U.S. patent application number 14/518419 was filed with the patent office on 2015-06-18 for metabolites of the janus kinase inhibitor (r)-3-(4-(7h-pyrrolo[2,3-d]pyrimidin-4-yl)-1h-pyrazol-1-yl)-3-cyclopentyl- propanenitrile.
The applicant listed for this patent is Incyte Corporation. Invention is credited to Argyrios G. Arvanitis, James D. Rodgers, Stacey Shepard, Jack Guoen Shi.
Application Number | 20150164900 14/518419 |
Document ID | / |
Family ID | 40029276 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150164900 |
Kind Code |
A1 |
Rodgers; James D. ; et
al. |
June 18, 2015 |
METABOLITES OF THE JANUS KINASE INHIBITOR
(R)-3-(4-(7H-PYRROLO[2,3-d]PYRIMIDIN-4-YL)-1H-PYRAZOL-1-YL)-3-CYCLOPENTYL-
PROPANENITRILE
Abstract
The present invention provides active metablites of
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylprop-
anenitrile that modulate the activity of Janus kinases and are
useful in the treatment of diseases related to activity of Janus
kinases including, for example, immune-related diseases, skin
disorders, myeloid proliferative disorders, cancer, and other
diseases.
Inventors: |
Rodgers; James D.;
(Landenberg, PA) ; Arvanitis; Argyrios G.;
(Kennett Square, PA) ; Shi; Jack Guoen;
(Wilmington, DE) ; Shepard; Stacey; (Wilmington,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Incyte Corporation |
Wilmington |
DE |
US |
|
|
Family ID: |
40029276 |
Appl. No.: |
14/518419 |
Filed: |
October 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12900180 |
Oct 7, 2010 |
8889697 |
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14518419 |
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12137883 |
Jun 12, 2008 |
7834022 |
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12900180 |
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60943695 |
Jun 13, 2007 |
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Current U.S.
Class: |
514/265.1 |
Current CPC
Class: |
A61K 31/519 20130101;
A61P 37/06 20180101; C07D 487/04 20130101; C07D 487/04 20130101;
C12N 9/99 20130101 |
International
Class: |
A61K 31/519 20060101
A61K031/519 |
Claims
1-53. (canceled)
54. A method of treating a myeloproliferative disorder in a patient
in need thereof, comprising administering to the patient a
metabolite selected from:
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclo-
pentyl)propanenitrile;
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclo-
pentyl)propanenitrile; and
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxycyclopent-
yl)propanenitrile, wherein the metabolite is generated as a result
of administration of
(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-
propanenitrile or a pharmaceutically acceptable salt thereof to the
patient.
55. The method of claim 54, wherein the myeloproliferative disorder
is polycythemia vera (PV).
56. The method of claim 54, wherein the myeloproliferative disorder
is essential thrombocythemia (ET).
57. The method of claim 54, wherein the myeloproliferative disorder
is myeloid metaplasia with myelofibrosis (MMM).
58. The method of claim 54, wherein the metabolite is
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclo-
pentyl)propanenitrile.
59. The method of claim 54, wherein the metabolite is
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclo-
pentyl)propanenitrile.
60. The method of claim 54, wherein the metabolite is
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxycyclopent-
yl)propanenitrile.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Ser. No.
60/943,695, filed Jun. 13, 2007, the disclosure of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides active metabolites of
(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-
propanenitrile that modulate the activity of Janus kinases and are
useful in the treatment of diseases related to activity of Janus
kinases including, for example, immune-related diseases, skin
disorders, myeloid proliferative disorders, cancer, and other
diseases.
BACKGROUND OF THE INVENTION
[0003] Protein kinases (PKs) are a group of enzymes that regulate
diverse, important biological processes including cell growth,
survival and differentiation, organ formation and morphogenesis,
neovascularization, tissue repair and regeneration, among others.
Protein kinases exert their physiological functions through
catalyzing the phosphorylation of proteins (or substrates) and
thereby modulating the cellular activities of the substrates in
various biological contexts. In addition to the functions in normal
tissues/organs, many protein kinases also play more specialized
roles in a host of human diseases including cancer. A subset of
protein kinases (also referred to as oncogenic protein kinases),
when dysregulated, can cause tumor formation and growth, and
further contribute to tumor maintenance and progression
(Blume-Jensen P et al, Nature 2001, 411(6835):355-365). Thus far,
oncogenic protein kinases represent one of the largest and most
attractive groups of protein targets for cancer intervention and
drug development.
