administering a
therapeutically effective
amount of a pharmaceutical composition comprising Compound 4 to patients
diagnosed with
Her2- breast cancer. Some methods include administering a therapeutically
effective amount of a
pharmaceutical composition comprising Compound 4 to patients diagnosed with ER-
, PR-, or ER-
/PR- breast cancer.
[0103] The present disclosure enables one of skill in the relevant art to make
and use the inventions
provided herein in accordance with multiple and varied embodiments. Various
alterations,
modifications, and improvements of the present disclosure that readily occur
to those skilled in the
art, including certain alterations, modifications, substitutions, and
improvements are also part of
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this disclosure. Accordingly, the foregoing description are by way of example
to illustrate the
discoveries provided herein. The present disclosure provides compounds which
are Selective CBP
Inhibitors.
EXAMPLES
Definitions used in the following Schemes and elsewhere herein are:
ACN acetonitrile
Ac20 acetic anhydride
(+)BINAP ( )-2,2'-Bis(diphenylphosphino)-1,1'-binaphthalen
Boc tert-butoxycarbonyl
n-BuOH butanol
cm centimeter
DCE 1,2-dichloroethane
DCM dichloromethane or methylene chloride
DEA diethylamine
DMC 2-Chloro-4,5-dihydro- 1,3 -dimethy1-1H-imidazolium chloride
DMP Dess-Martin periodinane
DMTMM 4-(4,6-Dimethoxy-1,3,5-triazin-2-y1)-4-methylmorpholinium chloride
DIEA N,N-diisopropylethylamine
DMAP 4-(dimethylamino)pyridine
DMF N,N-dimethylformamide
DMS 0 dimethylsulfoxide
DPPA diphenylphosphoryl azide
dppf bis(diphenylphosphino)ferrocene
ES electro spray ionization
Et3N triethylamine
Et0Ac ethyl acetate
Et0H ethanol
FA formic acid
FCC flash column chromatography
h hours
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HATU 2-(3H- [ 1,2,3 ] triazolo [4,5-b]pyridin-3 - y1)- 1, 1,3,3 -
tetramethylisouronium
hexafluorophosphate
HC1 hydrogen chloride
HOAc acetic acid
HPLC high performance liquid chromatography
(i-Pr)2NEt N,N-diisopropylethylamine
L liter
LC/MS liquid chromatography/mass spectrometry
LDA lithium diisopropylamine
K2CO3 potassium carbonate
Me0H methanol
mL milliliter
mmol millimole
mg milligram
MHz megahertz
MS mass spectrometry
m/z mass/charge ratio
NB S N-bromosuccinimide
nm nanometer
NMM 4-methylmorpholine
NMR nuclear magnetic resonance
Pd2(dba)3 tris(dibenzylideneacetone)dipalladium
Ph3P triphenylphosphine
PhCHO benzaldehyde
PhMe toluene
PPm parts per million
rt room temperature
RT rentention time
SFC supercritical fluid chromatography
STAB sodium triacetoxyborohydride
p-TSA para-toluenesulfonic anhydride
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p-Ts0H para-toluenesulfonic acid
TFA trifluoroacetic acid
TFAA trifluoroacetic anhydride
THF tetrahydrofuran
UV ultraviolet
XPhos 2-dic yclohexylpho sphino-2 1,4 ',6 '-triis oprop ylbiphenyl
Materials
[0104] Unless otherwise noted, all materials were obtained from
commercial suppliers and
were used without further purification. Anhydrous solvents were obtained from
Sigma-Aldrich
(Milwaukee, WI) and used directly. All reactions involving air- or
moisture¨sensitive reagents
were performed under a nitrogen atmosphere and all reactions utilizing
microwave irraditation
were run on a Biotage Initiator EXP EU instrument.
[0105] Unless otherwise noted, mass-triggered HPLC purification and/or
purity and low
resolution mass spectral data were measured using either: (1) Waters Acquity
ultra performance
liquid chromatography (UPLC) system (Waters Acquity UPLC with Sample Organizer
and
Waters Micromass ZQ Mass Spectrometer) with UV detection at 220 nm and a low
resonance
electrospray positive ion mode (ESI) (Column: Acquity UPLC BEH C18 1.7i.tm 2.1
X 50 mm;
gradient: 5-100% Solvent B (95/5/0.09%: Acetonitrile/Water/Formic Acid) in
Solvent A
(95/5/0.1%: 10mM Ammonium Formate/Acetonitrile/Formic Acid) for 2.2 min then
100-5%
Solvent B in Solvent A for 0.01 min then hold at 5% Solvent B in Solvent A for
0.29 min) or (2)
Waters HT2790 Alliance high performance liquid chromatography (HPLC) system
(Waters 996
PDA and Waters ZQ Single Quad Mass Spectrometer) with UV detection at 220 nm
and 254 nm
and a low resonance electrospray ionization (positive/negative) mode (ESI)
(Column: XBridge
Phenyl or C18, 5 iim 4.6x50 mm; gradient: 5-95% Solvent B (95% methanol/5%
water with 0.1%
Formic Acid) in Solvent A (95% water/5% methanol with 0.1% Formic Acid) for
2.5 min then
hold at 95% Solvent B in Solvent A for 1 min (purity and low resolution MS
only).
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General Methods of Compound Preparation
[0106] Described herein are methods of synthesizing the compounds of the
present disclosure.
Compounds of the present disclosure can be synthesized according to the
synthetic schemes
provided below. Preparation of the starting material for Schemes 1 and 2
("Intermediate 1") is
described below. Preparation of the starting material for Schemes 3 and 4 can
be found in Example
1, Part A of U.S. Patent No. 4,404,207.
[0107] Unless otherwise specified, the substituents R4 and R5 in the following
reaction schemes
are defined as follows.
( R2) ( R2),,
(1,3 (1
_ R4 = OH or
0
R5 = 1..._<:_!--OH
[0108] Scheme 1 provides methods useful for synthesizing compounds of Formula
I.
Scheme]
R4 R4
NH2 0NH 0NH
Br R4-00CI is Br NH3 NH2
,... _______________________________________________ ,...
N 40 ____________ N Cul, L-proline N
0 0 0 0 0 0
HOAc, H2SO4
R4 R4
Nr----( N.----
N-R5 NaH, R5-X NH
--c _________________________________________________
N N
0 0 0 0
[0109] Scheme 2 provides methods useful for synthesizing compounds of Formula
I.
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Scheme 2
R4
NH2 NH2 N=---(
0 Br NH2-R5 NH-R5 124-CHO N-R5
0
N Pd-based Catalyst N N
0 0 0 0 0 0
[NM Alternatively, Scheme 3 provides methods useful for synthesizing certain
compounds of
Formula I.
Scheme 3
NO2 NO2 NH2
F NH2-R5 NH-RS Fe, NH4CI NH-R5
/
N N N
cat
R4-CHO
. *
H2N-Ru-OTf
R4 R4 , R4
N--.--( N----r( pho=NTs
(s) (s) N----X
N-R5 CICO2CH3 N-Rs Ph /NR
s
4 __________________________________________ 4 _________ /
TJ
N N . FA, Et3N, Me0H N
H
0 0
[0111] Alternatively, Scheme 4 provides methods useful for synthesizing
certain compounds of
Formula I.
Scheme 4
1. H2, 12
NO2 (S)-(-)-Me0Biphep NO2 NO2
/ F [Ir(COD)C1]2 F CICO2CH3 F
_______________________ v.- ________________ )...-
N 2. D-CSA N N
H
0 0
,i R5-NH2
R4
N--=-- NH2 NO2
N-R5 R4-CHO s NH-R5 H2 NH-R5
N Pd/C
N' N
0 0 0 0 0 o
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Example 1: Syntheses of Compounds of the Disclosure
[0112] The compounds listed in Figure 1 were prepared using standard chemical
manipulations
and procedures similar to those described herein. In Figure 1, "Eluted Isomer"
refers to the order
in which the compound eluted by preparative HPLC.
Example 1.1: Preparation of Intermediate 1: methyl (S)-5-amino-6-bromo-2-
methyl-3,4-
dihydroquinoline-1(2H)-carboxylate
[0113] Figure 2(A) provides a synthetic scheme for the preparation of
Intermediate 1, as described
below.
Step 1. 8-chloro-5-methoxy-2-methylquinoline hydrochloride
[0114] Into a 5L 4-necked round-bottom flask purged and maintained with an
inert atmosphere of
nitrogen, 2-chloro-5-methoxyaniline (250 g, 1.59 mol) was dissolved in 1-
butanol (1200 mL).
Then hydrochloric acid (aq, 36.5%, 526.5 mL) and chloranil (456.5 g, 1.86 mol)
were added. The
resulting mixture was stirred for 1 h at 100 C under nitrogen atmosphere.
Then a solution of (E)-
but-2-enal (169 mL, 2.06 mol) in 1-butanol (300 mL) was added dropwise. The
resulting solution
was stirred for 1 h at 100 C under nitrogen atmosphere. The oil bath was
cooled to
70 C and tetrahydrofuran (1500mL) was added. Then the resulting mixture was
stirred for 1 h at
70 C. The reaction mixture was cooled to 0 C and the solids were filtered.
The solids were washed
with tetrahydrofuran (3L) at 0 C then dried in an oven to afford 8-chloro-5-
methoxy-2-
methylquinoline hydrochloride (83.0 g, 74%) as a yellow solid. MS (ES, m/z):
208 [M+H]t
Step 2. 5-methoxy-2-methylquinoline
[0115] Into a 1000-mL 3-necked round-bottom flask, 8-chloro-5-methoxy-2-
methylquinoline
hydrochloride (50 g, 204.82 mmol) was dissolved in methanol (300 mL). Then
sodium hydroxide
(3M, 205 mL) and 10% palladium on carbon (25 g) were added. Hydrogen (g) was
charged into
the reaction mixture. The reaction mixture was stirred under a hydrogen
atmosphere for 3 h at
room temperature. The reaction was vented to nitrogen and the solids were
filtered out over celite.
The filtered solution was concentrated under vacuum. The residue was subjected
to purification
by FCC eluting with ethyl acetate/petroleum ether (1:5). This afforded the
title compound (28.5 g,
80%) as a yellow oil. MS: (ES, m/z): 174 [M+H]t
Step 3. (2S)-5-methoxy-2-methyl-1,2,3,4-tetrahydroquinoline
[0116] Into a 30-mL pressure tank reactor (50 atm), 5-methoxy-2-
methylquinoline (4.0 g, 23.09
mmol) was dissolved in methanol (10 mL). Then Ru(0Tf)(0-
hexamethylbenzene)((S,S)-
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TsDPEN) 4N-R1S,25)-2-(amino-0)-1,2-diphenylethyl] -4-methylbenzene
sulfonamidato-
KATI [(1,2,3,4,5,6- q)-1,2,3,4,5,6-hexamethylbenzene] (1,1,1-trifluoromethane
sulfonato-K0)-
ruthenium, prepared according to the procedure in J. Am. Chem. Soc. 2011, 133,
9878-9891) (150
mg, 0.23 mmol) was added. To the above hydrogen was introduced in. The
resulting solution was
stirred for 6 h at room temperature. The resulting mixture was concentrated
under vacuum. The
residue was subjected to purification by FCC eluting with ethyl
acetate/petroleum ether (1:4). This
afforded the title compound (3.0 g, 73%) as a yellow oil. MS: (ES, m/z): 178
[M+H]t
Step 4. methyl (S)-5-methoxy-2-methyl-3,4-dihydroquinoline-1(2H)-carboxylate
[0117] Into a 250-mL round-bottom flask, (2S)-5-methoxy-2-methy1-1,2,3,4-
tetrahydroquinoline
(18 g, 99.52 mmol) was dissolved in dichloromethane (100 mL). Then pyridine
(23.6 g, 298.36
mmol) was added, followed by methyl carbonochloridate (9.4 g, 99.47 mmol). The
resulting
solution was stirred for 1 h at room temperature. The resulting solution was
diluted with 100 mL
of dichloromethane and washed with 3x200 mL of water. The organic layers were
combined, dried
over anhydrous sodium sulfate, filtered and concentrated under vacuum. The
residue was subjected
to purification by FCC eluting with ethyl acetate/petroleum ether (1:3). This
afforded the title
compound (21 g, 89%) as a yellow oil. MS: (ES, m/z): 236 [M+H]t
Step 5. methyl (S)-5-hydroxy-2-methyl-3,4-dihydroquinoline-1(2H)-carboxylate
[0118] Into a 500-mL 3-necked round-bottom flask, methyl (2S)-5-methoxy-2-
methy1-1,2,3,4-
tetrahydroquinoline-l-carboxylate (21 g, 89.36 mmol) was dissolved in
dichloromethane (150
mL). Then boron tribromide (150 mL, 0.15 mol, 1 M in CH2C12) was added. The
resulting solution
was stirred for 1 h at room temperature. The reaction was then quenched by the
addition of 300
mL of water. The resulting mixture was extracted with 3x300 mL of
dichloromethane. The organic
layers were combined, dried over anhydrous sodium sulfate, filtered and
concentrated under
vacuum. The residue was subjected to purification by FCC eluting with ethyl
acetate/petroleum
ether (1:2). This afforded the title compound (13.5 g, 68%) as a yellow solid.
