U.S. patent application number 16/158527 was filed with the patent office on 2019-02-07 for chemical compounds.
This patent application is currently assigned to AstraZeneca AB. The applicant listed for this patent is AstraZeneca AB. Invention is credited to Nadim Akhtar, Robert Hugh Bradbury, David Buttar, Gordon Stuart Currie, Christopher De Savi, Craig Samuel Donald, Richard Albert Norman, Matthew Osborne, Alfred Arthur Rabow, Heather Redfearn, Helen Elizabeth Williams, Neda Yavari.
Application Number | 20190038607 16/158527 |
Document ID | / |
Family ID | 50841880 |
Filed Date | 2019-02-07 |
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United States Patent
Application |
20190038607 |
Kind Code |
A1 |
Bradbury; Robert Hugh ; et
al. |
February 7, 2019 |
CHEMICAL COMPOUNDS
Abstract
The invention concerns compounds of Formula (I) ##STR00001## or
pharmaceutically-acceptable salts thereof, wherein R.sup.1 to
R.sup.5 have any of the meanings defined hereinbefore in the
description; processes for their preparation, pharmaceutical
compositions containing them and their use in the treatment of cell
proliferative disorders.
Inventors: |
Bradbury; Robert Hugh;
(Macclesfield, GB) ; Buttar; David; (Macclesfield,
GB) ; De Savi; Christopher; (Waltham, MA) ;
Donald; Craig Samuel; (Macclesfield, GB) ; Norman;
Richard Albert; (Macclesfield, GB) ; Rabow; Alfred
Arthur; (Macclesfield, GB) ; Currie; Gordon
Stuart; (Macclesfield, GB) ; Redfearn; Heather;
(Macclesfield, GB) ; Akhtar; Nadim; (Macclesfield,
GB) ; Williams; Helen Elizabeth; (Macclesfield,
GB) ; Osborne; Matthew; (Macclesfield, GB) ;
Yavari; Neda; (Macclesfield, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AstraZeneca AB |
Sodertalje |
|
SE |
|
|
Assignee: |
AstraZeneca AB
Sodertalje
SE
|
Family ID: |
50841880 |
Appl. No.: |
16/158527 |
Filed: |
October 12, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15445000 |
Feb 28, 2017 |
10130617 |
|
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16158527 |
|
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|
14840342 |
Aug 31, 2015 |
9616050 |
|
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15445000 |
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|
14287332 |
May 27, 2014 |
9155727 |
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14840342 |
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61915685 |
Dec 13, 2013 |
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61827951 |
May 28, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 487/04 20130101;
C07C 57/145 20130101; C07B 2200/13 20130101; C07D 471/04 20130101;
C07B 2200/07 20130101; A61K 45/06 20130101; A61P 15/00 20180101;
A61P 35/00 20180101; A61K 31/437 20130101; A61K 31/519
20130101 |
International
Class: |
A61K 31/437 20060101
A61K031/437; C07D 471/04 20060101 C07D471/04; C07C 57/145 20060101
C07C057/145; A61K 31/519 20060101 A61K031/519; A61K 45/06 20060101
A61K045/06; C07D 487/04 20060101 C07D487/04 |
Claims
1. A pharmaceutical composition comprising a compound and a
pharmaceutically-acceptable diluent or carrier, wherein the
compound is
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof.
2. A pharmaceutical composition according to claim 1, wherein the
compound is
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid.
3. A pharmaceutical composition according to claim 1, wherein the
compound is the maleic acid salt of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid.
4. A pharmaceutical composition according to claim 1, wherein the
pharmaceutical composition contains less than 5% w/w of
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.
5. A pharmaceutical composition according to claim 1, wherein the
pharmaceutical composition further comprises an anti-oxidant and
optionally further comprises a metal-chelating agent.
6. A method for making a compound of Formula (I): ##STR00075##
wherein: R.sup.1 and R.sup.2 are each independently H or F; R.sup.3
is H or methyl; and either: a) R.sup.4 is H and R.sup.5 is F; or b)
R.sup.4 is F and R.sup.5 is H, comprising hydrolysis of a compound
of Formula (II): ##STR00076## wherein R.sup.6 is (1-6C) alkyl.
7. A method according to claim 6, wherein the compound of Formula
(I) is
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid.
8. A method according to claim 7, wherein R.sup.6 is methyl.
9. (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate.
10. A method for making a compound of Formula (II): ##STR00077##
wherein: R.sup.1 and R.sup.2 are each independently H or F; R.sup.3
is H or methyl; either: a) R.sup.4 is H and R.sup.5 is F; or b)
R.sup.4 is F and R.sup.5 is H; R.sup.6 is (1-6C) alkyl, comprising
a reaction of a compound of Formula (III): ##STR00078## with a
compound of Formula (IV): ##STR00079##
11. A method according to claim 10, wherein the compound of Formula
(II) is (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/445,000 filed Feb. 28, 2017 which is a
continuation of U.S. patent application Ser. No. 14/840,342, filed
Aug. 31, 2015, granted Apr. 11, 2017 as U.S. Pat. No. 9,616,050,
which is a continuation of U.S. patent application Ser. No.
14/287,332, filed May 27, 2014, granted Oct. 13, 2015 as U.S. Pat.
No. 9,155,727, which claims the benefit under 35 U.S.C. .sctn.
119(e) of Provisional Patent Application No. 61/827,951, filed on
May 28, 2013, and Provisional Patent Application No. 61/915,685,
filed on Dec. 13, 2013, the entire contents of which are
incorporated herein by reference in their entirety.
[0002] The invention concerns certain novel indole derivatives, or
pharmaceutically-acceptable salts thereof, which possess
anti-cancer activity and are accordingly potentially useful in
methods of treatment of the human or animal body. The invention
also concerns processes for the manufacture of said indole
derivatives, pharmaceutical compositions containing them and their
use in therapeutic methods, for example in the manufacture of
medicaments for use in the prevention or treatment of cancers in a
warm-blooded animal such as man, including use in the prevention or
treatment of cancer.
[0003] The present invention also relates to indole derivatives
that are selective down-regulators of the estrogen receptor.
[0004] Estrogen receptor alpha (ER.alpha., ESR1, NR3A) and estrogen
receptor beta (ER.beta., ESR2, NR3b) are steroid hormone receptors
which are members of large nuclear receptor family. Structured
similarly to all nuclear receptors, ERa is composed of six
functional domains (named A-F) (Dahlman-Wright, et al., Pharmacol.
Rev., 2006, 58:773-781) and is classified as a ligand-dependent
transcription factor because after its association with the
specific ligand, the female sex steroid hormone 17b estradiol (E2),
the complex binds to genomic sequences, named Estrogen Receptor
Elements (ERE) and interacts with co-regulators to modulate the
transcription of target genes. The ER.alpha. gene is located on
6q25.1 and encodes a 595 AA protein and multiple isoforms can be
produced due to alternative splicing and translational start sites.
In addition to the DNA binding domain (Domain C) and the ligand
binding domain (Domain E) the receptor contains an N-terminal (A/B)
domain, a hinge (D) domain that links the C and E domains and a
C-terminal extension (F domain). While the C and E domains of ERa
and ERb are quite conserved (96% and 55% amino acid identity
respectively) conservation of the A/B, D and F domains is poor
(below 30% amino acid identity). Both receptors are involved in the
regulation and development of the female reproductive tract and in
addition play roles in the central nervous system, cardiovascular
system and in bone metabolism. The genomic action of ERs occurs in
the nucleus of the cell when the receptor binds EREs directly
(direct activation or classical pathway) or indirectly (indirect
activation or non-classical pathway). In the absence of ligand, ERs
are associated with heat shock proteins, Hsp90 and Hsp70, and the
associated chaperone machinery stabilizes the ligand binding domain
(LBD) making it accessible to ligand. Liganded ER dissociates from
the heat shock proteins leading to a conformational change in the
receptor that allows dimerisation, DNA binding, interaction with
co-w activators or co-repressors and modulation of target gene
expression. In the non-classical pathway, AP-1 and Sp-1 are
alternative regulatory DNA sequences used by both isoforms of the
receptor to modulate gene expression. In this example, ER does not
interact directly with DNA but through associations with other DNA
bound transcription factors e.g. c-Jun or c-Fos (Kushner et al.,
Pure Applied Chemistry 2003, 75:1757-1769). The precise mechanism
whereby ER affects gene transcription is poorly understood but
appears to be mediated by numerous nuclear factors that are
recruited by the DNA bound receptor. The recruitment of
co-regulators is primarily mediated by two protein surfaces, AF2
and AF1 which are located in E-domain and the A/B domain
respectively. AF1 is regulated by growth factors and its activity
depends on the cellular and promoter environment whereas AF2 is
entirely dependent on ligand binding for activity. Although the two
domains can act independently, maximal ER transcriptional activity
is achieved through synergistic interactions via the two domains
(Tzukerman, et al., Mol. Endocrinology, 1994, 8:21-30). Although
ERs are considered transcription factors they can also act through
non-genomic mechanisms as evidenced by rapid ER effects in tissues
following E2 administration in a timescale that is considered too
fast for a genomic action. It is still unclear if receptors
responsible for the rapid actions of estrogen are the same nuclear
ERs or distinct G-protein coupled steroid receptors (Warner, et
al., Steroids 2006 71:91-95) but an increasing number of E2 induced
pathways have been identified e.g. MAPK/ERK pathway and activation
of endothelial nitric oxide synthase and PI3K/Akt pathway. In
addition to ligand dependent pathways, ER.alpha. has been shown to
have ligand independent activity through AF-1 which has been
associated with stimulation of MAPK through growth factor
signalling e.g. insulin like growth factor 1 (IGF-1) and epidermal
growth factor (EGF). Activity of AF-1 is dependent on
phosphorylation of Ser118 and an example of cross-talk between ER
and growth factor signalling is the phosphorylation of Ser 118 by
MAPK in response to growth factors such as IGF-1 and EGF (Kato, et
al., Science, 1995, 270:1491-1494).
[0005] A large number of structurally distinct compounds have been
shown to bind to ER. Some compounds such as endogenous ligand E2,
act as receptor agonists whereas others competitively inhibit E2
binding and act as receptor antagonists. These compounds can be
divided into 2 classes depending on their functional effects.
Selective estrogens receptor modulators (SERMs) such as tamoxifen
have the ability to act as both receptor agonists and antagonists
depending on the cellular and promoter context as well as the ER
isoform targeted. For example tamoxifen acts as an antagonist in
breast but acts as a partial agonist in bone, the cardiovascular
system and uterus. All SERMs appear to act as AF2 antagonists and
derive their partial agonist characteristics through AF1. A second
group, fulvestrant being an example, are classified as full
antagonists and are capable of blocking estrogen activity via the
complete inhibition of AF1 and AF2 domains through induction of a
unique conformation change in the ligand binding domain (LBD) on
compound binding which is results in complete abrogation of the
interaction between helix 12 and the remainder of the LBD, blocking
co-factor recruitment (Wakeling, et al., Cancer Res., 1991,
51:3867-3873; Pike, et al., Structure, 2001, 9:145-153).
[0006] Intracellular levels of ER.alpha. are down-regulated in the
presence of E2 through the ubiquitin/proteosome (Ub/26S) pathway.
Polyubiquitinylation of liganded ER.alpha. is catalysed by at least
three enzymes; the ubiquitin-activating enzyme E1 activated
ubiquitin is conjugated by E2 with lysine residues through an
isopeptide bond by E3 ubiquitin ligase and polyubiquitinated
ER.alpha. is then directed to the proteosome for degradation.
Although ER-dependent transcription regulation and
proteosome-mediated degradation of ER are linked (Lonard, et al.,
Mol. Cell, 2000 5:939-948), transcription in itself is not required
for ER.alpha. degradation and assembly of the transcription
initiation complex is sufficient to target ER.alpha. for nuclear
proteosomal degradation. This E2 induced degradation process is
believed to necessary for its ability to rapidly activate
transcription in response to requirements for cell proliferation,
differentiation and metabolism (Stenoien, et al., Mol. Cell Biol.,
2001, 21:4404-4412). Fulvestrant is also classified as a selective
estrogen receptor down-regulator (SERD), a subset of antagonists
that can also induce rapid down-regulation of ER.alpha. via the 26S
proteosomal pathway. In contrast a SERM such as tamoxifen can
increase ER.alpha. levels although the effect on transcription is
similar to that seen for a SERD.
[0007] Approximately 70% of breast cancers express ER and/or
progesterone receptors implying the hormone dependence of these
tumour cells for growth. Other cancers such as ovarian and
endometrial are also thought to be dependent on ER.alpha.
signalling for growth. Therapies for such patients can inhibit ER
signalling either by antagonising ligand binding to ER e.g.
tamoxifen which is used to treat early and advanced ER positive
breast cancer in both pre and post menopausal setting; antagonising
and down-regulating ER.alpha. e.g. fulvestrant which is used to
treat breast cancer in women which have progressed despite therapy
with tamoxifen or aromatase inhibitors; or blocking estrogen
synthesis e.g. aromatase inhibitors which are used to treat early
and advanced ER positive breast cancer. Although these therapies
have had an enormously positive impact on breast cancer treatment,
a considerable number of patients whose tumours express ER display
de novo resistance to existing ER therapies or develop resistance
to these therapies over time. Several distinct mechanism have been
described to explain resistance to first-time tamoxifen therapy
which mainly involve the switch from tamoxifen acting as an
antagonist to an agonist, either through the lower affinity of
certain co-factors binding to the tamoxifen-ER.alpha. complex being
off-set by over-expression of these co-factors, or through the
formation of secondary sites that facilitate the interaction of the
tamoxifen-ER.alpha. complex with co-factors that normally do not
bind to the complex. Resistance could therefore arise as a result
of the outgrowth of cells expressing specific co-factors that drive
the tamoxifen-ER.alpha. activity. There is also the possibility
that other growth factor signalling pathways directly activate the
ER receptor or co-activators to drive cell proliferation
independently of ligand signalling.
[0008] More recently, mutations in ESR1 have been identified as a
possible resistance mechanism in metastatic ER-positive patient
derived tumour samples and patient-derived xenograft models (PDX)
at frequencies varying from 17-25%. These mutations are
predominantly, but not exclusively, in the ligand-binding domain
leading to mutated functional proteins; examples of the amino acid
changes include Ser463Pro, Val543Glu, Leu536Arg, Tyr537Ser,
Tyr537Asn and Asp538Gly, with changes at amino acid 537 and 538
constituting the majority of the changes currently described. These
mutations have been undetected previously in the genomes from
primary breast samples characterised in the Cancer Genome Atlas
database. Of 390 primary breast cancer samples positive for ER
expression not a single mutation was detected in ESR1 (Cancer
Genome Atlas Network, 2012 Nature 490: 61-70). The ligand binding
domain mutations are thought to have developed as a resistance
response to aromatase inhibitor endocrine therapies as these mutant
receptors show basal transcriptional activity in the absence of
estradiol. The crystal structure of ER, mutated at amino acids 537
and 538, showed that both mutants favoured the agonist conformation
of ER by shifting the position of helix 12 to allow co-activator
recruitment and thereby mimicking agonist activated wild type ER.
Published data has shown that endocrine therapies such as Tamoxifen
and Fulvestrant can still bind to ER mutant and inhibit
transcriptional activation to some extent and that Fulvestrant is
capable of degrading Try537Ser but that higher doses may be needed
for full receptor inhibition (Toy et al., Nat. Genetics 2013, 45:
1439-1445; Robinson et al., Nat. Genetics 2013, 45: 144601451; Li,
S. et al. Cell Rep. 4, 1116-1130 (2013). It is therefore feasible
that certain compounds of the formula (I), or
pharmaceutically-acceptable salts thereof will be capable of
down-regulating and antagonising mutant ER although it is not known
at this stage whether ESR1 mutations are associated with an altered
clinical outcome.
[0009] Regardless of which resistance mechanism or combination of
mechanisms takes place, many are still reliant on ER-dependent
activities and that removal of the receptor through a SERD
mechanism offers the best way of removing the ER.alpha. receptor
from the cell. Fulvestrant is currently the only SERD approved for
clinical use, yet despite its mechanistic properties, the
pharmacological properties of the drug have limited its efficacy
due to the current limitation of a 500 mg monthly dose which
results in less than 50% turnover of the receptor in patient
samples compared to the complete down-regulation of the receptor
seen in in-vitro breast cell line experiments (Wardell, et al.,
Biochem. Pharm., 2011, 82:122-130). Hence there is a need for new
ER targeting agents that have the required pharmaceutical
properties and SERD mechanism to provide enhanced benefit in the
early, metastatic and acquired resistance setting.
[0010] The compounds of the invention have been found to possess
potent anti-tumour activity, being useful in inhibiting the
uncontrolled cellular proliferation which arises from malignant
disease. The compounds of the invention provide an anti-tumour
effect by, as a minimum, acting as SERDs.
[0011] According to one aspect of the invention there is provided a
compound of the Formula (I) or a pharmaceutically-acceptable salt
thereof
##STR00002##
wherein: R.sup.1 and R.sup.2 are each independently H or F; R.sup.3
is H or methyl; and either: a) R.sup.4 is H and R.sup.5 is F; or b)
R.sup.4 is F and R.sup.5 is H.
[0012] In another aspect of the invention, there is provided a
compound of Formula (I) as defined above.
[0013] The compounds of formula (I) have one, two or three chiral
centres and the invention encompasses pure chiral forms or mixtures
thereof in any proportion. The synthesis of optically active forms
may be carried out by standard techniques of organic chemistry well
known in the art, for example by synthesis from optically active
starting materials or by resolution of a racemic form. Similarly,
the above-mentioned activity may be evaluated using the standard
laboratory techniques.
[0014] A particular enantiomer or diasteroeisomer of a compound
described herein may be more active than other enantiomers or
diastereoisomers of the same compound.
[0015] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, which is a single
enantiomer being in an enantiomeric excess (% ee) of .gtoreq.95,
.gtoreq.98% or .gtoreq.99%. Conveniently, the single enantiomer is
present in an enantiomeric excess (% ee) of .gtoreq.99%.
[0016] According to a further aspect of the invention there is
provided a pharmaceutical composition, which comprises a compound
of the Formula (I), which is a single enantiomer being in an
enantiomeric excess (% ee) of .gtoreq.95, .gtoreq.98% or
.gtoreq.99% or a pharmaceutically-acceptable salt thereof, in
association with a pharmaceutically-acceptable diluent or carrier.
Conveniently, the single enantiomer is present in an enantiomeric
excess (% ee) of .gtoreq.99%.
[0017] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, which is a single
diastereoisomer being in an diastereomeric excess (% de) of
.gtoreq.95, .gtoreq.98% or .gtoreq.99%. Conveniently, the single
diastereoisomer is present in an diastereomeric excess (% de) of
.gtoreq.99%.
[0018] According to a further aspect of the invention there is
provided a pharmaceutical composition, which comprises a compound
of the Formula (I), which is a single diastereoisomer being in an
diastereomeric excess (% de) of .gtoreq.95, .gtoreq.98% or
.gtoreq.99% or a pharmaceutically-acceptable salt thereof, in
association with a pharmaceutically-acceptable diluent or carrier.
Conveniently, the single diastereoisomer is present in an
diastereomeric excess (% de) of .gtoreq.99%.
[0019] In one particular aspect, the compound of Formula (I) is a
compound of Formula (IA):
##STR00003##
[0020] In another aspect, the compound of Formula (I) is a compound
of Formula (TB):
##STR00004##
[0021] Reference herein to compounds of Formula (I) is to be
understood as referring to compounds of Formula (IA) and/or (TB)
unless stated otherwise.
[0022] For Example, the compound of Example 1,
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid, is an
example of a compound of Formula (IA).
##STR00005##
[0023] Its isomer,
(E)-3-(3,5-difluoro-4-((1S,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid, is an
example of a compound of Formula (IB).
##STR00006##
[0024] Both of these two isomers are examples of
(E)-3-(3,5-difluoro-4-((3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9--
tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid, which is
an example of a compound of Formula (I).
##STR00007##
[0025] Some compounds of Formula (I) may be crystalline and may
have more than one crystalline form. It is to be understood that
the present invention encompasses any crystalline or amorphous
form, or mixtures thereof, which form possesses properties useful
in SERD activity, it being well known in the art how to determine
efficacy of a crystalline or amorphous form for the SERD activity
by the standard tests described hereinafter.
[0026] It is generally known that crystalline materials may be
analysed using conventional techniques such as X-Ray Powder
Diffraction (hereinafter XRPD) analysis, Differential Scanning
calorimetry (hereinafter DSC), Thermal Gravimetric Analysis
(hereinafter TGA), Diffuse Reflectance Infrared Fourier Transform
(DRIFT) spectroscopy, Near Infrared (NIR) spectroscopy, solution
and/or solid state nuclear magnetic resonance spectroscopy. The
water content of such crystalline materials may be determined by
Karl Fischer analysis.
[0027] As an example, the compound of Example 7 exhibits
crystallinity and one crystalline form has been identified.
[0028] Accordingly, a further aspect of the invention is Form A of
(E)-3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Example 7).
[0029] According to a further aspect of the present invention,
there is provided a crystalline form, Form A of Example 7 which has
an X-ray powder diffraction pattern with at least one specific peak
at about 2-theta=4.5.degree..
[0030] According to a further aspect of the present invention,
there is provided a crystalline form, Form A of Example 7 which has
an X-ray powder diffraction pattern with at least one specific peak
at about 2-theta=10.8.degree..
[0031] According to a further aspect of the present invention,
there is provided a crystalline form, Form A of Example 7 which has
an X-ray powder diffraction pattern with at least two specific
peaks at about 2-theta=4.5 and 10.8.degree..
[0032] According to a further aspect of the present invention,
there is provided a crystalline form, Form A of Example 7 which has
an X-ray powder diffraction pattern with at specific peaks at about
2-theta=4.5, 4.8, 6.1, 7.9, 9.9, 10.8, 13.4, 14.0, 14.3 and
18.5.degree..
[0033] According to the present invention there is provided
crystalline form, Form A of Example 7 which has an X-ray powder
diffraction pattern substantially the same as the X-ray powder
diffraction pattern shown in FIG. 1.
[0034] According to a further aspect of the present invention,
there is provided a crystalline form, Form A of Example 7 which has
an X-ray powder diffraction pattern with at least one specific peak
at about 2-theta=4.5.degree. plus or minus 0.2.degree. 2-theta.
[0035] According to a further aspect of the present invention,
there is provided a crystalline form, Form A of Example 7 which has
an X-ray powder diffraction pattern with at least one specific peak
at about 2-theta=10.8.degree. plus or minus 0.2.degree.
2-theta.
[0036] According to a further aspect of the present invention,
there is provided a crystalline form, Form A of Example 7 which has
an X-ray powder diffraction pattern with at least two specific
peaks at about 2-theta=4.5 and 10.8.degree. plus or minus
0.2.degree. 2-theta.
[0037] According to a further aspect of the present invention,
there is provided a crystalline form, Form A of Example 7 which has
an X-ray powder diffraction pattern with at specific peaks at about
2-theta=4.5, 4.8, 6.1, 7.9, 9.9, 10.8, 13.4, 14.0, 14.3 and
18.5.degree. plus or minus 0.2.degree. 2-theta.
[0038] Furthermore, Example 1 also shows crystallinity.
[0039] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with at least one specific
peak at about 2-theta=8.4.degree..
[0040] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with at least one specific
peak at about 2-theta=10.9.degree..
[0041] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with at least two specific
peaks at about 2-theta=8.4.degree. and 10.9.degree..
[0042] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with specific peaks at
about 2-theta=8.4, 10.9, 18.3, 24.0 and 14.0.degree..
[0043] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with specific peaks at
about 2-theta=8.4, 10.9, 18.3, 24.0, 14.0, 19.0, 14.4, 13.0, 15.3,
20.6.degree..
[0044] According to the present invention there is provided
crystalline form, Form B of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern substantially the same as
the X-ray powder diffraction pattern shown in FIG. 2.
[0045] According to the present invention there is provided
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with at least one specific
peak at 2-theta=8.4.degree. plus or minus 0.2.degree. 2-theta.
[0046] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with at least one specific
peak at 2-theta=10.9.degree. plus or minus 0.2.degree. 2-theta.
[0047] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with at least two specific
peaks at 2-theta=8.4.degree. and 10.9.degree. wherein said values
may be plus or minus 0.2.degree. 2-theta.
[0048] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with specific peaks at
about 2-theta=8.4, 10.9, 18.3, 24.0 and 14.0.degree. wherein said
values may be plus or minus 0.2.degree. 2-theta.
[0049] According to the present invention there is provided a
crystalline form, Form B, of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid which
has an X-ray powder diffraction pattern with specific peaks at
2-theta=8.4, 10.9, 18.3, 24.0, 14.0, 19.0, 14.4, 13.0, 15.3,
20.6.degree. wherein said values may be plus or minus 0.2.degree.
2-theta.
[0050] When it is stated that the present invention relates to a
crystalline form of Example 1 Form B, the degree of crystallinity
is conveniently greater than about 60%, more conveniently greater
than about 80%, preferably greater than about 90% and more
preferably greater than about 95%. Most preferably the degree of
crystallinity is greater than about 98%.
[0051] Furthermore, when it is stated that the present invention
relates to a crystalline form of Example 1 Form B, the material is
preferably substantially free of other crystalline forms or
amorphous material. By "substantially free", we mean conveniently
greater than about 60%, more conveniently greater than about 80%,
preferably greater than about 90%, more preferably greater than
about 95% and most preferably greater than about 98% of single
polymorph.
[0052] It will be understood that 2-theta values of the X-ray
powder diffraction patterns may vary slightly from one machine to
another or from one sample to another, and so the values quoted are
not to be construed as absolute.
[0053] It is known that an X-ray powder diffraction pattern may be
obtained which has one or more measurement errors depending on
measurement conditions (such as equipment or machine used). In
particular, it is generally known that intensities in an X-ray
powder diffraction pattern may fluctuate depending on measurement
conditions. Therefore it should be understood that the crystalline
Forms of the present invention described above, unless otherwise
stated, are not limited to the crystals that provide X-ray powder
diffraction patterns identical to the X-ray powder diffraction
pattern shown in the relevant Figures, and any crystals providing
X-ray powder diffraction patterns substantially the same as those
shown in these Figures fall within the scope of the present
invention. A person skilled in the art of X-ray powder diffraction
is able to judge the substantial identity of X-ray powder
diffraction patterns.
[0054] Persons skilled in the art of X-ray powder diffraction will
also realise that the relative intensity of peaks can be affected
by, for example, grains above 30 microns in size and non-unitary
aspect ratios, which may affect analysis of samples. The skilled
person will also realise that the position of reflections can be
affected by the precise height at which the sample sits in the
diffractometer and the zero calibration of the diffractometer. The
surface planarity of the sample may also have a small effect. Hence
the diffraction pattern data presented are not to be taken as
absolute values (see Jenkins, R & Snyder, R. L. `Introduction
to X-Ray Powder Diffractometry` John Wiley & Sons 1996; Bunn,
C. W. (1948), Chemical Crystallography, Clarendon Press, London;
Klug, H. P. & Alexander, L. E. (1974), X-Ray Diffraction
Procedures).
[0055] Generally, a measurement error of a diffraction angle in an
X-ray powder diffractogram is approximately plus or minus
0.2.degree. 2-theta, and such degree of a measurement error should
be taken into account when considering the X-ray powder diffraction
data. Furthermore, it should be understood that intensities might
fluctuate depending on experimental conditions and sample
preparation (preferred orientation).
[0056] Particular compounds of the invention are each of the
Examples, each of which provides a further independent aspect of
the invention. Further particular compounds of the invention are
pharmaceutically-acceptable salt(s) of each of the Examples, each
of which provides a further independent aspect of the
invention.
[0057] According to a further aspect of the invention there is
provided a compound of the Formula (I), which is obtainable by
following any of the Examples as disclosed herein.
[0058] A further feature is any of the scopes defined herein with
the proviso that specific Examples, such as Example 1, 2, 3 etc.
are individually disclaimed.
[0059] It will be appreciated by those skilled in the art that
certain compounds of Formula (I) contain asymmetrically substituted
carbon atoms, and accordingly may exist in, and be isolated in,
optically-active and racemic forms. Some compounds of Formula (I)
may exhibit polymorphism. It is to be understood that the present
invention encompasses any racemic, optically-active, polymorphic or
stereoisomeric form, or mixtures thereof, which form possesses
properties useful as SERDs, it being well known in the art how to
prepare optically-active forms (for example, by resolution of the
racemic form by recrystallization techniques, by synthesis from
optically-active starting materials, by chiral synthesis, by
enzymatic resolution, by biotransformation, or by chromatographic
separation using a chiral stationary phase) and how to determine
efficacy as SERDs by the standard tests described hereinafter.
[0060] It is to be understood that certain compounds of Formula (I)
defined above may exhibit the phenomenon of tautomerism. It is to
be understood that the present invention includes in its definition
any such tautomeric form, or a mixture thereof, which possesses
SERD activity and is not to be limited merely to any one tautomeric
form utilised within the formulae drawings or named in the
Examples. In general, just one of any such tautomeric forms is
named in the Examples that follow hereinafter or is presented in
any relevant formulae drawings that follow hereinafter.
[0061] The present invention is intended to include all isotopes of
atoms occurring in the present compounds. Isotopes will be
understood to include those atoms having the same atomic number but
different mass numbers. For example, isotopes of hydrogen include
tritium and deuterium. Isotopes of carbon include .sup.13C and
.sup.14C. A deuterated version of Example 1 is described in Example
10.
[0062] A suitable pharmaceutically-acceptable salt of a compound of
the Formula (I) is, for example, an alkali or alkaline earth metal
salt such as a sodium, calcium or magnesium salt, or an ammonium
salt, or a salt with an organic base such as methylamine,
dimethylamine, trimethylamine, piperidine, morpholine or
tris-(2-hydroxyethyl)amine. Further suitable
pharmaceutically-acceptable salts of a compound of the Formula (I)
may be other metal salts, such as potassium, zinc, or other such
metal cations known in the art. In one aspect of the invention, a
pharmaceutically-acceptable salt of a compound of Formula (I) is a
salt with a metal cation, an ammonium salt or a salt with an
organic base.
[0063] A further suitable pharmaceutically-acceptable salt of a
compound of the Formula (I) is, for example, a salt formed within
the human or animal body after administration of a compound of the
Formula (I).
[0064] A suitable pharmaceutically-acceptable salt of a compound of
the Formula (I) may also be, for example, an acid-addition salt of
a compound of the Formula (I), for example an acid-addition salt
with a strong inorganic or organic acid such as hydrochloric,
hydrobromic, sulphuric or trifluoroacetic acid. Other potential
suitable pharmaceutically-acceptable salt of a compound of the
Formula (I) may also be as described below for Example 1. In
another aspect of the invention, a pharmaceutically-acceptable salt
of a compound of Formula (I) is an acid-addition salt.
[0065] Experiments looking at formation of salts of the compounds
of Formula (I) examined the potential for Example 1
((E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,-
4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid) to
form crystalline salts. The following acids and bases were tried:
acetic acid, adipic acid, benzene sulfonic acid, benzoic acid,
cinnamic acid, citric acid, D,L-lactic acid, ethane disulfonic
acid, ethane sulfonic acid, fumaric acid, hydrochloric acid,
L-tartaric acid, maleic acid, malic acid, malonic acid, methane
sulfonic acid, napadisylic acid, phosphoric acid, saccharin,
succinic acid, sulphuric acid, toluene sulfonic acid, calcium
acetate, diethylamine, ethanolamine, ethylenediamine,
hydroxyethylpyrrolidine, magnesium acetate, meglumine, piperazine,
potassium hydroxide, sodium hydroxide, t-butylamine,
triethanolamine, tris(hydroxymethyl)aminomethane (Tris) and
N,N-diethylethanolamine.
[0066] Of the above acids and bases, isolatable solid salts were
not always obtainable, or not obtainable in crystalline form in the
experimental conditions employed. Preferred salts of Example 1
include those which may be isolated in crystalline form, for
example, benzene sulfonic acid salt (besylate salt), succinic acid
salt (succinate salt) and maleic acid salt (maleate salt).
[0067] In one aspect, suitable salts of Example 1 may include
besylate, succinate and maleate. In another aspect, a suitable salt
of Example 1 may be the maleate salt, which is described in Example
11.