[0004] The Janus Kinase (JAK) family plays a role in the
cytokine-dependent regulation of proliferation and function of
cells involved in immune response. Currently, there are four known
mammalian JAK family members: JAK1 (also known as Janus kinase-1),
JAK2 (also known as Janus kinase-2), JAK3 (also known as Janus
kinase, leukocyte; JAKL; L-JAK and Janus kinase-3) and TYK2 (also
known as protein-tyrosine kinase 2). The JAK proteins range in size
from 120 to 140 kDa and comprise seven conserved JAK homology (JH)
domains; one of these is a functional catalytic kinase domain, and
another is a pseudokinase domain potentially serving a regulatory
function and/or serving as a docking site for STATs (Scott,
Godshall et al. 2002, supra).
[0005] Blocking signal transduction at the level of the JAK kinases
holds promise for developing treatments for human cancers.
Inhibition of the JAK kinases is also envisioned to have
therapeutic benefits in patients suffering from skin immune
disorders such as psoriasis, and skin sensitization. Accordingly,
inhibitors of Janus kinases or related kinases are widely sought
and several publications report effective classes of compounds. For
example, certain JAK inhibitors, including
(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-
propanenitrile shown below, are reported in U.S. Ser. No.
11/637,545, filed Dec. 12, 2006.
##STR00001##
[0006] Thus, new or improved agents which inhibit kinases such as
Janus kinases are continually needed for developing new and more
effective pharmaceuticals to treat cancer and other diseases. The
metabolites, compositions and methods described herein are directed
toward these needs and other ends.
SUMMARY OF THE INVENTION
[0007] The present invention provides a compound selected from:
[0008]
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclo-
pentyl)propanenitrile; [0009]
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclo-
pentyl)propanenitrile; and [0010]
3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxocyclopent-
yl)propanenitrile;
[0011] or pharmaceutically acceptable salt thereof.
[0012] The present invention further provides one or more of the
above compounds, or pharmaceutically acceptable salts thereof, in
substantially isolated form.
[0013] The present invention further provides compositions
comprising a compound of the invention, or pharmaceutically
acceptable salt thereof, and at least one pharmaceutically
acceptable carrier.
[0014] The present invention further provides methods of modulating
an activity of JAK comprising contacting JAK with a compound of the
present invention, or pharmaceutically acceptable salt thereof.
[0015] The present invention further provides methods of treating a
disease in a patient, comprising administering to the patient a
therapeutically effective amount of a compound of the invention, or
pharmaceutically acceptable salt thereof.
DETAILED DESCRIPTION
[0016] The present invention provides, inter alia, compounds that
are active metabolites of the JAK inhibitor
(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-
propanenitrile. These metabolites modulate the activity of one or
more JAKs and are useful, for example, in the treatment of diseases
associated with JAK expression or activity. The metabolites of the
invention are indicated in Table 1 below. Structures are intended
to encompass all possible stereoisomers.
TABLE-US-00001 TABLE 1 Reference Name Structure Metabolite 1
3-(4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl)-3-(3-
hydroxycyclopentyl) propanenitrile ##STR00002## Metabolite 2
3-(4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl)-3-(2-
hydroxycyclopentyl) propanenitrile ##STR00003## Metabolite 3
3-(4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl)-3-(3-
oxocyclopentyl) propanenitrile ##STR00004##
[0017] The metabolites of the invention were isolated from rat or
dog urine samples collected from pharmacokinetic and toxicokinetic
studies of the JAK inhibitor
(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-
propanenitrile (Compound 1). As shown in Table 2 and detailed in
Example A, the metabolites are active and potent JAK inhibitors,
and have advantageous properties related to significantly higher
free fractions and higher metabolic stability in human microsomes
compared with Compound 1. This data suggests the present
metabolites may desirably have a longer elimination half-life in
humans than does Compound 1.