MS: (ES, m/z): 222
[M+H] .
Step 6. methyl (S)-2-methyl-5-(((trifluoromethyl)sulfonyl)oxy)-3,4-
dihydroquinoline-1(2H)-
carboxylate
[0119] Into a 250-mL round-bottom flask, methyl (2S)-5-hydroxy-2-methy1-
1,2,3,4-
tetrahydroquinoline-l-carboxylate (5 g, 18.08 mmol) was dissolved in
dichloromethane (50 mL).
Then pyridine (14.3 g, 180.78 mmol) and trifluoromethanesulfonic anhydride
(10.2 g, 36.15
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mmol) were added. The resulting solution was stirred for 1 h at room
temperature. The resulting
mixture was washed with 3x100 mL of water. The organic layers were combined,
dried over
anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue
was subjected to
purification by FCC eluting with ethyl acetate/petroleum ether (1:3). This
afforded the title
compound (5.5 g, 86%) as a yellow oil. MS: (ES, m/z): 354 [M+H]t
Step 7. methyl (S)-5-((diphenylmethylene)amino)-2-methyl-3,4-dihydroquinoline-
1(2H)-
carboxylate
[0120] Into a 500-mL round-bottom flask purged and maintained with an inert
atmosphere of
nitrogen, methyl (2S)-2-methy1-5-[(trifluoromethane)sulfonyloxy]-1,2,3,4-
tetrahydroquinoline -
1-carboxylate (23.5 g, 65.18 mmol) was dissolved in toluene (100 mL). Then
diphenylmethanimine (17.9 g, 97.78 mmol),
tris(dibenzylideneacetone)dipalladium-chloroform
adduct (1.19 g, 1.30 mmol), (+/-)-2,2'-Bis(diphenylphosphino)-1,1'-binaphthyl
(2.43 g, 3.90
mmol) and cesium carbonate (42.4 g, 130.13 mmol) were added. The resulting
solution was stirred
overnight at 100 C under nitrogen atmosphere. The reaction mixture was cooled
and the solids
were filtered out. The residue was subjected to purification by FCC eluting
with ethyl
acetate/petroleum ether (1:3). This afforded the title compound (33 g, 80%) as
a yellow oil. MS:
(ES, m/z): 385 [M+H]t
Step 8. methyl (S)-5-amino-2-methyl-3,4-dihydroquinoline-1(2H)-carboxylate
[0121] Into a 500-mL round-bottom flask, methyl (25)-5-
[(diphenylmethylidene)amino]-2-
methy1-1,2,3,4-tetrahydroquinoline-l-carboxylate (33 g, 85.93 mmol) was
dissolved in methanol
(200 mL). Then sodium acetate (17 g, 207.23 mmol) and hydroxylamine
hydrochloride (12.3 g,
177.00 mmol) were added. The resulting solution was stirred for 2 h at room
temperature. The
solids were filtered out. The resulting mixture was concentrated under vacuum.
The residue was
subjected to purification by FCC eluting with ethyl acetate/petroleum ether
(1:2). This afforded
the title compound (12.5 g, 66%) as a yellow solid. MS: (ES, m/z): 221 [M+H]t
Step 9. methyl (S)-5-amino-6-bromo-2-methyl-3,4-dihydroquinoline-1(2H)-
carboxylate
(Intermediate 1)
[0122] Into a 100-mL 3-necked round-bottom flask, methyl (25)-5-amino-2-methy1-
1,2,3,4-
tetrahydroquinoline-1-carboxylate (1 g, 4.09 mmol) was dissolved in
acetonitrile (20 mL). Then
N-bromosuccinimide (730 mg, 4.10 mmol) was added. The resulting solution was
stirred for 30
min at room temperature. The resulting mixture was concentrated under vacuum.
The residue was
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subjected to purification by FCC eluting with ethyl acetate/petroleum ether
(1:1). This afforded
the title compound (1.1 g, 90%) as a yellow solid. MS: (ES, m/z): 299, 301
[M+H]t
1H-NMR: (400 MHz, CD30D, ppm): 7.19(d, J= 8.8 Hz, 1H), 6.84(d, J= 8.8 Hz, 1H),
4.73-4.69(m,
1H), 3.74(s, 3H), 2.64-2.57(m, 1H), 2.55-2.44(m, 1H), 2.12-2.05(m, 1H), 1.82-
1.79(m, 1H),
1.17(d, J=6.9 Hz, 3H).
Example 1.2: Synthesis of
(1R,3R)-3-[(7S)-2-[(R)-(5-fluoro-2-
methoxyphenyl)(hydroxy)methy1]-6(methoxycarbony1)-7-methyl-3H,6H,7H,8H,9H-
imidazo [4,54] quinolin-3 -yl] cyclohexane-1 carboxylic acid (1); (1R,3R)-3 -
[(75 )- 2- [(S)- (5 -
fluoro-2-methoxyphenyl)(hydroxy)methy1]-6(methoxycarbony1)-7-methyl-
3H,6H,7H,8H,9H-imidazo[4,5-flquinolin-3-yl]cyclohexane-lcarboxylic acid (1')
[0123] Figure 2(B) provides a synthetic scheme for the preparation of Compound
1 and
Compound 1', as described below.
Synthesis of intermediate 2-(5-fluoro-2-methoxypheny1)-2-hydroxyacetic acid
Step 1. 2-(5-fluoro-2-methoxypheny1)-2-ktrimethylsily1)oxylacetonitrile
[0124] A solution of ZnI2 (1.6 mg, 0.01 mmol), 5-fluoro-2-methoxybenzaldehyde
(1.54 g, 9.99
mmol) in trimethylsilanecarbonitrile (1.5 mL, 11.25 mmol) was stirred for 1 h
at room temperature.
The resulting mixture was concentrated under vacuum. The resulting crude
product was purified
by silica gel chromatography (eluting with 1:1 ethyl acetate/petroleum ether)
to afford 2-(5-fluoro-
2-methoxypheny1)-2-[(trimethylsily1)oxy]acetonitrile as a white solid (2.0 g,
79%).
Step 2. 2-(5-fluoro-2-methoxypheny1)-2-hydroxyacetic acid
[0125] A solution of 2-(5-fluoro-2-methoxypheny1)-2-
[(trimethylsily1)oxy]acetonitrile (1.50 g,
5.92 mmol) in hydrochloric acid (10 mL, 12M was stirred for 1 h at 25 C, and
then stirred for 2
h at 70 C. The reaction mixture was cooled and concentrated under vacuum. The
crude product
was purified by reverse phase chromatography (Column: C18; Mobile phase, A:
water (containing
0.05% TFA) and B: ACN (5% to 20% over 30 min); Detector, UV 254 nm) to afford
2-(5-fluoro-
2-methoxypheny1)-2-hydroxyacetic acid as a white solid (1.10 g, 93%).
Step 3. 6-fluoro-2-methyl-5-nitroquinoline
[0126] A solution of trifluoromethanesulfonic acid (82.0 mL, 0.923 mol) in
HNO3 (19.6 mL, 0.437
mol) was stirred for 20 min at 0 C. This was followed by the addition of 6-
fluoro-2-
methylquinoline (50.0 g, 0.310 mol) in dichloromethane (300 mL) at 0 C. The
resulting mixture
was stirred for 15 h at room temperature (25 C). The reaction mixture was
diluted with water (300
38
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mL). The pH value of the solution was adjusted to 8 with sodium bicarbonate
(saturated aqueous
solution). The resulting solution was extracted with dichloromethane (3 x 300
mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered, and
concentrated under vacuum.
The residue was purified by silica gel chromatography (eluting with 1:4 ethyl
acetate/petroleum
ether) to afford 6-fluoro-2-methyl-5-nitroquinoline as a light yellow solid
(60.0 g, 94%). LCMS
(ES, m/z): 207 [M+H]t
Step 4. (28)-6-fluoro-2-methyl-5-nitro-1,2,3,4-tetrahydroquinoline
[0127] A solution of (S)-(-)-Me0-BIPHEP (1.03 g, 1.77 mmol), chloro(1,5-
cyclooctadiene)iridium(I) dimer (538 mg, 0.80 mmol) in toluene (100 mL) was
stirred for 30 min
at room temperature (25 C) under an atmosphere of nitrogen. This was followed
by the addition
of 12 (410 mg, 1.62 mmol) and 6-fluoro-2-methyl-5-nitroquinoline (33.0 g,
0.160 mol) in toluene
(100 mL). The resulting mixture was stirred for 20 h at room temperature (25
C) under hydrogen
(50 atm). The resulting mixture was concentrated under vacuum and purified by
silica gel
chromatography (eluting with 1:1 ethyl acetate/petroleum ether) to afford the
crude product (35.0
g). The crude product was dissolved in ethyl acetate (230 mL), followed by the
addition of D-
Camphorsulfonic acid (36.9 g, 0.158 mol). The resulting solution was stirred
for 1 h at 60 C and
then cooled to room temperature. The solids were collected by filtration, and
rinsed with ethyl
acetate (120 mL). The solids were dissolved in water (50 mL). The pH value of
the solution was
adjusted to 8 with sodium bicarbonate (saturated aqueous solution). The
resulting solution was
extracted with ethyl acetate (3 x 120 mL). The combined organic layers were
dried over anhydrous
sodium sulfate, filtered, and concentrated under vacuum to afford (25)-6-
fluoro-2-methy1-5-nitro-
1,2,3,4-tetrahydroquinoline as a red solid (25.5 g, 76%). LCMS (ES, m/z): 211
[M+H]
Step 5. methyl (28)-6-fluoro-2-methyl-5-nitro-1,2,3,4-tetrahydroquinoline-1-
carboxylate
[0128] A solution of (2S )-6-fluoro-2-methyl-5-nitro-1,2,3,4-
tetrahydroquinoline (25.3 g, 0.120
mol), pyridine (39.0 mL, 0.484 mol), methyl carbonochloridate (18.7 mL, 0.242
mol) in
dichloromethane (150 mL) was stirred for 3 h at room temperature (25 C). The
reaction was
washed with 1M hydrochloric acid (2 x 70 mL). The combined organic layers were
dried over
anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford
methyl (25)-6-fluoro-
2-methy1-5-nitro-1,2,3,4-tetrahydroquinoline-1-carboxylate as a yellow solid
(29.8 g, 92%).
LCMS (ES, m/z): 269 [M+H]t
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Step 6. methyl (2S)-6-[[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]amino]-2-methyl-
5-
nitro1,2,3,4-tetrahydroquinoline-1-carboxylate
[0129] A solution of methyl (2S )-6-fluoro-2-methy1-5-nitro-1,2,3,4-
tetrahydroquinoline-1-
carboxylate (29.6 g, 0.110 mol), pyridine (29.6 mL, 0.368 mol), potassium
carbonate (30.5 g, 0.220
mol), methyl (1R,3R)-3-aminocyclohexane- 1 -carboxylate (25.6 g, 162.84 mmol)
in DMSO (270
mL) was stirred for 15 h at 90 C and then cooled to room temperature. The
reaction was quenched
by the addition of water (200 mL) and extracted with ethyl acetate (3 x 300
mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered, and
concentrated under vacuum.