[0068] It is further to be understood that a suitable
pharmaceutically-acceptable co-crystal of a compound of the Formula
(I) also forms an aspect of the present invention. For the
avoidance of doubt, the term co-crystal (or cocrystal) refers to a
multicomponent system in which there exists a host API (active
pharmaceutical ingredient) molecule or molecules and a guest (or
co-former) molecule or molecules. In a co-crystal, both the API
molecule and the guest (or co-former) molecule exist as a solid at
room temperature when alone in their pure form (in order to
distinguish the co-crystal from solvates or hydrates). Salts, in
which significant or complete proton exchange occurs between the
API molecule and the guest molecule, are excluded from this
particular definition. In a co-crystal, the API and co-former
molecules interact by hydrogen bonding and possibly other
non-covalent interactions. Pharmaceutically acceptable co-formers
include neutral molecules such as nicotinamide, resorcinol and
xylenols, as well as ionisable molecules such as oxalic acid,
3,5-dihydroxybenzoic acid and isoquinoline (the extent of proton
exchange determining whether a salt or co-crystal is formed). It
may be noted that a co-crystal may itself form solvates, including
hydrates.
[0069] It is further to be understood that a suitable
pharmaceutically-acceptable solvate of a compound of the Formula
(I) also forms an aspect of the present invention. A suitable
pharmaceutically-acceptable solvate is, for example, a hydrate such
as a hemi-hydrate, a mono-hydrate, a di-hydrate or a tri-hydrate or
an alternative quantity thereof.
[0070] It is further to be understood that a suitable
pharmaceutically-acceptable pro-drug of a compound of the Formula
(I) also forms an aspect of the present invention. Accordingly, the
compounds of the invention may be administered in the form of a
pro-drug, which is a compound that is broken down in the human or
animal body to release a compound of the invention. A pro-drug may
be used to alter the physical properties and/or the pharmacokinetic
properties of a compound of the invention. A pro-drug can be formed
when the compound of the invention contains a suitable group or
substituent to which a property-modifying group can be attached.
Examples of pro-drugs include in-vivo cleavable ester derivatives
that may be formed at a carboxy group in a compound of the Formula
(I).
[0071] Accordingly, the present invention includes those compounds
of the Formula (I) as defined hereinbefore when made available by
organic synthesis and when made available within the human or
animal body by way of cleavage of a pro-drug thereof. Accordingly,
the present invention includes those compounds of the Formula (I)
that are produced by organic synthetic means and also such
compounds that are produced in the human or animal body by way of
metabolism of a precursor compound, that is a compound of the
Formula (I) may be a synthetically-produced compound or a
metabolically-produced compound.
[0072] A suitable pharmaceutically-acceptable pro-drug of a
compound of the Formula (I) is one that is based on reasonable
medical judgement as being suitable for administration to the human
or animal body without undesirable pharmacological activities and
without undue toxicity.
[0073] Various forms of pro-drug have been described, for example
in the following documents:-- [0074] a) Methods in Enzymology, Vol.
42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);
[0075] b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier,
1985); [0076] c) A Textbook of Drug Design and Development, edited
by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 "Design and
Application of Pro-drugs", by H. Bundgaard p. 113-191 (1991);
[0077] d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38
(1992); [0078] e) H. Bundgaard, et al., Journal of Pharmaceutical
Sciences, 77, 285 (1988); [0079] f) N. Kakeya, et al., Chem. Pharm.
Bull., 32, 692 (1984); [0080] g) T. Higuchi and V. Stella,
"Pro-Drugs as Novel Delivery Systems", A.C.S. Symposium Series,
Volume 14; and [0081] h) E. Roche (editor), "Bioreversible Carriers
in Drug Design", Pergamon Press, 1987.
[0082] A suitable pharmaceutically-acceptable pro-drug of a
compound of the Formula I that possesses a carboxy group is, for
example, an in vivo cleavable ester thereof. An in vivo cleavable
ester of a compound of the Formula I containing a carboxy group is,
for example, a pharmaceutically-acceptable ester which is cleaved
in the human or animal body to produce the parent acid. Suitable
pharmaceutically-acceptable esters for carboxy include (1-6C)alkyl
esters such as methyl, ethyl and tert-butyl, (1-6C)alkoxymethyl
esters such as methoxymethyl esters, (1-6C)alkanoyloxymethyl esters
such as pivaloyloxymethyl esters, 3-phthalidyl esters,
(3-8C)cycloalkylcarbonyloxy-(1-6C)alkyl esters such as
cyclopentylcarbonyloxymethyl and 1-cyclohexylcarbonyloxyethyl
esters, 2-oxo-1,3-dioxolenylmethyl esters such as
5-methyl-2-oxo-1,3-dioxolen-4-ylmethyl esters and
(1-6C)alkoxycarbonyloxy-(1-6C)alkyl esters such as
methoxycarbonyloxymethyl and 1-methoxycarbonyloxyethyl esters.
[0083] A suitable pharmaceutically-acceptable pro-drug of a
compound of the Formula I which have a carboxy group is for example
an in vivo cleavable amide such as a N--C.sub.1-6alkyl and
N,N-di-(C.sub.1-6alkyl)amide such as N-methyl, N-ethyl, N-propyl,
N,N-dimethyl, N-ethyl-N-methyl or N,N-diethylamide.
[0084] The in vivo effects of a compound of the Formula (I) may be
exerted in part by one or more metabolites that are formed within
the human or animal body after administration of a compound of the
Formula (I). As stated hereinbefore, the in vivo effects of a
compound of the Formula (I) may also be exerted by way of
metabolism of a precursor compound (a pro-drug).
[0085] Two isomeric active metabolites of Example 1 have been
identified from in-vitro human systems as shown below (where the
isomers are diastereomeric as a result of both configurations
existing at the carbon marked with a *), and synthesis of both
isomers is set out in Examples 14A and B herein:
##STR00008##
[0086] Additionally, the following compound is believed to be an
active metabolite in some species, such as in mouse:
##STR00009##
[0087] Such active metabolites form further independent aspects of
the invention.
[0088] For the avoidance of doubt it is to be understood that where
in this specification a group is qualified by `hereinbefore
defined` or `defined hereinbefore` the said group encompasses the
first occurring and broadest definition as well as each and all of
the particular definitions for that group.
[0089] Particular novel compounds of the invention include, for
example, compounds of the Formula (I), or
pharmaceutically-acceptable salts thereof, wherein, unless
otherwise stated, each of R.sup.1 and R.sup.2, has any of the
meanings defined hereinbefore or in the following statements:
[0090] In one aspect R.sup.1 is hydrogen. In another aspect R.sup.1
is fluoro.
[0091] In one aspect R.sup.2 is hydrogen. In another aspect R.sup.2
is fluoro.
[0092] In one aspect both R.sup.1 and R.sup.2 are hydrogen. In
another aspect both R.sup.1 and R.sup.2 are fluoro. In another
aspect R.sup.1 is hydrogen and R.sup.2 is fluoro.
[0093] In one aspect R.sup.3 is hydrogen. In another aspect R.sup.3
is methyl.
[0094] In one aspect R.sup.4 is hydrogen and R.sup.5 is fluoro. In
another aspect R.sup.5 is hydrogen and R.sup.4 is fluoro.
[0095] Particular compounds of the invention are, for example, the
compounds of the Formula (I) that are disclosed within the Examples
that are set out hereinafter.
[0096] For example, a particular compound of the invention is a
compound of the Formula (I) selected from any one of the
following:-- [0097]
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid;
[0098]
(E)-3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0099]
(E)-3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2-
,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid;
[0100]
(E)-3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrah-
ydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0101]
(E)-3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid;
[0102]
(E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0103]
(E)-3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0104]
(E)-3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0105]
(E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; or a
pharmaceutically-acceptable salt thereof.
[0106] A further particular compound of the invention is a compound
of the Formula (I) selected from any one of the following:-- [0107]
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid;
[0108]
(E)-3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0109]
(E)-3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2-
,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid;
[0110]
(E)-3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrah-
ydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0111]
(E)-3-(3,5-difluoro-4(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2-
,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid;
[0112]
(E)-3-(3,5-difluoro-4(1R)-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid;
[0113]
(E)-3-(4(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrah-
ydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0114]
(E)-3-(4(1R)-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; [0115]
(E)-3-(3-fluoro-4(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid; and
[0116]
(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-di-
hydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoic
acid; or a pharmaceutically-acceptable salt thereof.
[0117] A particular pharmaceutically-acceptable salt of the
invention is
(1R,3R)-1-{4-[(E)-2-carboxyethenyl]-2,6-difluorophenyl}-2-(2-fluoro-2-met-
hylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-beta-carbolin-2-ium
maleate.
[0118] Another aspect of the present invention provides a process
for preparing a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof. A suitable process is
illustrated by the following representative process variants in
which, unless otherwise stated, R.sup.1 to R.sup.5 have any of the
meanings defined hereinbefore. Necessary starting materials may be
obtained by standard procedures of organic chemistry. The
preparation of such starting materials is described in conjunction
with the following representative process variants and within the
accompanying Examples. Alternatively, necessary starting materials
are obtainable by analogous procedures to those illustrated which
are within the ordinary skill of an organic chemist.
[0119] Compounds of formula (I) are conveniently made by hydrolysis
of an ester derivative of formula (II), wherein R.sup.6 is (1-6C)
alkyl, such as methyl. Hydrolysis is conveniently carried out in
the presence of base such as using sodium hydroxide in a suitable
solvent (such as aqueous THF and MeOH (or another similar alcohol),
or such as an aqueous alcohol for example aqueous isopropanol) and
a suitable temperature, conveniently room temperature.
##STR00010##
[0120] Compounds of formula (II) may be made by, for example:
a) reaction of a compound of formula (III) with a compound of
formula (IV) under conditions known in the art as suitable for
Pictet-Spengler reactions (such as in the presence of acid (such as
acetic acid) and in a suitable solvent (for example toluene) and a
suitable temperature (such as 80.degree. C.)); or
##STR00011##
[0121] b) by reaction of a compound of formula (V) with a compound
of formula (VI), where LG is a leaving group known in the art such
as halide or trifluoromethanesulfonate (triflate), conveniently
triflate, in the presence of base (for example an amine base such
as N-ethyl-N-isopropylpropan-2-amine) and a suitable polar solvent
(such as dioxane) at a suitable temperature (such as from room
temperature to 90.degree. C.).
##STR00012##
[0122] Compounds of formula (III) may be prepared by reaction of a
compound of formula (VII) with a compound of formula (VI) under
conditions as described for the reaction of compounds of formulae
(V) and (VI) above.
##STR00013##
[0123] Compounds of formula (IV) may be prepared by reaction of a
compound of formula (VIII) with an alkyl acrylate ester (such as
methyl acrylate when R.sup.6 is methyl) under conditions known in
the art for a Heck reaction; that is in the presence of an aryl
phosphine (eg tri-o-tolylphosphine), a palladium catalyst (such as
palladium (II) acetate and base (such as triethylamine) in a
suitable solvent (such as DMA) and at a suitable temperature (eg
80.degree. C.).
##STR00014##
[0124] Compounds of formula (V) may be prepared by reaction of a
compound of formula (VII) with a compound of formula (IV) using
conditions similar to those described for the reaction of compounds
of formulae (III) and (IV) above.
[0125] Compounds of formula (VI) where LG is triflate may be
prepared as shown below in Schemes 1 and 2. Other compound of
formula (VI) where LG is other than triflate may be prepared by
similar methods known in the art.
##STR00015##
Step 1: Fluorinating agent, e.g.
N,N-diethyl-1,1,2,3,3,3-hexafluoropropan-1-amine/DCM/RT, then
reducing agent, e.g. lithium aluminium hydride/THF/RT Step 2:
Trifluoromethanesulfonic anhydride/base, e.g.
2,6-lutidine/DCM/0.degree. C.
##STR00016##
Step 1: Reducing agent, e.g. lithium aluminium
hydride/ether/0.degree. C.
[0126] Step 2: Trifluoromethanesulfonic anhydride/base, e.g.
2,6-lutidine/DCM/-10.degree. C.
[0127] Compounds of formula (I) are chiral. It will be understood
by the skilled person that stereoselective reactions may be used to
obtain the desired isomers. Alternatively, stereochemistry may be
adjusted by suitable means, such as by epimerisation from cis to
trans isomers via acidification of an intermediate with protected
amine group as illustrated in Example 4 herein (and described for
example in J. Org. Chem. 2009, 74, 2771-2779).
[0128] In a further aspect of the invention there is provided a
process for making a compound of formula (I) comprising hydrolysis
of a compound of formula (II), conveniently in the presence of
base.
[0129] It is to be understood that other permutations of the
process steps in the process variants described above are also
possible.
[0130] It is to be understood that any compound of Formula (I)
obtained by any of the processes described hereinbefore can be
converted into another compound of the Formula (I) if required.
[0131] When a pharmaceutically-acceptable salt of a compound of the
Formula (I) is required it may be obtained by, for example,
reaction of said compound with a suitable base.
[0132] When a pharmaceutically-acceptable pro-drug of a compound of
the Formula (I) is required, it may be obtained using a
conventional procedure. For example, an in vivo cleavable ester of
compound of the Formula (I) may be obtained by, for example,
reaction of a compound of the Formula (I) containing a carboxy
group with a pharmaceutically-acceptable alcohol. Further
information on pro-drugs has been provided hereinbefore.
[0133] It will also be appreciated that, in some of the reactions
mentioned hereinbefore, it may be necessary or desirable to protect
any sensitive groups in the compounds. The instances where
protection is necessary or desirable, and suitable methods for
protection, are known to those skilled in the art. Conventional
protecting groups may be used in accordance with standard practice
(for illustration see T. W. Green, Protective Groups in Organic
Synthesis, John Wiley and Sons, 1991). Thus, if reactants include
groups such as amino, carboxy or hydroxy, it may be desirable to
protect the group in some of the reactions mentioned herein.
[0134] A suitable protecting group for an amino or alkylamino group
is, for example, an acyl group, for example an alkanoyl group such
as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl,
ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl
group, for example benzyloxycarbonyl, or an aroyl group, for
example benzoyl. The deprotection conditions for the above
protecting groups necessarily vary with the choice of protecting
group. Thus, for example, an acyl group such as an alkanoyl or
alkoxycarbonyl group or an aroyl group may be removed for example,
by hydrolysis with a suitable base such as an alkali metal
hydroxide, for example lithium or sodium hydroxide. Alternatively
an acyl group such as a t-butoxycarbonyl group may be removed, for
example, by treatment with a suitable acid as hydrochloric,
sulphuric or phosphoric acid or trifluoroacetic acid and an
arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be
removed, for example, by hydrogenation over a catalyst such as
palladium-on-carbon, or by treatment with a Lewis acid for example
boron tris(trifluoroacetate). A suitable alternative protecting
group for a primary amino group is, for example, a phthaloyl group
which may be removed by treatment with an alkylamine, for example
dimethylaminopropylamine, or with hydrazine.
[0135] A suitable protecting group for a hydroxy group is, for
example, an acyl group, for example an alkanoyl group such as
acetyl, an aroyl group, for example benzoyl, or an arylmethyl
group, for example benzyl. The deprotection conditions for the
above protecting groups will necessarily vary with the choice of
protecting group. Thus, for example, an acyl group such as an
alkanoyl or an aroyl group may be removed, for example, by
hydrolysis with a suitable base such as an alkali metal hydroxide,
for example lithium or sodium hydroxide. Alternatively an
arylmethyl group such as a benzyl group may be removed, for
example, by hydrogenation over a catalyst such as
palladium-on-carbon.
[0136] A suitable protecting group for a carboxy group is, for
example, an esterifying group, for example a methyl or an ethyl
group which may be removed, for example, by hydrolysis with a base
such as sodium hydroxide, or for example a t-butyl group which may
be removed, for example, by treatment with an acid, for example an
organic acid such as trifluoroacetic acid, or for example a benzyl
group which may be removed, for example, by hydrogenation over a
catalyst such as palladium-on-carbon.
[0137] The protecting groups may be removed at any convenient stage
in the synthesis using conventional techniques well known in the
chemical art.
[0138] Certain of the intermediates (for example, compounds of the
Formulae II, III, IV, V, VI, VII and VIII, particularly compounds
of formula II, III and/or V) defined herein are novel and these are
provided as a further feature of the invention.
Biological Assays--
[0139] The following assays were used to measure the effects of the
compounds of the present invention.
ER.alpha. Binding Assay
[0140] The ability of compounds to bind to isolated Estrogen
Receptor Alpha Ligand binding domain (ER alpha--LBD (GST)) was
assessed in competition assays using a LanthaScreen.TM.
Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
detection end-point. For the LanthaScreen TR-FRET endpoint, a
suitable fluorophore (Fluormone ES2, Product code P2645) and
recombinant human Estrogen Receptor alpha ligand binding domain
(Product code PV4543) were purchased from Invitrogen and used to
measure compound binding. The assay principle is that ER alpha-LBD
(GST) is added to a fluorescent ligand to form a
receptor/fluorophore complex. A terbium-labelled anti-GST antibody
(Product code PV3551) is used to indirectly label the receptor by
binding to its GST tag, and competitive binding is detected by a
test compounds' ability to displace the fluorescent ligand
resulting in a loss of TR-FRET signal between the Tb-anti-GST
antibody and the tracer. The assay was performed as follows with
all reagent additions carried out using the Beckman Coulter
BioRAPTR FRD microfluidic workstation:--
1. Acoustic dispense 120 nl of the test compound into a black low
volume 384 well assay plates. 2. Prepare 1.times. ER
alpha--LBD/Tb-antiGST Ab in ES2 screening buffer and incubate for
20 minutes. 3. Add 1.times. fluorophore to the ER
alpha--LBD/Tb-antiGST Ab solution prior to use. 4. Dispense 12
.mu.l of the 1.times. AR-LBD/Tb-anti-GST Ab/Fluorophore reagent
into each well of the assay plate 5. Cover the assay plate to
protect the reagents from light and evaporation, and incubate at
room temperature for 1 hour. 6. Excite at 337 nm and measure the
fluorescent emission signal of each well at 490 nm and 520 nm using
the BMG PheraSTAR. Compounds were dosed directly from a compound
source microplate containing serially diluted compound (4 wells
containing 10 mM, 0.1 mM, 1 .mu.M and 10 nM final compound
respectively) to an assay microplate using the Labcyte Echo 550.
The Echo 550 is a liquid handler that uses acoustic technology to
perform direct microplate-to-microplate transfers of DMSO compound
solutions and the system can be programmed to transfer multiple
small nL volumes of compound from the different source plate wells
to give the desired serial dilution of compound in the assay which
is then back-filled to normalise the DMSO concentration across the
dilution range. In total 120 nL of compound plus DMSO is added to
each well and compounds were tested in a 12-point concentration
response format over a final compound concentration range of 100,
29.17, 10.42, 2.083, 1, 0.292, 0.104, 0.02083, 0.01, 0.002917,
0.001042, 0.0001 .mu.M, respectively. TR-FRET dose response data
obtained with each compound was exported into a suitable software
package (such as Origin or Genedata) to perform curve fitting
analysis. Competitive ER alpha binding was expressed as an
IC.sub.50 value. This was determined by calculation of the
concentration of compound that was required to give a 50% reduction
in tracer compound binding to ER alpha-LBD.
MCF-7 ER Down Regulation Assay
[0141] The ability of compounds to down-regulate Estrogen Receptor
(ER) numbers was assessed in a cell based immuno-fluorescence assay
using the MCF-7 human ductal carcinoma breast cell line. MCF-7
cells were revived directly from a cryovial (approx
5.times.10.sup.6 cells) in Assay Medium (phenol red free Dulbecco's
Modified Eagle's medium (DMEM) (Sigma D5921) containing 2 mM
L-Glutamine and 5% (v/v) Charcoal/Dextran treated foetal calf serum
Cells were syringed once using a sterile 18G.times.1.5 inch
(1.2.times.40 mm) broad gauge needle and cell density was measured
using a Coulter Counter (Beckman). Cells were further diluted in
Assay Medium to a density of 3.75.times.10.sup.4 cells per ml and
40 .mu.l per well added to transparent bottomed, black, tissue
culture treated 384 well plates (Costar, No. 3712) using a Thermo
Scientific Matrix WellMate or Thermo Multidrop. Following cell
seeding, plates were incubated overnight at 37.degree. C., 5%
CO.sub.2 (Liconic carousel incubator). Test data was generated
using the LabCyte Echo.RTM. model 555 compound reformatter which is
part of an automated workcell (Integrated Echo 2 workcell). 10 mM
compound stock solutions of the test compounds were used to
generate a 384 well compound dosing plate (Labcyte P-05525-CV1). 40
.mu.l of each of the 10 mM compound stock solutions was dispensed
into the first quadrant well and then 1:100 step-wise serial
dilutions in DMSO were performed using a Hydra II (MATRIX UK)
liquid handling unit to give 40 ul of diluted compound into
quadrant wells 2 (0.1 mM), 3 (1 .mu.M) and 4 (0.01 .mu.M),
respectively. 40 .mu.l of DMSO added to wells in row P on the
source plate allow for DMSO normalisation across the dose range. To
dose the control wells 40 .mu.l of DMSO was added to row 01 and 40
.mu.l of 10004 Faslodex.RTM. in DMSO was added to row O3 on the
compound source plate. The Echo uses acoustic technology to perform
direct microplate-to-microplate transfers of DMSO compound
solutions to assay plates. The system can be programmed to transfer
volumes as low as 2.5 nL in multiple increments between microplates
and in so doing generates a serial dilution of compound in the
assay plate which is then back-filled to normalise the DMSO
concentration across the dilution range. Compounds were dispensed
onto the cell plates with a compound source plate prepared as above
producing a 12 pt duplicate 3 .mu.M to 3 pM dose range with 3 fold
dilutions and one final 10 fold dilution using the Integrated Echo
2 workcell. The maximum signal control wells were dosed with DMSO
to give a final concentration of 0.3% and the minimum signal
control wells were dosed with Faslodex.RTM. to give a final
concentration of 100 nM accordingly. Plates were further incubated
for 18-22 hours at 37.degree. C., 5% CO.sub.2 and then fixed by the
addition of 20 .mu.l of 11.1% (v/v) formaldehyde solution (in
phosphate buffered saline (PBS)) giving a final formaldehyde
concentration of 3.7% (v/v). Cells were fixed at room temperature
for 20 mins before being washed two times with 250 .mu.l
PBS/Proclin (PBS with a Biocide preservative) using a BioTek
platewasher, 40 .mu.l of PBS/Proclin was then added to all wells
and the plates stored at 4.degree. C. The fixing method described
above was carried out on the Integrated Echo 2 workcell.
Immunostaining was performed using an automated AutoElisa workcell.
The PBS/Proclin was aspirated from all wells and the cells
permeabilised with 40 W PBS containing 0.5% Tween.TM. 20 (v/v) for
1 hour at room temperature. The plates were washed three times in
250 .mu.l of PBS/0.05% (v/v) Tween 20 with Proclin (PBST with a
Biocide preservative) and then 20 .mu.l of ER.alpha. (SP1) Rabbit
monoclonal antibody (Thermofisher) 1:1000 in PBS/Tween.TM./3% (w/v)
Bovine Serum Albumin was added. The plates were incubated overnight
at 4.degree. C. (Liconic carousel incubator) and then washed three
times in 250 .mu.l of PBS/0.05% (v/v) Tween.TM. 20 with Proclin
(PBST). The plates were then incubated with 20 .mu.l/well of a goat
anti-rabbit IgG AlexaFluor 594 or goat anti-rabbit AlexaFluor 488
antibody (Molecular Probes) with Hoechst at 1:5000 in
PBS/Tween.TM./3% (w/v) Bovine Serum Albumin for 1 hr at room
temperature. The plates were then washed three times in 250 .mu.l
of PBS/0.05% (v/v) Tween.TM. 20 with Proclin (PBST with a Biocide
preservative). 20 .mu.l of PBS was added to each well and the
plates covered with a black plate seal and stored at 4.degree. C.
before being read. Plates were read using a Cellomics Arrayscan
reading the 594 nm (24 hr time point) or 488 nm (5 hr timepoint)
fluorescence to measure the ER.alpha. receptor level in each well.
The mean total intensity was normalized for cell number giving the
total intensity per cell. The data was exported into a suitable
software package (such as Origin) to perform curve fitting
analysis. Down-regulation of the ER.alpha. receptor was expressed
as an IC.sub.50 value and was determined by calculation of the
concentration of compound that was required to give a 50% reduction
of the average maximum Total Intensity signal.
[0142] Although the pharmacological properties of the compounds of
the Formula (I) vary with structural change as expected, in general
activity possessed by compounds of the Formula (I) may be
demonstrated at the following concentrations or doses in one or
more of the above tests.
[0143] The following data were generated for the Examples (the data
below may be a result from a single experiment or an average of
multiple repeat experiments):
TABLE-US-00001 TABLE A Example ER binding IC.sub.50 value ER down
regulation IC.sub.50 value 1 <0.64 0.14 2 1 0.85 3 1 0.4 4 1.6
0.99 5 0.2 0.57 6 <1.3 0.44 7 5 1.7 8 2.2 3 9 <1.2 1.5
MCF-7 In-Vivo Xenograft Study with Example 1 as Single Agent and in
Combination with an mTOR Inhibitor.
[0144] MCF7 cells (5.times.10.sup.6 cells suspended in 100 .mu.l of
RPMI cell medium) were implanted subcutaneously in the hind flank
of immuno-compromised (SCID) mice the day after each mouse was
surgically implanted with a 0.5 mg/21 day oestrogen pellet
(Innovative Research, USA). Tumours were measured twice weekly and
changes in tumour volume and growth inhibition were determined by
bilateral Vernier calliper measurement (length.times.width) where
length was taken to be the longest diameter across the tumour and
width the corresponding perpendicular. Tumour volume was calculated
using the formula (length.times.width).times.
(length.times.width).times..pi./6).
[0145] Tumours were measured 13 days after cell implantation to
allow randomisation of mice into test groups. Treatment with
compounds began the day after (i.e. 14 days after cell
implantation).
[0146] The mTOR inhibitor AZD2014 was dosed to different groups of
mice at 15 mg/kg once daily every day orally (p.o.) at a volume of
0.1 ml per 10 g. Example 1 was dosed at 5 mg/kg once daily orally
at 0.1 ml/10 g. One group of animals was dosed with vehicle p.o. to
act as a control. Nine mice per group were used for active agents
for the control group.
[0147] The data obtained from this study are shown in FIG. 10.
[0148] The effect of a combination of a compound of Formula (I)
with an inhibitor of PI3K.alpha./.delta. may be studied in a
similar manner to the combination with the mTOR inhibitor
above.
HCC Long Term Estrogen Deprived (HCC1428 LTED) Xenograft Efficacy
Study
[0149] After a suitable period of cell culture, HCC1428 LTED cells
(1.times.10.sup.6) were implanted subcutaneously in the hind flank
of female immuno-compromised NSG mice (Jackson Labs, USA) that had
undergone overectomy. Tumours were measured twice weekly and
changes in tumour volume and growth inhibition were determined by
bilateral Vernier calliper measurement (length.times.width) where
length was taken to be the longest diameter across the tumour and
width the corresponding perpendicular. Tumour volume was calculated
using the formula (length.times.width).times.
/(length.times.width).times.(.pi.n/6). Tumours were measured once
weekly after cell implantation until the average size reached 150
mm.sup.3 at which point the mice were placed into randomised test
groups, each group containing 10 mice. Treatment with compounds
began the day after (day 62 in this study) and once weekly tumour
measurement continued. Example 1 was dosed 25 mg/kg once daily
every day orally (p.o.) at a volume of 0.1 ml per 10 g. Another
group of animals was dosed with vehicle p.o. to act as a
control.
[0150] After 28 days of dosing, control treated tumours grew on
average by 220 mm.sup.3 (using geometric mean values) while tumours
from mice treated by Example 1 decreased in size by 46 mm.sup.3
representing a 121% inhibition of tumour growth (P<0.001 by
unpaired t-test).
[0151] To measure the levels of estrogen receptor protein in
xenograft tumours, tumours samples were harvested 24 hrs post final
dose of vehicle or example 1 treatment and snap frozen in liquid
nitrogen. For protein extraction, tumour fragments were added to
700 ul of Invitrogen Cell Extraction buffer (FNN0011) with added
Sigma Phosphatase inhibitors (No. 2 (P5726) and 3 (P0044) 1 in 100
dilution) and Roche Complete (11836145001) protease inhibitor (1
tablet per 50 mls), 1 mM dithiothreitol (DTT) in 2 ml sample tubes
on wt ice. Homogenisation of the sample was done using a Mixermill
(level 27/sec) and 3.times.2 mins cycles of homogenisation. Samples
were spun briefly to ascertain complete homogenisation of the
tumours. Homogenate was sonicated for 10 seconds and then spun down
at top speed (13000 rpm) centrifuge for 15 mins. Levels of protein
in the supernatent were measured and approx 45 ug of protein were
run on a 15 well Bis-Tris Gels (4-12% Gels) using standard methods.
Following protein separation and transfer onto nitrocellulose
filter, estrogen receptor 68 kDa: ThermoFisher SP1 #9101S antibody
was added, diluted 1:400 in milk/PBS/T and incubated overnight at
4.degree. C. The filter was washed in 3.times.5 mins in .about.20
ml of TBS/T 0.05% and a secondary anti-rabbit detection antibody
was diluted 1:2000 in 5% marvel in TBS/T and incubate for 1 hr at
RT. Signal was detected using chemiluminescent SuperSignal West
Dura extended Duration substrate and quantified using Syngene
software. Vinculin protein levels were measured as a loading
control using V931 Sigma diluted 1:10,000 in marvel and an
anti-mouse detection antibody. The results in FIGS. 11 and 12 show
that a 60% decrease in ER levels were observed upon treatment with
Example 1 relative to vehicle control.
[0152] According to a further aspect of the invention there is
provided a pharmaceutical composition, which comprises a compound
of the Formula (I), or a pharmaceutically-acceptable salt thereof,
as defined hereinbefore in association with a
pharmaceutically-acceptable diluent or carrier.
[0153] Suitable pharmaceutically-acceptable excipients for a tablet
formulation include, for example, inert diluents, granulating and
disintegrating agents, binding agents, lubricating agents,
preservative agents and anti-oxidants. A further suitable
pharmaceutically-acceptable excipient may be a chelating-agent.
Tablet formulations may be uncoated or coated either to modify
their disintegration and the subsequent absorption of the active
ingredient within the gastrointestinal tract, or to improve their
stability and/or appearance, in either case, using conventional
coating agents and procedures well known in the art.
[0154] Compositions for oral use may alternatively be in the form
of hard gelatin capsules in which the active ingredient is mixed
with an inert solid diluent, or as soft gelatin capsules in which
the active ingredient is mixed with water or an oil.
[0155] Aqueous suspensions generally contain the active ingredient
in finely powdered form together with one or more suspending
agents, dispersing or wetting agents. The aqueous suspensions may
also contain one or more preservatives, anti-oxidants, colouring
agents, flavouring agents, and/or sweetening agents.
[0156] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil or in a mineral oil. The oily
suspensions may also contain a thickening agent. Sweetening agents
such as those set out above, and flavouring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant.
[0157] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water generally contain
the active ingredient together with a dispersing or wetting agent,
suspending agent and one or more preservatives. Additional
excipients such as sweetening, flavouring and colouring agents, may
also be present.
[0158] The pharmaceutical compositions of the invention may also be
in the form of oil-in-water emulsions. The oily phase may be a
vegetable oil or a mineral oil or a mixture of any of these. The
emulsions may also contain sweetening, flavouring and preservative
agents.
[0159] Syrups and elixirs may be formulated with sweetening agents,
and may also contain a demulcent, preservative, flavouring and/or
colouring agent.
[0160] The pharmaceutical compositions may also be in the form of a
sterile injectable aqueous or oily suspension, which may be
formulated according to known procedures using one or more of the
appropriate dispersing or wetting agents and suspending agents,
which have been mentioned above. A sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent system.
[0161] Compositions for administration by inhalation may be in the
form of a conventional pressurised aerosol arranged to dispense the
active ingredient either as an aerosol containing finely divided
solid or liquid droplets. Conventional aerosol propellants such as
volatile fluorinated hydrocarbons or hydrocarbons may be used and
the aerosol device is conveniently arranged to dispense a metered
quantity of active ingredient. Dry powder inhalers may also be
suitable.