[0018] In some embodiments, the metabolites of the invention are
substantially isolated. By "substantially isolated" is meant that
the compound is at least partially or substantially separated from
the environment in which it was formed or detected. Partial
separation can include, for example, a composition enriched in the
compound of the invention. Substantial separation can include
compositions containing at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least
about 95%, at least about 97%, or at least about 99% by weight of
the metabolite.
[0019] The present invention also includes pharmaceutically
acceptable salts of the compounds described herein. As used herein,
"pharmaceutically acceptable salts" refers to derivatives of the
disclosed compounds wherein the parent compound is modified by
converting an existing acid or base moiety to its salt form.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines; alkali or organic salts of acidic residues such as
carboxylic acids; and the like. The pharmaceutically acceptable
salts of the present invention include the conventional non-toxic
salts of the parent compound formed, for example, from non-toxic
inorganic or organic acids. The pharmaceutically acceptable salts
of the present invention can be synthesized from the parent
compound which contains a basic or acidic moiety by conventional
chemical methods. Generally, such salts can be prepared by reacting
the free acid or base forms of these compounds with a
stoichiometric amount of the appropriate base or acid in water or
in an organic solvent, or in a mixture of the two; generally,
nonaqueous media like ether, ethyl acetate, ethanol, isopropanol,
or acetonitrile are preferred. Lists of suitable salts are found in
Remington's Pharmaceutical Sciences, 17.sup.th ed., Mack Publishing
Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical
Science, 66, 2 (1977), each of which is incorporated herein by
reference in its entirety.
[0020] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0021] The metabolites are asymmetric (e.g., having one or more
stereocenters). All stereoisomers, such as enantiomers and
diastereomers, are intended unless otherwise indicated. Methods on
how to prepare optically active forms from optically active
starting materials are known in the art, such as by resolution of
racemic mixtures or by stereoselective synthesis.
[0022] Compounds of the invention also include all isotopes of
atoms occurring in the metabolites. Isotopes include those atoms
having the same atomic number but different mass numbers. For
example, isotopes of hydrogen include tritium and deuterium.
[0023] The term, "compound," as used herein is meant to include all
stereoisomers, geometric isomers, tautomers, and isotopes of the
structures depicted.
Synthesis
[0024] Compounds of the invention, including salts thereof, can be
prepared using known organic synthesis techniques and can be
synthesized according to any of numerous possible synthetic
routes.
[0025] The reactions for preparing compounds of the invention can
be carried out in suitable solvents which can be readily selected
by one of skill in the art of organic synthesis. Suitable solvents
can be substantially nonreactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, e.g., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected by the skilled artisan.
[0026] Preparation of compounds of the invention can involve the
protection and deprotection of various chemical groups. The need
for protection and deprotection, and the selection of appropriate
protecting groups, can be readily determined by one skilled in the
art. The chemistry of protecting groups can be found, for example,
in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic
Synthesis, 3rd. Ed., Wiley & Sons, Inc., New York (1999), which
is incorporated herein by reference in its entirety.
[0027] Reactions can be monitored according to any suitable method
known in the art. For example, product formation can be monitored
by spectroscopic means, such as nuclear magnetic resonance
spectroscopy (e.g., .sup.1H or .sup.13C), infrared spectroscopy,
spectrophotometry (e.g., UV-visible), or mass spectrometry, or by
chromatography such as high performance liquid chromatography
(HPLC) or thin layer chromatography.
[0028] Compounds of the invention can be prepared according to
numerous preparatory routes known in the literature. Example
synthetic methods for preparing compounds of the invention are
provided in the Schemes below.
[0029] As shown in Scheme 1, synthesis of the diastereomeric
mixture of the cis alcohols I starts with cyclopentene carboxylic
acid 1. Cyclopentene carboxylic acid 1 is bromolactonized following
a procedure described earlier (Hodgson, David M.; Witherington,
Jason; Moloney, Brian A., Journal of the Chemical Society, Perkin
Transactions 1: Organic and Bio-Organic Chemistry, 1994, 23, 3950)
to give the corresponding bromolactone 2. The bromolactone 2 is
debrominated with the use of a dehalogenating agent, such as
(Me.sub.3Si).sub.3SiH to give 3. The lactone 3 is reduced to the
corresponding hemiketal with the use of a reducing agent, such as
DIBAL-H; the hemiketal formed is treated directly with the ylid 3a
to give the crotonitrile derivative 4. The nitrile 4 then reacts
with the pyrazole 5 in the presence of a base such as DBU to give 6
as a mixture of diastereomers, which is converted to the alcohols I
after removal of the SEM group. The individual stereoisomers of
this mixture (I) can be separated by chiral chromatography to give
the enantiomerically pure alcohols (4 total stereoisomers).