The resulting crude product was purified by silica gel chromatography (eluting
with 1:1 ethyl
acetate/petroleum ether) to afford methyl
(2S )-64R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-methyl-5-nitro- 1,2,3 ,4-tetrahydro
quinoline-1-
carboxylate as a red oil (32 g, 72%). LCMS (ES, m/z): 406 [M+H]t
Step 7. methyl (2S)-5-amino-6-[[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]amino]-2-
methyl-
1,2,3,4-tetrahydroquinoline-1-carboxylate
[0130] A solution of methyl (2S )-64R1R,3R)-3-
(methoxycarbonyl)cyclohexyl]amino]-2-methyl-
5-nitro-1,2,3,4-tetrahydroquinoline- 1 -carboxylate (31.0 g, 76.46 mmol),
NH4C1 (24.3 g, 454.28
mmol), Fe (64.3 g, 1.15 mol) in tetrahydrofuran (300 mL), ethanol (300 mL),
and water (100 mL)
was stirred for 1 h at 80 C and then cooled to room temperature. The solids
were separated by
filtration. The resulting solution was diluted with water (300 mL) and
extracted with ethyl acetate
(3 x 400 mL). The combined organic layers were dried over anhydrous sodium
sulfate, filtered,
and
concentrated under vacuum to afford methyl (25)-5-amino-6- [R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-methyl- 1,2,3 ,4-tetrahydroquinoline-1-
c arboxylate as a
dark green solid (27.5 g, 92%). LCMS (ES, m/z): 376 [M+H]t
Step 8. methyl (2S)-5-[2-(5-fluoro-2-methoxypheny1)-2-hydroxyacetamido]-6-
[[(1R,3R)-
3(methoxycarbonyl)cyclohexyllamino]-2-methyl-1,2,3,4-tetrahydroquinoline-1-
carboxylate
[0131]
A solution of 2-(5-fluoro-2-methoxypheny1)-2-hydroxyacetic acid (240 mg, 1.20
mmol), HATU (228 mg, 0.60 mmol), methyl (25)-5-amino-64R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-methyl- 1,2,3 ,4-tetrahydroquinoline-1-
c arboxylate (150
mg, 0.40 mmol), DIEA (0.19 mL, 1.20 mmol) in N,N-dimethylformamide (10 mL) was
stirred for
1 h at 25 C. The resulting solution was diluted with H20 (10 mL). The
resulting solution was
extracted with ethyl acetate (3x15 mL) and the organic layers combined. The
resulting mixture
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was washed with brine (2x20 mL). The mixture was dried over anhydrous sodium
sulfate and
concentrated under vacuum. The resulting crude product was purified by silica
gel chromatography
(eluting with 3:2 ethyl acetate/petroleum ether) to afford methyl (2S)-542-
(5fluoro-2-
methoxypheny1)-2-hydroxyacetamido] -6- [ R1R,3R)-3 -(methoxyc arbonyl)c
yclohexyl] amino] -2-
methy1-1,2,3,4-tetrahydroquinoline-1-carboxylate as a yellow solid (180 mg,
81%). LCMS (ES,
m/z): 558 [M+H]t
Step 9. methyl (7S)-2-[(5-fluoro-2-methoxyphenyl)(hydroxy)methy1]-3-
[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]-7-methyl-3H,6H,7H,8H,9H-imidazo[4,5-
flquinoline-6-carboxylate.
[0132] A solution of methyl (2S)-5-[2-(5-fluoro-2-methoxypheny1)-2-
hydroxyacetamido]-6-
[R1R,3R)-3-(methoxycarbonyl)cyclohexyl] amino] -2-methy1-1,2,3 ,4-
tetrahydroquinoline- 1-
carboxylate (180 mg, 0.32 mmol) in AcOH (8 mL) was stirred overnight at 60 C.
The reaction
mixture was cooled and concentrated under vacuum. The resulting crude product
was purified by
silica gel chromatography (eluting with 1:1 ethyl acetate/petroleum ether) to
afford methyl (7S)-
2- R5-fluoro-2-methoxyphenyl)(hydroxy)methyl] -3- R1R,3R)-3 -(methoxyc
arbonyl)c yclohexyl] -
7-methyl-3H,6H,7H,8H,9H-imidazo[4,5-f]quinoline-6-carboxylate as a yellow
solid (120 mg,
69%). LCMS (ES, m/z): 540 [M+H]t
Step 10. (1R,3R)-3-[(7S)-2-[(R)-(5-fluoro-2-methoxyphenyl)(hydroxy)methyl]-6-
(methoxycarbony1)- 7- methy1-3H,6H,7H,8H,9H- imidazo [4,5 - f] quinolin-3 -yl]
cyclohexane-1 -
carboxylic acid (1); (1R,3R)-3-[(7S)-2-[(S)-(5-fluoro-2-
methoxyphenyl)(hydroxy)methyl]-6-
(methoxycarbony1)- 7- methyl-3H,6H,7H,8H,9H- imidazo [4,5 - f] quinolin-3 -yl]
cyclohexane-1 -
carboxylic acid (1')
[0133] A solution of methyl (7S)-24(5-fluoro-2-methoxyphenyl)(hydroxy)methyl]-
3-R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] -7-methyl-3H,6H,7H,8H,9H-imidazo [4,5-f]
quinoline-6-
carboxylate (120 mg, 0.22 mmol), and LiOH (16 mg, 0.67 mmol) in
tetrahydrofuran (2.0 mL),
methanol (2.0 mL) and water (2.0 mL) was stirred overnight at 25 C. The
resulting mixture was
concentrated under vacuum. The crude product was purified by Prep-HPLC
(Column, XBridge
Prep C18 OBD Column, 19x150 mm, Sum; Mobile phase, A: water (containing 10
mmol/L
NH4HCO3) and B: ACN (15.0% to 29.0% over 14 min); Detector, UV 220/254nm). The
product
was separated by Chiral-Prep-HPLC (Column, CHIRALPAK IE, 2x25cm, 5 um; Mobile
phase,
A: Hex (containing 0.1%FA) and B: ethanol (hold 50.0% ethanol over 12 min);
Detector, UV
41
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220/254 nm). The product fractions were concentrated to afford (1R,3R)-3-[(7S)-
2-[(R)-(5-fluoro-
2-methoxyphenyl)(hydroxy)methyll-6-(methoxycarbony1)-7-methyl-3H,6H,7H,8H,9H-
imidazo[4,5-fiquinolin-3-yl[cyclohexane-l-carboxylic acid (1) as a white solid
(23.6 mg, 20%);
and (1R,3R)-3 - [(7S )-2- RS )-(5-fluoro-2-methoxyphenyl)(hydroxy)methyll -6-
(methoxyc arbony1)-
7-methy1-3H,6H,7H,8H,9H-imidazo [4,5-f] quinolin-3 -y1] c yclohexane- 1-
carboxylic acid (1') as a
white solid (23.8 mg, 20%). Stereoisomeric purity was determined via HPLC:
Column:
CH1RALPAK 1E-3, Column size: 0.46 x 5 cm; 3 iim; Mobile phase: Hex (0.1%FA) :
Et0H =
50:50, Flow: 1.0 ml/min.
First eluting isomer (1): 1H-NMR (CD30D, 400 MHz) 6 (ppm): 7.56-7.47 (m, 1H),
7.47-7.31
(m, 1H), 7.21-7.09 (m, 1H), 7.09-6.89 (m, 2H), 6.53(s, 1H), 4.81-4.61(m, 2H),
3.85(s, 3H),
3.78(s, 3H), 3.31-3.18(m, 1H), 3.06-2.82 (m, 2H), 2.57-2.41 (m, 1H), 2.41-2.31
(m, 1H), 2.312.09
(m, 3H), 1.83-1.58 (m, 3H), 1.49-1.21 (m, 2H), 1.16 (d, J= 6.8 Hz, 3H). LCMS
(ES, m/z):
526 [M+H] .
Second eluting isomer (1'): 1H-NMR (CD30D, 400 MHz) 6 (ppm): 7.69-7.44 (m,
2H), 7.44-
7.29 (m, 1H), 7.12-6.99 (m, 1H), 6.98-6.82 (m, 1H), 6.37(s, 1H), 5.03-4.91(m,
1H), 4.81-4.69(m,
1H), 3.78(s, 3H), 3.61(s, 3H), 3.22-3.04(m, 1H), 3.02-2.87 (m, 2H), 2.54-2.41
(m, 1H), 2.41-2.27
(m, 1H), 2.27-2.08 (m, 3H), 1.82-1.58 (m, 3H), 1.58-1.41 (m, 2H), 1.14 (d, J=
6.4 Hz, 3H).
LCMS (ES, m/z): 526 [M+H]t
[0134] A composition of Formula (I) can comprise a compound of one or more of
Formula (II-a),
(II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-
k), (II-1), (II-m), (II-n), and/or (II-o).
For example, in some embodiments the disclosure provides a composition
comprising compound
1 of the foregoing structure or a pharmaceutically acceptable salt thereof at
a purity of at least 90%
wherein the composition comprises less than 10%, e.g. less than 9%, less than
8%, less than 7%,
less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less
than 1%, collectively
of one or more of the following stereoisomers of compound 1, represented as
Formulae (II-a) -
(II-o) below:
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F0/ F 0/ F 0/
OH OH OH
N¨
N N-...0
N N
0 OH N
fr¨OH in¨OH
0 0
0 0
I (II-a) o o
I (II-b) o o
I
(II-c)
/ / /
F 0
F 0 F 0
...,n0H
N¨ N¨
O H 1 N¨
N-...C1. N-...0
N N
.-- ¨OH N
t¨OH
/L 0
----
0 0 0 0 0 0
0 0
I (II-d) I (II-e) I
(II-f)
F 0/ F 0/ F 0/
OH OH OH
N-- N¨ N¨
N-...\3_ N-...0
_
OH l-- ¨OH )----
¨OH
/L 0
0 0 0 0 0 0 0 0
I (II-g) I (II-h) I
(II-i)
/
F 0 F 0 F 0
-.HON
N¨
N¨ N¨
N-....0
N-....9...
0 OH
/L 0
)---OH
----- 0
ri---- HO
0 0 0 0 0 0
I (II-j) I (II-k) I
(II-1)
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/ o/ F 0/
F 0 F
"...OH
N=rr N---
N-oun
N
OH OH 0
0 0
0 0
0 0 0 0 I
I (II-m) I (II-n)
(I1-0)/compound 1'
[0135] For instance, the disclosure provides a pharmaceutical composition
comprising compound
1 or a pharmaceutically acceptable salt thereof at a purity of at least 95% as
determined by the
above HPLC method of Example 1.7. The disclosure also provides a
pharmaceutical composition
comprising compound 1 at a purity of at least 95% as determined by the above
HPLC method.
[0136] The disclosure provides a compound of Formula II obtained by the
foregoing method
exemplified in Example 1.2:
/
F 0
OH
N ----
N
0 OH
0 0
I (II)
or a pharmaceutically acceptable salt, enantiomer, hydrate, solvate, isomer or
tautomer thereof.
[0137] It will be apparent to the skilled reader that each of the
stereoisomers of the compound of
Formula (II) can be obtained by varying the stereochemistry of the appropriate
reagents utilized in
the method of Example 1.2 above. For instance, by adjusting the reagent used
in Step 4 of Example
1.2, compounds such as those of Formulae (II-m) and (II-n) can be synthesized.
Similarly, in Step
6 of Example 1.2, the reagent methyl (1S ,3R)-3-aminocyclohexane-l-carboxylate
can be used in
place of methyl (1R,3R)-3-aminocyclohexane- 1-carboxylate to obtain compounds
of Formulae
(II-b) and (II-e). It will be apparent to the skilled reader that by making a
combination of these
types of modifications to the process set out in Example 1.2, each of
compounds (II-a) to (II-o)
depicted above can be synthesized.
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Example 1.3: (1R,3R)-3-[(78)-2-[(R)-hydroxy(phenyl)methyl]-6-(methoxycarbony1)-
7-
methyl-3H,6H,7H,8H,9H-imidazo[4,5-fiquinolin-3-yl]cyclohexane-1-carboxylic
acid (2)
[0138] Figure 2(C) provides a synthetic scheme for the preparation of Compound
2, as described
below.
Step]. 6-fluoro-2-methyl-5-nitroquinoline
[0139] A solution of trifluoromethanesulfonic acid (82.0 mL, 0.923 mol) in
HNO3 (19.6 mL, 0.437
mol) was stirred for 20 min at 0 C. This was followed by the addition of 6-
fluoro-2-
methylquinoline (50.0 g, 0.310 mol) in dichloromethane (300 mL) at 0 C. The
resulting mixture
was stirred for 15 h at room temperature (25 C). The reaction mixture was
diluted with water (300
mL). The pH value of the solution was adjusted to 8 with sodium bicarbonate
(saturated aqueous
solution). The resulting solution was extracted with dichloromethane (3 x 300
mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered, and
concentrated under vacuum.