[0162] For further information on formulation the reader is
referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal
Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon
Press 1990.
[0163] In one aspect of the invention, the pharmaceutical
composition described above comprises the compound of Example 1
[(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,-
4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid], or
a pharmaceutically acceptable salt thereof. Conveniently, the
compound of Example 1 is present in its polymorph described herein
as crystalline Form B.
[0164] The process for synthesising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid as set
out in Example 1, recommends that the process is carried out in the
absence of light and under a nitrogen atmosphere in order to avoid
the formation of a degradation product.
[0165] The degradation product referred to, which is
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate, has the
following structure:
##STR00017##
and is thought may be formed from Example 1
[(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,-
4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid] by
autooxidation in air via a free radical chain mechanism. For the
avoidance of doubt, this degradation product is not believed to
have significant SERD activity.
[0166] This compound could also be referred to as
(E)-3-[3,5-difluoro-4-[(3R)-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dih-
ydro-3H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate and a
synthetic method for making it is given in Example 13.
[0167] The skilled person will appreciate that control of the
formation of degradation products is essential to the safe
production and storage of pharmaceuticals. Also, the skilled person
will appreciate that certain compounds may degrade on storage, even
after formulation into a pharmaceutical composition, and that such
degradation may in some instances be controlled by the use of
appropriate excipients in the pharmaceutical composition and/or by
appropriate packaging of the final product. The skilled person will
further appreciate that a final formulation which is developed for
commercial use will need to be optimised for a number of
characteristics, including chemical stability, but also including
for instance physical stability and dissolution characteristics.
Therefore such formulations will be developed in order to balance a
number of different factors.
[0168] Suitably, compositions of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid, or a
pharmaceutically-acceptable salt thereof, include a compound which
acts as an anti-oxidant.
[0169] Anti-oxidant compounds which are suitable for use in
pharmaceutical compositions are known in the art, and include, for
example, acetone sodium bisulfite; alpha lipoic acid; alpha
tocopherol; ascorbic acid; ascorbyl palmitate; butylated
hydroxyanisole; butylated hydroxytoluene; carotenes; citric acid
monohydrate; dodecyl gallate; erythorbic acid; fumaric acid;
glutathione; histidine; hypophosphorous acid; lactobionic acid;
lipoic acid; malic acid; melatonin; methionine; d-mannose;
monothioglycerol; octyl gallate; potassium metabisulfite; propionic
acid; propyl gallate; sodium ascorbate; sodium bisulfite; sodium
formaldehyde sulfoxylate; sodium metabisulfite; sodium sulfite;
sodium thiosulfate; stannous chloride; sulfur dioxide; thymol;
tocopherol; tocotrienols; ubiquinol; uric acid; vitamin E; and
vitamin E polyethylene glycol succinate. Such compounds may exert
their anti-oxidant effect by a variety of mechanisms, and one or
more of these mechanisms may be more effective than others for any
particular compound. Some anti-oxidants compounds, such as BHA, act
as free-radical scavengers. Other anti-oxidants, such sodium
metabisulfite and ascorbic acid are easily oxidised and so may be
oxidised in preference to the active ingredient.
[0170] For example, where metal induced peroxide formation is
involved in the oxidation mechanism, use of a chelating agent, such
as for example EDTA (ethylenediaminetetraacetic acid), may be
useful to remove any metal contaminants and thereby indirectly
achieve a stabilising effect. Other metal-chelating agents are
known in the art and include, for example, betadex sulfobutyl ether
sodium; calcium acetate; citric acid monohydrate; cyclodextrins;
disodium edetate; edetic acid; fumaric acid; galactose; glutamic
acid; histidine; hydroxypropyl betadex; malic acid; pentetic acid;
phytochelatin; poly(methyl vinyl ether/maleic anhydride); potassium
citrate; sodium citrate dihydrate; sodium phosphate, dibasic;
sodium phosphate, monobasic; tartaric acid; and trehalose.
[0171] For example, propyl gallate, sodium metabisulfite, ascorbic
acid and butylated hydroxyanisole were included in exemplary
formulations of Example 1. EDTA was also included. An example of
such a formulation is provided as Example 12. Of these,
compositions containing sodium metabisulfite appeared to be less
stable than those with no anti-oxidant, after four weeks storage
under a number of different heat and humidity conditions.
Compositions containing ascorbic acid appeared to be the most
stable after 4 weeks storage under a number of different heat and
humidity conditions (as determined by Liquid Chromatography
analysis, eg UHPLC).
[0172] Further suitable additives for formulations comprising the
compound of Example 1, include using excipients with a low metal
content, excipients with a low peroxide content, excipients such as
mannitol which is a free-radical scavenger as well as a filler. The
process of production of such a formulation may also impact
stability. For example, for some active ingredients, ensuring
intimate mixing of the active ingredient with stability-inducing
excipients may be important in ensuring maximum stabilisation.
Intimate mixing may be influenced by, for example, mixing speed,
particle sizes and wet or dry mixing/granulation processes. An
active ingredient may be granulated with an antioxidant and then
mixed with other excipients. Antioxidants may also be added to any
coating on the outside of a pharmaceutical composition.
[0173] In one aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, in association with at
least one pharmaceutically-acceptable diluents or carrier.
[0174] In one aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, and also comprising an
anti-oxidant. Suitably, ascorbic acid may be used as the
anti-oxidant.
[0175] In one aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, and also comprising a
metal chelating agent. Suitably, EDTA may be used as a metal
chelating agent.
[0176] In another aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, an anti-oxidant and
optionally further comprising a metal chelating agent.
[0177] In another aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, in association with at
least one pharmaceutically-acceptable diluent or carrier, wherein
the composition contains less than 5% w/w of
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.
[0178] In another aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, in association with at
least one pharmaceutically-acceptable diluent or carrier, wherein
the composition contains less than 2% w/w of
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.
[0179] In another aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, in association with at
least one pharmaceutically-acceptable diluent or carrier, wherein
the composition contains less than 1% w/w of
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.
[0180] In another aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, in association with at
least one pharmaceutically-acceptable diluent or carrier, wherein
the composition contains less than 0.5% w/w of
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.
[0181] In another aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, in association with at
least one pharmaceutically-acceptable diluents or carrier, wherein
the composition contains less than 0.1% w/w of
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.
[0182] In another aspect of the invention, there is provided a
pharmaceutical composition comprising
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a
pharmaceutically-acceptable salt thereof, in association with at
least one pharmaceutically-acceptable diluents or carrier, wherein
the composition contains less than 0.05% w/w of
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.
[0183] In the above aspects, where the composition is described as
containing less than 5% w/w, 2% w/w, 1% w/w, 0.5% w/w, 0.1% w/w or
0.05% w/w of
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9--
dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate then the
skilled person will understand that this is intended to mean
percentage weight for weight in comparison to the weight of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
present in the composition.
[0184] The degradation product
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate may therefore be
used as a reference marker or reference standard in analytical
techniques such as HPLC to monitor the stability of the compound of
Example 1 or pharmaceutical compositions containing it.
[0185] The amount of active ingredient that is combined with one or
more excipients to produce a single dosage form will necessarily
vary depending upon the host treated and the particular route of
administration. For example, oral administration to humans will
generally require, for example, from 1 mg to 2 g of active agent
(more suitably from 100 mg to 2 g, for example from 250 mg to 1.8
g, such as from 500 mg to 1.8 g, particularly from 500 mg to 1.5 g,
conveniently from 500 mg to 1 g) to be administered compounded with
an appropriate and convenient amount of excipients which may vary
from about 3 to about 98 percent by weight of the total
composition. It will be understood that, if a large dosage is
required, multiple dosage forms may be required, for example two or
more tablets or capsules, with the dose of active ingredient
divided conveniently between them. Typically, unit dosage forms
will contain about 10 mg to 0.5 g of a compound of this invention,
although a unit dosage form may contain up to 1 g. Conveniently, a
single solid dosage form may contain between 1 and 300 mg of active
ingredient.
[0186] The size of the dose for therapeutic or prophylactic
purposes of compounds of the present invention will naturally vary
according to the nature and severity of the disease state, the age
and sex of the animal or patient and the route of administration,
according to well known principles of medicine.
[0187] In using compounds of the present invention for therapeutic
or prophylactic purposes it will generally be administered so that
a daily dose in the range, for example, 1 mg/kg to 100 mg/kg body
weight is received, given if required in divided doses. In general,
lower doses will be administered when a parenteral route is
employed. Thus, for example, for intravenous administration, a dose
in the range, for example, 1 mg/kg to 25 mg/kg body weight will
generally be used. Similarly, for administration by inhalation, a
dose in the range, for example, 1 mg/kg to 25 mg/kg body weight
will be used. Oral administration is however preferred,
particularly in tablet form.
[0188] In one aspect of the invention, compounds of the present
invention or pharmaceutically-acceptable salts thereof, are
administered as tablets comprising 10 mg to 100 mg of the compound
of Formula (I) (or a pharmaceutically-acceptable salt thereof),
wherein one or more tablets are administered as required to achieve
the desired dose.
[0189] As stated above, it is known that signalling through
ER.alpha. causes tumourigenesis by one or more of the effects of
mediating proliferation of cancer and other cells, mediating
angiogenic events and mediating the motility, migration and
invasiveness of cancer cells. We have found that the compounds of
the present invention possess potent anti-tumour activity which it
is believed is obtained by way of antagonism and down-regulation of
ER.alpha. that is involved in the signal transduction steps which
lead to the proliferation and survival of tumour cells and the
invasiveness and migratory ability of metastasising tumour
cells.
[0190] Accordingly, the compounds of the present invention may be
of value as anti-tumour agents, in particular as selective
inhibitors of the proliferation, survival, motility, dissemination
and invasiveness of mammalian cancer cells leading to inhibition of
tumour growth and survival and to inhibition of metastatic tumour
growth. Particularly, the compounds of the present invention may be
of value as anti-proliferative and anti-invasive agents in the
containment and/or treatment of solid tumour disease. Particularly,
the compounds of the present invention may be useful in the
prevention or treatment of those tumours which are sensitive to
inhibition of ER.alpha. and that are involved in the signal
transduction steps which lead to the proliferation and survival of
tumour cells and the migratory ability and invasiveness of
metastasising tumour cells. Further, the compounds of the present
invention may be useful in the prevention or treatment of those
tumours which are mediated alone or in part by antagonism and
down-regulation of ER.alpha., i.e. the compounds may be used to
produce an ER.alpha. inhibitory effect in a warm blooded animal in
need of such treatment.
[0191] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for use as a medicament in a warm-blooded animal such as man.
[0192] According to a further aspect of the invention, there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for use in the production of an anti-proliferative effect in a
warm-blooded animal such as man.
[0193] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for use in a warm-blooded animal such as man as an anti-invasive
agent in the containment and/or treatment of solid tumour
disease.
[0194] According to a further aspect of the invention, there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for the production of an anti-proliferative effect in a
warm-blooded animal such as man.
[0195] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
in the manufacture of a medicament for use in the production of an
anti-proliferative effect in a warm-blooded animal such as man.
[0196] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
in the manufacture of a medicament for use in a warm-blooded animal
such as man as an anti-invasive agent in the containment and/or
treatment of solid tumour disease.
[0197] According to a further aspect of the invention there is
provided a method for producing an anti-proliferative effect in a
warm blooded animal, such as man, in need of such treatment which
comprises administering to said animal an effective amount of a
compound of the Formula (I), or a pharmaceutically-acceptable salt
thereof, as defined hereinbefore.
[0198] According to a further aspect of the invention there is
provided a method for producing an anti-invasive effect by the
containment and/or treatment of solid tumour disease in a warm
blooded animal, such as man, in need of such treatment which
comprises administering to said animal an effective amount of a
compound of the Formula (I), or a pharmaceutically-acceptable salt
thereof, as defined hereinbefore.
[0199] According to a further aspect of the invention, there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for use in the prevention or treatment of cancer in a warm blooded
animal such as man.
[0200] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
in the manufacture of a medicament for use in the prevention or
treatment of cancer in a warm blooded animal such as man.
[0201] According to a further aspect of the invention there is
provided a method for the prevention or treatment of cancer in a
warm blooded animal, such as man, in need of such treatment which
comprises administering to said animal an effective amount of a
compound of the Formula (I), or a pharmaceutically-acceptable salt
thereof, as defined hereinbefore.
[0202] According to a further aspect of the invention, there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for use in the prevention or treatment of solid tumour disease in a
warm blooded animal such as man.
[0203] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
in the manufacture of a medicament for use in the prevention or
treatment of solid tumour disease in a warm blooded animal such as
man.
[0204] According to a further aspect of the invention there is
provided a method for the prevention or treatment of solid tumour
disease in a warm blooded animal, such as man, in need of such
treatment which comprises administering to said animal an effective
amount of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined
hereinbefore.
[0205] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for use in the prevention or treatment of those tumours which are
sensitive to inhibition of ER.alpha. that are involved in the
signal transduction steps which lead to the proliferation,
survival, invasiveness and migratory ability of tumour cells.
[0206] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
in the manufacture of a medicament for use in the prevention or
treatment of those tumours which are sensitive to inhibition of
ER.alpha. that are involved in the signal transduction steps which
lead to the proliferation, survival, invasiveness and migratory
ability of tumour cells.
[0207] According to a further aspect of the invention there is
provided a method for the prevention or treatment of those tumours
which are sensitive to inhibition of ER.alpha. that are involved in
the signal transduction steps which lead to the proliferation,
survival, invasiveness and migratory ability of tumour cells which
comprises administering to said animal an effective amount of a
compound of the Formula (I), or a pharmaceutically-acceptable salt
thereof, as defined hereinbefore.
[0208] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for use in providing an inhibitory effect on ER.alpha..
[0209] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
in the manufacture of a medicament for use in providing an
inhibitory effect on ER.alpha..
[0210] According to a further aspect of the invention there is also
provided a method for providing an inhibitory effect on ER.alpha.
which comprises administering an effective amount of a compound of
the Formula (I), or a pharmaceutically-acceptable salt thereof, as
defined hereinbefore.
[0211] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
for use in providing a selective inhibitory effect on
ER.alpha..
[0212] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined hereinbefore
in the manufacture of a medicament for use in providing a selective
inhibitory effect on ER.alpha..
[0213] According to a further aspect of the invention there is also
provided a method for providing a selective inhibitory effect on
ER.alpha. which comprises administering an effective amount of a
compound of the Formula (I), or a pharmaceutically-acceptable salt
thereof, as defined hereinbefore.
[0214] Described herein are compounds that can bind to ER.alpha.
ligand binding domain and are selective estrogen receptor
degraders. In biochemical and cell based assays the compounds of
the present invention are shown to be potent estrogen receptor
binders and reduce cellular levels of ER.alpha. and may therefore
be useful in the treatment of estrogen sensitive diseases or
conditions (including diseases that have developed resistance to
endocrine therapies), i.e. for use in the treatment of cancer of
the breast and gynaecological cancers (including endometrial,
ovarian and cervical) and cancers expressing ER.alpha. mutated
proteins which may be de novo mutations or have arisen as a result
of treatment with a prior endocrine therapy such as an aromatase
inhibitor.
[0215] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
for use in the treatment of breast or gynaecological cancers.
[0216] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
for use in the treatment of cancer of the breast, endometrium,
ovary or cervix.
[0217] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
for use in the treatment of cancer of the breast.
[0218] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
for use in the treatment of cancer of the breast, wherein the
cancer has developed resistance to one or more other endocrine
therapies.
[0219] According to a further aspect of the invention there is
provided a method for treating breast or gynaecological cancers,
which comprises administering an effective amount of a compound of
the Formula (I), or a pharmaceutically-acceptable salt thereof, as
defined hereinbefore.
[0220] According to a further aspect of the invention there is
provided a method for treating cancer of the breast, endometrium,
ovary or cervix, which comprises administering an effective amount
of a compound of the Formula (I), or a pharmaceutically-acceptable
salt thereof, as defined hereinbefore.
[0221] According to a further aspect of the invention there is
provided a method for treating breast cancer, which comprises
administering an effective amount of a compound of the Formula (I),
or a pharmaceutically-acceptable salt thereof, as defined
hereinbefore.
[0222] According to a further aspect of the invention there is
provided a method for treating breast cancer, wherein the cancer
has developed resistance to one or more other endocrine therapies,
which comprises administering an effective amount of a compound of
the Formula (I), or a pharmaceutically-acceptable salt thereof, as
defined hereinbefore.
[0223] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
in the manufacture of a medicament for use in the treatment of
breast or gynaecological cancers.
[0224] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
in the manufacture of a medicament for use in the treatment of
cancer of the breast, endometrium, ovary or cervix.
[0225] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
in the manufacture of a medicament for use in the treatment of
breast cancer.
[0226] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
in the manufacture of a medicament for use in the treatment of
breast cancer, wherein the cancer has developed resistance to one
or more other endocrine therapies.
[0227] In one feature of the invention, the cancer to be treated is
breast cancer. In a further aspect of this feature, the breast
cancer is Estrogen Receptor+ve (ER+ve). In one embodiment of this
aspect, the compound of Formula (I) is dosed in combination with
another anticancer agent, such as an anti-hormonal agent as defined
herein.
[0228] According to a further aspect of the invention there is
provided a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
for use in the treatment of ER+ve breast cancer.
[0229] According to a further aspect of the invention there is
provided a method for treating ER+ve breast cancer, which comprises
administering an effective amount of a compound of the Formula (I),
or a pharmaceutically-acceptable salt thereof, as defined
hereinbefore.
[0230] According to a further aspect of the invention there is
provided the use of a compound of the Formula (I), or a
pharmaceutically-acceptable salt thereof, as defined herein before
in the manufacture of a medicament for use in the treatment of
ER+ve breast cancer.
[0231] As stated hereinbefore, the in vivo effects of a compound of
the Formula (I) may be exerted in part by one or more metabolites
that are formed within the human or animal body after
administration of a compound of the Formula (I).
[0232] The present invention therefore also contemplates a method
for inhibiting ER-.alpha. in a patient, comprising administering to
a patient an amount of a compound of Formula (I), or a
pharmaceutically-acceptable salt thereof, effective in inhibiting
ER-.alpha. in the patient.
[0233] The present invention therefore also contemplates a method
for inhibiting ER-.alpha. in a patient, comprising administering to
a patient an amount of a compound of Formula (I), or a
pharmaceutically-acceptable salt thereof, effective in inhibiting
ER-.alpha. in the patient.
[0234] In all of the above uses and methods, a suitable compound of
the formula (I) is Example 1, or a pharmaceutically-acceptable salt
thereof. In one aspect, Example 1 is in crystalline form B as
described herein.
[0235] The anti-cancer treatment defined herein may be applied as a
sole therapy or may involve, in addition to the compounds of the
invention, conventional surgery or radiotherapy or chemotherapy.
Such chemotherapy may include one or more of the following
categories of anti-tumour agents:--
(i) other antiproliferative/antineoplastic drugs and combinations
thereof, as used in medical oncology, such as alkylating agents
(for example cis-platin, oxaliplatin, carboplatin,
cyclophosphamide, nitrogen mustard, melphalan, chlorambucil,
busulphan, temozolamide and nitrosoureas); antimetabolites (for
example gemcitabine and antifolates such as fluoropyrimidines like
5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine
arabinoside, and hydroxyurea); antitumour antibiotics (for example
anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin,
epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin);
antimitotic agents (for example vinca alkaloids like vincristine,
vinblastine, vindesine and vinorelbine and taxoids like taxol and
taxotere and polokinase inhibitors); and topoisomerase inhibitors
(for example epipodophyllotoxins like etoposide and teniposide,
amsacrine, topotecan and camptothecin); (ii) antihormonal agents
such as antioestrogens (for example tamoxifen, fulvestrant,
toremifene, raloxifene, droloxifene and iodoxyfene), progestogens
(for example megestrol acetate), aromatase inhibitors (for example
as anastrozole, letrozole, vorazole and exemestane); (iii)
inhibitors of growth factor function and their downstream
signalling pathways: included are Ab modulators of any growth
factor or growth factor receptor targets, reviewed by Stem et al.
Critical Reviews in Oncology/Haematology, 2005, 54, pp 11-29); also
included are small molecule inhibitors of such targets, for example
kinase inhibitors--examples include the anti-erbB2 antibody
trastuzumab [Herceptin.TM.], the anti-EGFR antibody panitumumab,
the anti-EGFR antibody cetuximab [Erbitux, C225] and tyrosine
kinase inhibitors including inhibitors of the erbB receptor family,
such as epidermal growth factor family receptor (EGFR/erbB1)
tyrosine kinase inhibitors such as gefitinib or erlotinib, erbB2
tyrosine kinase inhibitors such as lapatinib, and mixed erb1/2
inhibitors such as afatanib; similar strategies are available for
other classes of growth factors and their receptors, for example
inhibitors of the hepatocyte growth factor family or their
receptors including c-met and ron; inhibitors of the insulin and
insulin growth factor family or their receptors (IGFR, IR)
inhibitors of the platelet-derived growth factor family or their
receptors (PDGFR), and inhibitors of signalling mediated by other
receptor tyrosine kinases such as c-kit, AnLK, and CSF-1R; also
included are modulators which target signalling proteins in the
PI3-kinase signalling pathway, for example, inhibitors of
PI3-kinase isoforms such as PI3K-.alpha./.beta./.gamma. and ser/thr
kinases such as AKT, mTOR (such as AZD2014), PDK, SGK, PI4K or
PIPSK; also included are inhibitors of serine/threonine kinases not
listed above, for example raf inhibitors such as vemurafenib, MEK
inhibitors such as selumetinib (AZD6244), Abl inhibitors such as
imatinib or nilotinib, Btk inhibitors such as ibrutinib, Syk
inhibitors such as fostamatinib, aurora kinase inhibitors (for
example AZD1152), inhibitors of other ser/thr kinases such as JAKs,
STATs and IRAK4, and cyclin dependent kinase inhibitors such as
palbociclib; iv) modulators of DNA damage signalling pathways, for
example PARP inhibitors (e.g. Olaparib), ATR inhibitors or ATM
inhibitors; v) modulators of apoptotic and cell death pathways such
as Bcl family modulators (e.g. ABT-263/Navitoclax, ABT-199); (vi)
antiangiogenic agents such as those which inhibit the effects of
vascular endothelial growth factor, [for example the anti-vascular
endothelial cell growth factor antibody bevacizumab (Avastin.TM.)
and for example, a VEGF receptor tyrosine kinase inhibitor such as
sorafenib, axitinib, pazopanib, sunitinib and vandetanib (and
compounds that work by other mechanisms (for example linomide,
inhibitors of integrin .alpha.v.beta.3 function and angiostatin)];
(vii) vascular damaging agents, such as Combretastatin A4; (viii)
anti-invasion agents, for example c-Src kinase family inhibitors
like (dasatinib, J. Med. Chem., 2004, 47, 6658-6661) and bosutinib
(SKI-606), and metalloproteinase inhibitors like marimastat,
inhibitors of urokinase plasminogen activator receptor function or
antibodies to Heparanase]; (ix) immunotherapy approaches, including
for example ex-vivo and in-vivo approaches to increase the
immunogenicity of patient tumour cells, such as transfection with
cytokines such as interleukin 2, interleukin 4 or
granulocyte-macrophage colony stimulating factor, approaches to
decrease T-cell anergy, approaches using transfected immune cells
such as cytokine-transfected dendritic cells, approaches using
cytokine-transfected tumour cell lines and approaches using
anti-idiotypic antibodies. Specific examples include monoclonal
antibodies targeting PD-1 (e.g. BMS-936558) or CTLA4 (e.g.
ipilimumab and tremelimumab); (x) Antisense or RNAi based
therapies, for example those which are directed to the targets
listed. (xi) gene therapy approaches, including for example
approaches to replace aberrant genes such as aberrant p53 or
aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug
therapy) approaches such as those using cytosine deaminase,
thymidine kinase or a bacterial nitroreductase enzyme and
approaches to increase patient tolerance to chemotherapy or
radiotherapy such as multi-drug resistance gene therapy.
[0236] According to this aspect of the invention there is provided
a combination suitable for use in the treatment of cancer
comprising compounds of the present invention as defined herein or
a pharmaceutically acceptable salt thereof and another anti-tumour
agent, in particular any one of the anti tumour agents listed under
(i)-(xi) above. In particular, the anti-tumour agent listed under
(i)-(xi) above is the standard of care for the specific cancer to
be treated; the person skilled in the art will understand the
meaning of "standard of care". In one aspect, the compound of the
present invention is Example 1, or a pharmaceutically-acceptable
salt thereof.
[0237] Therefore in a further aspect of the invention there is
provided compounds of the present invention or a pharmaceutically
acceptable salt thereof in combination with another anti-tumour
agent, in particular an anti-tumour agent selected from one listed
under (i)-(xi) herein above.
[0238] In a further aspect of the invention there is provided
Example 1
[(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,-
4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid] or a
pharmaceutically acceptable salt thereof in combination with
another anti-tumour agent, in particular an anti-tumour agent
selected from one listed under (i)-(xi) herein above.
[0239] In a further aspect of the invention there is provided
compounds of the present invention or a pharmaceutically acceptable
salt thereof in combination with another anti-tumour agent, in
particular an anti-tumour agent selected from one listed under (i)
above.
[0240] In a further aspect of the invention there is provided a
compound of the present invention as defined herein before or a
pharmaceutically acceptable salt thereof and any one of the anti
tumour agents listed under (i) above.
[0241] In a further aspect of the invention there is provided
Example 1 or a pharmaceutically acceptable salt thereof in
combination with any one of the anti-tumour agents listed under (i)
above.
[0242] In a further aspect of the invention there is provided a
combination suitable for use in the treatment of cancer comprising
compounds of the present invention as defined hereinbefore or a
pharmaceutically acceptable salt thereof and a taxoid, such as for
example taxol or taxotere, conveniently taxotere. For example, a
suitable compound of the invention in combination with a taxoid,
such as for example taxol or taxotere, conveniently taxotere, is
Example 1, or a pharmaceutically-acceptable salt thereof.
[0243] In a further aspect of the invention there is provided
compounds of the present invention or a pharmaceutically acceptable
salt thereof in combination with another anti-tumour agent, in
particular an anti-tumour agent selected from one listed under (ii)
herein above.
[0244] In a further aspect of the invention there is provided a
combination suitable for use in the treatment of cancer comprising
compounds of the present invention as defined herein before or a
pharmaceutically acceptable salt thereof and any one of
antihormonal agents listed under (ii) above, for example any one of
the anti-oestrogens listed in (ii) above, or for example an
aromatase inhibitor listed in (ii) above.
[0245] In a further aspect of the invention there is provided a
combination suitable for use in the treatment of cancer comprising
Example 1 or a pharmaceutically acceptable salt thereof and any one
of antihormonal agents listed under (ii) above, for example any one
of the anti-oestrogens listed in (ii) above, or for example an
aromatase inhibitor listed in (ii) above.
[0246] In a further aspect of the invention there is provided a
combination suitable for use in the treatment of cancer comprising
Example 1 or a pharmaceutically acceptable salt thereof and an mTOR
inhibitor, such as AZD2014 (see for example WO2008/023161).
##STR00018##
[0247] In a further aspect of the invention there is provided a
combination suitable for use in the treatment of cancer comprising
Example 1, or a pharmaceutically-acceptable salt thereof and a
PI3K.alpha.-inhibitor, such as those PI3K .alpha./.delta.
inhibitors in our co-pending PCT application PCT/GB2014/050163. One
example of a suitable PI3K .alpha./.delta. inhibitor is Example 3
from PCT/GB2014/050163, which is the compound
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, or
a pharmaceutically-acceptable salt thereof. A process to make
Example 3 of PCT/GB2014/050163 is set out in Reference Example 1
herein.
[0248] In a further aspect of the invention there is provided a
combination suitable for use in the treatment of cancer comprising
Example 1 or a pharmaceutically acceptable salt thereof and
palbociclib.
[0249] In one aspect the above combination of a compound of formula
(I) or a pharmaceutically acceptable salt thereof, particularly
Example 1 or a pharmaceutically acceptable salt thereof, with an
anti-tumour agent listed in (ii) above, or an mTOR inhibitor (such
as AZD2014), or a PI3K-.alpha. inhibitor (such as those PI3K
.alpha./.delta. inhibitors in our co-pending PCT application
PCT/GB2014/050163, particularly Example 3 therein) or palbociclib,
is suitable for use in the treatment of breast or gynaecological
cancers, such as cancer of the breast, endometrium, ovary or
cervix, particularly breast cancer, such as ER+ve breast
cancer.
[0250] Herein, where the term "combination" is used it is to be
understood that this refers to simultaneous, separate or sequential
administration. In one aspect of the invention "combination" refers
to simultaneous administration. In another aspect of the invention
"combination" refers to separate administration. In a further
aspect of the invention "combination" refers to sequential
administration. Where the administration is sequential or separate,
the delay in administering the second component should not be such
as to lose the beneficial effect of the combination. Where a
combination of two or more components is administered separately or
sequential, it will be understood that the dosage regime for each
component may be different to and independent of the other
components. Conveniently, the compounds of the present invention
are dosed once daily.
[0251] According to a further aspect of the invention there is
provided a pharmaceutical composition which comprises a compound of
Formula (I) or a pharmaceutically acceptable salt thereof in
combination with an anti-tumour agent selected from one listed
under (i)-(xi) herein above, in association with a pharmaceutically
acceptable diluent or carrier.
[0252] According to a further aspect of the invention there is
provided a pharmaceutical composition which comprises Example 1
[(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,-
4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid] or a
pharmaceutically acceptable salt thereof in combination with an
anti-tumour agent selected from one listed under (i)-(xi) herein
above, in association with a pharmaceutically acceptable diluent or
carrier.
[0253] According to a further aspect of the invention there is
provided a pharmaceutical composition which comprises a compound of
Formula (I) or a pharmaceutically acceptable salt thereof in
combination with any one of antihormonal agents listed under (ii)
above, for example any one of the anti-oestrogens listed in (ii)
above, or for example an aromatase inhibitor listed in (ii) above
in association with a pharmaceutically acceptable diluent or
carrier.
[0254] In a further aspect of the invention there is provided a
pharmaceutical composition which comprises Example 1 or a
pharmaceutically acceptable salt thereof and any one of
antihormonal agents listed under (ii) above, for example any one of
the anti-oestrogens listed in (ii) above, or for example an
aromatase inhibitor listed in (ii) above; in association with a
pharmaceutically acceptable diluent or carrier.
[0255] In a further aspect of the invention there is provided a
pharmaceutical composition comprising Example 1 or a
pharmaceutically acceptable salt thereof and an mTOR inhibitor,
such as AZD2014 (see for example WO2008/023161); in association
with a pharmaceutically acceptable diluent or carrier.
[0256] In a further aspect of the invention there is provided a
pharmaceutical composition comprising Example 1, or a
pharmaceutically-acceptable salt thereof and a
PI3K.alpha.-inhibitor, such as those PI3K .alpha./.delta.
inhibitors in our co-pending PCT application PCT/GB2014/050163, in
association with a pharmaceutically acceptable diluent or carrier.
One example of a suitable PI3K .alpha./.delta. inhibitor is Example
3 from PCT/GB2014/050163, as described hereinbefore.
[0257] In a further aspect of the invention there is provided a
pharmaceutical composition comprising Example 1 or a
pharmaceutically acceptable salt thereof and palbociclib in
association with a pharmaceutically acceptable diluent or
carrier.
[0258] In one aspect the above pharmaceutical compositions of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof, particularly Example 1 or a pharmaceutically acceptable
salt thereof, with an anti-tumour agent listed in (ii) above, or an
mTOR inhibitor (such as AZD2014), or a PI3K-.alpha. inhibitor (such
as those PI3K .alpha./.delta. inhibitors in our co-pending PCT
application PCT/GB2014/050163, particularly Example 3 therein) or
palbociclib, is suitable for use in the treatment of breast or
gynaecological cancers, such as cancer of the breast, endometrium,
ovary or cervix, particularly breast cancer, such as ER+ve breast
cancer.
[0259] According to a further aspect of the invention there is
provided a pharmaceutical composition which comprises a compound of
Formula (I) or a pharmaceutically acceptable salt thereof in
combination with an anti-tumour agent selected from one listed
under (i)-(xi) herein above, in association with a pharmaceutically
acceptable diluent or carrier for use in treating cancer.