##STR00005##
[0030] As shown in Scheme 2, the synthesis of the trans alcohols II
starts with the diastereomeric mixture of alcohols 6. The
diastereomeric mixture of alcohols 6 is treated with benzoic acid
under the Mitsunobu conditions to give a mixture of the trans
benzoates 7 with complete inversion. The mixture of the benzoates 7
is hydrolyzed by treatment with a base such as LiOH to give a
mixture of the trans alcohols 8. The SEM group within the alcohols
8 is then removed to give the diastereomeric mixture of the trans
alcohols II, which is separated by chiral chromatography to give
individual stereoisomers (4 total stereoisomers).
##STR00006##
[0031] The synthesis of the ketones III is described in Scheme 3. A
mixture of the cis alcohols 6 can be oxidized under Swern
conditions to give the corresponding mixture of ketones 9. The SEM
group within the ketones 9 is removed to give a mixture of the
ketone III, which can be separated by chiral chromatography to give
the individual stereoisomers (4 total stereoisomers).
##STR00007##
Methods
[0032] Compounds of the invention can modulate activity of one or
more Janus kinases (JAKs). The term "modulate" is meant to refer to
an ability to increase or decrease the activity of one or more
members of the JAK family of kinases. Accordingly, compounds of the
invention can be used in methods of modulating a JAK by contacting
the JAK with any one or more of the compounds or compositions
described herein. In some embodiments, compounds of the present
invention can act as inhibitors of one or more JAKs. In some
embodiments, compounds of the present invention can act to
stimulate the activity of one or more JAKs. In further embodiments,
the compounds of the invention can be used to modulate activity of
a JAK in an individual in need of modulation of the receptor by
administering a modulating amount of a compound of the
invention.
[0033] JAKs to which the present compounds bind and/or modulate
include any member of the JAK family. In some embodiments, the JAK
is JAK1, JAK2, JAK3 or TYK2. In some embodiments, the JAK is JAK1
or JAK2. In some embodiments, the JAK is JAK2. In some embodiments,
the JAK is JAK3.
[0034] The compounds of the invention can be selective. By
"selective" is meant that the compound binds to or inhibits a JAK
with greater affinity or potency, respectively, compared to at
least one other JAK. In some embodiments, the compounds of the
invention are selective inhibitors of JAK1 or JAK2 over JAK3 and/or
TYK2. In some embodiments, the compounds of the invention are
selective inhibitors of JAK2 (e.g., over JAK1, JAK3 and TYK2).
Without wishing to be bound by theory, because inhibitors of JAK3
can lead to immunosuppressive effects, a compound which is
selective for JAK2 over JAK3 and which is useful in the treatment
of cancer (such as multiple myeloma, for example) can offer the
additional advantage of having fewer immunosuppressive side
effects. Selectivity can be at least about 5-fold, 10-fold, at
least about 20-fold, at least about 50-fold, at least about
100-fold, at least about 200-fold, at least about 500-fold or at
least about 1000-fold. Selectivity can be measured by methods
routine in the art. In some embodiments, selectivity can be tested
at the Km of each enzyme. In some embodiments, selectivity of
compounds of the invention for JAK2 over JAK3 can be determined by
the cellular ATP concentration.
[0035] Another aspect of the present invention pertains to methods
of treating a JAK-associated disease or disorder in an individual
(e.g., patient) by administering to the individual in need of such
treatment a therapeutically effective amount or dose of a compound
of the present invention or a pharmaceutical composition thereof. A
JAK-associated disease can include any disease, disorder or
condition that is directly or indirectly linked to expression or
activity of the JAK, including overexpression and/or abnormal
activity levels. A JAK-associated disease can also include any
disease, disorder or condition that can be prevented, ameliorated,
or cured by modulating JAK activity.
[0036] Examples of JAK-associated diseases include diseases
involving the immune system including, for example, organ
transplant rejection (e.g., allograft rejection and graft versus
host disease).