The residue was purified by silica gel chromatography (eluting with 1:4 ethyl
acetate/petroleum
ether) to afford 6-fluoro-2-methyl-5-nitroquinoline as a light yellow solid
(60.0 g, 94%). LCMS
(ES, m/z): 207 [M+H]t
Step 2. (28)-6-fluoro-2-methyl-5-nitro-1,2,3,4-tetrahydroquinoline
[0140] A solution of (S)-(-)-Me0-BIPHEP (1.03 g, 1.77 mmol), chloro(1,5-
cyclooctadiene)iridium(I) dimer (538 mg, 0.80 mmol) in toluene (100 mL) was
stirred for 30 min
at room temperature (25 C) under an atmosphere of nitrogen. This was followed
by the addition
of 12 (410 mg, 1.62 mmol), 6-fluoro-2-methyl-5-nitroquinoline (33.0 g, 0.160
mol) in toluene (100
mL). The resulting mixture was stirred for 20 h at room temperature (25 C)
under hydrogen (50
atm). The resulting mixture was concentrated under vacuum and purified by
silica gel
chromatography (eluting with 1:1 ethyl acetate/petroleum ether) to afford the
crude product (35.0
g). The crude product was dissolved in ethyl acetate (230 mL), followed by the
addition of D-
Camphorsulfonic acid (36.9 g, 0.158 mol). The resulting solution was stirred
for 1 h at 60 C and
then cooled to room temperature. The solids were collected by filtration, and
rinsed with ethyl
acetate (120 mL). The solids were dissolved in water (50 mL). The pH value of
the solution was
adjusted to 8 with sodium bicarbonate (saturated aqueous solution). The
resulting solution was
extracted with ethyl acetate (3 x 120 mL). The combined organic layers were
dried over anhydrous
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sodium sulfate, filtered, and concentrated under vacuum to afford (2S)-6-
fluoro-2-methy1-5-nitro-
1,2,3,4-tetrahydroquinoline as a red solid (25.5 g, 76%). LCMS (ES, m/z): 211
[M+H]t
Step 3. methyl (2S)-6-fluoro-2-methyl-5-nitro-1,2,3,4-tetrahydroquinoline-1-
carboxylate
[0141] A solution of (2S )-6-fluoro-2-methyl-5-nitro-1,2,3,4-
tetrahydroquinoline (25.3 g, 0.120
mol), pyridine (39.0 mL, 0.484 mol), methyl carbonochloridate (18.7 mL, 0.242
mol) in
dichloromethane (150 mL) was stirred for 3 h at room temperature (25 C). The
reaction was
washed with 1M hydrochloric acid (2 x 70 mL). The combined organic layers were
dried over
anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford
methyl (25)-6-fluoro-
2-methy1-5-nitro-1,2,3,4-tetrahydroquinoline-1-carboxylate as a yellow solid
(29.8 g, 92%).
LCMS (ES, m/z): 269 [M+H]t
Step 4. methyl (2S)-6-[[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]amino]-2-methyl-
5-nitro-
1,2,3,4-tetrahydroquinoline-1-carboxylate
[0142] A solution of methyl (2S )-6-fluoro-2-methy1-5-nitro-1,2,3,4-
tetrahydroquinoline-1-
carboxylate (29.6 g, 0.110 mol), pyridine (29.6 mL, 0.368 mol), potassium
carbonate (30.5 g, 0.220
mol), methyl (1R,3R)-3-aminocyclohexane- 1 -carboxylate (25.6 g, 162.84 mmol)
in DMSO (270
mL) was stirred for 15 h at 90 C and then cooled to room temperature. The
reaction was quenched
by the addition of water (200 mL) and extracted with ethyl acetate (3 x 300
mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered, and
concentrated under vacuum.
The resulting crude product was purified by silica gel chromatography (eluting
with 1:1 ethyl
acetate/petroleum ether) to afford methyl
(25)-64R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-methyl-5-nitro- 1,2,3 ,4-tetrahydro
quinoline-1-
carboxylate as a red oil (32 g, 72%). LCMS (ES, m/z): 406 [M+H]t
Step 5. methyl (2S)-5-amino-6-[[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]amino]-2-
methyl-
1,2,3,4-tetrahydroquinoline-1-carboxylate
101431 A solution of (2S )-64R1R,3R)-3-(methoxycarbonyl)cyclohexyllamino]-2-
methy1-5-nitro-
1,2,3,4-tetrahydroquinoline- 1 -carboxylate (31.0 g, 76.46 mmol), NH4C1 (24.3
g, 454.28 mmol),
Fe (64.3 g, 1.15 mol) in tetrahydrofuran (300 mL), ethanol (300 mL), water
(100 mL) was stirred
for 1 h at 80 C and then cooled to room temperature. The solids were filtered
out by filtration.
The resulting solution was diluted with water (300 mL) and extracted with
ethyl acetate (3 x 400
mL). The combined organic layers were dried over anhydrous sodium sulfate,
filtered, and
concentrated under vacuum to afford
methyl (2S )-5-amino-6-[R1R,3R)-3 -
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(methoxycarbonyl)cyclohexyl] amino] -2-methyl- 1,2,3 ,4-tetrahydroquinoline-1-
c arboxylate as a
dark green solid (27.5 g, 92%). LCMS (ES, m/z): 376 [M+H]t
Step 6. methyl
(2S)-5-((R)-2-hydroxy-2-phenylacetamido)-6-[[(1R,3R)-3-
(methoxycarbonyl)cyclohexyllamino]-2-methyl-1,2,3,4-tetrahydroquinoline-1-
carboxylate
101441 A solution of (R)-2-hydroxy-2-phenylacetic acid (972 mg, 6.39 mmol),
HATU (1.20 g,
3.16 mmol), methyl (2S )-5-amino-6- [R1R,3R)-3-(methoxycarbonyl)cyclohexyl]
amino] -2-
methy1-1,2,3,4-tetrahydroquinoline-1-carboxylate (800 mg, 2.13 mmol), DIEA
(1.08 mL, 6.20
mmol) in N,N-dimethylformamide (10 mL) was stirred for 5 h at room temperature
(25 C). The
resulting solution was diluted with water (30 mL), and extracted with ethyl
acetate (3 x 50 mL).
The organic layers were combined and washed with brine (2 x 25 mL). The
combined organic
layers were dried over anhydrous sodium sulfate, filtered, and concentrated
under vacuum. The
resulting crude product was purified by silica gel chromatography (eluting
with 1:1 ethyl
acetate/petroleum ether) to afford methyl (25)-54(R)-2-hydroxy-2-
phenylacetamido)-6-
[R1R,3R)-3-(methoxycarbonyl)cyclohexyl] amino] -2-methy1-1,2,3 ,4-
tetrahydroquinoline- 1-
carboxylate as a colorless oil (600 mg, 55%). LCMS (ES, m/z): 510 [M+H]t
Step 7. methyl
(7S)-2-[(R)-hydroxy(phenyl)methy1]-3-[(1R,3R)-3-
(methoxycarbonyl)cyclohexyl]-7-methyl-3H,6H,7H,8H,9H-imidazo[4,5-flquinoline-6-
carboxylate
[0145] A solution of methyl (2S)-5-((R)-2-hydroxy-2-phenylacetamido)-6-
[R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-methyl- 1,2,3 ,4-tetrahydroquinoline-1-
c arboxylate (600
mg, 1.18 mmol) in glacial acetic acid (5 mL, 98%) was stirred for overnight at
40 C and then
cooled to room temperature. The reaction mixture was diluted with water (10
mL). The pH value
of the solution was adjusted to 8 with sodium bicarbonate (saturated aqueous
solution). The
resulting solution was extracted with ethyl acetate (3 x 15 mL). The organic
layers were combined
and dried over anhydrous sodium sulfate, filtered, and concentrated under
vacuum. The resulting
crude product was purified by silica gel chromatography (eluting with 1:1
ethyl acetate/petroleum
ether) to afford methyl
(7S )-2-RR)-hydroxy(phenyl)methyl] -3-R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] -7-methyl-3H,6H,7H,8H,9H-imidazo [4,5-f]
quinoline-6-
carboxylate(400 mg, 69%) as a colorless oil. LCMS (ES, m/z): 492 [M+H]t
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Step 8. (1R,3R)-3-[(7S)-2-[(R)-hydroxy(phenyl)methyl]-6-(methoxycarbony1)-7-
methyl-
3H,6H,7H,8H,9H-imidazo-[4,541-quinolin-3-yl]cyclohexane-1-carboxylic acid (2)
[0146] A solution of methyl (7S)-2-[(R)-
hydroxy(phenyl)methy11-3-[(1R,3R)-3-
(methoxycarbonyl)cyclohexyll -7-methyl-3H,6H,7H,8H,9H-imidazo [4,5-fl
quinoline-6-
carboxylate (400 mg, 0.81 mmol), LiOH (100 mg, 4.17 mmol) in tetrahydrofuran
(5 mL) and water
(2 mL) was stirred for overnight at room temperature (25 C). The resulting
mixture was
concentrated under vacuum. The crude product was purified by Prep-HPLC
(Column: XBridge
Shield RP18 OBD Column, Sum, 19 x 150 mm; Mobile Phase, A: water (containing
10 mmol/L
NH4HCO3) and B: ACN (3% to 30% over 21 min); Detector: UV 254 nm). The product
fractions
were lyophilized to afford (1R,3R)-3-[(7S)-2-[(R)-
hydroxy(phenyl)methyll-6-
(methoxycarbony1)-7-methyl-3H,6H,7H,8H,9H-imidazo- [4,5-f] -quinolin-3-yl] c
yclohexane- 1-
carboxylic acid as a white solid (83.7 mg, 22%). Stereoisomeric purity was
determined via HPLC:
Column: CHIRALPAK 1E-3, Column size: 0.46 x 5 cm; 3 iim; Mobile phase: Hex
(0.1%FA):
Et0H = 85:15, Flow :1.0m1/min.
1H-NMR (CD30D, 400 MHz) 6 (ppm): 7.47-7.28 (m, 7H), 6.12(s, 1H), 4.84-4.74(m,
2H), 3.79(s,
3H), 3.33-3.25(m, 1H), 3.03-2.96 (m, 1H), 2.86-2.82 (m, 1H), 2.38-2.25 (m,
2H), 2.25-2.07 (m,
3H), 1.79-1.72 (m, 1H), 1.64-1.57 (m, 2H), 1.40-1.29 (m, 2H), 1.16 (d, J = 6.8
Hz, 3H). LCMS
(ES, m/z): 478 [M+H]; 99.13% ee.
[0147] A composition of Formula (I) can comprise a compound of one or more of
Formula (M-
a), (III-b), (III-c), (III-d), (III-e), (III-f), (III-g), (III-h), (III-i),
(III-j), (III-k), (III-1), (III-m),
(III-n), and/or (III-o). For example, in some embodiments the disclosure
provides a composition
comprising compound 2 of the foregoing structure or a pharmaceutically
acceptable salt thereof at
a purity of at least 90% wherein the composition comprises less than 10%, e.g.
less than 9%, less
than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,
less than 2% or less
than 1%, collectively of one or more of the following stereoisomers of
compound 2, represented
as Formulae (III-a) ¨ (III-o) below:
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. . .
OH
OH OH N¨
N¨ N¨ N-...0
N ,
0 2)-OH
¨OH OH
0 0 0 P
0 0 0 0 I
(III-
( 111-a) I (III-b)
c)
=,10H .,10H
..10H
N¨ N¨ N¨
N N N
02)¨OH 0/2---
OH
0 OH
0 0 0 0 0 0
I (III-d) 1 (III-e) I
(III-0
OH OH OH
N¨ N¨ N¨
N-....C.,. N.õg
N¨Q,
7.
o----OH
0 OH
0 0 0 0 0 0
I (III-g) I (III-h) I
(III-i)
. . .
-10H ..10H .,10H
N¨ N¨ N¨
N, N¨<?, 0 N-....0
00
N
02)-- ¨OH
crOH
0 OH
0 0 0 0 0 0
I (III-j) I (III-k) 1
(III-1)
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ilfr . ilfr
OH ..10H -10H
N-
1\1,,Q
0)-OH 0' N OH N
0 0 0
0)-OH
0 0 0 0
I I (III-n) I (III-m) (III-o)
[0148] For instance, the disclosure provides a pharmaceutical composition
comprising compound
2 or a pharmaceutically acceptable salt thereof at a purity of at least 95% as
determined by the
above HPLC method of Example 1.7. The disclosure also provides a
pharmaceutical composition
comprising compound 2 at a purity of at least 95% as determined by the above
HPLC method.
Example 1.4: (1R,3R)-3-[(7S)-2-[(S)-[2-(difluoromethoxy)-5-
fluorophenyl](hydroxy)
methy1]-6-(methoxycarbony1)-7-methyl-3H,6H,7H,8H,9H-imidazo[4,54]quinolin-3-
yl]cyclohexane-1-carboxylic acid (452), (1R,3R)-3-[(7S)-2-[(R)-[2-
(difluoromethoxy)-5-
fluoropheny1](hydroxy)methy1]-6-(methoxycarbony1)-7-methyl-3H,6H,7H,8H,9H-
imidazo
[4,54]quinolin-3-yl]cyclohexane-1-carboxylic acid (3)
[0149] Figure 2(D) provides a synthetic scheme for the preparation of Compound
3 and
Compound 452, as described below.