[0260] According to a further aspect of the invention there is
provided a pharmaceutical composition which comprises a compound of
Formula (I) or a pharmaceutically acceptable salt thereof in
combination with any one of antihormonal agents listed under (ii)
above, for example any one of the anti-oestrogens listed in (ii)
above, or for example an aromatase inhibitor listed in (ii) above
in association with a pharmaceutically acceptable diluent or
carrier for use in treating cancer.
[0261] According to a further aspect of the invention there is
provided a pharmaceutical composition which comprises Example 1 or
a pharmaceutically acceptable salt thereof in combination with an
anti-tumour agent selected from one listed under (i)-(xi) herein
above, in association with a pharmaceutically acceptable diluent or
carrier for use in treating cancer.
[0262] In a further aspect of the invention there is provided a
pharmaceutical composition which comprises Example 1 or a
pharmaceutically acceptable salt thereof and any one of
antihormonal agents listed under (ii) above, for example any one of
the anti-oestrogens listed in (ii) above, or for example an
aromatase inhibitor listed in (ii) above; in association with a
pharmaceutically acceptable diluent or carrier for use in treating
cancer.
[0263] In a further aspect of the invention there is provided a
pharmaceutical composition comprising Example 1 or a
pharmaceutically acceptable salt thereof and an mTOR inhibitor,
such as AZD2014 (see for example WO2008/023161); in association
with a pharmaceutically acceptable diluent or carrier for use in
treating cancer.
[0264] In a further aspect of the invention there is provided a
pharmaceutical composition comprising Example 1, or a
pharmaceutically-acceptable salt thereof and a
PI3K.alpha.-inhibitor, such as those PI3K .alpha./.delta.
inhibitors in our co-pending PCT application PCT/GB2014/050163, in
association with a pharmaceutically acceptable diluent or carrier
for use in treating cancer. One example of a suitable PI3K
.alpha./.delta. inhibitor is Example 3 from PCT/GB2014/050163, as
described hereinbefore.
[0265] In a further aspect of the invention there is provided a
pharmaceutical composition comprising Example 1 or a
pharmaceutically acceptable salt thereof and palbociclib in
association with a pharmaceutically acceptable diluent or carrier
for use in treating cancer.
[0266] In one aspect the above pharmaceutical compositions of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof, particularly Example 1 or a pharmaceutically acceptable
salt thereof, with an anti-tumour agent listed in (ii) above, or an
mTOR inhibitor (such as AZD2014), or a PI3K-.alpha. inhibitor (such
as those PI3K .alpha./.delta. inhibitors in our co-pending PCT
application PCT/GB2014/050163, particularly Example 3 therein) or
palbociclib, is suitable for use in the treatment of breast or
gynaecological cancers, such as cancer of the breast, endometrium,
ovary or cervix, particularly breast cancer, such as ER+ve breast
cancer.
[0267] According to another feature of the invention there is
provided the use of a compound of the Formula (I) or a
pharmaceutically acceptable salt thereof in combination with an
anti-tumour agent selected from one listed under (i)-(xi) herein
above, in the manufacture of a medicament for use in the treatment
of cancer in a warm-blooded animal, such as man.
[0268] According to another feature of the invention there is
provided the use of Example 1 or a pharmaceutically acceptable salt
thereof in combination with an anti-tumour agent selected from one
listed under (i)-(xi) herein above, in the manufacture of a
medicament for use in cancer in a warm-blooded animal, such as
man.
[0269] According to a further aspect of the invention there is
provided the use of a compound of Formula (I) or a pharmaceutically
acceptable salt thereof in combination with any one of antihormonal
agents listed under (ii) above, for example any one of the
anti-oestrogens listed in (ii) above, or for example an aromatase
inhibitor listed in (ii) above in the manufacture of a medicament
for use in the treatment of cancer in a warm-blooded animal, such
as man.
[0270] In a further aspect of the invention there is provided the
use of Example 1 or a pharmaceutically acceptable salt thereof in
combination with any one of antihormonal agents listed under (ii)
above, for example any one of the anti-oestrogens listed in (ii)
above, or for example an aromatase inhibitor listed in (ii) above;
in the manufacture of a medicament for use in the treatment of
cancer in a warm-blooded animal, such as man.
[0271] In a further aspect of the invention there is provided the
use of Example 1 or a pharmaceutically acceptable salt thereof in
combination with an mTOR inhibitor, such as AZD2014 (see for
example WO2008/023161); in the manufacture of a medicament for use
in the treatment of cancer in a warm-blooded animal, such as
man.
[0272] In a further aspect of the invention there is provided the
use of Example 1, or a pharmaceutically-acceptable salt thereof in
combination with a PI3K.alpha.-inhibitor, such as those PI3K
.alpha./.delta. inhibitors in our co-pending PCT application
PCT/GB2014/050163, in the manufacture of a medicament for use in
the treatment of cancer in a warm-blooded animal, such as man. One
example of a suitable PI3K .alpha./.delta. inhibitor is Example 3
from PCT/GB2014/050163, as described hereinbefore.
[0273] In a further aspect of the invention there is provided the
use of Example 1 or a pharmaceutically acceptable salt thereof in
combination with palbociclib in the manufacture of a medicament for
use in the treatment of cancer in a warm-blooded animal, such as
man.
[0274] In one aspect the above uses of a compound of formula (I) or
a pharmaceutically acceptable salt thereof, particularly Example 1
or a pharmaceutically acceptable salt thereof, in combination with
an anti-tumour agent listed in (ii) above, or an mTOR inhibitor
(such as AZD2014), or a PI3K-.alpha. inhibitor (such as those PI3K
.alpha./.delta. inhibitors in our co-pending PCT application
PCT/GB2014/050163, particularly Example 3 therein) or palbociclib,
is suitable for use in the manufacture of a medicament for the
treatment of breast or gynaecological cancers, such as cancer of
the breast, endometrium, ovary or cervix, particularly breast
cancer, such as ER+ve breast cancer.
[0275] Therefore in an additional feature of the invention, there
is provided a method of treating cancer in a warm-blooded animal,
such as man, in need of such treatment which comprises
administering to said animal an effective amount of a compound of
Formula (I) or a pharmaceutically acceptable salt thereof in
combination with an anti-tumour agent selected from one listed
under (i)-(xi) herein above.
[0276] According to a further aspect of the invention, there is
provided a method of treating cancer in a warm-blooded animal, such
as man, in need of such treatment which comprises administering to
said animal an effective amount of Example 1 or a pharmaceutically
acceptable salt thereof in combination with an anti-tumour agent
selected from one listed under (i)-(xi) herein above.
[0277] According to a further aspect of the invention there is
provided a method of treating cancer in a warm-blooded animal, such
as man, in need of such treatment which comprises administering to
said animal an effective amount of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof in combination with any
one of antihormonal agents listed under (ii) above, for example any
one of the anti-oestrogens listed in (ii) above, or for example an
aromatase inhibitor listed in (ii) above.
[0278] In a further aspect of the invention there is provided a
method of treating cancer in a warm-blooded animal, such as man, in
need of such treatment which comprises administering to said animal
an effective amount of Example 1 or a pharmaceutically acceptable
salt thereof in combination with any one of antihormonal agents
listed under (ii) above, for example any one of the anti-oestrogens
listed in (ii) above, or for example an aromatase inhibitor listed
in (ii) above.
[0279] In a further aspect of the invention there is provided a
method of treating cancer in a warm-blooded animal, such as man, in
need of such treatment which comprises administering to said animal
an effective amount of Example 1 or a pharmaceutically acceptable
salt thereof in combination with an mTOR inhibitor, such as AZD2014
(see for example WO2008/023161).
[0280] In a further aspect of the invention there provided a method
of treating cancer in a warm-blooded animal, such as man, in need
of such treatment which comprises administering to said animal an
effective amount of Example 1, or a pharmaceutically-acceptable
salt thereof in combination with a PI3K.alpha.-inhibitor, such as
those PI3K .alpha./.delta. inhibitors in our co-pending PCT
application PCT/GB2014/050163. One example of a suitable PI3K
.alpha./.delta. inhibitor is Example 3 from PCT/GB2014/050163, as
described hereinbefore.
[0281] In a further aspect of the invention there is provided a
method of treating cancer in a warm-blooded animal, such as man, in
need of such treatment which comprises administering to said animal
an effective amount of Example 1 or a pharmaceutically acceptable
salt thereof in combination with palbociclib.
[0282] In one aspect the above methods of treating cancer, are
methods for the treatment of breast or gynaecological cancers, such
as cancer of the breast, endometrium, ovary or cervix, particularly
breast cancer, such as ER+ve breast cancer.
[0283] According to a further aspect of the present invention there
is provided a kit comprising a compound of Formula (I) or a
pharmaceutically acceptable salt thereof in combination with an
anti-tumour agent selected from one listed under (i)-(xi) herein
above.
[0284] According to a further aspect of the present invention there
is provided a kit comprising a compound of Formula (I) or a
pharmaceutically acceptable salt thereof in combination with an
anti-tumour agent selected from one listed under (i) or (ii) herein
above.
[0285] According to a further aspect of the present invention there
is provided a kit comprising:
a) a compound of Formula (I) or a pharmaceutically acceptable salt
thereof in a first unit dosage form; b) an anti-tumour agent
selected from one listed under (i)-(xi) herein above in a second
unit dosage form; and c) container means for containing said first
and second dosage forms.
[0286] According to a further aspect of the present invention there
is provided a kit comprising:
a) a compound of Formula (I) or a pharmaceutically acceptable salt
thereof in a first unit dosage form; b) an anti-tumour agent
selected from one listed under (i)-(ii) herein above in a second
unit dosage form; and c) container means for containing said first
and second dosage forms.
[0287] According to a further aspect of the present invention there
is provided a kit comprising Example 1 or a pharmaceutically
acceptable salt thereof in combination with an anti-tumour agent
selected from one listed under (i)-(xi) herein above.
[0288] According to a further aspect of the present invention there
is provided a kit comprising Example 1 or a pharmaceutically
acceptable salt thereof in combination with an anti-tumour agent
selected from one listed under (i) or (ii) herein above.
[0289] According to a further aspect of the present invention there
is provided a kit comprising:
a) Example 1 or a pharmaceutically acceptable salt thereof in a
first unit dosage form; b) an anti-tumour agent selected from one
listed under (i)-(xi) herein above in a second unit dosage form;
and c) container means for containing said first and second dosage
forms.
[0290] According to a further aspect of the present invention there
is provided a kit comprising:
a) Example 1 or a pharmaceutically acceptable salt thereof in a
first unit dosage form; b) an anti-tumour agent selected from one
listed under (i)-(ii) herein above in a second unit dosage form;
and c) container means for containing said first and second dosage
forms.
[0291] According to a further aspect of the present invention there
is provided a kit comprising:
a) Example 1 or a pharmaceutically acceptable salt thereof in a
first unit dosage form; b) an anti-tumour agent selected from
AZD2014, a PI3K.alpha.-inhibitor (such as those PI3K
.alpha./.delta. inhibitors in our co-pending PCT application
PCT/GB2014/050163) and palbociclib in a second unit dosage form;
and c) container means for containing said first and second dosage
forms.
[0292] In all of the above methods, uses and other aspects, where
the compound of Example 1 is used, it is suitably used as
crystalline Form B.
[0293] Combination therapy as described above may be added on top
of standard of care therapy typically carried out according to its
usual prescribing schedule.
[0294] Although the compounds of the Formula (I) are primarily of
value as therapeutic agents for use in warm-blooded animals
(including man), they are also useful whenever it is required to
inhibit ER-.alpha.. Thus, they are useful as pharmacological
standards for use in the development of new biological tests and in
the search for new pharmacological agents.
Personalised Healthcare
[0295] Another aspect of the present invention is based on
identifying a link between the status of the gene encoding
ER.alpha. and potential susceptibility to treatment with a compound
of Formula (I). In particular, ER.alpha. gene status may indicate
that a patient is less likely to respond to exisiting hormone
therapy (such as aromatase inhibitors), in part at least because
some ER.alpha. mutations are though to arise as resistance
mechanisms to existing treatments. A SERD, particularly a SERD
which can be administered orally in potentially larger doses
without excessive inconvenince, may then advantageously be used to
treat patents with ER.alpha. mutations who may be resistant to
other therapies. This therefore provides opportunities, methods and
tools for selecting patients for treatment with a compound of
Formula (I), particularly cancer patients. The present invention
relates to patient selection tools and methods (including
personalised medicine). The selection is based on whether the
tumour cells to be treated possess wild-type or mutant ER.alpha.
gene. The ER.alpha. gene status could therefore be used as a
biomarker to indicate that selecting treatment with a SERD may be
advantageous. For the avoidance of doubt, compounds of the formula
(I) as described herein are thought to be similarly active against
wild-type and mutant ER.alpha. genes, at least those mutations in
ER.alpha. gene identified at the date of filing this
application.
[0296] There is a clear need for biomarkers that will enrich for or
select patients whose tumours will respond to treatment with a
SERD, such as a compound of Formula (I). Patient selection
biomarkers that identify the patients most likely to respond to one
agent over another are ideal in the treatment of cancer, since they
reduce the unnecessary treatment of patients with non-responding
tumours to the potential side effects of such agents.
[0297] A biomarker can be described as "a characteristic that is
objectively measured and evaluated as an indicator of normal
biologic processes, pathogenic processes, or pharmacologic
responses to a therapeutic intervention". A biomarker is any
identifiable and measurable indicator associated with a particular
condition or disease where there is a correlation between the
presence or level of the biomarker and some aspect of the condition
or disease (including the presence of, the level or changing level
of, the type of, the stage of, the susceptibility to the condition
or disease, or the responsiveness to a drug used for treating the
condition or disease). The correlation may be qualitative,
quantitative, or both qualitative and quantitative. Typically a
biomarker is a compound, compound fragment or group of compounds.
Such compounds may be any compounds found in or produced by an
organism, including proteins (and peptides), nucleic acids and
other compounds.
[0298] Biomarkers may have a predictive power, and as such may be
used to predict or detect the presence, level, type or stage of
particular conditions or diseases (including the presence or level
of particular microorganisms or toxins), the susceptibility
(including genetic susceptibility) to particular conditions or
diseases, or the response to particular treatments (including drug
treatments). It is thought that biomarkers will play an
increasingly important role in the future of drug discovery and
development, by improving the efficiency of research and
development programs. Biomarkers can be used as diagnostic agents,
monitors of disease progression, monitors of treatment and
predictors of clinical outcome. For example, various biomarker
research projects are attempting to identify markers of specific
cancers and of specific cardiovascular and immunological diseases.
It is believed that the development of new validated biomarkers
will lead both to significant reductions in healthcare and drug
development costs and to significant improvements in treatment for
a wide variety of diseases and conditions.
[0299] In order to optimally design clinical trials and to gain the
most information from these trials, a biomarker may be required.
The marker may be measurable in surrogate and tumour tissues.
Ideally these markers will also correlate with efficacy and thus
could ultimately be used for patient selection.
[0300] Thus, the technical problem underlying this aspect of the
present invention is the identification of means for stratification
of patients for treatment with a compound of Formula (I). The
technical problem is solved by provision of the embodiments
characterized in the claims and/or description herein.
[0301] Tumours which contain wild type ER.alpha. are believed to be
susceptible to treatment with a compound of formula (I), for
example as a first-line treatment. Tumours may also respond to
treatment with a compound of formula (I) as a second-line,
third-line or subsequent therapy and this may be useful, in
particular, where the tumours contain mutant ER.alpha. and may thus
be resistant to existing therapies such as AIs. A higher dosage of
a compound of formula (I) may be required in the resistant setting
than in wild type tumours).
[0302] The invention provides a method of determining sensitivity
of cells to a compound of Formula (I). The method comprises
determining the status of ER.alpha. gene in said cells. A cell is
defined as sensitive to a compound of Formula (I) if it inhibits
the increase in cell number in a cell growth assay (either through
inhibition of cell proliferation and/or through increased cell
death). Methods of the invention are useful for predicting which
cells are more likely to respond to a compound of Formula (I) by
growth inhibition.
[0303] A sample "representative of the tumour" can be the actual
tumour sample isolated, or may be a sample that has been further
processed, e.g. a sample of PCR amplified nucleic acid from the
tumour sample.
Definitions
[0304] In this Personalised Healthcare section:
[0305] "Allele" refers to a particular form of a genetic locus,
distinguished from other forms by its particular nucleotide or
amino acid sequence.
[0306] "Amplification reactions" are nucleic acid reactions which
result in specific amplification of target nucleic acids over
non-target nucleic acids. The polymerase chain reaction (PCR) is a
well known amplification reaction.
[0307] "Cancer" is used herein to refer to neoplastic growth
arising from cellular transformation to a neoplastic phenotype.
Such cellular transformation often involves genetic mutation.
[0308] "Gene" is a segment of DNA that contains all the information
for the regulated biosynthesis of an RNA product, including a
promoter, exons, introns, and other sequence elements which may be
located within 5' or 3' flanking regions (not within the
transcribed portions of the gene) that control expression.
[0309] "Gene status" refers to whether the gene is wild type or not
(i.e. mutant).
[0310] "Label" refers to a composition capable of producing a
detectable signal indicative of the presence of the target
polynucleotide in an assay sample. Suitable labels include
radioisotopes, nucleotide chromophores, enzymes, substrates,
fluorescent molecules, chemiluminescent moieties, magnetic
particles, bioluminescent moieties, and the like. As such, a label
is any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical
means.
[0311] "Non-synonymous variation" refers to a variation (variance)
in or overlapping the coding sequence of a gene that result in the
production of a distinct (altered) polypeptide sequence. These
variations may or may not affect protein function and include
missense variants (resulting in substitution of one amino acid for
another), nonsense variants (resulting in a truncated polypeptide
due to generation of a premature stop codon) and insertion/deletion
variants.
[0312] "Synonymous variation" refers to a variation (variance) in
the coding sequence of a gene that does not affect sequence of the
encoded polypeptide. These variations may affect protein function
indirectly (for example by altering expression of the gene), but,
in the absence of evidence to the contrary, are generally assumed
to be innocuous.
[0313] "Nucleic acid" refers to single stranded or double stranded
DNA and RNA molecules including natural nucleic acids found in
nature and/or modified, artificial nucleic acids having modified
backbones or bases, as are known in the art.
[0314] "Primer" refers to a single stranded DNA oligonucleotide
sequence capable of acting as a point of initiation for synthesis
of a primer extension product which is complementary to the nucleic
acid strand to be copied. The length and sequence of the primer
must be such that they are able to prime the synthesis of extension
products. A typical primer contains at least about 7 nucleotides in
length of a sequence substantially complementary to the target
sequence, but somewhat longer primers are preferred. Usually
primers contain about 15-26 nucleotides, but longer or shorter
primers may also be employed.
[0315] "Polymorphic site" is a position within a locus at which at
least two alternative sequences are found in a population.
[0316] "Polymorphism" refers to the sequence variation observed in
an individual at a polymorphic site. Polymorphisms include
nucleotide substitutions, insertions, deletions and microsatellites
and may, but need not, result in detectable differences in gene
expression or protein function. In the absence of evidence of an
effect on expression or protein function, common polymorphisms,
including non-synonomous variants, are generally considered to be
included in the definition of wild-type gene sequence. A catalog of
human polymorphisms and associated annotation, including
validation, observed frequencies, and disease association, is
maintained by NCBI (dbSNP:
http://www.ncbi.nlm.nih.gov/projects/SNP/). Please note that the
term "polymorphism" when used in the context of gene sequences
should not be confused with the term "polymorphism" when used in
the context of solid state form of a compound, that is the
crystalline or amorphous nature of a compound. The skilled person
will understand the intended meaning by its context.
[0317] "Probe" refers to single stranded sequence-specific
oligonucleotides which have a sequence that is exactly
complementary to the target sequence of the allele to be
detected.
[0318] "Response" is defined by measurements taken according to
Response Evaluation Criteria in Solid Tumours (RECIST) involving
the classification of patients into two main groups: those that
show a partial response or stable disease and those that show signs
of progressive disease.
[0319] "Stringent hybridisation conditions" refers to an overnight
incubation at 42.degree. C. in a solution comprising 50% formamide,
5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran
sulphate, and 20 pg/mI denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0320] "Survival" encompasses a patients' overall survival and
progression-free survival.
[0321] "Overall survival" (OS) is defined as the time from the
initiation of drug administration to death from any cause.
"Progression-free survival" (PFS) is defined as the time from the
initiation of drug administration to first appearance of
progressive disease or death from any cause.
[0322] According to one aspect of the invention there is provided a
method for selecting a patient for treatment with a compound of
Formula (I), the method comprising providing a tumour cell
containing sample from a patient; determining whether the ER.alpha.
gene in the patient's tumour cell containing sample is wild type or
mutant; and selecting a patient for treatment with a compound of
Formula (I) based thereon.
[0323] The method may include or exclude the actual patient sample
isolation step. Thus, according to one aspect of the invention
there is provided a method for selecting a patient for treatment
with a compound of Formula (I), the method comprising determining
whether the ER.alpha. gene in a tumour cell containing sample
previously isolated from the patient is wild type or mutant; and
selecting a patient for treatment with a compound of Formula (I)
based thereon.
[0324] In one embodiment, the patient is selected for treatment
with a compound of Formula (I) if the tumour cell DNA has a mutant
ER.alpha. gene. In other embodiments, a patient whose tumour cell
DNA possesses a wild type ER.alpha. gene is selected for treatment
with a compound of Formula (I).
[0325] For the purpose of this invention, a gene status of
wild-type is meant to indicate normal or appropriate expression of
the gene and normal function of the encoded protein. In contrast,
mutant status is meant to indicate expression of a protein with
altered function, consistent with the known roles of mutant
ER.alpha. genes in cancer (as described herein). Any number of
genetic or epigenetic alterations, including but not limited to
mutation, amplification, deletion, genomic rearrangement, or
changes in methylation profile, may result in a mutant status.
However, if such alterations nevertheless result in appropriate
expression of the normal protein, or a functionally equivalent
variant, then the gene status is regarded as wild-type. Examples of
variants that typically would not result in a functional mutant
gene status include synonomous coding variants and common
polymorphisms (synonymous or non-synonymous). As discussed below,
gene status can be assessed by a functional assay, or it may be
inferred from the nature of detected deviations from a reference
sequence.
[0326] In certain embodiments the wild-type or mutant status of the
ER.alpha. gene is determined by the presence or absence of
non-synonymous nucleic acid variations in the genes. Observed
non-synonymous variations corresponding to known common
polymorphisms with no annotated functional effects do not
contribute to a gene status of mutant.
[0327] Other variations in the ER.alpha. gene that signify mutant
status include splice site variations that decrease recognition of
an intron/exon junction during processing of pre-mRNA to mRNA. This
can result in exon skipping or the inclusion of normally intronic
sequence in spliced mRNA (intron retention or utilization of
cryptic splice junctions). This can, in turn, result in the
production of aberrant protein with insertions and/or deletions
relative to the normal protein. Thus, in other embodiments, the
gene has a mutant status if there is a variant that alters splice
site recognition sequence at an intron/exon junction.
[0328] For ESR1, reference sequences are available for the gene
(GenBank accession number: NG_008493), mRNA (GenBank accession
number: NM_000125), and protein (GenBank accession number:
NP_000116 or Swiss-Prot accession: P03372). A person of skill in
the art will be able to determine the ESR1 gene status, i.e.
whether a particular ESR1 gene is wild type or mutant, based on
comparison of DNA or protein sequence with wild type.
[0329] It will be apparent that the gene and mRNA sequences
disclosed for ER.alpha. gene are representative sequences. In
normal individuals there are two copies of each gene, a maternal
and paternal copy, which will likely have some sequence
differences, moreover within a population there will exist numerous
allelic variants of the gene sequence. Other sequences regarded as
wild type include those that possess one or more synonymous changes
to the nucleic acid sequence (which changes do not alter the
encoded protein sequence), non-synonymous common polymorphisms
(e.g. germ-line polymorphisms) which alter the protein sequence but
do not affect protein function, and intronic non-splice-site
sequence changes.
[0330] There are numerous techniques available to the person
skilled in the art to determine the gene status of ER.alpha.. The
gene status can be determined by determination of the nucleic acid
sequence. This could be via direct sequencing of the full-length
gene or analysis of specific sites within the gene, e.g. commonly
mutated sites.
Samples
[0331] The patient's sample to be tested for the gene status can be
any tumour tissue or tumour-cell containing sample obtained or
obtainable from the individual. The test sample is conveniently a
sample of blood, mouth swab, biopsy, or other body fluid or tissue
obtained from an individual. Particular examples include:
circulating tumour cells, circulating DNA in the plasma or serum,
cells isolated from the ascites fluid of ovarian cancer patients,
lung sputum for patients with tumours within the lung, a fine
needle aspirate from a breast cancer patient, urine, peripheral
blood, a cell scraping, a hair follicle, a skin punch or a buccal
sample.
[0332] It will be appreciated that the test sample may equally be a
nucleic acid sequence corresponding to the sequence in the test
sample, that is to say that all or a part of the region in the
sample nucleic acid may firstly be amplified using any convenient
technique e.g. polymerase chain reaction (PCR), before analysis.
The nucleic acid may be genomic DNA or fractionated or whole cell
RNA. In particular embodiments the RNA is whole cell RNA and is
used directly as the template for labelling a first strand cDNA
using random primers or poly A primers. The nucleic acid or protein
in the test sample may be extracted from the sample according to
standard methodologies (see Green & Sambrook, Eds., Molecular
Cloning: A Laboratory Manual, (2012, 4th edition, Vol. 1-3, ISBN
9781936113422), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[0333] The diagnostic methods of the invention can be undertaken
using a sample previously taken from the individual or patient.
Such samples may be preserved by freezing or fixed and embedded in
formalin-paraffin or other media. Alternatively, a fresh tumour
cell containing sample may be obtained and used.
[0334] The methods of the invention can be applied using cells from
any tumour. Suitable tumours for treatment with a compound of
Formula (I) have been described hereinbefore.
Methods for Detection of Nucleic Acids
[0335] The detection of mutant ER.alpha. nucleic acids can be
employed, in the context of the present invention, to select drug
treatment. Since mutations in these genes occur at the DNA level,
the methods of the invention can be based on detection of mutations
or variances in genomic DNA, as well as transcripts and proteins
themselves. It can be desirable to confirm mutations in genomic DNA
by analysis of transcripts and/or polypeptides, in order to ensure
that the detected mutation is indeed expressed in the subject.
[0336] It will be apparent to the person skilled in the art that
there are a large number of analytical procedures which may be used
to detect the presence or absence of variant nucleotides at one or
more positions in a gene. In general, the detection of allelic
variation requires a mutation discrimination technique, optionally
an amplification reaction (such as one based on polymerase chain
reaction) and optionally a signal generation system. There are a
multitude of mutation detection techniques available in the art and
these may be used in combination with a signal generation system,
of which there are numerous available in the art. Many methods for
the detection of allelic variation are reviewed by Nollau et al.,
Clin. Chem., 1997, 43, 1114-1120; Anderson S M. Expert Rev Mol
Diagn., 2011, 11, 635-642; Meyerson M. et al., Nat Rev Genet.,
2010, 11, 685-696; and in standard textbooks, for example
"Laboratory Protocols for Mutation Detection", Ed. by U. Landegren,
Oxford University Press, 1996 and "PCR", 2.sup.nd Edition by Newton
& Graham, BIOS Scientific Publishers Limited, 1997.
[0337] As noted above, determining the presence or absence of a
particular variance or plurality of variances in the ER.alpha. gene
in a patient with cancer can be performed in a variety of ways.
Such tests are commonly performed using DNA or RNA collected from
biological samples, e.g., tissue biopsies, urine, stool, sputum,
blood, cells, tissue scrapings, breast aspirates or other cellular
materials, and can be performed by a variety of methods including,
but not limited to, PCR, hybridization with allele-specific probes,
enzymatic mutation detection, chemical cleavage of mismatches, mass
spectrometry or DNA sequencing, including minisequencing.
[0338] Suitable mutation detection techniques include amplification
refractory mutation system (ARMS.TM.), amplification refractory
mutation system linear extension (ALEX.TM.) competitive
oligonucleotide priming system (COPS), Taqman, Molecular Beacons,
restriction fragment length polymorphism (RFLP), and restriction
site based PCR and fluorescence resonance energy transfer (FRET)
techniques.
[0339] In particular embodiments the method employed for
determining the nucleotide(s) within a biomarker gene is selected
from: allele-specific amplification (allele specific PCR)--such as
amplification refractory mutation system (ARMS), sequencing,
allelic discrimination assay, hybridisation, restriction fragment
length polymorphism (RFLP) or oligonucleotide ligation assay
(OLA).
[0340] In particular embodiments, hybridization with allele
specific probes can be conducted by: (1) allele specific
oligonucleotides bound to a solid phase (e.g. glass, silicon, nylon
membranes) with the labelled sample in solution, for example as in
many DNA chip applications; or, (2) bound sample (often cloned DNA
or PCR amplified DNA) and labelled oligonucleotides in solution
(either allele specific or short so as to allow sequencing by
hybridization). Diagnostic tests may involve a panel of variances,
often on a solid support, which enables the simultaneous
determination of more than one variance. Such hybridization probes
are well known in the art (see, e.g., Green & Sambrook, Eds.,
Molecular Cloning: A Laboratory Manual, (2012, 4th edition, Vol.
1-3, ISBN 9781936113422), Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.) and may span two or more variance sites.
[0341] Thus, in one embodiment, the detection of the presence or
absence of at least one mutation provides for contacting ER.alpha.
nucleic acid containing a putative mutation site with at least one
nucleic acid probe. The probe preferentially hybridizes with a
nucleic acid sequence including a variance site and containing
complementary nucleotide bases at the variance site under selective
hybridization conditions. Hybridization can be detected with a
detectable label using labels known to one skilled in the art. Such
labels include, but are not limited to radioactive, fluorescent,
dye, and enzymatic labels.
[0342] In another embodiment, the detection of the presence or
absence of at least one mutation provides for contacting ER.alpha.
nucleic acid containing a putative mutation site with at least one
nucleic acid primer. The primer preferentially hybridizes with a
nucleic acid sequence including a variance site and containing
complementary nucleotide bases at the variance site under selective
hybridization conditions.
[0343] Oligonucleotides used as primers for specific amplification
may carry the complementary nucleotide base to the mutation of
interest in the centre of the molecule (so that amplification
depends on differential hybridization; see, e.g., Gibbs, et al.,
1989. Nucl. Acids Res., 17, 2437-248) or at the extreme 3'-terminus
of one primer where, under appropriate conditions, mismatch can
prevent, or reduce polymerase extension (see, e.g., Prossner, 1993,
Tibtech, 11 238).
[0344] In yet another embodiment, the detection of the presence or
absence of at least one mutation comprises sequencing at least one
nucleic acid sequence and comparing the obtained sequence with the
known wild type nucleic acid sequence.
[0345] Alternatively, the presence or absence of at least one
mutation comprises mass spectrometric determination of at least one
nucleic acid sequence.
[0346] In one embodiment, the detection of the presence or absence
of at least one nucleic acid variance comprises performing a
polymerase chain reaction (PCR). The target nucleic acid sequence
containing the hypothetical variance is amplified and the
nucleotide sequence of the amplified nucleic acid is determined.
Determining the nucleotide sequence of the amplified nucleic acid
comprises sequencing at least one nucleic acid segment.
Alternatively, amplification products can be analyzed using any
method capable of separating the amplification products according
to their size, including automated and manual gel electrophoresis,
and the like.
[0347] Mutations in genomic nucleic acid are advantageously
detected by techniques based on mobility shift in amplified nucleic
acid fragments. For instance, Chen et al., Anal Biochem 1996, 239,
61-9, describe the detection of single-base mutations by a
competitive mobility shift assay. Moreover, assays based on the
technique of Marcelino et al., BioTechniques 1999, 26, 1134-1148
are available commercially.
[0348] In a particular example, capillary heteroduplex analysis may
be used to detect the presence of mutations based on mobility shift
of duplex nucleic acids in capillary systems as a result of the
presence of mismatches.