[0037] Further examples of JAK-associated diseases include
autoimmune diseases such as multiple sclerosis, rheumatoid
arthritis, juvenile arthritis, type I diabetes, lupus, psoriasis,
inflammatory bowel disease, ulcerative colitis, Crohn's disease,
myasthenia gravis, immunoglobulin nephropathies, autoimmune thyroid
disorders, and the like. In some embodiments, the autoimmune
disease is an autoimmune bullous skin disorder such as pemphigus
vulgaris (PV) or bullous pemphigoid (BP).
[0038] Further examples of JAK-associated diseases include allergic
conditions such as asthma, food allergies, atopic dermatitis and
rhinitis. Further examples of JAK-associated diseases include viral
diseases such as Epstein Barr Virus (EBV), Hepatitis B, Hepatitis
C, HIV, HTLV 1, Varicella-Zoster Virus (VZV) and Human Papilloma
Virus (HPV).
[0039] Further examples of JAK-associated diseases or conditions
include skin disorders such as psoriasis (for example, psoriasis
vulgaris), atopic dermatitis, skin rash, skin irritation, skin
sensitization (e.g., contact dermatitis or allergic contact
dermatitis). For example, certain substances including some
pharmaceuticals when topically applied can cause skin
sensitization. In some embodiments, co-administration or sequential
administration of at least one JAK inhibitor of the invention
together with the agent causing unwanted sensitization can be
helpful in treating such unwanted sensitization or dermatitis. In
some embodiments, the skin disorder is treated by topical
administration of at least one JAK inhibitor of the invention.
[0040] In further embodiments, the JAK-associated disease is cancer
including those characterized by solid tumors (e.g., prostate
cancer, renal cancer, hepatic cancer, pancreatic cancer, gastric
cancer, breast cancer, lung cancer, cancers of the head and neck,
thyroid cancer, glioblastoma, Kaposi's sarcoma, Castleman's
disease, melanoma etc.), hematological cancers (e.g., lymphoma,
leukemia such as acute lymphoblastic leukemia, acute myelogenous
leukemia (AML) or multiple myeloma), and skin cancer such as
cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma.
Example cutaneous T-cell lymphomas include Sezary syndrome and
mycosis fungoides.
[0041] JAK-associated diseases can further include those
characterized by expression of a mutant JAK2 such as those having
at least one mutation in the pseudo-kinase domain (e.g.,
JAK2V617F).
[0042] JAK-associated diseases can further include
myeloproliferative disorders (MPDs) such as polycythemia vera (PV),
essential thrombocythemia (ET), myeloid metaplasia with
myelofibrosis (MMM), chronic myelogenous leukemia (CML), chronic
myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES),
systemic mast cell disease (SMCD), and the like.
[0043] Further JAK-associated diseases include inflammation and
inflammatory diseases. Example inflammatory diseases include
inflammatory diseases of the eye (e.g., iritis, uveitis, scleritis,
conjunctivitis, or related disease), inflammatory diseases of the
respiratory tract (e.g., the upper respiratory tract including the
nose and sinuses such as rhinitis or sinusitis or the lower
respiratory tract including bronchitis, chronic obstructive
pulmonary disease, and the like), inflammatory myopathy such as
myocarditis, and other inflammatory diseases. Other inflammatory
diseases treatable by the compounds of the invention include
systemic inflammatory response syndrome (SIRS) and septic
shock.
[0044] The JAK inhibitors described herein can further be used to
treat ischemia reperfusion injuries or a disease or condition
related to an inflammatory ischemic event such as stroke or cardiac
arrest. The JAK inhibitors described herein can further be used to
treat anorexia, cachexia, or fatigue such as that resulting from or
associated with cancer. The JAK inhibitors described herein can
further be used to treat restenosis, sclerodermitis, or fibrosis.
The JAK inhibitors described herein can further be used to treat
conditions associated with hypoxia or astrogliosis such as, for
example, diabetic retinopathy, cancer, or neurodegeneration. See,
e.g., Dudley, A. C. et al. Biochem. J. 2005, 390(Pt 2):427-36 and
Sriram, K. et al. J. Biol. Chem. 2004, 279(19):19936-47. Epub 2004
Mar. 2.