Step]. 2-(difluoromethoxy)-5-fluorobenzaldehyde
[0150] A solution of 5-fluoro-2-hydroxybenzaldehyde (2.0 g, 14.3 mmol),
diethyl
(bromodifluoromethyl)phosphonate (5.69 g, 21.3 mmol), potassium hydroxide
(16.0 g, 285 mmol)
in MeCN (100 mL) and water(50 mL) was stirred for 1 h at -30 C. The reaction
mixture was
diluted with water (20 mL). The resulting solution was extracted with ethyl
acetate (3x100 mL)
and the organic layers combined and dried over anhydrous sodium sulfate. The
solids were filtered
out. The resulting mixture was concentrated under vacuum. The resulting crude
product was
purified by silica gel chromatography (eluting with 1:1 ethyl
acetate/petroleum ether) to afford 2-
(difluoromethoxy)-5-fluorobenzaldehyde as a yellow solid (1.46 g, 54%). LCMS
(ES, m/z): 191
[M+H] .
Step 2. 2-[2-(difluoromethoxy)-5-fluoropheny1]-2-
[(trimethylsilypoxy]acetonitrile
[0151] A solution of 2-(difluoromethoxy)-5-fluorobenzaldehyde (1.46 g, 7.68
mmol), TMSCN
(760 mg, 7.66 mmol), ZnI2 (50 mg, 0.16 mmol) in dichloromethane (3 mL) was
stirred for 2 h at
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room temperature (25 C). The resulting mixture was concentrated under vacuum.
The resulting
crude product was purified by silica gel chromatography (eluting with 1:1
ethyl acetate/petroleum
ether) to afford 2[2-(difluoromethoxy)-5-fluoropheny1]-2-[(trimethylsily1)
oxy]acetonitrile as a
yellow solid (800 mg, 36%) . LCMS (ES, m/z):290 [M+H]
Step 3. 242-(difluoromethoxy)-5-fluorophenyl]-2-hydroxyacetic acid
[0152] A solution of 2-[2-(difluoromethoxy)-5-fluoropheny1]-2-
[(trimethylsily1)oxy] acetonitrile
(800 mg, 2.77 mmol), 1,4-dioxane (2.0 mL), hydrogen chloride (1.0 mL, 12M) in
water (2 mL)
was stirred for 12 h at 70 C and then cooled to room temperature. The
resulting solution was
concentrated under vacuum. The crude product was purified by reverse phase
column
chromatography (water (containing 0.05%TFA)/MeCN) to afford 242-
(difluoromethoxy)-5-
fluoropheny1]-2-hydroxyacetic acid (400 mg, 61%). LCMS (ES, m/z): 237 [M+H]t
Step 4. 6-fluoro-2-methyl-5-nitroquinoline
[0153] A solution of trifluoromethanesulfonic acid (82.0 mL, 0.923 mol) in
HNO3 (19.6 mL, 0.437
mol) was stirred for 20 min at 0 C. This was followed by the addition of 6-
fluoro-2-
methylquinoline (50.0 g, 0.310 mol) in dichloromethane (300 mL) at 0 C. The
resulting mixture
was stirred for 15 h at room temperature (25 C). The reaction mixture was
diluted with water (300
mL). The pH value of the solution was adjusted to 8 with sodium bicarbonate
(saturated aqueous
solution). The resulting solution was extracted with dichloromethane (3 x 300
mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered, and
concentrated under vacuum.
The residue was purified by silica gel chromatography (eluting with 1:4 ethyl
acetate/petroleum
ether) to afford 6-fluoro-2-methyl-5-nitroquinoline as a light yellow solid
(60.0 g, 94%). LCMS
(ES, m/z): 207 [M+H]t
Step 5. (2S)-6-fluoro-2-methy1-5-nitro-1,2,3,4-tetrahydroquinoline
[0154] A solution of (S)-(-)-Me0-BIPHEP (1.03 g, 1.77 mmol), chloro(1,5-
cyclooctadiene)iridium(I) dimer (538 mg, 0.80 mmol) in toluene (100 mL) was
stirred for 30 min
at room temperature (25 C) under an atmosphere of nitrogen. This was followed
by the addition
of 12 (410 mg, 1.62 mmol), 6-fluoro-2-methyl-5-nitroquinoline (33.0 g, 0.160
mol) in toluene (100
mL). The resulting mixture was stirred for 20 h at room temperature (25 C)
under hydrogen (50
atm). The resulting mixture was concentrated under vacuum and purified by
silica gel
chromatography (eluting with 1:1 ethyl acetate/petroleum ether) to afford the
crude product (35.0
g). The crude product was dissolved in ethyl acetate (230 mL), followed by the
addition of D-
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Camphorsulfonic acid (36.9 g, 0.158 mol). The resulting solution was stirred
for 1 h at 60 C and
then cooled to room temperature. The solids were collected by filtration, and
rinsed with ethyl
acetate (120 mL). The solids were dissolved in water (50 mL). The pH value of
the solution was
adjusted to 8 with sodium bicarbonate (saturated aqueous solution). The
resulting solution was
extracted with ethyl acetate (3 x 120 mL). The combined organic layers were
dried over anhydrous
sodium sulfate, filtered, and concentrated under vacuum to afford (2S)-6-
fluoro-2-methy1-5-nitro-
1,2,3,4-tetrahydroquinoline as a red solid (25.5 g, 76%). LCMS (ES, m/z): 211
[M+H]t
Step 6. methyl (2S)-6-fluoro-2-methyl-5-nitro-1,2,3,4-tetrahydroquinoline-1-
carboxylate
[0155] A solution of (2S )-6-fluoro-2-methyl-5-nitro-1,2,3,4-
tetrahydroquinoline (25.3 g, 0.120
mol), pyridine (39.0 mL, 0.484 mol), methyl carbonochloridate (18.7 mL, 0.242
mol) in
dichloromethane (150 mL) was stirred for 3 h at room temperature (25 C). The
reaction was
washed with 1M hydrogen chloride (2 x 70 mL). The combined organic layers were
dried over
anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford
methyl (25)-6-fluoro-
2-methy1-5-nitro-1,2,3,4-tetrahydroquinoline-1-carboxylate as a yellow solid
(29.8 g, 92%).
LCMS (ES, m/z): 269 [M+H]t
Step 7. methyl (2S)-6-[[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]amino]-2-methyl-
5-nitro-
1,2,3,4-tetrahydroquinoline-1-carboxylate
[0156] A solution of methyl (2S )-6-fluoro-2-methy1-5-nitro-1,2,3,4-
tetrahydroquinoline-1-
carboxylate (29.6 g, 0.110 mol), pyridine (29.6 mL, 0.368 mol), potassium
carbonate (30.5 g, 0.220
mol), methyl (1R,3R)-3-aminocyclohexane- 1 -carboxylate (25.6 g, 162.84 mmol)
in DMSO (270
mL) was stirred for 15 h at 90 C and then cooled to room temperature. The
reaction was quenched
by the addition of water (200 mL) and extracted with ethyl acetate (3 x 300
mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered, and
concentrated under vacuum.
The resulting crude product was purified by silica gel chromatography (eluting
with 1:1 ethyl
acetate/petroleum ether) to afford methyl (2S )-6- [R1R,3R)-3-
(methoxycarbonyl)cyclohexyl]
amino]-2-methy1-5-nitro-1,2,3,4-tetrahydroquinoline-l-carboxylate as a red oil
(32 g, 72%).
LCMS (ES, m/z): 406 [M+H]t
Step 8. methyl (2S)-5-amino-6-[[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]amino]-2-
methyl-
1,2,3,4-tetrahydroquinoline-1-carboxylate
[0157] A solution of methyl (2S )-64R1R,3R)-3-(methoxycarbonyl)cyclohexyl]
amino]-2-methyl-
5-nitro-1,2,3,4-tetrahydroquinoline- 1 -carboxylate (31.0 g, 76.46 mmol),
NH4C1 (24.3 g, 454.28
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mmol), Fe (64.3 g, 1.15 mol) in tetrahydrofuran (300 mL), ethanol (300 mL),
water (100 mL) was
stirred for 1 h at 80 C and then cooled to room temperature. The solids were
filtered out by
filtration. The resulting solution was diluted with water (300 mL) and
extracted with ethyl acetate
(3 x 400 mL). The combined organic layers were dried over anhydrous sodium
sulfate, filtered,
and
concentrated under vacuum to afford methyl (2S )-5-amino-6- [R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-methyl- 1,2,3 ,4-tetrahydroquinoline-1-
c arboxylate as a
dark green solid (27.5 g, 92%). LCMS (ES, m/z): 376 [M+H]t
Step 9. methyl (28)-542-[2-(difluoromethoxy)-5-fluoropheny1]-2-
hydroxyacetamido]-6-
[[(1R,3R)-3-(methoxycarbonyl)cyclohexyllamino]-2-methyl-1,2,3,4-
tetrahydroquinoline-1-
carboxylate
[0158] A solution of methyl (2S )-5-amino-6-[[(1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino]-
2-methy1-1,2,3,4-tetrahydroquinoline-l-carboxylate (200 mg,
0.53 mmol), 2- [2-
(difluoromethoxy)-5-fluoropheny1]-2-hydroxyacetic acid (220 mg, 0.93 mmol),
DMTMM (350
mg, 1.26 mmol) in dichloromethane (5 mL) was stirred for 1 h room temperature
(25 C). The
resulting solution was concentrated under vacuum. The resulting crude product
was purified by
silica gel chromatography (eluting with 1:1 ethyl acetate/petroleum ether) to
afford methyl (2S)-
5- [2- [2-(difluoromethoxy)-5-fluoropheny1]-2-hydroxyacetamido]-6- [R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-methyl- 1,2,3 ,4-tetrahydroquinoline-1-
c arboxylate as a
yellow solid (70.0 mg, 22%). LCMS (ES, m/z): 594 [M+H]t
Step 10. methyl (78)-2-[[2-(difluoromethoxy)-5-fluorophenyl](hydroxy)methy1]-3-
[(1R,3R)-
3-(methoxycarbonyl)cyclohexyl]-7-methyl-3H,6H,7H,8H,9H-imidazo[4,5-flquinoline-
6-
carboxylate
[0159] A solution of methyl
(2S )-5- [2- [2-(difluoromethoxy)-5 -fluorophenyl] -2-
hydroxyacetamido] -6- [R1R,3R)-3-(methoxycarbonyl)cyclohexyl] amino] -2-methy1-
1,2,3,4-
tetrahydroquinoline-l-carboxylate (70.0 mg, 0.12 mmol) in glacial acetic acid
(2.0 mL) was stirred
for overnight at 40 C and then cooled to room temperature. The resulting
solution was
concentrated under vacuum.
The resulting crude product was purified by silica gel
chromatography (eluting with 1:2 ethyl acetate/petroleum ether) to afford
methyl (75)-24[2-
(difluoromethoxy)-5-fluorophenyl] (hydroxy)methyl] -3- R1R,3R)-3-
(methoxycarbonyl)
cyclohexyl]-7-methy1-3H,6H,7H,8H,9H-imidazo[4,5-fiquinoline-6-carboxylate as a
yellow solid
(50.0 mg, 74%). LCMS (ES, m/z): 576 [M+H]t
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Step 11. (1R,3R)-3-[(7S)-2-[(S)-[2-(difluoromethoxy)-5-
fluorophenyl](hydroxy)methy1]-6-
(methoxycarbony1)- 7-methy1-3H,6H,7H,8H,9H- imidazo [4,54] quinolin-3-yl]
cyclohexane-1 -
carboxylic acid (452); (1R,3R)-3- [(7S)-2-[(R)- [2-
(difluoromethoxy)-5-
fluorophenyl](hydroxy)methy1]-6-(methoxycarbony1)-7-methyl-3H,6H,7H,8H,9H-
imidazo [4,54] quinolin-3-yl] cyclohexane-1 -carboxylic acid (3)
[0160] A solution of methyl (7S)-2-[[2-(difluoromethoxy)-5-
fluorophenyl](hydroxy)methy1]-3-
[(1R,3R)-3-(methoxycarbonyl)cyclohexyl] -7-methyl-3H,6H,7H,8H,9H-imidazo [4,5-
f] quinoline
-6-carboxylate (50.0 mg, 0.09 mmol), LiOH (10.0 mg, 0.42 mmol) in
tetrahydrofuran (2.0 mL)
and water (2.0 mL) was stirred for overnight at room temperature (25 C). The
resulting mixture
was concentrated under vacuum. The crude product was purified by Prep-HPLC
(Column,
XBridge Shield RP18 OBD Column, 30x150 mm, 5 um; Mobile phase, A: water
(containing 10
mmol/L NH4HCO3) and B: ACN (25.0% to 35.0% over 8 min); Detector, UV 254/220
nm). The
product fractions were concentrated to afford (1R,3R)-3-[(7S)-2-[(S)-[2-
(difluoromethoxy)-5-
fluorophenyl](hydroxy)methyl] -6-(methoxyc arbony1)-7-methy1-3H,6H,7H,8H,9H-
imidazo [4,5-
f]quinolin-3-yl]cyclohexane- 1-carboxylic acid (452) as a white solid (4.50
mg, 9%), and (1R,3R)-
3 - [(7S )-2- [(R)- [2-(difluoromethoxy)-5-fluorophenyl] (hydroxy)methyl] -6-
(methoxyc arbony1)-7 -
methyl-3H,6H,7H,8H,9H-imidazo [4,5-f] quinolin-3 -yl] c yclohexane- 1-
carboxylic acid (515) as a
white solid (4.30 mg, 9%). Enantiomeric excess was determined via HPLC:
Column:
CH1RALPAK 1E-3, Column size: 0.46 x 5 cm; 3 iim; Co-Solvent: IPA (20 mM NH3)
Gradient
(B%) : 10% to 50% in 4.0min, hold 2.0 min at 50%.