[0349] Generation of nucleic acids for analysis from samples
generally requires nucleic acid amplification. Many amplification
methods rely on an enzymatic chain reaction (such as a polymerase
chain reaction, a ligase chain reaction, or a self-sustained
sequence replication) or from the replication of all or part of the
vector into which it has been cloned. Preferably, the amplification
according to the invention is an exponential amplification, as
exhibited by for example the polymerase chain reaction.
[0350] Many target and signal amplification methods have been
described in the literature, for example, general reviews of these
methods in Landegren, U., et al., Science, 1988 242, 229-237 and
Lewis, R., Genetic Engineering News 1990, 10, 54-55. These
amplification methods can be used in the methods of our invention,
and include polymerase chain reaction (PCR), PCR in situ, ligase
amplification reaction (LAR), ligase hybridisation, Q.beta.
bacteriophage replicase, transcription-based amplification system
(TAS), genomic amplification with transcript sequencing (GAWTS),
nucleic acid sequence-based amplification (NASBA) and in situ
hybridisation. Primers suitable for use in various amplification
techniques can be prepared according to methods known in the
art.
[0351] Polymerase Chain Reaction (PCR) PCR is a nucleic acid
amplification method described inter alia in U.S. Pat. Nos.
4,683,195 and 4,683,202. PCR consists of repeated cycles of DNA
polymerase generated primer extension reactions. The target DNA is
heat denatured and two oligonucleotides, which bracket the target
sequence on opposite strands of the DNA to be amplified, are
hybridised. These oligonucleotides become primers for use with DNA
polymerase. The DNA is copied by primer extension to make a second
copy of both strands. By repeating the cycle of heat denaturation,
primer hybridisation and extension, the target DNA can be amplified
a million fold or more in about two to four hours. PCR is a
molecular biology tool, which must be used in conjunction with a
detection technique to determine the results of amplification. An
advantage of PCR is that it increases sensitivity by amplifying the
amount of target DNA by 1 million to 1 billion fold in
approximately 4 hours. PCR can be used to amplify any known nucleic
acid in a diagnostic context (Mok et al., Gynaecologic Oncology,
1994, 52: 247-252,).
[0352] An allele specific amplification technique such as
Amplification Refractory Mutation System (ARMS.TM.) (Newton et al.,
Nucleic Acids Res., 1989, 17, 2503-2516) can also be used to detect
single base mutations. Under the appropriate PCR amplification
conditions a single base mismatch located at the 3'-end of the
primer is sufficient for preferential amplification of the
perfectly matched allele (Newton et al., 1989, supra), allowing the
discrimination of closely related species. The basis of an
amplification system using the primers described above is that
oligonucleotides with a mismatched 3'-residue will not function as
primers in the PCR under appropriate conditions. This amplification
system allows genotyping solely by inspection of reaction mixtures
after agarose gel electrophoresis.
[0353] Analysis of amplification products can be performed using
any method capable of separating the amplification products
according to their size, including automated and manual gel
electrophoresis, mass spectrometry, and the like.
[0354] The methods of nucleic acid isolation, amplification and
analysis are routine for one skilled in the art and examples of
protocols can be found, for example, Green & Sambrook, Eds.,
Molecular Cloning: A Laboratory Manual, (2012, 4th edition, Vol.
1-3, ISBN 9781936113422), Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.) Particularly useful protocol source for
methods used in PCR amplification is PCR (Basics: From Background
to Bench) by M. J. McPherson, S. G. Mailer, R. Beynon, C. Howe,
Springer Verlag; 1st edition (Oct. 15, 2000), ISBN: 0387916008.
[0355] The present invention also provides predictive and
diagnostic kits comprising degenerate primers to amplify a target
nucleic acid in the ER.alpha. gene and instructions comprising;
amplification protocol and analysis of the results. The kit may
alternatively also comprise buffers, enzymes, and containers for
performing the amplification and analysis of the amplification
products. The kit may also be a component of a screening, or
diagnostic kit comprising other tools such as DNA microarrays, or
other supports. Preferably, the kit also provides one or more
control templates, such as nucleic acids isolated from normal
tissue sample, and/or a series of samples representing different
variances in the reference genes.
[0356] In one embodiment, the kit provides two or more primer
pairs, each pair capable of amplifying a different region of the
reference (ER.alpha.) gene (each region a site of potential
variance) thereby providing a kit for analysis of expression of
several gene variances in a biological sample in one reaction or
several parallel reactions.
[0357] Primers in the kits may be labelled, for example
fluorescently labelled, to facilitate detection of the
amplification products and consequent analysis of the nucleic acid
variances. The kit may also allow for more than one variance to be
detected in one analysis. A combination kit will therefore comprise
of primers capable of amplifying different segments of the
reference gene. The primers may be differentially labelled, for
example using different fluorescent labels, so as to differentiate
between the variances.
[0358] In another aspect, the invention provides a method of
treating a patient suffering from cancer comprising: determining
the mutant or wild type status of the ER.alpha. gene in the
patient's tumour cells and if the ER.alpha. gene is mutant,
administering to the patient an effective amount of a compound of
Formula (I).
[0359] As used herein, the terms "effective" and "effectiveness"
includes both pharmacological effectiveness and physiological
safety. Pharmacological effectiveness refers to the ability of the
treatment to result in a desired biological effect in the patient.
Physiological safety refers to the level of toxicity, or other
adverse physiological effects at the cellular, organ and/or
organism level (often referred to as side-effects) resulting from
administration of the treatment. "Less effective" means that the
treatment results in a therapeutically significant lower level of
pharmacological effectiveness and/or a therapeutically greater
level of adverse physiological effects.
[0360] According to another aspect of the invention there is
provided the use of a compound of Formula (I) or a
pharmaceutically-acceptable salt thereof to treat a cancer patient
whose tumour cells have been identified as possessing a mutant
ER.alpha. gene. In one embodiment the compound of Formula (I) is
Example 1.
[0361] According to another aspect of the invention there is
provided a compound of Formula (I) or a pharmaceutically-acceptable
salt thereof for treating cancers with tumour cells identified as
harbouring mutant ER.alpha. gene. In one embodiment the compound of
Formula (I) is Example 1.
[0362] According to another aspect of the invention there is
provided a method of treating cancers with tumour cells identified
as harbouring mutant ER.alpha. gene comprising administering an
effective amount of a compound of Formula (I) or a
pharmaceutically-acceptable salt thereof. In one embodiment the
compound of Formula (I) is Example 1.
[0363] In still further embodiments, the invention relates to
pharmaceutical composition comprising a compound of Formula (I) for
use in the prevention and treatment of cancer with tumour cells
identified as harbouring a mutant ER.alpha. gene. In one embodiment
the compound of Formula (I) is Example 1.
[0364] For all the aspects above, mutant forms of ER.alpha.
determined/identified are at all positions across the gene.
[0365] For all the aspects above, using tumours such as breast
cancer as an example, particular mutant forms of ER.alpha.
determined/identified are those at positions Ser463Pro, Val543Glu,
Leu536Arg, Tyr537Ser, Tyr537Asn and Asp538Gly.
BRIEF DESCRIPTION OF THE FIGURES
[0366] FIG. 1 shows an X-Ray Powder Diffraction Pattern of Example
7 Form A
[0367] FIG. 2 shows an X-Ray Powder Diffraction Pattern of Example
1 Form B
[0368] FIG. 3 shows DSC Thermogram of Example 1 Form B
[0369] FIG. 4 shows X-Ray Powder Diffraction Pattern of Example 1
Form A
[0370] FIG. 5 shows TGA Thermogram of Example 1 Form A
[0371] FIG. 6 shows X-Ray Powder Diffraction Pattern of Example 1
Form C
[0372] FIG. 7 shows TGA Thermogram of Example 1 Form C
[0373] FIG. 8 shows an X-Ray Powder Diffraction Pattern of Example
11
[0374] FIG. 9 shows a DSC trace of Example 11.
[0375] FIG. 10 shows the results of an MCF-7 xenograft study with
Example 1 and AZD2014.
[0376] FIGS. 11 and 12 show the results of an HCC1428 long term
estrogen deprived (LTED) xenograft efficacy study with Example
1.
EXAMPLES
[0377] The invention will now be illustrated in the following
Examples in which, generally: [0378] (i) operations were carried
out at ambient temperature, i.e. in the range 17 to 25.degree. C.
and under an atmosphere of an inert gas such as nitrogen unless
otherwise stated; [0379] (ii) evaporations were carried out by
rotary evaporation or utilising Genevac equipment or Biotage v10
evaporator in vacuo and work-up procedures were carried out after
removal of residual solids by filtration; [0380] (iii) flash
chromatography purifications were performed on an automated
Teledyne Isco CombiFlash.RTM. Rf or Teledyne Isco CombiFlash.RTM.
Companion.RTM. using prepacked RediSep Rf Gold.TM. Silica Columns
(20-40 .mu.m, spherical particles), GraceResolv.TM. Cartridges
(Davisil.RTM. silica) or Silicycle cartridges (40-63 .mu.m). [0381]
(iv) preparative chromatography was performed on a Gilson prep HPLC
instrument with UV collection; [0382] (v) chiral preparative
chromatography was performed on a Gilson instrument with UV
collection (233 injector/fraction collector, 333 & 334 pumps,
155 UV detector) or a Varian Prep Star instrument (2.times.SD1
pumps, 325 UV detector, 701 [0383] fraction collector) pump running
with Gilson 305 injection; [0384] (vi) yields, where present, are
not necessarily the maximum attainable; [0385] (vii) in general,
the structures of end-products of the Formula I were confirmed by
nuclear magnetic resonance (NMR) spectroscopy; NMR chemical shift
values were measured on the delta scale [proton magnetic resonance
spectra were determined using a Bruker Avance 500 (500 MHz) or
Bruker Avance 400 (400 MHz) instrument]; measurements were taken at
ambient temperature unless otherwise specified; the following
abbreviations have been used: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of
doublet of doublet; dt, doublet of triplets; bs, broad signal
[0386] (viii) in general, end-products of the Formula I were also
characterised by mass spectroscopy following liquid chromatography
(LCMS or UPLC); UPLC was carried out using a Waters UPLC fitted
with Waters SQ mass spectrometer (Column temp 40, UV=220-300 nm,
Mass Spec=ESI with positive/negative switching) at a flow rate of 1
ml/min using a solvent system of 97% A+3% B to 3% A to 97% B over
1.50 mins (total runtime with equilibration back to starting
conditions etc 1.70 min), where A=0.1% formic acid in water (for
acid work) or 0.1% ammonia in water (for base work) B=acetonitrile.
For acid analysis the column used was Waters Acquity HSS T3 1.8
.mu.m 2.1.times.50 mm, for base analysis the column used was Waters
Acquity BEH 1.7 .mu.m 2.1.times.50 mm; LCMS was carried out using a
Waters Alliance HT (2795) fitted with a Waters ZQ ESCi mass
spectrometer and a Phenomenex Gemini--NX (50.times.2.1 mm 5 .mu.m)
column at a flow rate of 1.1 ml/min 95% A to 95% B over 4 min with
a 0.5 min hold. The modifier is kept at a constant 5% C (50:50
acetonitrile:water 0.1% formic acid) or D (50:50 acetonitrile:water
0.1% ammonium hydroxide (0.88 SG) depending on whether it is an
acidic or basic method. [0387] (ix) ion exchange purification was
generally preformed using a SCX-2 (Biotage, Propylsulfonic acid
functionalized silica. Manufactured using a trifunctional silane.
Non end-capped) cartridge. [0388] (x) intermediates purity was
assessed by thin layer chromatographic, mass spectral, HPLC (high
performance liquid chromatography) and/or NMR analysis; [0389] (xi)
For XRPD analysis of Example 7, samples were mounted on zero
background silicon wafers and analysed using the PANalytical CubiX
Pro diffractometer (1=1.5418 .ANG.). Samples were spun to improve
counting statistics. Data was collected in reflection geometry in
theta -2theta configuration over the scan range 2.degree. to
40.degree. 2-theta with 25 second exposure per 0.025067.degree.
increment. X-rays were generated by a copper long-fine focus tube
operated at 45 kV and 40 mA. Persons skilled in the art of X-ray
powder diffraction will realise that the relative intensity of
peaks can be affected by, for example, grains above 30 microns in
size and non-unitary aspect ratios that may affect analysis of
samples. The skilled person will also realise that the position of
reflections can be affected by the precise height at which the
sample sits in the diffractometer and the zero calibration of the
diffractometer. The surface planarity of the sample may also have a
small effect. Hence the diffraction pattern data presented are not
to be taken as absolute values; [0390] (xii) For XRPD analysis of
Example 1, The X-ray powder diffractogram was determined by
mounting a sample of the crystalline material on a Panalytical
single silicon crystal (SSC) wafer mount and spreading out the
sample into a thin layer with the aid of a microscope slide. The
sample was spun at 30 revolutions per minute (to improve counting
statistics) and irradiated with X-rays generated by a copper
long-fine focus tube operated at 45 kV and 40 mA with a wavelength
of 1.5418 angstroms. The X-ray beam was passed through a 0.04 rad
soller slit, then an automatic variable divergence slit set at 20
mm and finally a 20 mm beam mask. The reflected radiation was
directed through a 20 mm antiscatter slit and a 0.04 rad soller
slit. The sample was exposed for 1.905 seconds per
0.0025067.degree. 2-theta increment (continuous scan mode) over the
range 2 degrees to 40 degrees 2-theta in theta-theta mode. The
running time was 3 minutes and 36 seconds. The instrument was
equipped X-Celerator detector. Control and data capture was by
means of a Dell Pentium 4HT Workstation operating with X'Pert
Industry software. [0391] (xiii) Differential Scanning calorimetry:
Analytical Instrument: TA Instruments Q1000 DSC. Typically less
than 5 mg of material contained in a standard aluminium pan fitted
with a lid was heated over the temperature range 25.degree. C. to
300.degree. C. at a constant heating rate of 10.degree. C. per
minute. A purge gas using nitrogen was used--flow rate 50 ml per
minute. [0392] (xiv) Thermogravimetric Analysis Analytical
Instrument: TA Instruments Q5000 TGA. Typically less than 10 mg of
material is placed on a 100 .mu.l platinum pan and heated over the
temperature range 30.degree. C. to 150.degree. C. at a constant
heating rate of 10.degree. C. per minute. [0393] (xv) the following
abbreviations have been used:-- [0394] aq. aqueous [0395]
CDCl.sub.3 deutero-chloroform [0396] Conc. concentrated [0397] DCM
dichloromethane [0398] DMA N,N-dimethylacetamide [0399] DMSO
dimethyl sulphoxide [0400] DSC differential scanning calorimetry,
[0401] EtOH ethanol [0402] EtOAc ethyl acetate [0403] IPA/iPrOH
isopropyl alcohol [0404] MeCN acetonitrile [0405] MTBE
methyltertbutyl ether [0406] rt/RT room temperature [0407] sat.
saturated [0408] sol. solution [0409] THF tetrahydrofuran [0410]
TFA trifluoroacetic acid [0411] TGA Thermogravimetric analysis
Example 1:
(E)-3-(3,5-Difluoro-44(1R,3R)-2-(2-fluoro-2-methylpropyl)-3-met-
hyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic
acid
##STR00019##
[0413] The following processes should be carried out under an
atmosphere of nitrogen in the absence of light as a light
degradation product
[(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydr-
o-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate] may be
formed.
[0414] (E)-Methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (350 g, 766.69
mmol) was charged to a 5 L fixed vessel. Isopropyl alcohol (2.80 L)
was added to the vessel. Sodium hydroxide (5M, 460 ml, 2.30 mol)
was added in one portion and the mixture was stirred at 21.degree.
C. for 16 hrs. The dark solution was screened through a filter to
remove particulates. The filtrate was returned to the reactor
vessel. The filter and filtrate collection vessel were washed with
isopropanol (700 ml) and the washings were added to the reactor
vessel. The reaction mixture was agitated and water (1.75 L) w
added. Concentrated hydrochloric acid (37% w/w, 165 ml, 1.92 mol)
was charged to the vessel. Further hydrochloric acid (21.5 ml) was
added to the vessel to adjust the pH between 4.0 and 4.5. The
solution was heated to 50.degree. C. Water (1.92 L) added to the
vessel over 1 hour maintaining the internal temperature between
50-53.degree. C. The jacket temperature was raised to 70.degree. C.
to maintain the reactor temperature in this range during the
addition. Within 10 minutes of completion of the water addition,
the mixture self seeded and started to crystallise. The mixture was
held at 50-52.degree. C. for 1.5 hours (jacket set temperature
58.degree. C.). The resulting yellow suspension was cooled to
5.degree. C. (jacket temperature) over 6 hours. The slurry was held
at 5.degree. C. (jacket temperature) for 11 hrs. The resultant
yellow solid was isolated by filtration. The cake was pasted with a
spatula to prevent cracking of the cake. The vessel was washed with
water (1.05 L). The washings were used to wash the cake. The cake
was pulled dry in air then dried in vacuo to constant weight over 4
days (oven temperature=30.degree. C.).
(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid was
thus isolated as a yellow crystalline solid, "Form B" (319.45 g,
94%). .sup.1H NMR (500 MHz, DMSO, 27.degree. C.) 1.05 (3H, d),
1.08-1.28 (6H, m), 2.35 (1H, dd), 2.58 (1H, dd), 2.8-2.97 (2H, m),
3.47-3.57 (1H, m), 5.22 (1H, s), 6.67 (1H, d), 6.91-7.06 (2H, m),
7.19 (1H, d), 7.41 (1H, d), 7.46 (2H, d), 7.54 (1H, d), 10.58 (1H,
s), 12.62 (1H, s).
[0415] An alternative method for synthesising Example 1, which
results in formation of Form B crystalline material, is as
follows:
[0416] The following processes should be carried out under an
atmosphere of nitrogen in the absence of light as a light
degradation product (as described above) may be formed. (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (50.0 g;
109.53 mmol) was stirred in isopropyl alcohol (450 ml). Sodium
hydroxide (68.34 g, 65.72 ml, 328.58 mmol) was added in one portion
and the mixture stirred at 20.degree. C. for 16 hrs. The reaction
mixture was diluted with water (250 ml), the pH adjusted to pH4
with conc. hydrochloric acid (27.28 ml, 317.63 mmol) and the
mixture heated to 50.degree. C. Further water (225 ml) was added
over 30 minutes, maintaining the temperature above 45.degree. C.
During the addition, the material began to crystallise. The mixture
was cooled from 50.degree. C. to 5.degree. C. over 5 hours then the
suspension was held at 0.degree. C. for a further 11 hours. The
yellow solid was isolated by filtration. The filter cake was washed
with water (100 ml), dried on the filter for a further 20 minutes
then dried in a vacuum oven for 16 hours to constant weight
(30.degree. C., air bleed) to give the title compound (46.52 g) as
a crystalline solid (Form B).
[0417] .sup.1H NMR (500 MHz, DMSO, 27.degree. C.) 1.02-1.09 (3H,
m), 1.17 (6H, dd), 2.37 (1H, dd), 2.59 (1H, dd), 2.8-2.98 (2H, m),
3.47-3.58 (1H, m), 5.24 (1H, s), 6.68 (1H, d), 6.9-7.06 (2H, m),
7.20 (1H, d), 7.38-7.51 (3H, m), 7.55 (1H, d), 10.59 (1H, s), 12.60
(1H, br). Crystalline form B may also be isolated from
ethanol/water mixtures and ethanol/MTBE mixtures.
[0418] In a further aspect of the invention there is provided
crystalline form B of Example 1, isolated from isopropanol/water
mixture.
[0419] In a further aspect of the invention, there is provided a
process for isolation of crystalline form B of Example 1 which
comprises hydrolysis of (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate in isopropyl
alcohol and base, followed by acidification and isolation of
crystalline product from aqueous isopropanol.
[0420] Example 1 Form B is characterised in providing at least one
of the following 20 values measured using CuK.alpha. radiation: 8.4
and 10.9. Example 1 Form B is characterised in providing an X-ray
powder diffraction pattern, substantially as shown in FIG. 2. Ten
X-Ray powder diffraction peaks are shown in Table A:
TABLE-US-00002 TABLE A Ten X-Ray Powder Diffraction peaks for
Example 1 Form B Angle 2- Theta (2.theta.) Intensity % 8.4 100 10.9
88.5 18.3 84.5 24.0 78.5 14.0 66.4 19.0 55.9 14.4 54.3 13.0 45 15.3
44.7 20.6 44.2
[0421] DSC analysis of Example 1 Form B shows it to be a high
melting solid with an endotherm showing onset of melting at
188.6.degree. C. (FIG. 3). Example 1 shows degradation through the
melt which may lead to variation in melting onset, thus the value
of 188.6.degree. C. should not be taken as absolute.
[0422] Two solvated forms of Example 1 have also been observed.
[0423] Form A is a methyl tertiary butyl ether mono-solvate. The
X-ray powder diffraction pattern is shown in FIG. 4. TGA shows an
associated weight loss of 14.7% w/w between 55-150.degree. C. (FIG.
5). A theoretical loss for a mono methyl tertiary butyl ether
solvate is calculated to be 16.6%.
[0424] Form C is an Acetone mono-solvate, the X-ray powder
diffraction pattern is shown in FIG. 6. TGA shows an associated
weight loss of 10.0% w/w 50-150.degree. C. %. (FIG. 7). The
theoretical loss for a mono-acetone solvate is calculated to be
11.0%.
[0425] The (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as
starting material was prepared as follows:--
Preparation of 2-fluoro-2-methylpropyl
trifluoromethanesulfonate
##STR00020##
[0427] Under an atmosphere of nitrogen,
2-fluoro-2-methyl-propan-1-ol (1000 g, 10.86 moles) was charged to
a 20 L vessel. DCM (8.5 L) was added to the vessel. The mixture was
agitated and cooled to 1.degree. C. 2,6-Lutidine (1395 g, 13.02
moles) was added to the mixture. A solution of
trifluoromethanesulfonic anhydride (3220 g; 11.41 moles) in DCM (1
L) was added over 1 hour maintaining the temperature of the
reaction mixture below 5.degree. C. (the jacket set temperature was
lowered to -20.degree. C. during the addition). The addition vessel
was and lines were washed with DCM (0.5 L) and the washings were
added to the vessel in one portion. The mixture was agitated at
0.degree. C. for 1 hour affording a red solution.
[0428] A solution of concentrated hydrochloric acid (1.23 L, 37%
w/w, 16.3 moles) was added to water (7 L). The dilute hydrochloric
acid solution was added to the red solution and the stirred mixture
was warmed to 25.degree. C. The layers were allowed to separate and
the upper aqueous layer was discarded. The organic layer was washed
with water (2.times.5 L). The organic solution was concentrated
under reduced pressure to afford a red oil. The red oil was
purified by distillation using a wiped film evaporator (4.5 mbar,
jacket temperature 50.degree. C., condenser temperature 4.degree.
C.) to afford 2-fluoro-2-methylpropyl trifluoromethanesulfonate
(1.69 Kg, 69%) as a pale red oil. .sup.1H NMR (500 MHz, DMSO-d6,
27.degree. C.) .delta. 1.40 (6H, d), 4.79 (2H, d).
Preparation of
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine
##STR00021##
[0430] (2R)-1-(1H-Indol-3-yl)propan-2-amine (3.81 kg, 21.21 moles)
was added to a 100 L glass lined jacketed vessel under an
atmosphere of nitrogen. 1,4-dioxane (23 L) was added, and the
agitator was switched on. Diisopropylethylamine (5.55 L; 31.82
moles) was added to the stirred suspension followed by
(2-fluoro-2-methyl-propyl)trifluoromethanesulfonate (5.55 kg, 23.77
moles). 1,4-Dioxane (4 L) was added to the vessel, and the mixture
was heated to 75.degree. C. Heating was continued for 24 hours
before cooling the mixture to 25.degree. C. Water (30.5 L) was
added to the vessel, followed by toluene (30.5 L). After 40 minutes
the agitator was switched off and the layers were allowed to
separate. The aqueous layer was removed and water (30.5 L) was
added to the organic solution. The mixture was agitated for 15
minutes before allowing the layers to separate. The aqueous layer
was removed from the vessel. The organic solution was concentrated
by vacuum distillation (jacket temperature 65.degree. C., 110 mbar
pressure) until approximately 27 L of distillate had been removed.
The remaining solution in the vessel was cooled to afford
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine
as a solution in toluene (33% w/w) (15.4 Kg, 97%). .sup.1H NMR (500
MHz, DMSO, 27.degree. C.) 0.98 (3H, d), 1.26 (3H, d), 1.30 (3H, d),
2.57-2.75 (3H, m), 2.81 (1H, dd), 2.84-2.92 (1H, m), 6.97 (1H, t),
7.06 (1H, t), 7.11-7.22 (1H, multiplet obscured by toluene
signals), 7.34 (1H, d), 7.52 (1H, d), 10.80 (1H, s).
Preparation of sodium
{2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}(hydroxy)metha-
nesulfonate
##STR00022##
[0432] 2,6-Difluoro-4-bromobenzaldehyde (1000 g, 4.39 mol) and
1,1-bis(di-tert-butylphosphino)ferrocene palladium dichloride (57.2
g; 87.76 mmol) were charged to a 20 L vessel. Tetra-n-butylammonium
chloride (122 g, 438.97 mmol) was added followed by
dimethylacetamide (5 L). The vessel was purged with a stream of
nitrogen gas. Diisopropylethylamine (1.5 L, 8.78 mol) was added to
the vessel followed by methyl acrylate (0.435 L, 4.82 mol). The
mixture was agitated and heated to 60.degree. C. The mixture was
held at this temperature for 20 hours. Ethyl acetate (10 L) was
added to the mixture and the heating was switched off. Water (5 L)
was added to the vessel. The stirred mixture was cooled to
25.degree. C. and stirring was continued for 10 minutes. Agitation
was stopped and the layers were allowed to separate. The aqueous
layer was removed and discarded. The organic layer was washed
sequentially with hydrochloric acid (2.2M, 6 L) and water (5 L).
Phosphonics SPM32 Scavenger (1050 g, 1050 mol) was added to the
vessel and the mixture was stirred for 3 days at 25.degree. C. The
solid material was removed by filtration. The cake was washed with
ethanol (5 L) and the combined filtrates were concentrated under
reduced pressure to afford a solid. The solid was dissolved in
ethanol (9 L) and the solution was agitated in a 20 L vessel. The
solution was heated to 50.degree. C. A solution of sodium bisulfite
(460 g, 4.42 mol) in water (2.5 L) was added over 30 minutes. A
thick slurry resulted which was stirred for 4 hours at 50.degree.
C. The slurry was cooled to 20.degree. C. over 2 hours. The solid
was isolated by filtration and the vessel and filter cake were
washed with MTBE (2.times.3 L). The resulting solid was dried in
vacuo to afford sodium
{2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}(hydroxy)metha-
nesulfonate (1035 g, 71%) as alight brown solid. .sup.1H NMR (500
MHz, DMSO, 27.degree. C.) .delta. 3.73 (3H, s), 5.32 (1H, d), 5.94
(1H, d), 6.76 (1H, d), 7.40 (2H, d), 7.60 (1H, d).
Preparation of (E)-methyl
3-(3,5-difluoro-4-formylphenyl)acrylate
##STR00023##
[0434] Under an atmosphere of nitrogen, sodium
{2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}(hydroxy)metha-
nesulfonate (1.211 kg, 3.52 mol) was to a 100 L vessel followed by
potassium carbonate (0.974 kg, 7.05 mol). Water (9.1 L) was added
and the agitator was started. Ethyl acetate (9.1 L) was added. The
mixture was agitated at 25.degree. C. for 5 hours. The agitator was
stopped and the mixture was allowed to stand for 14 hours at
25.degree. C. The lower aqueous phase was removed and discarded.
The upper organic phase was concentrated under reduced pressure to
afford a pale brown solid. The solid was dried in vacuo to afford
(E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate as a brown solid
(608 g, 76%). .sup.1H NMR (500 MHz, DMSO, 27.degree. C.) .delta.
3.77 (3H, s), 6.94 (1H, d), 7.66 (1H, d), 7.71 (2H, d), 10.20 (1H,
s).
Preparation of (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
##STR00024##
[0436] Under an atmosphere of nitrogen, (E)-Methyl
3-(3,5-difluoro-4-formylphenyl)acrylate (0.606 kg, 2.65 mol) and
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine
(33% w/w solution in toluene, 2.0 kg, 2.65 mol) were charged to a
20 L vessel followed by toluene (4.22 L). Acetic acid (304 ml, 5.31
mol) was added to the vessel. The mixture was agitated and heated
to 80.degree. C. The mixture was agitated at 80.degree. C.
overnight before being cooled to 20.degree. C. A solution of
potassium carbonate (0.916 kg, 6.63 mol) in water (3.3 L) was added
to the mixture. The mixture was stirred for 10 minutes before the
agitator was switched off and the layers were allowed to separate.
The aqueous layer was removed and discarded. Water (3.3 L) was
charged to the reactor. The mixture was agitated for 10 minutes
then allowed to stand for 10 minutes. The lower aqueous phase was
removed and the organic layer was allowed to stand at room
temperature overnight. The batch was heated to 80.degree. C.
Heptane (4.61 L) was added to the hot solution over 35 minutes. The
stirred mixture was held at approximately 80.degree. C. for 1 hour.
The mixture was cooled to 30.degree. C. over 2 hours during which
time the product crystallised. The slurry was stirred at 30.degree.
C. for 2.5 hours. The solid was isolated by filtration. The reactor
vessel walls were washed with heptane and the washings were used to
wash the filter cake. The solid was dried in vacuo to afford
(E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate as a pink
solid (0.763 kg, 61%). .sup.1H NMR (400 MHz, DMSO, 27.degree. C.)
.delta. 1.06 (3H, d), 1.13 (3H, d), 1.21 (3H, d), 2.35 (1H, dd),
2.58 (1H, dd), 2.8-2.98 (2H, m), 3.44-3.61 (1H, m), 3.74 (3H, s),
5.24 (1H, s), 6.80 (1H, d), 6.9-7.05 (2H, m), 7.19 (1H, d), 7.41
(1H, d), 7.50 (2H, d), 7.63 (1H, d), 10.58 (1H, s). m/z: ES+ [M+H]+
457.
Alternative Preparation of Example 1:
(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
##STR00025##
[0438] 7.5M Sodium hydroxide (32.9 ml, 247.10 mmol) was added to a
solution of (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (11.28 g,
24.71 mmol) in THF (143 ml) and methanol (71.4 ml). The reaction
was stirred at room temperature for 4 h. The pH of the aqueous was
adjusted to .about.6.5 by addition of 2N HCl solution, then the
solution was extracted with diethyl ether (3.times.150 ml). The
combined organics were dried over Na.sub.2SO.sub.4 and
concentrated. The crude product was purified by flash silica
chromatography, elution gradient 0 to 20% methanol in DCM which
afforded a yellow solid. Attempted trituration with acetone/heptane
failed due to higher than expected solubility. The solvents were
removed to give a yellow solid which was triturated in isohexane
(50 ml) with a few drops of diethyl ether, the resulting solid was
filtered off and dried to give crude product (11.14 g) as a yellow
powder. The solid was dissolved in ethanol (100 ml), under nitrogen
and in the dark. The solution was then evaporated to 5mBar using a
vacuum pump at 62.degree. C. in the dark. This procedure was
repeated twice and the resulting yellow glass scratched with a
spatula into a fine powder and subjected to 5mBar using a vacuum
pump at 62.degree. C. for 60 min, to afford a yellow powder. The
powder was then left in a vacuum oven over P.sub.2O.sub.5 at
62.degree. C. at 300 mBar overnight to afford the title product
(9.77 g, 89%) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO,
27.degree. C.) .delta. 1.07-1.16 (3H, m), 1.18-1.29 (6H, m), 2.39
(1H, dd), 2.62 (1H, dd), 2.92 (2H, dd), 3.56 (1H, d), 5.26 (1H, s),
6.70 (1H, d), 7.02 (2H, dd), 7.22 (1H, d), 7.47 (3H, dd), 7.58 (1H,
d), 10.60 (1H, s), 12.60 (1H, s). m/z: ES+ (ElectroSpray+) [M+H]+
443.