[0045] The JAK inhibitors described herein can further be used to
treat gout and increased prostate size due to, e.g., benign
prostatic hypertrophy or benign prostatic hyperplasia.
[0046] As used herein, the term "contacting" refers to the bringing
together of indicated moieties in an in vitro system or an in vivo
system. For example, "contacting" a JAK with a compound of the
invention includes the administration of a compound of the present
invention to an individual or patient, such as a human, having a
JAK, as well as, for example, introducing a compound of the
invention into a sample containing a cellular or purified
preparation containing the JAK.
[0047] As used herein, the term "individual" or "patient," used
interchangeably, refers to any animal, including mammals,
preferably mice, rats, other rodents, rabbits, dogs, cats, swine,
cattle, sheep, horses, or primates, and most preferably humans.
[0048] As used herein, the phrase "therapeutically effective
amount" refers to the amount of active compound or pharmaceutical
agent that elicits the biological or medicinal response that is
being sought in a tissue, system, animal, individual or human by a
researcher, veterinarian, medical doctor or other clinician.
[0049] As used herein, the term "treating" or "treatment" refers to
one or more of (1) preventing the disease; for example, preventing
a disease, condition or disorder in an individual who may be
predisposed to the disease, condition or disorder but does not yet
experience or display the pathology or symptomatology of the
disease; (2) inhibiting the disease; for example, inhibiting a
disease, condition or disorder in an individual who is experiencing
or displaying the pathology or symptomatology of the disease,
condition or disorder; and (3) ameliorating the disease; for
example, ameliorating a disease, condition or disorder in an
individual who is experiencing or displaying the pathology or
symptomatology of the disease, condition or disorder (i.e.,
reversing the pathology and/or symptomatology) such as decreasing
the severity of disease.
Combination Therapies
[0050] One or more additional pharmaceutical agents such as, for
example, chemotherapeutics, anti-inflammatory agents, steroids,
immunosuppressants, as well as Bcr-Abl, Flt-3, RAF and FAK kinase
inhibitors such as, for example, those described in WO 2006/056399,
or other agents can be used in combination with the compounds of
the present invention for treatment of JAK-associated diseases,
disorders or conditions. The one or more additional pharmaceutical
agents can be administered to a patient simultaneously or
sequentially.
[0051] Example chemotherapeutics include proteosome inhibitors
(e.g., bortezomib), thalidomide, revlimid, and DNA-damaging agents
such as melphalan, doxorubicin, cyclophosphamide, vincristine,
etoposide, carmustine, and the like.
[0052] Example steroids include coriticosteroids such as
dexamethasone or prednisone.
[0053] Example Bcr-Abl inhibitors include the compounds, and
pharmaceutically acceptable salts thereof, of the genera and
species disclosed in U.S. Pat. No. 5,521,184, WO 04/005281,
EP2005/009967, EP2005/010408, and U.S. Ser. No. 60/578,491.
[0054] Example suitable Flt-3 inhibitors include compounds, and
their pharmaceutically acceptable salts, as disclosed in WO
03/037347, WO 03/099771, and WO 04/046120.
[0055] Example suitable RAF inhibitors include compounds, and their
pharmaceutically acceptable salts, as disclosed in WO 00/09495 and
WO 05/028444.
[0056] Example suitable FAK inhibitors include compounds, and their
pharmaceutically acceptable salts, as disclosed in WO 04/080980, WO
04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO
01/014402.
[0057] In some embodiments, one or more of the metabolites of the
invention can be used in combination with one or more other kinase
inhibitors including imatinib, particularly for treating patients
resistant to imatinib or other kinase inhibitors.
[0058] In some embodiments, one or more JAK inhibitors of the
invention can be used in combination with a chemotherapeutic in the
treatment of cancer, such as multiple myeloma, and may improve the
treatment response as compared to the response to the
chemotherapeutic agent alone, without exacerbation of its toxic
effects. Examples of additional pharmaceutical agents used in the
treatment of multiple myeloma, for example, can include, without
limitation, melphalan, melphalan plus prednisone [MP], doxorubicin,
dexamethasone, and Velcade (bortezomib). Further additional agents
used in the treatment of multiple myeloma include Bcr-Abl, Flt-3,
RAF and FAK kinase inhibitors. Additive or synergistic effects are
desirable outcomes of combining a JAK inhibitor of the present
invention with an additional agent. Furthermore, resistance of
multiple myeloma cells to agents such as dexamethasone may be
reversible upon treatment with a JAK inhibitor of the present
invention. The agents can be combined with the present compounds in
a single or continuous dosage form, or the agents can be
administered simultaneously or sequentially as separate dosage
forms.