First eluting isomer (452): 1H-NMR (CD30D, 400 MHz) 6 (ppm): 7.63-7.61 (m,
1H), 7.53 (d, J
= 8.8 Hz, 1H), 7.41(d, J= 9.2Hz, 1H) 7.20-7.13 (m, 2H), 6.67-6.30 (m, 2H),
4.98-4.95 (m, 1H),
4.76-4.71 (m, 1H), 3.78 (s, 3H), 3.15-2.86 (m, 3H), 2.46-2.20 (m, 5H), 1.81-
1.53 (m, 5H), 1.13 (d,
J= 6.8 Hz, 3H). LCMS (ES, m/z): 562 [M+H]t
Second eluting isomer (3): 1H-NMR (CD30D, 400 MHz) 6 (ppm): 7.55-7.53 (m, 1H),
7.47-7.42
(m, 2H), 7.40-7.12 (m, 2H), 6.85-6.44 (m, 2H), 4.94-4.91 (m, 1H), 4.76-4.71
(m, 1H), 3.78 (s, 3H),
3.22-2.84 (m, 3H), 2.46-2.23 (m, 5H), 1.84-1.61 (m, 5H), 1.14 (d, J = 6.4 Hz,
3H). LCMS (ES,
m/z): 562 [M+H[ ; >99.99% ee.
[0161] In some embodiments, the disclosure provides the first eluting isomer
obtained from Step
11 of the process described in Example 1.4. In some embodiments, the
disclosure provides the
second eluting isomer obtained from Step 11 of the process described in
Example 1.4.
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[0162] A composition of Formula (I) can comprise a compound of one or more of
Formula (IV-
a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), (IV-g), (IV-h), (IV-i), (IV-j),
(IV-k), (IV-1), (IV-m), (IV-
n), and/or (IV-o). For example, in some embodiments the disclosure provides a
composition
comprising compound 3 of the foregoing structure or a pharmaceutically
acceptable salt thereof at
a purity of at least 90% wherein the composition comprises less than 10%, e.g.
less than 9%, less
than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,
less than 2% or less
than 1%, collectively of one or more of the following stereoisomers of
compound 3, represented
as Formulae (IV-a) ¨ (IV-o) below:
F F
F 40 0)¨F F . 0)¨F F 441 F0)¨F
OH OH OH
N¨
N-..<3..
N."0 N-.C7
101 ,
N N ==;- N
0
OH
ofp-
OH0.70H----
0 0 0 0 0 0
I (IV-a) I (IV-b) I
(IV-c)
F
F 40 0 F 4. 0)_F F 40 0
-10H ..10H .,10H
N79. OH 0 OH N-...,C7
i
N N "--- N
0
ol)--OH
0 0 0 0 0 0
I (IV-d) I (IV-e) I
(IV-f)
F F F
F . 0 F 411 0 F 41 0
OH OH OH
N¨
N-.C....
..
0
OH
o;)-- 0
OH OH
0 0 0 0 0 0
I (IV-g) I (IV-h) I
(IV-i)
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F F F
F * 0
>-F
F * 0
)-F
F * 0
>-F
-10H ..10H -10H
N-
N-.<1._
..
0
OH
o2-- - 0 OH
---OH
0 0 0 0 0 0
I (IV-j) I (IV-k)
I (IV-1)
F F
F)-F F * 0)-F F afr 0 F afr 0)-F
OH -10H ..10H
N-
N,,,9_
0 ' N \`µµ. N la N
1.0 OH
0 OH
0 OH
0 0 0 0 0 0
I (IV-m) I (IV-n)
I (IV-o)
[0163] For instance, the disclosure provides a pharmaceutical composition
comprising compound
3 or a pharmaceutically acceptable salt thereof at a purity of at least 95% as
determined by the
above HPLC method of Example 1.7. The disclosure also provides a
pharmaceutical composition
comprising compound 3 at a purity of at least 95% as determined by the above
HPLC method.
Example 1.5: (1R,3R)-34(S)-2-benzy1-6-(methoxycarbony1)-7-methyl-6,7,8,9-
tetrahydro-
3H-imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic acid (4)
[0164] Figure 2(E) provides a synthetic scheme for the preparation of Compound
4, as described
below.
Step 1. methyl (S)-5-amino-6-(41R,3R)-3-(methoxycarbonyl)cyclohexyl)amino)-2-
methyl-
3,4-dihydroquinoline-1-(2H)-carboxylate
[0165] Into a 10-mL round-bottom flask purged and maintained with an inert
atmosphere of
nitrogen, methyl (1R,3R)-3-aminocyclohexane-1-carboxylate hydrochloride (130
mg, 0.67 mmol)
was dissolved in dioxane (4 mL). Then methyl (2S)-5-amino-6-bromo-2-methy1-
1,2,3,4-
tetrahydroquinoline-l-carboxylate (100 mg, 0.33 mmol, Intermediate 1),
Brettphos (72 mg, 0.13
mmol), 3rd Generation Brettphos precatalyst (61 mg, 0.07 mmol) and sodium tert-
butoxide (97
mg, 1.01 mmol) were added. The resulting solution was stirred for 1 h at 100
C under nitrogen
atmosphere. The reaction mixture was cooled and the solids were filtered out.
The resulting
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mixture was concentrated under vacuum. The residue was subjected to
purification by FCC eluting
with ethyl acetate/petroleum ether (2:1). This afforded the title compound
(41.3 mg, 33%) as a
dark green solid. MS: (ES, m/z): 376 [M+H]t
Step 2. methyl (S)-2-benzy1-3-41R,3R)-3-(methoxycarbonyl)cyclohexyl)-7-methyl-
3,7,8,9-
tetrahydro-6H-imidazo[4,5-fiquinoline-6-carboxylate
[0166] Into a 25-mL round-bottom flask, methyl methyl (S)-5-amino-6-(((lR,3R)-
3-
(methoxyc arbonyl)c yclohexyl)amino)-2-methy1-3 ,4-dihydroquinoline- 1(2H)-c
arboxylate (165.4
mg, 0.44 mmol) was dissolved in dichloromethane (5 mL). Then 2-
phenylacetaldehyde (158.8 mg,
1.32 mmol) was added. The resulting solution was stirred for 2 h at room
temperature. The
resulting mixture was concentrated under vacuum. The residue was subjected to
purification by
FCC eluting with ethyl acetate/petroleum ether (2:1). This afforded the title
compound (122.9 mg,
59%) as a yellow solid. MS: (ES, m/z): 476 [M+H]t
Step 3.
(1R,3R)-3-[(78)-2-benzy1-6-(methoxycarbony1)-7-methyl-3H,6H,7H,8H,9H-
imidazo [4,5-f] quinolin-3-yl] cyclohexane-1 -carboxylic acid (4)
[0167] Into a 25-mL round-bottom flask,
methyl (S)-2-benzy1-34(1R,3R)-3-
(methoxycarbonyl)cyclohexyl)-7-methy1-3,7,8,9-tetrahydro-6H-imidazo [4,5-f]
quinoline-6-
carboxylate (30 mg, 0.06 mmol) was dissolved in tetrahydrofuran (0.5 mL). Then
water (0.5 mL)
was added, followed by lithium hydroxide (7.0 mg, 0.29 mmol). The resulting
solution was stirred
for 3 h at 85 C. The pH value of the solution was adjusted to 5-6 with
hydrochloric acid (1 mol/L).
The resulting solution was extracted with ethyl acetate (3x20 mL). The organic
layers were
combined, dried over anhydrous sodium sulfate, filtered and concentrated under
vacuum. The
crude product was purified by Prep-HPLC with the following conditions: Column,
XBridge C18
OBD Prep Column, 100A, 5 iim, 19 mm X 250 mm; mobile phase, A: Water
(containing 10
mmol/L NH4HCO3) and B: ACN (10.0% to 30.0% ACN over 10 min); UV Detector:
254nm. This
afforded the title compound (15.2 mg, 52%) as a white solid.
1H NMR (CD30D, 400 MHz) 6 (ppm): 7.47 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 8.9
Hz, 1H), 7.35-
7.19 (m, 5H), 4.84-4.68 (m, 2H), 4.45-4.25 (m, 2H), 3.79 (s, 3H), 3.22-3.14
(m, 1H), 2.98-2.85
(m, 2H), 2.40-2.02 (m, 5H), 1.83-1.70 (m, 1H), 1.64-1.54 (m, 2H), 1.33-1.13
(m, 5H). MS: (ES,
m/z): 462 [M+H]t
[0168] Compound 17, Compound 18, and Compound 19 were prepared using standard
chemical
manipulations and procedures similar to those used for the preparation of
Example 16.
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Example 1.6: 3-((7S)-2-((4-chlorophenyl)(hydroxy)methyl)-6-(methoxycarbonyl)-7-
methyl-
6,7,8,9-tetrahydro-3H-imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic
acid (413); and
(1R,3R)-34(S)-2-((S)-(4-chlorophenyl)(hydroxy)methyl)-6-(methoxycarbonyl)-7-
methyl-
6,7,8,9-tetrahydro-3H-imidazo[4,5-fiquinolin-3-y1)cyclohexane-1-carboxylic
acid (501)
[0169] Figure 2(F) provides a synthetic scheme for the preparation of Compound
413 and
Compound 501, as described below.
Step]. 6-fluoro-2-methyl-5-nitroquinoline
[0170] A solution of trifluoromethanesulfonic acid (82.0 mL, 0.923 mol) in
HNO3 (19.6 mL, 0.437
mol) was stirred for 20 min at 0 C. This was followed by the addition of 6-
fluoro-2-
methylquinoline (50.0 g, 0.310 mol) in dichloromethane (300 mL) at 0 C. The
resulting mixture
was stirred for 15 hours at room temperature (25 C). The reaction mixture was
diluted with water
(300 mL). The pH value of the solution was adjusted to 8 with sodium
bicarbonate (saturated
aqueous solution). The resulting solution was extracted with dichloromethane
(3 x 300 mL). The
combined organic layers were dried over anhydrous sodium sulfate, filtered,
and concentrated
under vacuum. The residue was purified by silica gel chromatography (eluting
with 1:4 ethyl
acetate/petroleum ether) to afford 6-fluoro-2-methyl-5-nitroquinoline as a
light yellow solid (60.0
g, 94%). LCMS (ES, m/z): 207 [M+H]t
Step 2. (2S)-6-fluoro-2-methyl-5-nitro-1,2,3,4-tetrahydroquinoline
[0171] A solution of (S)-(-)-Me0-BIPHEP (1.03 g,
1.77 mmol), chloro(1,5-
cyclooctadiene)iridium(I) dimer (538 mg, 0.80 mmol) in toluene (100 mL) was
stirred for 30 min
at room temperature (25 C) under an atmosphere of nitrogen. This was followed
by the addition
of 12 (410 mg, 1.62 mmol), and 6-fluoro-2-methyl-5-nitroquinoline (33.0 g,
0.160 mol) in toluene
(100 mL). The resulting mixture was stirred for 20 h at room temperature (25
C) under hydrogen
(50 atm). The resulting mixture was concentrated under vacuum and purified by
silica gel
chromatography (eluting with 1:1 ethyl acetate/petroleum ether) to afford the
crude product (35.0
g). The crude product was dissolved in ethyl acetate (230 mL), followed by the
addition of D-
Camphorsulfonic acid (36.9 g, 0.158 mol). The resulting solution was stirred
for 1 h at 60 C and
then cooled to room temperature. The solids were collected by filtration, and
rinsed with ethyl
acetate (120 mL). The solids were dissolved in water (50 mL). The pH value of
the solution was
adjusted to 8 with sodium bicarbonate (saturated aqueous solution). The
resulting solution was
extracted with ethyl acetate (3 x 120 mL). The combined organic layers was
dried over anhydrous
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sodium sulfate, filtered, and concentrated under vacuum to afford (2S)-6-
fluoro-2-methy1-5-nitro-
1,2,3,4-tetrahydroquinoline as a red solid (25.5 g, 76%). LCMS (ES, m/z): 211
[M+H]t
Step 3. methyl (2S)-6-fluoro-2-methyl-5-nitro-1,2,3,4-tetrahydroquinoline-1-
carboxylate
[0172] A solution of (2S )-6-fluoro-2-methyl-5-nitro-1,2,3,4-
tetrahydroquinoline (25.3 g, 0.120
mol), pyridine (39.0 mL, 0.484 mol), and methyl carbonochloridate (18.7 mL,
0.242 mol) in
dichloromethane (150 mL) was stirred for 3 h at room temperature (25 C). The
reaction was
washed with 1N hydrogen chloride (aq., 2 x 70 mL). The combined organic layers
were dried over
anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford
methyl (25)-6-fluoro-
2-methy1-5-nitro-1,2,3,4-tetrahydroquinoline-1-carboxylate as a yellow solid
(29.8 g, 92%).