[0439] The (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as
starting material was prepared as follows:--
Preparation of 2-fluoro-2-methylpropan-1-ol
##STR00026##
[0441] Lithium aluminium hydride (3.37 g, 88.56 mmol) was added
portionwise over 15 min to a cooled solution of ethyl
2-fluoro-2-methylpropanoate (9.9 g, 73.80 mmol) in diethyl ether
(184 ml) at 0.degree. C. The reaction was stirred for 1 hr, then
water (3.3 ml), followed by 15% NaOH solution (3.3 ml) and water
(6.7 ml) were added sequentially. The suspension was stirred for 15
min, then filtered and the solids washed with diethyl ether. The
filtrate was evaporated to give 2-fluoro-2-methylpropan-1-ol (5.90
g, 87%) as a colourless oil.
[0442] .sup.1H NMR (400 MHz, CDCl.sub.3, 27.degree. C.) .delta.
1.37 (6H, d), 3.56 (2H, d), OH not observed.
Alternative Preparation of 2-fluoro-2-methylpropyl
trifluoromethanesulfonate
##STR00027##
[0444] Trifluoromethanesulfonic anhydride (12.06 ml, 71.24 mmol),
followed by 2,6-lutidine (11.42 ml, 81.42 mmol) were added to a
solution of 2-fluoro-2-methylpropan-1-ol (6.25 g, 67.85 mmol) in
DCM (146 ml) at -10.degree. C. The reaction was stirred for 1 hr,
then washed with 2N HCl (2.times.100 ml) and saturated NaHCO.sub.3
solution (2.times.100 ml). The organic phase was then dried over
Na.sub.2SO.sub.4 and concentrated to give 2-fluoro-2-methylpropyl
trifluoromethanesulfonate (12.89 g, 85%) as a red oil.
[0445] .sup.1H NMR (400 MHz, CDCl.sub.3, 27.degree. C.) .delta.
1.46 (6H, d), 4.41 (2H, d).
[0446] This intermediate may be purified by vacuum distillation.
Analysis by DSC showed the material had the potential to self heat.
For reasons of process safety a wiped film evaporator or similar
may be preferable to a batch distillation.
Alternative Preparation of
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine
##STR00028##
[0448] 2-Fluoro-2-methylpropyl trifluoromethanesulfonate (8.04 g,
35.87 mmol) was added to a solution of
(R)-1-(1H-indol-3-yl)propan-2-amine (5.00 g, 28.70 mmol) and
N-ethyl-N-isopropylpropan-2-amine (7.44 ml, 43.04 mmol) in dioxane
(50 ml). The reaction was heated to 90.degree. C. for 3 h. After
cooling to room temperature, the reaction was diluted with EtOAc
(200 ml) and washed with saturated. NaHCO.sub.3 solution
(2.times.100 ml). The aqueous phase was extracted with EtOAc (150
ml), then the combined organics were dried over MgSO.sub.4 and
concentrated. The crude product was purified by flash silica
chromatography, elution gradient 100% EtOAc. Pure fractions were
evaporated to dryness to afford
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine
(6.49 g, 91%) as a brown oil. .sup.1H NMR (400 MHz, CDCl.sub.3,
27.degree. C.) .delta. 1.14 (3H, d), 1.31 (3H, d), 1.37 (3H, d),
1.94 (1H, s), 2.63-2.87 (3H, m), 2.92 (1H, dd), 3.07 (1H, h), 7.07
(1H, d), 7.08-7.15 (1H, m), 7.16-7.24 (1H, m), 7.37 (1H, d), 7.62
(1H, d), 8.04 (1H, s). m/z: ES+ [M+H]+ 249
Alternative Preparation of (E)-methyl
3-(3,5-difluoro-4-formylphenyl)acrylate
##STR00029##
[0450] 4-Bromo-2,6-difluorobenzaldehyde (9.99 g, 45.20 mmol) and
methyl acrylate (6.14 ml, 67.81 mmol) were taken up in thoroughly
degassed DMA (100 ml) and tri-o-tolylphosphine (1.376 g, 4.52
mmol), palladium(II) acetate (0.507 g, 2.26 mmol) and triethylamine
(12.60 ml, 90.41 mmol) added. The reaction was stirred and heated
to 80.degree. C. for 6 hours. The reaction mixture was cooled and
filtered through a layer of celite, and washed with methanol (50
ml). The crude product was pre-absorbed onto silica and purified by
suction chromatography eluting with 0-10% diethyl
ether/dichloromethane. Fractions containing the desired product
were evaporated and triturated with diethyl ether (50 ml) to afford
a yellow solid which was triturated with water (50 ml) and dried
under high vacuum at 50.degree. C. to afford (E)-methyl
3-(3,5-difluoro-4-formylphenyl)acrylate (8.85 g, 87%) as a yellow
solid. .sup.1H NMR (400 MHz, DMSO, 27.degree. C.) .delta. 3.75 (3H,
s), 6.93 (1H, d), 7.52-7.81 (3H, m), 10.18 (1H, s). No mass ion
observed in LCMS.
Alternative Preparation of (E)-methyl
3-(3,5-difluoro-44(1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-te-
trahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
##STR00030##
[0452] (E)-Methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (6.58 g,
29.09 mmol) was added to a suspension of
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine
(6.02 g, 24.24 mmol) in toluene (51.1 ml) and acetic acid (2.78 ml,
48.48 mmol). The reaction was heated to 80.degree. C. for 5 hours.
The reaction mixture was purified by ion exchange chromatography,
using an SCX-2 column. The desired product was eluted from the
column using 7M NH.sub.3/methanol and pure fractions were
evaporated to dryness to afford a brown solid. The crude product
was purified by flash silica chromatography, elution gradient 0 to
30% EtOAc in heptane. Pure fractions were evaporated to dryness to
afford the title product (7.52 g, 68.0%) as a yellow solid. .sup.1H
NMR (400 MHz, DMSO, 100.degree. C.) .delta. 1.10 (3H, d), 1.12-1.31
(6H, m), 2.28-2.72 (2H, m), 2.84-3.09 (2H, m), 3.52-3.69 (1H, m),
3.76 (3H, s), 5.30 (1H, s), 6.64 (1H, d), 6.9-7.11 (2H, m), 7.21
(1H, d), 7.32 (2H, d), 7.42 (1H, d), 7.58 (1H, d), 10.14 (1H, s).
m/z: ES+ [M+H]+ 457
Example 2:
(E)-3-(4-((1R,3R)-2-(2-Fluoro-2-methylpropyl)-3-methyl-2,3,4,9--
tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
##STR00031##
[0454] 7.5M Sodium hydroxide solution (0.983 ml, 7.37 mmol) was
added to a solution of (E)-methyl
3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylate (310 mg, 0.74 mmol) in
methanol (5 ml). The mixture was stirred at 20.degree. C. for 2
hours. The reaction mixture was purified by ion exchange
chromatography, using an SCX-2 column. Fractions containing the
desired product were eluted from the column using 7M
NH.sub.3/methanol and pure fractions were evaporated to dryness to
afford a yellow solid. The crude product was purified by
preparative HPLC (Waters SunFire column, 5.mu. silica, 50 mm
diameter, 100 mm length), using decreasingly polar mixtures of
water (containing 0.1% formic acid) and MeCN as eluents. Fractions
containing the desired compound were evaporated to dryness and then
loaded onto SCX-2 column and eluted with 7N ammonia in methanol to
afford the title product (63.0 mg, 21.02%) as a yellow solid.
.sup.1H NMR (400 MHz, DMSO, 30.degree. C.) .delta. 1.06 (3H, d),
1.30 (3H, d), 1.47 (3H, d), 2.53-2.64 (2H, m), 2.79 (2H, s), 3.10
(1H, d), 5.08 (1H, s), 6.47 (1H, d), 6.98 (1H, t), 7.06 (1H, t),
7.19-7.37 (3H, m), 7.44 (1H, d), 7.56 (1H, d), 7.63 (2H, d), 10.81
(1H, s), 12.30 (1H, s). m/z: ES+ [M+H]+ 407.
[0455] The (E)-methyl
3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as starting material
was prepared as follows:--
Preparation of (E)-methyl 3-(4-formylphenyl)acrylate
##STR00032##
[0457] 4-Bromobenzaldehyde (30 g, 162.15 mmol) and methyl acrylate
(20.94 g, 243.22 mmol) were taken up in thoroughly degassed DMA
(300 ml) and treated with tri-o-tolylphosphine (4.94 g, 16.21
mmol), palladium(II) acetate (1.820 g, 8.11 mmol) and triethylamine
(45.2 ml, 324.29 mmol) and heated to 110.degree. C. for 16 hours.
The reaction appeared complete after this time. The reaction
mixture was poured into water (4 L) and the resulting precipitate
was filtered and dried. The solid was chromatographed on silica,
eluting with 100% heptane to 30% EtOAc in heptane. Relevant
fractions were combined and evaporated to dryness to afford a
yellow solid product which was triturated with heptane, filtered
and washed with cold heptane. The solid was dried to afford
(E)-methyl 3-(4-formylphenyl)acrylate (25.6 g, 83%) as a yellow
crystalline product. .sup.1H NMR (400 MHz, DMSO, 30.degree. C.)
.delta. 3.75 (3H, s), 6.79 (1H, d), 7.72 (1H, d), 7.93 (4H, s),
10.03 (1H, s). No mass ion observed in LCMS.
Preparation of (E)-methyl
3-(44(1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-p-
yrido[3,4-b]indol-1-yl)phenyl)acrylate
##STR00033##
[0459]
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-ami-
ne (obtained as described in Example 1, preparation of starting
materials) (450 mg, 1.81 mmol) and (E)-methyl
3-(4-formylphenyl)acrylate (345 mg, 1.81 mmol) were dissolved in
toluene (15 ml), acetic acid (5 ml) and molecular sieves were
added. The reaction was stirred at 110.degree. C. for 16 hours
under nitrogen then cooled to room temperature. The crude product
was purified by ion exchange chromatography, using an SCX-2 column.
The desired product was eluted from the column using 7M
NH.sub.3/methanol and pure fractions were evaporated to dryness to
afford crude product. The crude product was purified by flash
silica chromatography, elution gradient 0 to 30% EtOAc in heptane.
Pure fractions were evaporated to dryness to afford (E)-methyl
3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylate (317 mg, 41.6%) as a yellow
solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30.degree. C.) .delta.
1.09 (3H, d), 1.30 (3H, d), 1.47 (3H, d), 2.48-2.78 (4H, m), 3.30
(1H, m), 3.79 (3H, s), 5.09 (1H, s), 6.40 (1H, d), 7.12 (1H, td),
7.17 (1H, td), 7.29 (1H, d), 7.34 (2H, d), 7.43 (2H, d), 7.54 (1H,
d), 7.66 (2H, m). m/z: ES+ [M+H]+ 421.
Example 3:
(E)-3-(3,5-Difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)--
3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic
acid
##STR00034##
[0461] 2M Sodium hydroxide (3.0 ml, 6.00 mmol) was added to a
solution of (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (275 mg,
0.60 mmol) in THF (1.5 ml)/methanol (1.5 ml). The reaction was
stirred at room temperature for 3 h. EtOAc (15 ml) and water (15
ml) were added, then the pH of the aqueous was adjusted to .about.7
by addition of 2N HCl. The layers were separated and the aqueous
was extracted with EtOAc (15 ml). The combined organics were dried
over Na.sub.2SO.sub.4 and concentrated. The crude product was
purified by flash silica chromatography, elution gradient 0 to 10%
methanol in DCM. Pure fractions were evaporated to dryness to
afford the title product (250 mg, 94%) as a pale yellow solid.
.sup.1H NMR (400 MHz, DMSO, 30.degree. C.) .delta. 0.77 (3H, d),
1.06 (3H, d), 1.93 (1H, m), 2.18 (1H, dd), 2.58 (1H, dd), 2.65 (1H,
dd), 2.84 (1H, dd), 3.35 (1H, dd), 4.32 (1H, d), 4.44 (1H, d), 5.16
(1H, s), 6.67 (1H, d), 6.93-7.04 (2H, m), 7.21 (1H, d), 7.42 (1H,
d), 7.46 (2H, m), 7.54 (1H, d), 10.57 (1H, s), 12.51 (1H, s). m/z:
ES+ [M+H]+ 443.
[0462] The (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as
starting material was prepared as follows:--
Preparation of (S)-3-fluoro-2-methylpropan-1-ol
##STR00035##
[0464] N,N-Diethyl-1,1,2,3,3,3-hexafluoropropan-1-amine (18.25 ml,
100.57 mmol) was added dropwise to a solution of (R)-methyl
3-hydroxy-2-methylpropanoate (9.25 ml, 83.81 mmol) in DCM (77 ml)
(reaction warms to .about.40.degree. C.). The reaction was stirred
for 1 h at ambient temperature, then warmed to reflux for 4 h,
before cooling to room temperature overnight. The reaction mixture
was poured onto ice, and the layers separated. The aqueous was
extracted with DCM (2.times.150 ml), then the combined organics
were dried and carefully concentrated. The residue was dissolved in
THF (200 ml) and cooled in an ice-bath. Lithium aluminium hydride
(6.45 g, 167.61 mmol) was added in portions over 15 min. The
reaction was stirred at 0.degree. C. for 1 h and warmed to room
temperature for a further 1 h. After cooling in an ice-bath, the
reaction was quenched by addition of water (7 ml), followed by 15%
NaOH (7 ml), and finally water (21 ml). MgSO.sub.4 was added until
a granular solid was formed. The solid was filtered through celite
and the solids washed with diethyl ether (50 ml). The filtrate was
washed with 2N HCl (2.times.100 ml), then the organic phase was
dried over Na.sub.2SO.sub.4 and concentrated. The crude product was
purified by flash silica chromatography, elution gradient 0 to 10%
EtOAc in DCM. Pure fractions were evaporated to dryness to afford
(S)-3-fluoro-2-methylpropan-1-ol (6.42 g, 83%) as a straw coloured
oil. .sup.1H NMR (400 MHz, CDCl.sub.3, 30.degree. C.) .delta. 0.97
(3H, dd), 1.96-2.14 (1H, m), 3.64 (2H, d), 4.3-4.42 (1H, m),
4.42-4.54 (1H, m), OH not observed.
Preparation of (S)-3-fluoro-2-methylpropyl
trifluoromethanesulfonate
##STR00036##
[0466] To a stirred solution of (S)-3-fluoro-2-methylpropan-1-ol
(7.9 g, 85.77 mmol) in DCM (140 ml) at 0.degree. C. was added
trifluoromethanesulfonic anhydride (17.31 ml, 102.92 mmol) dropwise
followed by dropwise addition of 2,6-dimethylpyridine (11.95 ml,
102.92 mmol). The reaction mixture was stirred at 0.degree. C. for
45 min and room temperature for 30 min. The reaction mixture was
diluted with DCM (60 ml), and washed sequentially with 1M HCl
(3.times.100 ml), saturated sodium bicarbonate solution (100 ml)
and saturated brine (50 ml). The organic layer was dried over
Na.sub.2SO.sub.4, filtered and evaporated to near dryness. The
solution was filtered through a plug of silica and washed through
with DCM (50 ml) and evaporated to give (S)-3-fluoro-2-methylpropyl
trifluoromethanesulfonate (14.38 g, 74.8%) as a brown oil. .sup.1H
NMR (400 MHz, CDCl.sub.3, 27.degree. C.) .delta. 1.09 (3H, dd),
2.24-2.44 (1H, m), 4.30 (0.5H, dd), 4.37-4.46 (1H, m), 4.52 (2.5H,
tt).
Preparation of
(S)--N--((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amin-
e
##STR00037##
[0468] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (666
mg, 2.97 mmol) was added to a solution of
(R)-1-(1H-indol-3-yl)propan-2-amine (470 mg, 2.7 mmol) and
N-ethyl-N-isopropylpropan-2-amine (0.700 ml, 4.05 mmol) in
1,4-dioxane (6.05 ml). The reaction was stirred at room temperature
for 1 h. The reaction mixture was diluted with EtOAc (20 ml) and
washed with water (20 ml). The aqueous was extracted with EtOAc
(2.times.20 ml), then the combined organics were dried (MgSO.sub.4)
and concentrated. The crude product was purified by flash silica
chromatography, elution gradient 0 to 10% methanol in DCM. Pure
fractions were evaporated to dryness to afford
(S)--N--((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amin-
e (590 mg, 88%) as a brown oil. .sup.1H NMR (400 MHz, CDCl.sub.3,
30.degree. C.) .delta. 0.86 (3H, dd), 1.20 (3H, d), 1.94-2.11 (1H,
m), 2.64-2.74 (2H, m), 2.85-2.98 (2H, m), 3.05-3.15 (1H, m),
4.13-4.39 (2H, m), 7.09 (1H, d), 7.12 (2H, ddd), 7.20 (1H, ddd),
7.33-7.41 (1H, m), 7.56-7.65 (1H, m), 8.10 (1H, s). m/z: ES+ [M+H]+
249
Preparation of (E)-methyl
3-(3,5-difluoro-44(1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,-
9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
##STR00038##
[0470] Acetic acid (2.0 ml) was added to a solution of
(S)--N--((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amin-
e (273 mg, 1.10 mmol) and (E)-methyl
3-(3,5-difluoro-4-formylphenyl)acrylate (obtained as described in
Example 1, preparation of starting materials) (226 mg, 1 mmol) in
toluene (8.0 ml). The reaction was warmed to 95.degree. C. for 2.5
h. The volatiles were removed under vacuum, then the residue was
passed through an SCX-2 column. The column was then eluted with 7M
NH.sub.3/methanol to liberate the product. The filtrate was
concentrated and the crude product was purified by flash silica
chromatography, elution gradient 0 to 10% methanol in DCM. Pure
fractions were evaporated to dryness to afford (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (282 mg,
61.8%) as a pale yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3,
30.degree. C.) .delta. 0.82 (3H, d), 1.12 (3H, d), 1.81-1.99 (1H,
m), 2.24 (1H, ddd), 2.64 (1H, ddd), 2.71 (1H, dd), 2.93-3.01 (1H,
ddd), 3.42 (1H, dq), 3.81 (3H, s), 4.25-4.39 (1H, m), 4.38-4.53
(1H, m), 5.20 (1H, s), 6.39 (1H, d), 6.99 (2H, m), 7.06-7.16 (2H,
m), 7.21-7.25 (1H, m), 7.52 (3H, m). m/z: ES+ [M+H]+ 457.
Example 4:
(E)-3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,-
4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
##STR00039##
[0472] 7.5M Sodium hydroxide solution (0.904 ml, 6.78 mmol) was
added to a solution of (E)-methyl
3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (285 mg, 0.68 mmol) in
methanol (3 ml). The mixture was stirred at 20.degree. C. for 2
hours. The reaction mixture was purified by ion exchange
chromatography, using an SCX-2 column. Fractions containing the
desired product were eluted from the column using 7M
NH.sub.3/methanol and pure fractions were evaporated to dryness to
afford a yellow solid. The crude product was purified by
preparative LCMS (Phenomenex Gemini-NX axia Prep C18 OBD column,
5.mu. silica, 50 mm diameter, 100 mm length), using decreasingly
polar mixtures of water (containing 1% NH.sub.3) and MeCN as
eluents. Fractions containing the desired compound were evaporated
to dryness to afford the title product (80 mg, 29.0%) as a yellow
solid. .sup.1H NMR (500 MHz, DMSO, 33.degree. C.) .delta. 0.87 (3H,
d), 1.04 (3H, d), 1.91-2.27 (2H, m), 2.50 (1H, p), 2.57-2.75 (2H,
m), 3.13 (1H, s), 4.51 (2H, dd), 4.86 (1H, s), 6.47 (1H, d),
6.92-6.99 (1H, m), 7-7.09 (1H, m), 7.15-7.35 (3H, m), 7.35-7.5 (2H,
m), 7.57 (2H, d), 10.64 (1H, d), CO.sub.2H not observed. m/z: ES+
[M+H]+ 407.
[0473] The (E)-methyl
3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as starting
material was prepared as follows:--
Preparation of (E)-methyl
3-(44(1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pheny-
l)acrylate
##STR00040##
[0475] (E)-Methyl 3-(4-formylphenyl)acrylate (obtained as described
in Example 2, preparation of starting materials) (18.56 g, 97.57
mmol) was added to a stirred solution of
(R)-1-(1H-indol-3-yl)propan-2-amine (17 g, 97.57 mmol) in acetic
acid (250 ml) at 23.degree. C. under nitrogen. The resulting
solution was stirred at 80.degree. C. for 2 hours. The reaction
mixture was evaporated to dryness and redissolved in DCM (500 ml),
and washed sequentially with saturated NaHCO.sub.3 (300
ml.times.2), 2M NaOH (aq) (300 ml), water (300 ml), and saturated
brine (300 ml). The organic layer was dried over MgSO.sub.4,
filtered and evaporated to afford crude product. The crude product
was purified by flash silica chromatography, elution gradient 1 to
7% methanol in DCM. Pure fractions were evaporated to dryness to
afford (E)-methyl
3-(4-((1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phen-
yl)acrylate (25.1 g, 74.3%) as a beige foam. The product was mostly
cis isomer, containing about 12% trans isomer which was
inseparable. .sup.1H NMR (400 MHz, DMSO, 30.degree. C.) .delta.
1.25 (3H, d), 2.37-2.48 (1H, m), 2.74 (1H, d), 3.12 (1H, s), 3.73
(3H, s), 5.18 (1H, s), 6.64 (1H, d), 6.97 (2H, dd), 7.19 (1H, d),
7.36-7.46 (3H, m), 7.64-7.75 (3H, m), 10.19 (1H, s), no NH
observed. m/z: ES+ [M+H]+ 347.
Preparation of (E)-methyl
3-(44(1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1--
yl)phenyl)acrylate
##STR00041##
[0477] (E)-Methyl
3-(4-((1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phen-
yl)acrylate (35 g, 101.03 mmol), 3-bromoprop-1-ene (9.62 ml, 111.14
mmol) and N-ethyl-N-isopropylpropan-2-amine (19.36 ml, 111.14 mmol)
were suspended in acetonitrile (160 ml), nitrogen was bubbled
through for 5 min and then the mixture was sealed into a microwave
tube. The reaction was heated to 140.degree. C. for 3.5 hours in
the microwave reactor and cooled to room temperature.
[0478] The reaction mixture was evaporated to dryness and
redissolved in DCM (100 ml), and washed sequentially with 1M citric
acid (100 ml), water (100 ml), and saturated brine (100 ml). The
organic layer was dried over MgSO.sub.4, filtered and evaporated to
afford crude product. The crude product was purified by flash
silica chromatography, elution gradient 0 to 20% EtOAc in heptane.
Pure fractions were evaporated to dryness to afford a 50:50 mixture
of (E)-methyl
3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
-yl)phenyl)acrylate: (E)-methyl
3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
-yl)phenyl)acrylate (10.00 g, 25.6%) as a pale yellow solid. m/z:
ES+ [M+H]+ 387.
Preparation of (E)-methyl
3-(44(1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1--
yl)phenyl)acrylate
##STR00042##
[0480] Trifluoroacetic acid (5.59 ml, 75.29 mmol) was added to
(E)-methyl
3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
(9.7 g, 25.10 mmol) in DCM (100 ml) at 20.degree. C. under
nitrogen. The resulting solution was stirred at 20.degree. C. for 3
days. The reaction mixture was diluted cautiously with saturated
NaHCO.sub.3 solution (250 ml), and the DCM layer washed
sequentially with water (250 ml) and saturated brine (250 ml). The
organic layer was dried over MgSO.sub.4, filtered and evaporated to
afford crude product. The crude product was purified by flash
silica chromatography, elution gradient 10 to 20% EtOAc in heptane.
Fractions were evaporated to dryness to afford a 65:35 mixture of
(E)-methyl
3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
-yl)phenyl)acrylate: (E)-methyl
3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
-yl)phenyl)acrylate (7.99 g, 82%) as a pale yellow solid. m/z: ES+
[M+H]+ 387
Preparation of (E)-methyl
3-(44(1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pheny-
l)acrylate
##STR00043##
[0482] 7 Separate batches of 65:35 (E)-methyl
3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
-yl)phenyl)acrylate: (E)-methyl
3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
-yl)phenyl)acrylate (2.00 g, 5.17 mmol) were reacted as
follows.
[0483] (E)-methyl
3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
-yl)phenyl)acrylate (2.00 g, 5.17 mmol) and
chlorotris(triphenylphosphine)rhodium(I) (Wilkinson's catalyst)
(2.346 g, 2.54 mmol) were suspended in acetonitrile (12 ml) and
water (2.4 ml) and nitrogen was bubbled through for 5 min before
being sealed into a microwave tube. The reaction was heated to
100.degree. C. for 60 min in the microwave reactor and cooled to
room temperature. The reaction mixtures were combined and
evaporated to dryness and redissolved in DCM (200 ml) and saturated
NaHCO.sub.3 solution (200 ml) added. The organic layer was washed
sequentially with water (200 ml) and saturated brine (200 ml)
before being dried over MgSO.sub.4, filtered and evaporated to
afford crude product. The crude product was purified by flash
silica chromatography, elution gradient 0 to 5% methanol in DCM.
Pure fractions were evaporated to dryness to afford (E)-methyl
3-(4-((1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phen-
yl)acrylate (7.02 g, 55.9%) as a pale yellow solid. .sup.1H NMR
(400 MHz, DMSO, 30.degree. C.) .delta. 1.14 (3H, d), 2.27-2.4 (1H,
m), 2.81 (1H, dd), 2.94-3.05 (1H, m), 3.72 (3H, s), 5.19 (1H, s),
6.60 (1H, d), 6.94-7 (1H, m), 7.01-7.09 (1H, m), 7.26 (3H, d), 7.43
(1H, d), 7.59-7.68 (3H, m), 10.70 (1H, s), NH not observed. m/z:
ES+ [M+H]+ 347
Preparation of (E)-methyl
3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
##STR00044##
[0485] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate
(obtained as described in Example 3, preparation of starting
materials) (291 mg, 1.30 mmol) was added to a solution of
(E)-methyl
3-(4-((1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phen-
yl)acrylate (300 mg, 0.87 mmol) and N,N-diisopropylethylamine
(0.226 ml, 1.30 mmol) in 1,4-dioxane (5 ml). The mixture was
stirred at 90.degree. C. for 1 hour then the mixture was evaporated
to dryness and the residue was partitioned between DCM (30 ml) and
water (30 ml). The aqueous layer was extracted with DCM (30 ml) and
the extracts combined with the organic layer. The combined extracts
were filtered through a phase-separating paper and evaporated. The
residue was purified by flash silica chromatography, elution
solvent 15% EtOAc in heptane. Pure fractions were evaporated to
dryness to afford (E)-methyl
3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-
-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (314 mg, 86%) as an
off-white solid. .sup.1H NMR (400 MHz, DMSO, 30.degree. C.) .delta.
0.87 (3H, d), 1.06 (3H, d), 1.9-2.28 (2H, m), 2.55-2.8 (3H, m),
2.97-3.21 (1H, m), 3.72 (3H, s), 4.31-4.69 (2H, m), 4.88 (1H, s),
6.60 (1H, dd), 6.98 (1H, t), 7.04 (1H, t), 7.17-7.35 (3H, m), 7.44
(1H, d), 7.55-7.76 (3H, m), 10.65 (1H, s). m/z: ES+ [M+H]+ 421.
Example 5:
(E)-3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dime-
thyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic
acid (Isomer 1)*
##STR00045##
[0487] Sodium hydroxide (184 mg, 4.61 mmol) was added to (E)-methyl
3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1)
(217 mg, 0.46 mmol) in THF (1 ml)/methanol (1 ml). The resulting
solution was stirred at 20.degree. C. for 16 hours. The reaction
was diluted with water (10 ml) and the pH was adjusted to 7 by the
addition of 2N HCl. The solution was extracted with EtOAc
(2.times.20 ml). The combined organics were dried over
Na.sub.2SO.sub.4 and concentrated. The crude product was purified
by flash silica chromatography, elution gradient 0 to 50% EtOAc in
heptane. The pure fractions were evaporated to give a crude
material. The crude product was triturated using a diethyl
ether/isohexane mixture to give the title product (53.0 mg, 25.2%)
as a yellow solid. .sup.1H NMR (400 MHz, DMSO, 27.degree. C.)
.delta. 0.54 (3H, d), 1.06 (3H, s), 1.34 (3H, s), 2.14 (1H, dd),
2.66 (1H, d), 2.83 (1H, d), 3.03 (1H, dd), 4.21 (1H, t), 4.33 (1H,
t), 5.12 (1H, s), 6.73 (1H, d), 7.01 (2H, dtd), 7.14-7.28 (1H, m),
7.43 (1H, d), 7.54 (2H, s), 7.59 (1H, d), 10.50 (1H, s), 12.61 (1H,
s), CO.sub.2H not observed. m/z: ES+ [M+H]+ 457
[0488] Stereochemistry inferred to be (R) at the undefined centre
by analogy with other examples, ie compound inferred to be:
(E)-3-(3,5-difluoro-4-(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl--
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic
acid
[0489] The
3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
(isomer 1) used as starting material was prepared as follows:--
Preparation of (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-dif-
luorophenyl)acrylate
##STR00046##
[0491] 1-(1H-Indol-3-yl)-2-methylpropan-2-amine (807 mg, 4.29 mmol)
and (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (obtained as
described in Example 1, preparation of starting materials) (970 mg,
4.29 mmol) were combined in acetic acid (15 ml) and the mixture
heated to 80.degree. C. for 2 hours. The reaction mixture was
purified by ion exchange chromatography, using an SCX-2 column. The
desired product was eluted from the column using 2M NH.sub.3 in
methanol and product-containing fractions were evaporated to
dryness to afford (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-dif-
luorophenyl)acrylate (1610 mg, 95%) as a yellow foam. .sup.1H NMR
(500 MHz, DMSO, 20.degree. C.) .delta. 1.15 (3H, s), 1.27 (3H, s),
2.23 (1H, s), 2.61 (2H, s), 3.75 (3H, s), 5.46 (1H, s), 6.84 (1H,
d), 6.97 (2H, dtd), 7.18 (1H, d), 7.39 (1H, d), 7.58 (2H, s), 7.67
(1H, d), 10.60 (1H, s). m/z: ES- [M-H]- 395.
Preparation of (E)-methyl
3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)
##STR00047##
[0493] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate
(obtained as described in Example 3, preparation of starting
materials) (0.339 g, 1.51 mmol) was added to a solution of
(E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-dif-
luorophenyl)acrylate (0.3 g, 0.76 mmol) and
N-ethyl-N-isopropylpropan-2-amine (0.458 ml, 2.65 mmol) in
1,4-dioxane (2 ml). The stirring was continued for 24 hours then
the volatiles were removed under vacuum and the crude product was
purified by flash silica chromatography, elution gradient 0 to 25%
EtOAc in heptane. Pure fractions were evaporated to dryness to
afford (E)-methyl
3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (0.202 g, 36%)
as a white solid. The material was combined with another batch
(0.36 g) and purified by preparative HPLC (Chiralpak IA column, 20
.mu.m silica, 20 mm diameter, 250 mm length), Heptane:IPA 70:30 at
80 ml/min (4 injections). Fractions containing the desired
compounds were evaporated to yield (E)-methyl
3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1,
first eluted, 217 mg) and (E)-methyl
3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 2,
second eluted, 165 mg). Analysis was done on Chiralpak IA column, 5
.mu.m silica, 4.6 mm diameter, 50 mm length, Heptane:IPA 70:30 at 2
ml/min. .sup.1H NMR (400 MHz, DMSO, 30.degree. C.) .delta. 0.50
(3H, d), 1.02 (3H, s), 1.30 (3H, s), 2.11 (1H, dd), 2.62 (2H, d),
2.80 (1H, d), 2.99 (1H, dd), 3.74 (3H, s), 4.09-4.23 (1H, m), 4.29
(1H, d), 5.09 (1H, s), 6.81 (1H, d), 6.97 (2H, dt), 7.16 (1H, d),
7.39 (1H, d), 7.53 (2H, d), 7.64 (1H, d), 10.44 (1H, s). m/z: ES+
[M+H]+ 471.
Example 6:
(E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
(Isomer 1)*
##STR00048##
[0495] 2M Sodium hydroxide (1.6 ml, 3.20 mmol) was added to a
solution of (E)-methyl
3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (148
mg, 0.31 mmol) in THF (0.8 ml)/methanol (0.8 ml). The reaction was
stirred at room temperature for 3 h. EtOAc (15 ml) and water (15
ml) were added, and the pH of the aqueous was adjusted to .about.7
by addition of 2N HCl. The layers were separated, and the aqueous
was extracted with EtOAc (15 ml). The combined organics were dried
over Na.sub.2SO.sub.4 and concentrated. The crude product was
purified by flash silica chromatography, elution gradient 25 to
100% EtOAc in heptane. Pure fractions were evaporated to dryness to
afford the title product (isomer 1) (124 mg, 86%) as a pale yellow
solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30.degree. C.) .delta.
1.03 (3H, d), 1.08 (3H, s), 1.16 (3H, d), 1.35 (3H, s), 2.56 (1H,
dd), 2.66 (1H, d), 3.02 (1H, d), 3.17 (1H, dd), 5.24 (1H, s), 6.39
(1H, d), 7.00 (2H, d), 7.05-7.16 (2H, m), 7.20 (1H, dd), 7.28 (1H,
s), 7.49 (1H, dd), 7.61 (1H, d), CO.sub.2H not observed. m/z: ES+
[M+H]+ 457.
[0496] Stereochemistry inferred to be (R) at the undefined centre
by analogy with other examples.
[0497] The (E)-methyl
3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) used as
starting material was prepared as follows:--
Preparation of (E)-methyl
3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)
##STR00049##
[0499] 2-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained
as described in Example 1, preparation of starting materials) (679
mg, 3.03 mmol) was added to a solution of (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-dif-
luorophenyl)acrylate (obtained as described in Example 5,
preparation of starting materials) (600 mg, 1.51 mmol) and
N-ethyl-N-isopropylpropan-2-amine (0.915 ml, 5.30 mmol) in
1,4-dioxane (2.5 ml). The reaction was stirred at room temperature
for 1 h. The reaction was then heated to 105.degree. C. for 88
hours. The volatiles were removed under vacuum and the crude
product was purified by flash silica chromatography, elution
gradient 0 to 50% EtOAc in heptane. Pure fractions were evaporated
to dryness to afford (E)-methyl
3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (430 mg, 60.4%) as
a white solid. The racemic product was purified by preparative HPLC
(Chiralpak AD column, 20 .mu.m silica, 50 mm diameter, 250 mm
length), Heptane:Ethanol 90:10 90 ml/min. Fractions containing the
desired compounds were evaporated to dryness to afford (E)-methyl
3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1, first
eluted, 149 mg, 34.6%) and (E)-methyl
3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 2, second
eluted, 143 mg, 33.3%) as cream coloured solids. .sup.1H NMR (400
MHz, CDCl.sub.3, 30.degree. C.) .delta. 1.01 (3H, d), 1.08 (3H, s),
1.15 (3H, d), 1.34 (3H, s), 2.55 (1H, dd), 2.66 (1H, d), 2.98-3.06
(1H, m), 3.17 (1H, dd), 3.80 (3H, s), 5.23 (1H, s), 6.38 (1H, d),
6.97 (2H, d), 7.07-7.12 (2H, m), 7.17-7.22 (1H, m), 7.32 (1H, s),
7.46-7.5 (1H, m), 7.52 (1H, d). m/z ES- [M-H]- 469.
Example 7:
(E)-3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9--
tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Isomer
1)*
##STR00050##
[0501] 2M Sodium hydroxide solution (20.42 ml, 40.85 mmol) was
added to a solution of (E)-methyl
3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (3.55 g, 8.17
mmol) in methanol (100 ml), THF (100 ml) and water (75 ml). The
mixture was heated at 40.degree. C. for 16 hours. The mixture was
diluted with water (100 ml) and concentrated to a volume such that
the organic solvents had been removed. The resulting aqueous
solution was acidified to pH 6 with 2M HCl. The resulting aqueous
suspension was extracted with DCM (500 ml) (adding brine to help
separate the emulsion which formed), filtered through a
phase-separating paper, dried over MgSO.sub.4 then filtered through
celite and evaporated to afford .about.3.5 g of a pale yellow
solid. The crude product was treated with diethyl ether/DCM (1:1,
150 ml) and sonicated. The fine suspension which formed was passed
through a pad of silica (.about.100 g) and the silica was eluted
with diethyl ether (.about.2 L). Product-containing fractions were
combined, evaporated and dried under vacuum at 50.degree. C. to
afford the title product (2.305 g, 64.5%) as a beige solid. .sup.1H
NMR (400 MHz, DMSO, 30.degree. C.) .delta. 0.52 (3H, d), 1.03 (3H,
s), 1.05-1.16 (1H, m), 1.23 (3H, s), 2.21 (1H, dd), 2.65 (1H, d),
2.84 (1H, d), 2.89 (1H, dd), 4.19 (1H, ddd), 4.31 (1H, ddd), 4.66
(1H, s), 6.50 (1H, d), 6.93 (1H, ddd), 6.98 (1H, ddd), 7.18 (1H,
d), 7.38 (2H, d), 7.40 (1H, d), 7.58 (1H, d), 7.63 (2H, d), 10.18
(1H, s), 12.30 (1H, s). m/z: ES+ [M+H]+ 421.
[0502] Stereochemistry inferred to be (R) at the undefined centre
by analogy with other examples.
[0503] The product (9.0 g, 21.40 mmol) was slurried in acetonitrile
(150 ml) under nitrogen in the dark for 1 hour in a stoppered 250
ml round bottomed flask. The mixture was stirred over the weekend
at room temperature then filtered and washed with cold acetonitrile
(60 ml) to afford a white solid which was dried under high vacuum
at 40.degree. C. for 5 hours to yield crystalline form A of the
title product (7.81 g, 87%).
[0504] An XRPD trace of Crystalline form A includes the following
peaks and is shown in FIG. 1.
TABLE-US-00003 2-Theta .degree. % 4.48 100 10.76 42.2 9.88 21.4
6.13 20.8 13.41 18.9 14.01 18.2 14.31 14.7 18.46 13.2 7.92 12.2
4.76 9.3
[0505] The (E)-methyl
3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) used as starting
material was prepared as follows:--
Preparation of (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)a-
crylate (racemate)
##STR00051##
[0507] (E)-Methyl 3-(4-formylphenyl)acrylate (41.8 g, 219.62 mmol)
(obtained as described in Example 2, preparation of starting
materials) was added in one portion to
1-(1H-indol-3-yl)-2-methylpropan-2-amine (43.8 g, 219.62 mmol) in
acetic acid (314 ml) under nitrogen. The resulting solution was
stirred at 80.degree. C. for 5 hours. The reaction mixture was
concentrated in vacuo. Toluene (200 ml) was added and the residue
evaporated to dryness. The azeotrope treatment was repeated twice
more to give a brown solid. This was stirred in 1:1 EtOAc/heptane
(500 ml) for 30 min before filtering and washing with 1:1
EtOAc/heptane. The compound was air dried to give a white solid.
The crude material was suspended in 2-methyl tetrahydrofuran (750
ml), and saturated sodium bicarbonate solution was added over 10
min to the stirred mixture (effervescence), the mixture was stirred
until the material dissolved and the aqueous phase remained basic.
The phases were separated and the organic phase washed with brine,
dried over MgSO.sub.4, filtered and concentrated in vacuo to give a
pale yellow foam (.about.78 g). The material was dissolved in
diethyl ether (200 ml) and concentrated to dryness (repeated
twice). On the second addition a proper solid was obtained. This
was stirred in diethyl ether and evaporated to dryness to give
(E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)a-
crylate (racemate) (73.6 g, 89%). .sup.1H NMR (400 MHz, CDCl.sub.3,
30.degree. C.) .delta. 1.26 (3H, s), 1.35 (3H, s), 1.42 (1H, br s),
2.69-2.82 (2H, m), 3.80 (3H, s), 5.12 (1H, s), 6.41 (1H, d),
7.06-7.16 (2H, m), 7.21 (1H, dd), 7.37 (2H, d), 7.46-7.54 (4H, m),
7.67 (1H, d). m/z: ES+ [M+H]+ 361.
Preparation of (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)a-
crylate (Isomer 1)
##STR00052##
[0509] (E)-Methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)a-
crylate (racemate) (65 g) was purified in seven injections as
follows.
[0510] The racemic material was purified by preparative HPLC
(Chiralpak OD column, 20 .mu.m silica, 100 mm diameter, 250 mm
length), Heptane:IPA 50:50. Fractions containing the desired
compounds were evaporated to dryness to afford (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)a-
crylate (isomer 1, first eluted, 30.3 g, 93%) and (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)a-
crylate (isomer 2, second eluted, 28.2 g, 86%). .sup.1H NMR (400
MHz, CDCl.sub.3, 30.degree. C.) .delta. 1.27 (3H, s), 1.36 (3H, s),
2.69-2.82 (2H, m), 3.80 (3H, s), 5.14 (1H, s), 6.43 (1H, d), 7.12
(2H, pd), 7.2-7.24 (1H, m), 7.39 (3H, d), 7.51 (3H, d), 7.68 (1H,
d), NH not observed. m/z: ES+ [M+H]+ 361.
Preparation of (E)-methyl
3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)
##STR00053##
[0512] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate
(obtained as described in Example 3, preparation of starting
materials) (5.32 g, 21.36 mmol) was added to a solution of
(E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)a-
crylate (isomer 1) (3.5 g, 9.71 mmol) and
N-ethyl-N-isopropylpropan-2-amine (6.34 ml, 36.41 mmol) in
1,4-dioxane (17.5 ml). The mixture was stirred at 22.degree. C. for
3 days. The mixture was evaporated and the residue was partitioned
between DCM (150 ml) and water (150 ml). The aqueous layer was
extracted with DCM (50 ml) and the extracts combined with the
organic layer. The combined extracts were filtered through a
phase-separating paper and evaporated. The residue was purified by
flash silica chromatography, elution solvent 15% EtOAc in heptane.
Fractions containing significant amounts of product began to form
crystals; the tubes were agitated to encourage further
crystallisation. The crystals were collected by filtration and
washed with a small amount of 15% EtOAc in heptane to afford
(E)-methyl
3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (2.91 g, 69.0%)
as a white crystalline solid. Liquors from the crystallisation and
other product-containing fractions were combined and evaporated.
The residue was recrystallised from EtOAc/heptane to afford more
3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H--
pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) as a white
crystalline solid (635 mg, 15.1%). 1H NMR (400 MHz, CDCl.sub.3,
30.degree. C.) .delta. 0.53 (3H, d), 0.95-1.07 (1H, m), 1.09 (3H,
s), 1.32 (3H, s), 2.16 (1H, dd), 2.66 (1H, d), 2.94 (1H, d), 2.97
(1H, d), 3.80 (3H, s), 4.14 (1H, ddd), 4.31 (1H, ddd), 4.59 (1H,
s), 6.42 (1H, d), 7.05-7.11 (2H, m), 7.13 (1H, s), 7.17 (1H, dd),
7.35 (2H, d), 7.45 (2H, d), 7.50 (1H, dd), 7.67 (1H, d). m/z: ES+
[M+H]+ 435.
Example 8:
(E)-3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetr-
ahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Isomer
1)*
##STR00054##
[0514] 2M Sodium hydroxide solution (0.782 ml, 1.56 mmol) was added
to a solution of (E)-methyl
3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyri-
do[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (68 mg, 0.16 mmol)
in methanol (5 ml), THF (5.00 ml) and water (5.00 ml) and the
mixture stirred for 40 hours at 22.degree. C. The mixture was
concentrated to a volume such that all of the organic solvent had
been removed and was acidified to pH6 with 2M HCl. Concentrated
aqueous ammonia (2 drops) was added, followed by methanol (.about.2
ml), giving a pale yellow solution. The crude product was purified
by preparative HPLC (Waters XBridge Prep C18 OBD column, 5.mu.
silica, 50 mm diameter, 100 mm length), using decreasingly polar
mixtures of water (containing 1% NH.sub.3) and MeCN as eluents.
Fractions containing the desired compound were evaporated to
dryness to afford the title product (isomer 1) (50.0 mg, 76%) as a
yellow solid. .sup.1H NMR (500 MHz, DMSO, 30.degree. C.) .delta.
0.93 (3H, s), 1.19 (3H, d), 1.23 (3H, s), 1.51 (3H, d), 2.56-2.64
(2H, m), 2.75 (1H, d), 3.04 (1H, dd), 5.06 (1H, s), 6.40 (1H, d),
7.03 (1H, ddd), 7.09-7.15 (2H, m), 7.35 (1H, dd), 7.44-7.51 (5H,
m), 10.87 (1H, s), CO.sub.2H not observed. m/z: ES+ [M+H]+ 421.
[0515] Stereochemistry inferred to be (R) at the undefined centre
by analogy with other examples.
[0516] The (E)-methyl
3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyri-
do[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) used as starting
material was prepared as follows:--
Preparation of (E)-methyl
3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyri-
do[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)
##STR00055##
[0518] 2-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained
as described in Example 1, preparation of starting materials) (389
mg, 1.73 mmol) was added to a solution of (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)a-
crylate (racemate) (obtained as described in Example 7, preparation
of starting materials) (250 mg, 0.69 mmol) and
N,N-diisopropylethylamine (0.453 ml, 2.60 mmol) in 1,4-dioxane
(1.25 ml). The mixture was stirred at 95.degree. C. for 64 hours
and then partitioned between DCM (30 ml) and water (30 ml). The
aqueous layer was extracted with DCM (20 ml) and the extracts
combined with the organic layer. The combined extracts were
filtered through a phase-separating paper and evaporated. The
residue was purified by flash silica chromatography, elution
solvent 15% EtOAc in heptane. Pure fractions were evaporated to
dryness to afford (E)-methyl
3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyri-
do[3,4-b]indol-1-yl)phenyl)acrylate (racemate) (178 mg, 59.1%) as a
beige solid. The racemic product was purified by preparative HPLC
(Chiralpak AD column, 20 .mu.m silica, 50 mm diameter, 250 mm
length), Heptane:Ethanol: 80:20. Fractions containing the desired
compounds were evaporated to dryness to afford (E)-methyl
3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyri-
do[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1, first eluted, 70 mg)
and (E)-methyl
3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyri-
do[3,4-b]indol-1-yl)phenyl)acrylate (isomer 2, second eluted, 66
mg). .sup.1H NMR (400 MHz, CDCl.sub.3, 30.degree. C.) .delta. 1.00
(3H, s), 1.13 (3H, d), 1.20 (3H, s), 1.44 (3H, d), 2.57-2.78 (3H,
m), 2.93 (1H, dd), 3.80 (3H, s), 5.05 (1H, s), 6.41 (1H, d), 7.13
(1H, ddd), 7.18 (1H, ddd), 7.32 (1H, d), 7.43 (2H, d), 7.51 (2H,
d), 7.54 (1H, d), 7.64 (1H, s), 7.67 (1H, d). m/z: ES+ [M+H]+
435.
Example 9:
(E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
(Isomer 1)*
##STR00056##
[0520] 2M Sodium hydroxide (3.31 ml, 6.63 mmol) was added to a
solution of (E)-methyl
3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (600 mg, 1.33
mmol) (mixture of diastereoisomers) in methanol (5 ml), and THF (20
ml) and the mixture stirred at ambient temperature for 4 hours. The
mixture was concentrated to a volume such that all of the organic
solvent had been removed, diluted with water (50 ml), acidified
with dilute HCl to pH 6 and extracted with ethyl acetate
(2.times.50 ml). The extracts were combined and evaporated under
reduced pressure. The residue was purified by preparative HPLC
(Chiralpak IA column, 20 .mu.m silica, 20 mm diameter, 250 mm
length), Heptane:IPA 90:10, 0.2% acetic acid at 80 ml/min.
Fractions containing the desired compounds were combined and
analysed to yield
(E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (isomer 1,
first eluted, 130 mg, 22.36%) and
(E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-t-
etrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (isomer 2,
second eluted, 30.0 mg, 5.16%). .sup.1H NMR (500 MHz, DMSO,
30.degree. C.) .delta. 0.49 (3H, d), 1.03 (3H, s), 1.14-1.21 (1H,
m), 1.28 (3H, s), 2.17 (1H, dd), 2.59-2.71 (1H, m), 2.8-2.99 (2H,
m), 4.09-4.34 (2H, m), 4.99 (1H, s), 6.59 (1H, d), 6.86-7.07 (2H,
m), 7.1-7.19 (1H, m), 7.18-7.29 (1H, m), 7.36-7.49 (2H, m),
7.49-7.66 (2H, m), 10.31 (1H, s), CO.sub.2H not observed. m/z: ES+
[M+H]+ 439.
[0521] Stereochemistry inferred to be (R) at the undefined centre
by analogy with other examples.
[0522] The (E)-methyl
3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (mixture of
diastereomers) used as starting material was prepared as
follows:--
Preparation of (E)-methyl 3-(3-fluoro-4-formylphenyl)acrylate
##STR00057##
[0524] 4-Bromo-2-fluorobenzaldehyde (20.88 g, 102.87 mmol) and
methyl acrylate (13.98 ml, 154.30 mmol) were taken up in thoroughly
degassed DMA (150 ml) and tri-o-tolylphosphine (3.13 g, 10.29
mmol), palladium(II) acetate (1.155 g, 5.14 mmol) and triethylamine
(28.7 ml, 205.74 mmol) added. The reaction was stirred and heated
to 100.degree. C. for 16 hours. More tri-o-tolylphosphine (3.13 g,
10.29 mmol) and palladium(II) acetate (1.155 g, 5.14 mmol) were
added and the reaction mixture was heated to 110.degree. C. for a
further 2 hours. Water (1 L) was added and the reaction mixture
extracted with DCM (2.times.500 ml). Combined organics were dried
(MgSO.sub.4), filtered and evaporated to give a brown solid. The
crude product was purified by flash silica chromatography, elution
gradient 0 to 25% EtOAc in heptane. Pure fractions were evaporated
to dryness to afford (E)-methyl 3-(3-fluoro-4-formylphenyl)acrylate
(15.20 g, 71.0%) as a yellow solid. .sup.1H NMR (400 MHz,
CDCl.sub.3, 30.degree. C.) .delta. 3.83 (3H, s), 6.53 (1H, d), 7.31
(1H, dd), 7.41 (1H, d), 7.65 (1H, d), 7.79-8 (1H, m), 10.25-10.41
(1H, m). No mass ion observed in LCMS.
Preparation of (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluor-
ophenyl)acrylate (racemate)
##STR00058##
[0526] 1-(1H-Indol-3-yl)-2-methylpropan-2-amine (1 g, 5.31 mmol)
and (E)-methyl 3-(3-fluoro-4-formylphenyl)acrylate (1.106 g, 5.31
mmol) in acetic acid (15 ml) were stirred at 80.degree. C. for 2
hours under nitrogen. The crude product was purified by ion
exchange chromatography, using an SCX-2 column. The desired product
was eluted from the column using 7M NH.sub.3/methanol and pure
fractions were evaporated to dryness to afford (E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluor-
ophenyl)acrylate (racemate) (2.000 g, 99%). .sup.1H NMR (400 MHz,
DMSO, 30.degree. C.) .delta. 1.14 (3H, s), 1.27 (3H, s), 2.52-2.74
(2H, m), 3.74 (3H, s), 5.12 (1H, s), 6.69 (1H, d), 6.86-7.05 (2H,
m), 7.20 (1H, d), 7.25-7.33 (2H, m), 7.39 (1H, d), 7.73 (1H, d),
7.85 (1H, t), 10.29 (1H, s), NH not observed. m/z: ES+ [M+H]+
379.
Preparation of (E)-methyl
3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (mixture of
diastereomers)
##STR00059##
[0528] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate
(obtained as described in Example 3, preparation of starting
materials) (1.259 g, 5.62 mmol) was added to a solution of
(E)-methyl
3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluor-
ophenyl)acrylate (racemate) (850 mg, 2.25 mmol) and
N-ethyl-N-isopropylpropan-2-amine (1.467 ml, 8.42 mmol) in
1,4-dioxane (5 ml). The mixture was heated at 60.degree. C. for 4
hours, then stirred at ambient temperature for 12 hours. The
mixture was partitioned between ethyl acetate (25 ml) and water (25
ml). The aqueous layer was extracted with ethyl acetate (2.times.25
ml) and the extracts combined with the organic layer. The combined
extracts were evaporated under vacuum. The residue was purified by
flash silica chromatography, elution solvent 10% EtOAc in heptane.
Pure fractions were evaporated to dryness to afford (E)-methyl
3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetra-
hydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (mixture of
diastereoisomers) (600 mg, 59.0%). m/z: ES+ [M+H]+ 453.
Example 10:
(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-di-
hydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoic
acid
##STR00060##
[0530] Methyl
(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-di-
hydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate
(4.70 g, 10.27 mmol) was dissolved in iPrOH (42.8 ml) and 5M sodium
hydroxide solution (6.16 ml, 30.82 mmol) was added in one portion,
the reaction was then stirred at room temperature for 4 hours.
Water was added (100 ml) and the pH was brought to .about.5 by
addition of 2N HCl. The solution was extracted with EtOAc
(.times.2) and the combined organics were dried (MgSO.sub.4) and
concentrated in vacuo. The residue was passed through a silica
plug, eluting first with DCM, then up to 5% MeOH in DCM. Fractions
containing product were evaporated to a yellow solid (.about.4.2
g). The residue (4.2 g) was dissolved in EtOH (20 ml) and warmed to
35.degree. C. Water (30 ml) was added slowly over .about.40 mins.
The mixture was then stirred for another 30 minutes, then slowly
cooled to room temperature. Additional water (30 ml) was added, and
the reaction was then cooled to 0.degree. C. The mixture was
filtered and the solids were washed with water before being dried
under vacuum at 35.degree. C. overnight to afford
(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-
-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoic
acid (3.34 g, 73.3%) as a pale yellow solid. .sup.1H NMR (400 MHz,
CDCl.sub.3, 30.degree. C.) 1.12 (3H, d), 1.19 (3H, d), 1.26 (3H,
d), 2.43 (1H, dd), 2.63 (1H, dd), 2.87 (1H, dd), 3.07 (1H, dd),
3.65 (1H, q), 6.41 (1H, d), 7.02 (2H, d), 7.06-7.16 (2H, m),
7.19-7.25 (1H, m), 7.41 (1H, s), 7.48-7.57 (1H, m), 7.63 (1H, d),
CO.sub.2H not observed. m/z: ES+ [M+H]+ 444.
[0531] This compound could alternatively be named
(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-di-
hydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]acrylic
acid.
Preparation of
(4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol
##STR00061##
[0533] Lithium borodeuteride (0.497 g, 17.25 mmol) was added
portionwise to a solution of methyl 4-bromo-2,6-difluorobenzoate
(2.89 g, 11.5 mmol) in THF (46.0 ml). The reaction was heated to
50.degree. C. for 2 hours. After cooling, (30 ml) 2N HCl was
carefully added. The layers were separated, and the aqueous was
extracted with EtOAc (2.times.50 ml). The combined organics were
washed with brine, dried (MgSO.sub.4) and concentrated to afford
(4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol (2.250 g, 87%) as
a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30.degree. C.)
1.96 (1H, s), 7.07-7.13 (2H, m).
Preparation of 4-bromo-2,6-difluoro-1-deuterobenzaldehyde
##STR00062##
[0535] Dess-Martin reagent (4.98 g, 11.73 mmol) was added to
(4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol (2.20 g, 9.78
mmol) in DCM (39.1 ml) at room temperature. The reaction was
stirred for 1 hour, then was quenched by addition of (50 ml) sat.
NaHCO.sub.3 containing 10% sodium thiosulfate. The layers were
separated and the aqueous phase was extracted with DCM (2.times.50
ml). The organics were dried (MgSO.sub.4) and concentrated, then
the crude product was purified by flash silica chromatography,
elution gradient 0 to 25% EtOAc in heptane. Pure fractions were
evaporated to dryness to afford
4-bromo-2,6-difluoro-1-deuterobenzaldehyde (2.040 g, 94%) as a
white solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30.degree. C.)
7.18-7.25 (2H, m). No mass ion observed.
Preparation of methyl
(E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate
##STR00063##
[0537] 4-Bromo-2,6-difluoro-1-deuterobenzaldehyde (3.33 g, 15.0
mmol), triethylamine (4.18 ml, 30.00 mmol), palladium (II) acetate
(0.168 g, 0.75 mmol) and tritolylphosphine (0.457 g, 1.50 mmol)
were dissolved in DMF (36.6 ml), which was degassed. Methyl
acrylate (2.026 ml, 22.50 mmol) was then added and the reaction was
heated to 80.degree. C. for 4 hours. After cooling, the mixture was
added to water (150 ml) and extracted with EtOAc (2.times.150 ml).
The combined organics were washed with 2N HCl (100 ml) then brine
(100 ml), then dried (MgSO4) and concentrated. The crude product
was purified by flash silica chromatography, elution gradient 0 to
40% EtOAc in heptane. Pure fractions were evaporated to dryness to
afford methyl
(E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate (2.93
g, 86%) as a yellow solid. 1H NMR (400 MHz, CDCl3, 30.degree. C.)
3.83 (3H, d), 6.51 (1H, d), 7.12 (2H, m), 7.57 (1H, d). m/z (ES+),
[M+H]+=228.
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine
##STR00064##
[0539] (2R)-1-(1H-indol-3-yl)propan-2-amine (3.81 kg, 21.21 moles)
was added to a 100 L glass lined jacketed vessel under an
atmosphere of nitrogen. 1,4-dioxane (23 L) was added, and the
agitator was switched on. Diisopropylethylamine (5.55 L; 31.82
moles) was added to the stirred suspension followed by
(2-fluoro-2-methyl-propyl)trifluoromethanesulfonate (5.55 kg, 23.77
moles). 1,4-Dioxane (4 L) was added to the vessel, and the mixture
was heated to 75.degree. C. Heating was continued for 24 hours
before cooling the mixture to 25.degree. C. Water (30.5 L) was
added to the vessel, followed by toluene (30.5 L). After 40 minutes
the agitator was switched off and the layers were allowed to
separate. The aqueous layer was removed and water (30.5 L) was
added to the organic solution. The mixture was agitated for 15
minutes before allowing the layers to separate. The aqueous layer
was removed from the vessel. The organic solution was concentrated
by vacuum distillation (jacket temperature 65.degree. C., 110 mbar
pressure) until approximately 27 L of distillate had been removed.
The remaining solution in the vessel was cooled to afford
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine
as a solution in toluene (33% w/w) (15.4 Kg, 97%). 1H NMR (500 MHz,
DMSO, 27.degree. C.) 0.98 (3H, d), 1.26 (3H, d), 1.30 (3H, d),
2.57-2.75 (3H, m), 2.81 (1H, dd), 2.84-2.92 (1H, m), 6.97 (1H, t),
7.06 (1H, t), 7.11-7.22 (1H, multiplet obscured by toluene
signals), 7.34 (1H, d), 7.52 (1H, d), 10.80 (1H, s).
Preparation of methyl
(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-di-
hydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate
##STR00065##
[0541]
(R)--N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-ami-
ne [33% w/w in toluene] (11.26 g, 14.97 mmol) and
(E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate (3.40
g, 14.97 mmol) were heated in toluene (55.6 ml)/acetic acid (4.28
ml, 74.83 mmol) at 80.degree. C. for 5 hr. After cooling, the
volatiles were removed under vacuum. The residue was taken-up in
DCM (200 ml) and washed with sat. NaHCO.sub.3 solution (200 ml).
The aqueous phase was extracted with DCM (100 ml) then the combined
organics were washed with brine, dried and concentrated in vacuo.
The crude material was loaded to an SCX-2 column, eluting with
methanol to remove unreacted aldehyde. The column was then eluted
with 7M NH.sub.3-MeOH to liberate the product. The basic filtrate
was evaporated and the crude product was purified by flash silica
chromatography, elution gradient 0 to 40% EtOAc in heptane. Pure
fractions were evaporated to dryness to afford methyl
(E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-di-
hydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate
(4.70 g, 68.6%) as a pale yellow solid. .sup.1H NMR (400 MHz,
CDCl.sub.3, 30.degree. C.) 1.11 (3H, d), 1.19 (3H, d), 1.25 (3H,
d), 2.42 (1H, dd), 2.62 (1H, dd), 2.87 (1H, dd), 3.07 (1H, dd),
3.65 (1H, q), 3.81 (3H, s), 6.39 (1H, d), 6.99 (2H, d), 7.06-7.17
(2H, m), 7.23 (1H, dd), 7.45 (1H, s), 7.49-7.6 (2H, m). m/z: ES+
[M+H]+ 458.
Example 11: Preparation of
(1R,3R)-1-{4-[(E)-2-carboxyethenyl]-2,6-difluorophenyl}-2-(2-fluoro-2-met-
hylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-beta-carbolin-2-ium
maleate
##STR00066##
[0543] A solution of maleic acid (1.31 g, 11.29 mmol) in acetone
(15 ml) was stirred under nitrogen. A solution of
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4-
,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
(Example 1) (5.00 g, 11.3 mmol) in acetone (25 ml) was added to the
maleic acid solution to give a yellow solution. The reaction vessel
was covered in foil to protect from light and purged with a stream
of nitrogen gas overnight until the solvent evaporated. A solid was
obtained which was dried in vacuo for 2 hours to give the title
compound as a cream solid (6.23 g, 98%). .sup.1H NMR (500 MHz,
DMSO, 27.degree. C.) 0.95-1.34 (9H, m), 2.24-2.45 (1H, m),
2.54-2.66 (1H, m), 2.8-2.99 (2H, m), 3.52 (1H, s), 5.22 (1H, s),
6.26 (2H, s), 6.67 (1H, d), 6.89-7.07 (2H, m), 7.19 (1H, d),
7.39-7.51 (3H, m), 7.55 (1H, d), 10.59 (1H, s).
[0544] An XRPD trace of this maleate salt is shown in FIG. 8 and a
DSC trace shown in FIG. 9.
Example 12
[0545] Exemplary compositions of Example 1 were manufactured at the
75 g scale using a wet granulation process. The active ingredient,
mannitol, microcrystalline cellulose and sodium starch glycolate
were weighed in the quantities tabulated below and transferred to a
Diosna P1-6mixer-granulator and mixed (with chopping) at 600 rpm
for 6 minutes. For Composition A, mixing was continued while 30 mL
water was added in two aliquots at a rate of approximately 1 mL per
second, pausing mixing in between aliquots, while for Composition B
a solution prepared by stirring the required amounts of EDTA and
ascorbic acid with 20 mL water at 50.degree. C. for 20 minutes
(protected from light) was added using an analogous process, the
second aliquot in this case comprising approximately 10 mL of rinse
liquor. Wet mixing was continued for a total of 1.5 minutes. The
wet granules were passed through a 1.5 mm screen then dried under
vacuum at 50-60.degree. C. to a moisture content of <2% w/w. The
resulting granules were milled using a 1 mm screen then mixed with
the lubricant for 5 minutes at 32 rpm using a Turbula blender.
Tablets containing 10 mg of the active ingredient were formed by
compressing the granules to a nominal 100 mg compression weight
using a Riva Mini-press equipped with 6 mm normal concave
tooling.
TABLE-US-00004 Composition A Composition B Component Function % w/w
Amount (g) % w/w Amount (g) Example 1 Form B Active ingredient 10.0
7.50 10.0 7.50 EDTA Chelating agent Not present 0.1 0.075 Ascorbic
acid Anti-oxidant Not present 0.5 0.375 Mannitol Diluent 68.0 51.00
67.4 50.55 Microcrystalline Diluent 15.0 11.25 15.0 11.25 cellulose
Sodium starch Disintegrant 5.0 3.75 5.0 3.75 glycolate Stearic acid
Lubricant 2.0 1.50 2.0 1.50 Total 100 75.00 100 75.00
[0546] The stability of Compositions A and B with regard to
degradant formation (wherein "degradant" means
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate) was evaluated
for tablets packed in induction sealed 75 cc HDPE bottles or
exposed to the atmosphere in petri dishes, stored in the dark,
under controlled temperature and humidity as tabulated below. It is
apparent that Composition B, which contains both a chelating agent
and anti-oxidant, is more stable to chemical degradation than
Composition A which is a standard tablet formulation.