LCMS (ES, m/z): 269 [M+H]t
Step 4. methyl (2S)-6-[[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]amino]-2-methyl-
5-nitro-
1,2,3,4-tetrahydroquinoline-1-carboxylate
[0173] A solution of methyl (2S )-6-fluoro-2-methy1-5-nitro-1,2,3,4-
tetrahydroquinoline-1-
carboxylate (29.6 g, 0.110 mol), pyridine (29.6 mL, 0.368 mol), potassium
carbonate (30.5 g, 0.220
mol), and methyl (1R,3R)-3-aminocyclohexane- 1 -carboxylate (25.6 g, 162.84
mmol) in DMSO
(270 mL) was stirred for 15 h at 90 C and then cooled to room temperature.
The reaction was
quenched by the addition of water (200 mL) and extracted with ethyl acetate (3
x 300 mL). The
combined organic layers were dried over anhydrous sodium sulfate, filtered,
and concentrated
under vacuum. The resulting crude product was purified by silica gel
chromatography (eluting
with 1:1 ethyl acetate/petroleum ether) to afford methyl (25)-6-[R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-methyl-5-nitro- 1,2,3 ,4-tetrahydro
quinoline-1-
carboxylate as a red oil (32 g, 72%). LCMS (ES, m/z): 406 [M+H]t
Step 5. methyl (2S)-5-amino-6-[[(1R,3R)-3-(methoxycarbonyl)cyclohexyl]amino]-2-
methyl-
1,2,3,4-tetrahydroquinoline-1-carboxylate
101741 A solution of methyl
(2S )-2-methy1-5-nitro-64R1R,3R)-4-
(methoxycarbonyl)cyclohexyl] amino] -1,2,3 ,4-tetrahydroquinoline- 1 -
carboxylate (31.0 g, 76.46
mmol), NH4C1 (24.3 g, 454.28 mmol), and Fe (powder, 64.3 g, 1.15 mol) in
tetrahydrofuran (300
mL), ethanol (300 mL), water (100 mL) was stirred for 1 h at 80 C and then
cooled to room
temperature. The solids were filtered out by filtration. The resulting
solution was diluted with
water (300 mL) and extracted with ethyl acetate (3 x 400 mL). The combined
organic layers were
dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum
to afford methyl
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(2S )-5-((R)-2-hydroxy-2-phenylacetamido)-6- [R1R,3R)-3-
(methoxycarbonyl)cyclohexyl]
amino]-2-methy1-1,2,3,4-tetrahydroquinoline-1-carboxylate as a dark green
solid (27.5 g, 92%).
LCMS (ES, m/z): 376 [M+H]t
Step 6. methyl
(2S)-542-(4-chloropheny1)-2-hydroxyacetamido]-6-[[(1R,3R)-3-
(methoxycarbonyl)cyclohexyliamino]-2-methyl-1,2,3,4-tetrahydroquinoline-1-
carboxylate
101751 A solution of 2-(4-chloropheny1)-2-hydroxyacetic acid (112 mg, 0.60
mmol), HATU (304
mg, 0.80 mmol), methyl (25 )-5- amino-6- [R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] amino] -2-
methy1-1,2,3,4-tetrahydroquinoline-1-carboxylate (150 mg, 0.40 mmol), and DlEA
(155 mg, 1.20
mmol) in N,N-dimethylformamide (2 mL) was stirred for 15 h at room temperature
(25 C). The
resulting solution was diluted with water (30 mL), and extracted with ethyl
acetate (3 x 50 mL).
The organic layers were combined and washed with brine (2 x 25 mL). The
combined organic
layers were dried over anhydrous sodium sulfate, filtered, and concentrated
under vacuum. The
resulting crude product was purified by silica gel chromatography (eluting
with 1:1 ethyl
acetate/petroleum ether) to afford methyl (2S)-5-[2-(4-chloropheny1)-2-
hydroxyacetamido]-6-
[R1R,3R)-3-(methoxycarbonyl)cyclohexyl] amino] -2-methy1-1,2,3 ,4-
tetrahydroquinoline- 1-
carboxylate as yellow oil (70.0 mg, 32%). LCMS (ES, m/z): 544 [M+H]t
Step 7. methyl
(7S)-2-[(4-chlorophenyl)(hydroxy)methy11-3-[(1R,3R)-3-
(methoxycarbonyl)cyclohexy11-7-methyl-3H,6H,7H,8H,9H-imidazo[4,5-flquinoline-6-
carboxylate
[01176]
A solution of methyl (2S)-5- [2-(4-chloropheny1)-2-hydroxyacetamido]-6-
[R1R,3R)-3 -(methoxyc arbonyl)c yclohexyl] amino] -2-methy1-1,2,3 ,4-
tetrahydroquinoline- 1-
carboxylate (60.0 mg, 0.11 mmol) in AcOH (2 mL) was stirred for 15 h at 40 C
and then cooled
to room temperature. The reaction mixture was diluted with water (10 mL). The
pH value of the
solution was adjusted to 8 with sodium bicarbonate (saturated aqueous
solution). The resulting
solution was extracted with ethyl acetate (3 x 15 mL). The organic layers were
combined and dried
over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The
resulting crude
product was purified by silica gel chromatography (eluting with 1:1 ethyl
acetate/petroleum ether)
to afford methyl
(7S )-2-R4-chlorophenyl)(hydroxy)methyl] -3- R1R,3R)-3-
(methoxycarbonyl)cyclohexyl] -7-methyl-3H,6H,7H,8H,9H-imidazo [4,5-f]
quinoline-6-
carboxylate as yellow oil (46.0 mg, 79%). LCMS (ES, m/z): 526 [M+H]t
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Step 8. (1R,3R)-3-[(7S)-2-[(R)-(4-chlorophenyl)(hydroxy)methyl]-6-
(methoxycarbony1)-7-
methy1-3H,6H,7H,8H,9H-imidazo[4,5-fiquinolin-3-yl]cyclohexane-1-carboxylic
acid (413);
(1R,3R)-3-[(7S)-2-[(S)-(4-chlorophenyl)(hydroxy)methyl]-6-(methoxycarbony1)-7-
methy1-
3H,6H,7H,8H,9H-imidazo[4,5-fiquinolin-3-yl]cyclohexane-1-carboxylic acid (501)
[0177] A solution of methyl (7S)-2-[(4-chlorophenyl)(hydroxy)methyll -3-
[(1R,3R)-3-
(methoxyc arbonyl)c yclohexyl] -7-methyl-3H,6H,7H,8H,9H-imidazo [4,5-f]
quinoline-6-
carboxylate (50.0 mg, 0.10 mmol), and LiOH (11.4 mg, 0.48 mmol) in
tetrahydrofuran (1 mL) and
water (1 mL) was stirred for 15 h at 25 C. The resulting mixture was
concentrated under vacuum.
The crude product was purified by Prep-HPLC (Column: XBridge Shield RP18 OBD
Column,
Sum, 19 x 150 mm; Mobile Phase, A: water (containing 10 mmol/L NH4HCO3) and B:
ACN (10%
to 37% over 12 min); Detector: UV 254 nm). The product fractions were
lyophilized to afford
(1R,3R)-3-[(7S)-2-[(R)-(4-chlorophenyl)(hydroxy)methyl] -6-(methoxyc arb ony1)-
7 -methyl-
3H,6H,7H,8H,9H-imidazo[4,5-fiquinolin-3-yl[cyclohexane- 1-carboxylic acid
(413) as a white
solid (10.5 mg, 43%); and (1R,3R)-3-[(75)-2-[(S)-(4-
chlorophenyl)(hydroxy)methyll-6-
(methoxyc arbony1)-7-methy1-3H,6H,7H,8H,9H-imidazo [4,5-f] quinolin-3 -yl] c
yclohexane- 1-
carboxylic acid (501) as a white solid (7.0 mg, 29%).
First eluting isomer (413): 1H-NMR (CD30D, 400 MHz) 6 (ppm): 7.49 (d, J= 9.0
Hz, 1H), 7.42-
7.33 (m, 5H), 6.19 (s, 1H), 4.92-4.90 (m, 1H), 4.82-4.72 (m, 1H), 3.79 (s,
3H), 3.34-3.20 (m, 1H),
3.02-2.94 (m, 1H), 2.90-2.87 (m, 1H), 2.36-2.09 (m, 4H), 1.99-1.96 (m, 1H),
1.80-1.42 (m, 5H),
1.16 (d, J= 6.6 Hz, 3H). LCMS (ES, m/z): 512 [M+H]t
Second eluting isomer (501): 1H-NMR (CD30D, 400 MHz) 6 (ppm): 7.52-7.33 (m,
6H), 6.22 (s,
1H), 4.84-4.73 (m, 2H), 3.78 (s, 3H), 3.27-3.16 (m, 1H), 3.04-2.92 (m, 1H),
2.90-2.88 (m, 1H),
2.46-2.35 (m, 2H), 2.30-2.22 (m, 1H), 2.15-2.02 (m, 2H), 1.82-1.71 (m, 1H),
1.63-1.55 (m, 2H),
1.40-1.28 (m, 1H), 1.15 (d, J= 6.6 Hz, 4H). LCMS (ES, m/z): 512 [M+H]t
Example 1.7: HPLC Conditions
[0178] In any of the foregoing embodiments, the percentage purity recited may
be determined by
HPLC. In some embodiments the percentage purity is determined using the
following HPLC
method:
Parameters Values
Mobile Phase A 10 mM Ammonium Acetate
Mobile Phase B Acetonitrile
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Parameters Values
Column Waters XSelect Phenyl-Hexyl, 3.5 p.m, 4.6x150 mm
Column Temperature 35 C
0 min 10%B
min 30%B
min 45%B
LC Gradient
21 min 80%B
22 min 80%B
22.1 min 10%B
Runtime 25 min
LC Flow Rate 1 mL/min
UV Wavelength 238 nm
Ionization Mode Electrospray Ionization- Positive Mode
Injection Volume 8 jut
Example 2: HTRF biochemical assay for CBP and BRD4 activity
[0179] The ability of compounds of Formula (I) to selectively inhibit CBP was
determined using
the following HTRF biochemical assay for CBP and BRD4 activity. The assay was
performed in
a final volume of 6 i.iL in assay buffer containing 50 mM Hepes pH 7.5, 0.5 mM
GSH, 0.01%
BGG, 0.005% BSA and 0.01% Triton X-100. Nanoliter quantities of 10-point, 3-
fold serial
dilution in DMSO were pre-dispensed into 1536 assay plates with a top
concentration of 33 i.tM
and half log dilutions. 3 i.iL of 2x Protein and 3 i.iL of 2 x Peptide Ligand
were added to assay
plates (pre-stamped with compound). Plates were incubated for varying times up
to 4 hours at
room temperature prior to measuring the TR-FRET signal. IC50 values are shown
in Figure 1. As
set forth in Figure 1, an IC50 value of greater than or equal to 0.001 11M and
less than or equal to
0.01 11M is marked "++++"; a value greater than 0.01 11M and less than or
equal to 0.1 11M is
marked "+++"; a value greater than 0.111M and less than or equal to 111M is
marked "++"; and a
value greater than 111M and less than 100011M is marked "+".
Example 3: Compounds 1 and Compound 1 demonstrated in vitro activity against
CBP
[0180] The potency and selectivity of CBP/P300 inhibitor compounds including
Compound 1
were determined in biochemical time resolved fluorescence assays using GST
fusions of the
bromodomains of CBP and BRD4. Briefly, CBP inhibitors were pre-dispensed into
1536 assay
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plates for a final test concentration of 33 i.tM to 1.7 nM. Plates and
incubated for 4h. Data were
reported as percent inhibition compared with control wells. IC5o values were
determined by curve
fitting of the standard 4 parameter logistic fitting algorithm. In these
conditions, Compound 1 was
determined to be a potent inhibitor of CBP with an IC5o < 2 nM (N=16). In a
similar assay, BRD4
potency was determined and Compound 1 showed an IC5o of <500 nM (N=15),
indicating >200-
fold selectivity.