[0547] Stability of Exemplary Compositions with Regard to Degradant
Formation (%)
TABLE-US-00005 4 Week Time Point (storage condition) Initial
(25.degree. C./ (25.degree. C./ (40.degree. C./ Time (5.degree. C.,
60% RH, 60% RH, 75% RH, Formulation Point packed) packed) exposed)
exposed) Composition A 0.05 0.06 0.08 0.27 0.28 Composition B ND
<0.05 <0.05 0.05 0.11 ND Not detected (<0.02% w/w)
Example 13:
(E)-3-[3,5-difluoro-4-[(3R)-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dih-
ydro-3H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate
##STR00067##
[0549]
(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
(200 mg, 0.45 mmol) was added to a solution of cerium ammonium
nitrate (248 mg, 0.45 mmol) in acetonitrile (6 ml)/water (1.500 ml)
at room temperature. The reaction was stirred for 2 hr and further
cerium ammonium nitrate (248 mg, 0.45 mmol) was added. The solution
was stirred at 25.degree. C. for a further 15 minutes. The reaction
mixture was acidified with 2M HCl (3 ml) and extracted with DCM
(2.times.10 ml). The organics were then concentrated in vacuo and
the crude product was purified by preparative HPLC (Waters SunFire
column, 5.mu. silica, 50 mm diameter, 100 mm length), using
decreasingly polar mixtures of water (containing 0.1% formic acid)
and MeCN as eluents. Fractions containing the desired compound were
evaporated to dryness to afford
(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-
-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate (35.0 mg, 17.58%)
as an orange glass. 1H NMR (500 MHz, DMSO, 30.degree. C.) 1.27 (3H,
d), 1.43-1.55 (6H, m), 3.51 (1H, d), 3.72 (1H, dd), 3.96 (1H, dd),
4.18-4.32 (1H, m), 4.67 (1H, s), 6.59 (1H, s), 7.09-7.3 (2H, m),
7.42 (1H, t), 7.52-7.73 (3H, m), 7.78 (1H, d), NH not observed.
HRMS (ESI): [M+H].sup.+, found 441.17831,
C.sub.23H.sub.24F.sub.2N.sub.2O.sub.2 requires 441.17844.
Preparation of
(E)-3-[3,5-difluoro-4-[2-(2-fluoro-2-methyl-propyl)-3-methyl-9H-pyrido[3,-
4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate
##STR00068##
[0551]
(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid
(0.500 g, 1.13 mmol) was dissolved in DMSO (10 mL) and heated to
120.degree. C. in air and light for 16 h. The reaction was then
heated to 180.degree. C. for 2.5 h. The reaction mixture was cooled
and purified by preparative HPLC (Waters XBridge Prep C18 OBD
column, 5.mu. silica, 50 mm diameter, 100 mm length), using
decreasingly polar mixtures of water (containing 1% NH.sub.3) and
MeCN as eluents. Fractions containing the desired compound were
evaporated to dryness to afford
(E)-3-[3,5-difluoro-4-[2-(2-fluoro-2-methyl-propyl)-3-methyl-9H-pyrido[3,-
4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate (0.047 g, 9.40%) as an
orange solid. 1H NMR (400 MHz, DMSO, 27.degree. C.) 1.27 (3H, s),
1.33 (3H, d), 3.09 (3H, s), 4.59-4.8 (1H, m), 5.14-5.37 (1H, m),
6.54 (1H, d), 7.19 (1H, d), 7.39 (1H, t), 7.63 (2H, d), 7.74 (1H,
t), 7.91 (1H, d), 8.45 (1H, d), 8.88 (1H, s), 10.79 (1H, s). m/z:
ES+ [M+H]+ 439.
Example 14A: Preparation of
(E)-3-[3,5-difluoro-4-[(1R,3R)-2-(2-fluoro-3-hydroxy-2-methyl-propyl)-3-m-
ethyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]prop-2-enoic
acid (Isomer 1)
##STR00069##
[0553] 2M Sodium hydroxide (1.27 mL, 2.54 mmol) was added to a
solution of (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate--Isomer
1 (120 mg, 0.25 mmol) in THF (0.635 mL)/methanol (0.635 mL). The
reaction was stirred at room temperature for 1 h, then diluted with
EtOAc and water. The aqueous was adjusted to pH 6 by addition of 2M
HCl, and the layers were separated. The aqueous layer was extracted
with EtOAc, then the combined organics were dried (MgSO.sub.4) and
concentrated in vacuo. The crude product was purified by flash
silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure
fractions were evaporated to dryness to afford
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpr-
opyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acryli-
c acid--Isomer 1 (85 mg, 72.9%) as a beige solid. 1H NMR (400 MHz,
DMSO, 27.degree. C.) 0.97 (3H, d), 1.05 (3H, d), 2.32-2.4 (1H, m),
2.44-2.54 (1H, m), 2.73-2.93 (3H, m), 3-3.14 (2H, m), 3.32-3.49
(2H, m), 4.73 (1H, s), 5.14 (1H, s), 6.58 (1H, d), 6.82-6.96 (2H,
m), 7.10 (1H, d), 7.33 (1H, d), 7.36 (1H, d), 7.45 (1H, d), 10.49
(1H, s). m/z (ES+), [M+H]+=459.
Example 14B: Preparation of
(E)-3-[3,5-difluoro-4-[(1R,3R)-2-(2-fluoro-3-hydroxy-2-methyl-propyl)-3-m-
ethyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]prop-2-enoic
acid (Isomer 2)
##STR00070##
[0555] 2M Sodium hydroxide (1.27 mL, 2.54 mmol) was added to a
solution of (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate--Isomer
2 (110 mg, 0.23 mmol) in THF (0.529 mL)/methanol (0.529 mL). The
reaction was stirred at room temperature for 1 h, then diluted with
EtOAc and water. The aqueous was adjusted to pH 6 by addition of 2M
HCl, and the layers were separated. The aqueous layer was extracted
with EtOAc, then the combined organics were dried (MgSO.sub.4) and
concentrated in vacuo. The crude product was purified by flash
silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure
fractions were evaporated to dryness to afford
(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpr-
opyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acryli-
c acid--Isomer 2 (81 mg, 76%) as a beige solid. 1H NMR (400 MHz,
DMSO, 27.degree. C.) 1.02 (2H, s), 1.05 (3H, d), 1.23 (1H, s), 1.90
(3H, s), 2.28-2.46 (1H, m), 2.53-2.7 (1H, m), 2.86-3.03 (2H, m),
3.56 (1H, d), 4.83 (1H, s), 5.17 (1H, s), 6.66 (1H, d), 6.86-7.08
(2H, m), 7.17 (1H, d), 7.40 (1H, d), 7.44 (2H, d), 7.53 (1H, d),
10.54 (1H, s). m/z (ES+), [M+H]+=459.
[0556] The (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
(Isomer 1 and 2) used as starting materials were prepared as
follows:--
2-Fluoro-2-methylpropane-1,3-diol
##STR00071##
[0558] LiAlH.sub.4 (0.741 g, 19.25 mmol) was added portionwise to a
cooled solution of diethyl 2-fluoro-2-methylmalonate (1.345 g, 7.00
mmol) in THF (35.0 ml). The reaction was allowed to warm to room
temperature over 1 h. After cooling to 0.degree. C., the reaction
was quenched by addition of water (0.75 mL), 15% NaOH (0.75 mL),
then water (1.5 mL). The suspension was stirred for 30 min, then
filtered and the solids were washed with THF. The filtrate was
evaporated to afford 2-fluoro-2-methylpropane-1,3-diol (0.745 g,
98%) as a colourless oil. 1H NMR (400 MHz, CDCl3, 27.degree. C.)
1.34 (3H, d), 2.12-2.27 (2H, m), 3.75 (4H, d).
3-(((R)-1-(1H-Indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-ol
##STR00072##
[0560] Trifluoromethanesulfonic anhydride (1.151 ml, 6.80 mmol) was
added to a solution of 2-fluoro-2-methylpropane-1,3-diol (0.70 g,
6.47 mmol) in DCM (17.85 ml) at 0.degree. C., followed by
2,6-lutidine (0.908 ml, 7.77 mmol). The reaction was allowed to
warm to room temperature over 30 min, then was washed with 2M HCl.
The organic phase was passed through a phase separator cartridge
and concentrated in vacuo. The residue was dissolved in dioxane (12
mL), then (R)-1-(1H-indol-3-yl)propan-2-amine (1.128 g, 6.47 mmol)
and DIPEA (1.678 ml, 9.71 mmol) were added and the reaction was
heated to 90.degree. C. for 2 h. After cooling, the reaction was
diluted with DCM and washed with water. The aqueous was extracted
with DCM, then the organics were concentrated in vacuo. The crude
product was purified by flash silica chromatography, elution
gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to
dryness to afford
3-(((R)-1-(1H-indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-ol
(0.815 g, 47.6%) as a brown gum. m/z (ES+), [M+H]+=265.
(E)-methyl
3-(3,5-difluoro-44(1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-
-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
(Isomer 1 and
##STR00073##
[0562] (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (565 mg,
2.50 mmol) was added to a suspension of
3-(((R)-1-(1H-indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-01
(661 mg, 2.50 mmol) in toluene (11.3 ml)/acetic acid (1.25 ml). The
reaction was heated to 90.degree. C. for 5 h. After cooling, the
volatiles were removed under vacuum, then the residue was passed
through an SCX-2 column, eluting with methanol to remove unreacted
aldehyde. The column was then eluted with NH.sub.3/MeOH to liberate
the products. The basic filtrate was evaporated then the crude
product was purified by flash silica chromatography, elution
gradient 0 to 50% EtOAc in heptane. Pure fractions were evaporated
to dryness to afford (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
(Isomer 1-122 mg, 10.3%) as a yellow solid and (E)-methyl
3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-
-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
(Isomer 2-129 mg, 11% as a yellow/orange solid. Isomer 1-1H NMR
(400 MHz, CDCl3, 27.degree. C.) 1.10 (3H, d), 1.14 (3H, d), 2.54
(1H, dd), 2.62-2.73 (1H, m), 3.06-3.3 (2H, m), 3.40 (1H, dd), 3.56
(1H, t), 3.80 (3H, s), 3.87-4.08 (1H, m), 4.27 (1H, s), 5.16 (1H,
s), 6.37 (1H, d), 7.01 (2H, d), 7.07-7.15 (2H, m), 7.14-7.24 (1H,
m), 7.42-7.56 (2H, m), 7.71 (1H, s). m/z (ES+), [M+H]+=473. Isomer
2-1H NMR (400 MHz, CDCl3, 27.degree. C.) 1.15 (3H, d), 1.20 (3H,
d), 2.65 (1H, dd), 2.79 (1H, t), 2.93-3.09 (2H, m), 3.57 (1H, dt),
3.70 (1H, dd), 3.78 (3H, s), 4.2-4.67 (1H, m), 5.42 (1H, s), 6.32
(1H, d), 6.94 (2H, d), 7.06-7.15 (2H, m), 7.19-7.27 (2H, m), 7.42
(1H, s), 7.51 (1H, dd), 8.02 (1H, s). m/z (ES+), [M+H]+=473.
Reference Example 1:
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one
##STR00074##
[0564] Pyridine 4-methylbenzenesulfonate (11.62 g, 46.24 mmol) was
added to a suspension of
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)pr-
opan-1-one (128 g, 231.19 mmol) in methanol (1 L) under nitrogen.
The mixture was stirred at 60.degree. C. for 1.5 hours. The mixture
was soluble after 5 minutes. The mixture was held at 50.degree. C.
overnight during which time a precipitate formed. The solid
material was isolated by filtration and washed with water and
acetonitrile. This material still contained minor impurities from
the previous stage and required further purification. The material
was dissolved in dichloromethane and purified by flash
chromatography on silica gel (0% methanol/DCM to 10% methanol/DCM).
The desired product,
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one
(Reference Example 1) (92 g, 85%), was thus isolated as a cream
solid (Form A): .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.4-1.51 (12H,
m), 1.51-1.78 (2H, m), 1.89-2.05 (2H, m), 2.72-2.86 (1H, m),
2.91-3.05 (1H, m), 3.12-3.24 (1H, m), 3.64 (2H, q), 3.83-4.01 (1H,
m), 4.29-4.41 (1H, m), 4.47 (1H, t), 4.58 (2H, q), 8.26 (2H, s),
8.85 (1H, s); Mass Spectrum [M+H].sup.+=470.
[0565]
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yL)-
-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yl-
oxy)propan-1-one was prepared as follows:
[0566] 1,8-Diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) was
added to a suspension of tert-butyl
4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-
-triazol-3-yl)piperidine-1-carboxylate (150 g, 319.46 mmol) in
2-methylTHF (1.2 L). Iodoethane (46 mL, 575.03 mmol) was added and
the mixture was stirred for 16 hours at 35.degree. C. Further
iodoethane (46 mL, 575.03 mmol) and
1,8-diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) were added
and stirring was continued for 24 hours at 35.degree. C. The
mixture was poured into water and the insoluble material was
isolated by filtration, washed with water and MTBE and dried in
vacuo to afford tert-butyl
4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl--
1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (116 g, 73.0%) as a
yellow solid. The filtrate was extracted with DCM and the organic
solution was dried with magnesium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica using gradient elution (30%
MTBE/heptane to 100% MTBE). A second crop of the desired product
(12 g, 24.12 mmol, 7.55%), was thus isolated as a yellow solid
which was later combined with the first crop: .sup.1H NMR Spectrum:
(DMSO-d.sub.6) 1.41 (9H, s), 1.44 (9H, s), 1.48 (3H, t), 1.52-1.69
(2H, m), 1.87-2.04 (2H, m), 2.79-3.03 (3H, m), 3.86-4.03 (2H, m),
4.59 (2H, q), 7.89 (2H, s), 8.85 (1H, s); Mass Spectrum
[M+H]+=498.
[0567] A suspension of tert-butyl
4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl--
1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (3009.5 g, 6.05 mol)
in DCM (9 L) was cooled to 5-10.degree. C. under N.sub.2. TFA (9 L)
was added portionwise to the suspension whilst maintaining the
temperature <30.degree. C. The reaction mixture was stirred at
room temperature for 1 h. The mixture was concentrated, the
resulting residue was dissolved in water (30 L) and added slowly to
a 35% aqueous ammonia solution (12 L) at 0-5.degree. C. The
suspension was stirred for 30 min then the product was filtered off
and washed with water (2.times.6 L). The product was dried at
50.degree. C. in vacuo for 2 days. to afford
3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,-
2,4-triazol-5-yl)pyrazin-2-amine ((2496 g): .sup.1H NMR Spectrum:
(DMSO-d.sub.6) 1.4-1.52 (12H, m), 1.57-1.73 (2H, m), 1.83-1.93 (2H,
m), 2.57-2.7 (2H, m), 2.71-2.84 (1H, m), 2.96-3.09 (2H, m), 4.58
(2H, q), 8.06 (2H, s), 8.84 (1H, s); Mass Spectrum
[M+H].sup.+=398.
[0568] To a solution of 3-(tetrahydro-2H-pyran-2-yloxy) propanoic
acid (HATU, 48.80 g 0.2774 mol) and
N-ethyl-N-isopropylpropan-2-amine (86.96 mL, 0.4993 mol) in THF
(552 mL) was added
O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium
hexafluorophosphate (115.73 g, 0.3051 mol) portionwise at RT under
nitrogen. The resulting mixture was stirred for 20 min then
3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,-
2,4-triazol-5-yl)pyrazin-2-amine (122.5 g (110.25 g active), 0.2774
mol) was added portionwise over 1 h. After 3.5 h, the mixture was
concentrated and the residue was slurried in MeCN (275 mL) for 15
min at room temperature. The product was filtered off, washed with
MeCN (3.times.110 mL) and dried overnight at 50.degree. C. in
vacuo. This gave
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)pr-
opan-1-one (131.9 g, 96%). .sup.1H NMR Spectrum: (DMSO-d.sub.6)
1.29-1.48 (16H, m), 1.48-1.75 (4H, m), 1.83-1.99 (2H, m), 2.48-2.68
(2H, m), 2.68-2.79 (1H, m), 2.87-2.99 (1H, m), 3.07-3.19 (1H, m),
3.32-3.42 (1H, m), 3.47-3.6 (1H, m), 3.64-3.75 (1H, m), 3.75-3.84
(1H, m), 3.84-3.95 (1H, m), 4.24-4.39 (1H, m), 4.47-4.6 (3H, m),
7.84 (2H, s), 8.79 (1H, s): Mass Spectrum [M+Na].sup.+=577.
Alternative Preparation of Reference Example 1
[0569] To a suspension of
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)pr-
opan-1-one (131.9 g, 0.2382 mol) in methanol (1045 mL) was added
pyridinium p-toluenesulfonate (11.97 g, 47.7 mmol) under N2. The
reaction mixture was stirred at 60.degree. C. for 5.5 h then at
50.degree. C. overnight. The reaction mixture was cooled to
0.degree. C. and the solid was filtered off. The product was
slurried in water (250 mL) for 20 min at room temperature, filtered
off, washed with water (3.times.40 mL) and dried at 50.degree. C.
in vacuo. This gave
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one
(21.4 g) as Form A (see below).
[0570] The methanol liquors were concentrated and the resulting
solid was slurried in water (0.6 L) for 20 min at room temperature.
The solid was isolated by filtration and washed with water
(3.times.100 mL). The filter cake was slurried for a second time in
water (0.5 L) for a further 20 minutes. The product was isolated by
filtration, washed with water (100 mL) and dried at 50.degree. C.
in vacuo. This gave 81.9 g
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one
(81.9 g) as Form A.
[0571] Both crops were combined (103.3 g), seeded with Form B
(16.68 g) and slurried in MeCN (826 mL) at room temperature
overnight. This gave 117.4 g of
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one as a
pale yellow solid (117.4 g), Form B (see below). This material was
further purified by slurrying in heptane (7.5 rel vols) for 1 hour.
The mixture was filtered, pulled dry on the filter, and dried at
50.degree. C. in a vacuum oven overnight to afford
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one
(102.5 g) as Form B.
[0572] Form B may also be made by slurrying Form A in MeCN without
seeding.
[0573] Form A or B may also be converted to Form C as follows:
[0574] A suspension of
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (eg
Form B made by the processes outlined above) in IPA (12 vol) was
heated at reflux until the solid dissolved. The solution was hot
filtered then cooled to room temperature. This gave
1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-eth-
yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one as a
pale yellow solid (99.3 g, 97%) as Form C.
[0575] Form C may also be converted to Form B as follows:
[0576] In a 10 L flange flask, Form C (377.8 g portion 1) in MIBK
(7900 mL) was heated to 110-115.degree. C. to give a solution. The
solution was allowed to cool to 97-103.degree. C. and immediately
polish filtered into a SOL vessel containing a seed of Form B (0.8
g) in acetonitrile (8220 mL) stirring at -15.degree. C. During the
addition the temperature in the 50 L vessel was maintained between
-15 and 25.degree. C. by means of jacket cooling. Three further
portions of the compound dissolved in MIBK were added by a similar
method. To the resulting slurry was added a seed of form B (0.8 g)
and the mixture was then stirred at 10-20.degree. C. overnight.
In-process analysis confirmed the desired form (Form B) with no
Form C or amorphous visible. The mixture was filtered and washed
with acetonitrile (3340 mL). The solid was oven dried for 2 days
(solid was broken up during the drying to a powder and a mixture of
small lumps .about.1 mm to .about.3-4 mm size) until constant
weight was obtained. Yield=1532.8 g (93.5%)
[0577] 3-(Tetrahydro-2H-pyran-2-yloxy)propanoic acid was prepared
as follows:
[0578] To a stirred solution of methanol (2.4 L) and concentrated
sulfuric acid (44.4 mL, 832.61 mmol) at 0.degree. C. under nitrogen
was added, dropwise, beta-propiolactone (175 mL, 2.78 mol). This
solution was allowed to stir at room temperature for 2 days. The
reaction mixture was cooled to 10.degree. C. before adding,
portionwise, sodium bicarbonate (145 g, 1.72 mol), the resulting
suspension was left to stir at room temperature for 75 minutes.
This solution was filtered, the filter-cake was washed with
methanol (800 mL). The filtrate was evaporated to an oil which was
redissolved in dichloromethane (1.2 L) and stirred for 60 minutes
before refiltering. This solution was filtered before evaporating
to give methyl 3-hydroxypropanoate (219 g, 76%) as an oil. .sup.1H
NMR Spectrum: (CDCl.sub.3) 2.50 (2H, t), 3.63 (3H, s), 3.78 (2H,
t).
[0579] Pyridinium p-toluenesulfonate (7.65 g, 30.45 mmol) was added
to a clear solution of methyl 3-hydroxypropanoate (63.4 g, 609.00
mmol) and 3,4-dihydro-2H-pyran (78 mL, 852.60 mmol) in
dichloromethane (650 mL) at room temperature under nitrogen to give
a cloudy solution. This was allowed to stir at room temperature
overnight. The reaction mixture was washed with water (250 mL) and
brine (250 mL) before drying (MgSO.sub.4) and evaporating to an
oil. This crude product was purified by flash silica
chromatography, elution gradient 15 to 30% EtOAc in heptane. Pure
fractions were evaporated to dryness to afford methyl
3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.7 g, 59.0%) as a
colourless oil: .sup.1H NMR Spectrum: (CDCl.sub.3) 1.47 (4H, dddd),
1.55-1.84 (2H, m), 2.55 (2H, t), 3.33-3.53 (1H, m), 3.53-3.7 (4H,
m), 3.78 (1H, ddd), 3.93 (1H, dt), 4.42-4.72 (1H, m); Mass Spectrum
[MH].sup.+=189.
[0580] Sodium hydroxide (2M, 349 mL, 697.58 mmol) was added to a
solution of methyl 3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.68
g, 359.58 mmol) in THF (680 mL) at room temperature. The reaction
mixture was stirred at room temperature for 3 hours. The THF was
removed in vacuo, the aqueous layer was then washed with ethyl
acetate (260 mL), before cooling to 0.degree. C. and careful
acidification to pH 5 by the addition of hydrochloric acid (2M).
The product was extracted with ethyl acetate (3.times.250 mL)
before drying (MgSO.sub.4) and evaporation to give
3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (57.0 g, 91%) as a
clear oil. This material was dissolved in ethyl acetate (750 mL)
then washed with water (3.times.250 mL) and brine (250 mL) to
remove remaining acetic acid. The organic solution was dried
(MgSO.sub.4) and evaporated to give
3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (45.67 g, 72.9%) as a
colourless oil: .sup.1H NMR Spectrum: .sup.1H NMR (CDCl.sub.3)
1.43-1.67 (4H, m), 1.65-1.95 (2H, m), 2.68 (2H, t), 3.48-3.58 (1H,
m), 3.73 (1H, dt), 3.88 (1H, ddd), 4.02 (1H, dt), 4.59-4.7 (1H, m);
Mass Spectrum [M-H].sup.-=173.
[0581] The tert-butyl
4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-
-triazol-3-yl)piperidine-1-carboxylate was prepared as follows:
[0582] Hydrazine hydrate (23.59 mL, 480.75 mmol) was added dropwise
to a stirred mixture of methyl
3-amino-6-bromopyrazine-2-carboxylate (100 g, 418.04 mmol) in EtOH
(2 L). The mixture was heated at 50.degree. C. under nitrogen. The
resulting thick suspension was stirred at 50.degree. C. for 16
hours. Further hydrazine (2.5 mL) was added in one portion and the
suspension was stirred at 50.degree. C. for a further 24 hours.
Ethanol (500 mL) was charged to the thick reaction mixture and the
mixture was allowed to cool to room temperature. The resulting
suspension was filtered and the solid washed with ethanol (1 L) and
dried in vacuo to give 3-amino-6-bromopyrazine-2-carbohydrazide (98
g, quantitative) as a cream solid: .sup.1H NMR Spectrum
(DMSO-d.sub.6) 4.52 (2H, s), 7.59 (2H, s), 8.30 (1H, s), 9.74 (1H,
s); Mass Spectrum [M+H].sup.+=232.
[0583] Pivalic anhydride (165 mL, 815.38 mmol) was added to a
stirred mixture of 3-amino-6-bromopyrazine-2-carbohydrazide (172 g,
741.26 mmol) in acetonitrile (1.8 L) and the mixture was heated at
80.degree. C. for 1 hour. The reaction was left to stir for 16
hours. The required yellow solid material was isolated by
filtration. The filtrate was partitioned between EtOAc (2 L) and
aqueous sodium bicarbonate (2 L). The organic layer was washed with
saturated brine and dried over MgSO.sub.4. The solution was
filtered and concentrated to give an orange sticky solid which was
triturated with MTBE (250 mL). The insoluble yellow solid was
isolated by filtration and this material was shown to be identical
to the first solid. The combined solids were dried in the vacuum
oven at 50.degree. C. for 3 days to afford
3-amino-6-bromo-N'-pivaloylpyrazine-2-carbohydrazide (224 g, 96%)
as a yellow solid: .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.17 (9H,
s), 7.62 (2H, s), 8.37 (1H, s), 9.42-9.56 (1H, m), 10.09-10.23 (1H,
m); Mass Spectrum [M+H].sup.+=318.
[0584] To 3-amino-6-bromo-N'-pivaloylpyrazine-2-carbohydrazide
(2301 g, 7.28 mol) in MeCN (10.8 L) was added DIPEA (3.044 L, 17.48
mol) and p-toluenesulfonyl chloride (1665 g, 8.73 mol) portion-wise
(.about.280 g.times.6) at 50.degree. C. over a period of 30 mins.
The reaction temperature was maintained between 65-70.degree. C. by
controlling the rate of addition. After the addition was complete,
the reaction mixture was stirred at 70.degree. C. for 1 h. The
mixture was cooled to room temperature and quenched with 5%
NaHCO.sub.3 (aqueous, 24.2 L). The resulting suspension was stirred
for 30 min then filtered. The product was washed with water (14.8
L), pulled dry and dried at 50.degree. C. for 16 h. The product was
dissolved in DCM (12 L) and the phases separated. The organic phase
was loaded onto a silica pad (6 kg) and the product was eluted with
20% EtOAc/DCM (8.times.10 L). Concentration of the product
containing fractions gave 1987 g (92% yield) with a purity of 99.8%
by HPLC of
5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (36 g,
17%): .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.43 (9H, s), 7.70 (2H,
s), 8.39 (1H, s); Mass Spectrum [M+H].sup.+=298.
[0585] A stream of nitrogen gas was passed through a solution of
5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (89.35
g, 239.75 mmol) in DMA (1.2 L) for 20 minutes.
Dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl)phosphine (11.43 g,
23.98 mmol), tris(dibenzylideneacetone)dipalladium(0) (5.49 g, 5.99
mmol), zinc (1.568 g, 23.98 mmol) and dicyanozinc (16.89 g, 143.85
mmol) were added sequentially to the stirred mixture. The mixture
was heated to 100.degree. C. and stirred for 1 hour. The mixture
was cooled and partitioned between DCM (3 L) and water (1 L). The
black mixture was filtered through celite and the organic layer was
separated. The solution was washed with water then brine. The
solution was dried with magnesium sulfate and concentrated under
reduced pressure. The residue was triturated with MTBE and isolated
by filtration, washing with MTBE. The filter cake was dried in
vacuo to afford
5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile
(55.7 g, 95%) as a pale orange solid: .sup.1H NMR Spectrum:
(DMSO-d.sub.6) 1.46 (9H, s), 6.02 (1H, s), 8.38 (2H, s); Mass
Spectrum [M-H].sup.-=242.
[0586] Hydrazine hydrate (82 mL, 1.69 mol) was added to
5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile
(55 g, 225.18 mmol) in IPA (200 mL) and the mixture was heated at
50.degree. C. under nitrogen for 16 hours. The mixture was cooled
in an ice bath. The resulting precipitate was collected by
filtration, washed with IPA and diethyl ether and dried to a
constant weight to afford
(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazon-
amide (49.2 g, 79%) as a yellow solid: .sup.1H NMR Spectrum:
(DMSO-d.sub.6) 1.45 (9H, s), 5.26 (2H, s), 5.58 (2H, s), 7.56 (2H,
s), 8.75 (1H, s); Mass
[0587] Spectrum [M+H].sup.+=277.
[0588] O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (74.3 g, 195.44 mmol) was added to a solution
of N-Boc-isonipecotic acid (41.1 g, 179.15 mmol) and
4-methylmorpholine (35.9 mL, 325.74 mmol) in DMA (800 mL). The
mixture was stirred for 10 minutes then
(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazon-
amide (45 g, 162.87 mmol) was added to the solution in one portion
(exotherm observed from 22.degree. C. to 27.degree. C.). After a
few minutes the product crystallised from the reaction mixture. The
reaction mixture was removed from the vessel and filtered through a
sinter. Additional DMA was added to wash product from the sides of
the vessel (150 mL) and this was poured onto the filter cake.
Isopropanol (600 mL) was added to the vessel and the remainder of
the product in the vessel was suspended in this solvent using
vigorous agitation. The isopropanol suspension was used to wash the
filter cake once the DMA had been removed by suction. The filter
cake was sucked dry then washed with MTBE and sucked dry once
again. The filter cake was dried in vacuo to afford (Z)-tert-butyl
4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)met-
hylene)hydrazinecarbonyl)piperidine-1-carboxylate (76 g, 95%) as a
yellow solid:
[0589] .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.40 (9H, s), 1.46 (9H,
s), 1.63-1.9 (2H, m), 2.33-2.6 (2H, m, obscured by DMSO signal),
2.63-3.03 (2H, m), 3.18-3.48 (4H, m, obscured by water signal),
3.88-4.11 (2H, m), 6.43 (2H, s), 7.76 (2H, br), 8.84 (0.5H, s),
8.87 (0.5H, s), 9.85 (1H, s); Mass Spectrum [M+H].sup.+=488
[0590] In an alternative preparation, the N-Boc-isonipecotic acid
may be made in situ as follows: Isonipecotic acid (858 g, 3.74 mol)
was dissolved in DMA (25.3 L) and 4-methylmorpholine (393 mL, 3.74
mol) added. Stirred for 5 mins and isobutyl chloroformate (489 mL,
3.74 mol) added. The reaction mixture was stirred at 25.degree. C.
for 2 h and cooled to 15.degree. C. before
(Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazon-
amide (940 g, 3.4 mol) was added portionwise over 10 mins. The
reaction mixture was stirred for 1-2 h at 15.degree. C. Water (20.5
L) was added portionwise over 1 h and stirred for a further 1 h
before being filtered. The filtercake was then washed with water
(4.times.4 L) and pulled dry on the filter before being dried in a
vacuum oven at 50.degree. C. until dry to give the desired
product.
[0591] Acetic acid (200 mL) was added to dioxane (500 mL) in a 3 L
fixed double jacketed vessel and the solution was heated to
70.degree. C. under nitrogen. (Z)-tert-butyl
4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)met-
hylene)hydrazinecarbonyl)-piperidine-1-carboxylate (74.5 g, 152.80
mmol) was added portionwise to the warm mixture. After 10 minutes
the temperature was increased to 100.degree. C. (slight reflux).
The reaction mixture was stirred at 100.degree. C. for 1.5 hours
(suspension) then held at 80.degree. C. overnight (solution formed
after overnight hold). The resulting solution was concentrated
under reduced pressure, then diluted with toluene, evaporated to
dryness, taken up with toluene and concentrated again. The residual
oil was mixed with some ethyl acetate and concentrated to dryness.
A solid crystallised from solution which was triturated with MTBE
(200 mL) and isolated by filtration. The filter cake was washed
with water and MTBE to afford tert-butyl
4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-
-triazol-3-yl)piperidine-1-carboxylate (50 g, 70%) as a grey
solid.
[0592] The filtrate was concentrated under reduced pressure to give
a yellow solid. This material was triturated with MTBE and
filtered. The filter cake was washed with ethyl acetate and then
MTBE to give a second crop as a pale yellow solid (4.93 g, 7%).
This material was identical to the first crop: .sup.1H NMR
Spectrum: (DMSO-d.sub.6) 1.17 (9H, s), 1.22 (9H, s), 1.29-1.47 (2H,
m), 1.67-1.78 (2H, m), 2.57-2.87 (3H, m), 3.57-3.92 (2H, m), 7.56
(2H, br), 8.56 (1H, s), 13.47 (2H, br s); Mass Spectrum
[M+H]+=470.
* * * * *
References