[0181] Selectivity of Compound 1 was evaluated in screening assays for kinase
inhibition and
BRD binding. Compound 1 showed no to low binding affinity for the human
kinases and disease-
relevant mutant variants evaluated in a KINOMEscanTm screen. A panel of 10 BRD
representing
the various branches or the bromodomain tree were tested using an AlphaScreen.
Of the 10
bromodomains surveyed, Compound 1 was inactive against 8. Compound 1 IC5o
values for
bromodomains of CREBBP and BRD4 (tandem BD1/BD2) were 0.1 and >10 [tM,
respectively,
confirming the high selectivity of Compound 1 for CBP.
[0182] The ability of Compound 1 and Compound 1 'to selectively inhibit CBP
was determined
using the biochemical assay of Example 2 for CBP and BRD4 activity. Results
are shown in Table
1 below:
Table 1
Compound CBP (IC5o) Selectivity Ratio of BRD4 (IC5o)/CBP (IC5o)
1 <10 nM >240
l' <20 nM >76
2 <10 nM 530
4 <10 nM 742
[0183] Both Compound 1 and Compound 1' potently inhibited (e.g., IC5o < 100
nM) CBP in the
HTRF biochemical assay of Example 2, while Compound 1 was about 3.5-times more
selective
for CBP inhibition compared to BRD4 using this assay.
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Example 4: Compound 1 demonstrates modulation of H3K27Ac in vitro in breast
cancer
cells
[0184] AR-positive breast cancer cells were exposed to increasing
concentrations of Compound 1 for
24 hours. Lysates were prepared in E-PAGE loading buffer (Invitrogen) and
analyzed by western blot
with antibodies diluted 1:1000 for anti-H3K27Ac, 1:2000 for anti-total H3 and
1:10,000 for anti-13
actin. The blots were scanned and analyzed on a LI-COR Odyssey image analyzer.
H3K27Ac levels
were normalized to 13-Actin.
[0185] Compound 1 induced a concentration-dependent reduction of H3K27Ac, a
mark specific
to CBP/P300, in an AR positive breast cancer cell line. (Figure 3). Fifty
percent reduction was
seen between 0.03 and 0.1 M with maximal reduction of approximately 60% seen
at 0.3 M.
Example 5: Compound 1, 2 and 4 reduce AR target gene TMPRSS2 and ER target
gene
XBP1 in AR-positive breast cancer cells
[0186] AR-positive breast cancer cells were exposed to increasing
concentrations of compounds for
24 hours. RNA was extracted and gene expression measured using Taqman assays
for TMPRSS2
and XBP1.
[0187] All compounds reduced the mRNA expression of TMPRSS2 and XBP1 in an
AR-positive breast cancer cell line.
[0188] As set forth in Table 2, below, an IC50 value less than 100 nM is
marked "+++"; a value
greater than 100 nM and less than 500 nM is marked "++"; and a value greater
than 500 nM is
marked "+".
Table 2
Compound 4 Compound 1 Compound 2
TMPRSS2 ++ +++ +
XBP1 ++ +++ +++
Example 6: Compounds 1, 2 and 4 have antiproliferative activity against AR+
breast cancer
cell lines in vitro
[0189] Breast cell lines were cultured according to the distributor's
recommendations. The
following day, cells were exposed to compounds continuously for 10 days. Cell
viability was
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assessed using a CellTiter-Glo assay (Promega) at the end of the incubation
period. The growth
inhibitory effect was assessed by the concentration inhibiting growth by 50%
using a nonlinear
regression equation and a variable slope (Graphpad Prism).
[0190] Compound 1, 2 and 4 inhibit proliferation of breast cancer cell lines
after 10 days
continuous exposure to the drug. The cell lines with high expression of AR
mRNA are more
sensitive than those with low expression.
[0191] As set forth in Table 3, below, an IC50 value of less than 0.2 11M is
marked "++++"; a
value greater than 0.211M and less than 0.5 11M is marked "+++"; a value
greater than 0.5 11M and
less than 111M is marked "++"; and a value greater than 111M is marked "+".
Table 3
AR
Compound 4 Compound 1 Compound 2
MDA-MB-453 High ++++ ++++ +++
CAMA1 High ++++ ++++ ++++
HCC1187 High +++ +++ +++
HCC1500 High + ++ ++
BT549 Low + + +
CAL148 Low + + +
MFM223 Low + + +
MDA-MB-231 Low + + +
Example 7: Compound 1 demonstrated in vivo efficacy in AR-positive human
derived breast
cancer xenografts (TNBC)
[0192] The antitumor activity of Compound 1 was tested in an AR-positive cell
line derived
xenograft model of AR+ triple negative breast cancer (Robinson et al.,
"Androgen receptor driven
transcription in molecular apocrine breast cancer is mediated by FoxAl," The
EMBO Journal
(2011) 30, 3019-3027 (2011), incorporated herein by reference in its
entirety). Briefly, AR-
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positive breast cancer tumor cells (1x107) were implanted subcutaneously in
the flank of 6-8-week-
old NOD SCID mice. Mice were randomized, and treatment started when mean tumor
size reached
160 mm3 (8 mice per cohort). 50 mg/kg Compound 1 was administered orally daily
for the duration
of the experiment. Tumor volume (TV) was measured twice weekly by caliper and
the tumor
volume (mm3) calculated as follows: TV=a x b x b/2, where "a" and "b" are long
and short
diameters of a tumor, respectively.
[0193] Results are shown in Figure 4. At the end of the treatment period,
Compound 1 treatment
produced a tumor growth inhibition (TGI) of 104% (p<0.001) compared with
vehicle control
(TGI,[1-(TreatedTVfinal-TreatedTVinitial)/(VehicleTVfinal-
VehicleTVinitial)]*100 where
"TVfinal" and "TVinitial" are the mean tumor volumes on the final day and
initial day of dosing).
The average body weight loss was 3.7%.
Example 8: Compound 2 modulates the level of AR and variants at the protein
level.
[0194] AR-v7+ Prostate cancer cells were exposed to Compound 2 for 24 hours at
which time
lysates were prepared and the impact of the compound on the protein level of
AR was assessed by
western blot. The results are shown in Figure 5. Treatment of prostate cancer
cells with Compound
2 led to the reduction of both full length and variant forms of the AR
including AR-v7.
Example 9: Compounds 1, 2 and 4 demonstrate modulation of AR target genes
TMPRSS2
and KLK3 as well as MYC in an AR-v7+ prostate cancer cell line
[0195] AR-v7+ prostate cancer cells were exposed to compounds 1, 2, and
4 for 24h. RNA was extracted using Qiacube RNAeasy Mini (Qiagen). For all
genes tested, the
qPCR reactions were carried out in triplicates with 250 ng RNA per reaction
and Taqman
primer-probes. GAPDH was used as a housekeeping gene.
[0196] In the conditions tested, all 3 compounds reduced AR target genes
TMPRSS2 and KLK3,
as well as MYC in a concentration-dependent manner in the AR-v7+ prostate
cancer cells.
[0197] As set forth in Table 4, below, an IC50 value of less than 10 nM is
marked "++++"; a value
greater than 10 nM and less than 50 nM is marked "+++"; a value greater than
50 nM and less than
100 nM is marked "++"; and a value greater than 100 nM is marked "+".
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Table 4
Target gene Compound 4 Compound 1 Compound 2
KLK3 +++ +++ ++
TMPRS S2 +++ ++++ ++
MYC N/A + +
Example 10: Compounds 1,2 and 4 demonstrate antiproliferative activity in
prostate cancer
cell lines
[0198] Prostate cancer cells, androgen-dependent (LnCaP and VCaP)
and androgen-
independent (22Rv1), were plated and incubated overnight. The following day,
cells were exposed
to Compounds 1, 2, 4, and enzalutamide (final top concentration 10 p.M, half-
log dilutions)
continuously. Cell viability was assessed using a CellTiter-Glo assay
(Promega) after 10 days
drug exposure. The growth inhibitory effect was assessed by the concentration
inhibiting growth
by 50% using a nonlinear regression equation and a variable slope (Graphpad
Prism).
[0199] Enzalutamide was active against androgen-dependent cell lines LnCaP and
VCaP but was
inactive in AR negative (PC3 and DU145) and the AR-v7 expressing 22Rv 1 cell
line.
Conversely, all compounds had a potent and concentration-dependent growth
inhibitory effect in
all AR+ cell lines including 22Rv1 cells. All compounds were inactive in AR¨
cell lines.
[0200] As set forth in Table 5, below, an IC50 value of less than 0.511M is
marked "+++"; a value
greater than 0.5 11M and less than 111M is marked "++"; and a value greater
than 1 11M is marked
4,1_,,.
Table 5
Cell Line AR status Compound 4 Compound 1 Compound 2 Enzalutamide
LNCaP Androgen- +++ + +
dependent
VCAP Androgen- +++ ++ + +++
dependent
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Cell Line AR status Compound 4 Compound 1 Compound 2 Enzalutamide
22Rv1 Androgen- ++ ++ ++ +
independent
AR-v7+
PC-3 AR negative + + +
DU 145 AR negative + + +
Example 11: Compound 4 demonstrates antitumor activity in a patient-derived
xenograft
model of prostate cancer resistant to enzalutamide
[0201] Male NOG mice (6-8 weeks of age) were implanted with prostate PDX tumor
fragments.
When tumors reached an average tumor volume of 100-300 mm3 animals were
randomized into
different cohort groups and dosing initiated on the same day (Day 0). Compound
4 was formulated
in (0.5 CMC/0.5% Tween 80) pH8 and administered as a solution. Animals were
weighed twice
weekly. Tumors were measured twice weekly. The maximum tumor volume of control
animals
was 1500 mm3.
[0202] Compound 4 was administered by oral gavage at a dose and schedule of 40
mg/kg/dose
daily Mon-Thu repeated weekly or 80 mg/kg/dose Mo and Thu (twice weekly)
repeated weekly.
[0203] Treatment with Compound 4 at 40 mg/kg/dose daily Mon-Thu repeated
weekly or 80
mg/kg/dose Mo and Thu (twice weekly) repeated weekly resulted in a strong
antitumor responss
(Figure 6) with tumor growth inhibition values of 84% and 82%, respectively.
Enzalutamide had
modest activity (TGI = 21%).
Example 12: Pharmaceutical Composition comprising Compound 1 and Compound 1'
[0204] A pharmaceutical composition can comprise one or more compounds of
formula (I), as
provided herein, including Compound 1 and/or Compound F.
[0205] In one example, an active pharmaceutical ingredient (API) can comprise
about 90% or
more of Compound 1 and up to about 10% (preferably up to about 5%, most
preferably up to about
2.5% including about 1.5%) of Compound 1'.
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[0206] Oral dosage forms comprising Compound 1 can be prepared as a drug-in-
capsule (DiC),
encapsulated simple dry-blend granulation, and lipid-based solution in hard
shell capsule. The
capsules can contain pharmaceutically acceptable excipients, and encapsulated
capsules can be
packaged in high-density polyethylene induction sealed bottles.
[0207] Further embodiments of the disclosure are set out in the following
numbered clauses:
1. A method of treating a patient diagnosed with an enzalutamide-resistant
form of AR+
cancer, the method comprising administering to a patient in need thereof a
therapeutically effective
amount of a pharmaceutical composition comprising Compound 1, or a
pharmaceutically
acceptable salt thereof:
11 0/
OH
N-
0 OH
0 0
1.
2. A method of treating a patient diagnosed with an enzalutamide-resistant
form of AR+
cancer, the method comprising administering to a patient in need thereof a
therapeutically effective
amount of a pharmaceutical composition comprising (1R,3R)-3-[(7S)-2-[(R)-(5-
fluoro-2-
methoxyphenyl)(hydroxy)methyl] -6-(methoxycarbony1)-7 -methyl-
3H,6H,7H,8H,9Himidazo [4,5-f] quinolin-3 -yl] cyclohexane-l-carboxylic
acid or a
pharmaceutically acceptable salt thereof.
3. The method of clause 1 or clause 2, wherein Compound 1 is administered
to the patient in
an oral unit dosage form.
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4. The method of any one of clauses 1-3, wherein the pharmaceutical
composition further
comprises Compound l' or a pharmaceutically acceptable salt thereof:
0/
..10H
N-
N
0 OH
0 0
(1).
5. The method of clause 4, wherein the pharmaceutical composition comprises
about 95% or
more (by HPLC) of Compound 1 and up to 5% by weight of Compound 1'.
6. The method of one of clauses 1-5, wherein the AR+ cancer is an AR+
castrate resistant
prostate cancer (CRPC).
7. The method of any one of clauses 1-5, wherein the AR+ cancer is an AR+
breast cancer.
8. The method of clause 7, wherein the patient is diagnosed with TNBC.
9. The method of any one of clauses 1-8, wherein the patient is diagnosed
with a cancer
harboring the AR-v7 spliceform of the Androgen Receptor.