[0059] In some embodiments, a corticosteroid such as dexamethasone
is administered to a patient in combination with at least one JAK
inhibitor where the dexamethasone is administered intermittently as
opposed to continuously.
[0060] In some further embodiments, combinations of one or more JAK
inhibitors of the invention with other therapeutic agents can be
administered to a patient prior to, during, and/or after a bone
marrow transplant or stem cell transplant.
Pharmaceutical Formulations and Dosage Forms
[0061] When employed as pharmaceuticals, the compounds of the
invention can be administered in the form of pharmaceutical
compositions. These compositions can be prepared in a manner well
known in the pharmaceutical art, and can be administered by a
variety of routes, depending upon whether local or systemic
treatment is desired and upon the area to be treated.
Administration may be topical (including transdermal, epidermal,
ophthalmic and to mucous membranes including intranasal, vaginal
and rectal delivery), pulmonary (e.g., by inhalation or
insufflation of powders or aerosols, including by nebulizer;
intratracheal or intranasal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal, intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Parenteral administration can be in the form of a
single bolus dose, or may be, for example, by a continuous
perfusion pump. Pharmaceutical compositions and formulations for
topical administration may include transdermal patches, ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids and
powders. Conventional pharmaceutical carriers, aqueous, powder or
oily bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful.
[0062] This invention also includes pharmaceutical compositions
which contain, as the active ingredient, one or more of the
compounds of the invention above in combination with one or more
pharmaceutically acceptable carriers (excipients). In making the
compositions of the invention, the active ingredient is typically
mixed with an excipient, diluted by an excipient or enclosed within
such a carrier in the form of, for example, a capsule, sachet,
paper, or other container. When the excipient serves as a diluent,
it can be a solid, semi-solid, or liquid material, which acts as a
vehicle, carrier or medium for the active ingredient. Thus, the
compositions can be in the form of tablets, pills, powders,
lozenges, sachets, cachets, elixirs, suspensions, emulsions,
solutions, syrups, aerosols (as a solid or in a liquid medium),
ointments containing, for example, up to 10% by weight of the
active compound, soft and hard gelatin capsules, suppositories,
sterile injectable solutions, and sterile packaged powders.
[0063] In preparing a formulation, the active compound can be
milled to provide the appropriate particle size prior to combining
with the other ingredients. If the active compound is substantially
insoluble, it can be milled to a particle size of less than 200
mesh. If the active compound is substantially water soluble, the
particle size can be adjusted by milling to provide a substantially
uniform distribution in the formulation, e.g. about 40 mesh.
[0064] The compounds of the invention may be milled using known
milling procedures such as wet milling to obtain a particle size
appropriate for tablet formation and for other formulation types.
Finely divided (nanoparticulate) preparations of the compounds of
the invention can be prepared by processes known in the art, for
example see International Patent Application No. WO
2002/000196.
[0065] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, and methyl cellulose. The formulations can
additionally include: lubricating agents such as talc, magnesium
stearate, and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and
propylhydroxy-benzoates; sweetening agents; and flavoring agents.
The compositions of the invention can be formulated so as to
provide quick, sustained or delayed release of the active
ingredient after administration to the patient by employing
procedures known in the art.
[0066] The compositions can be formulated in a unit dosage form,
each dosage containing from about 5 to about 1000 mg (1 g), more
usually about 100 to about 500 mg, of the active ingredient. The
term "unit dosage forms" refers to physically discrete units
suitable as unitary dosages for human subjects and other mammals,
each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient.
[0067] The active compound can be effective over a wide dosage
range and is generally administered in a pharmaceutically effective
amount. It will be understood, however, that the amount of the
compound actually administered will usually be determined by a
physician, according to the relevant circumstances, including the
condition to be treated, the chosen route of administration, the
actual compound administered, the age, weight, and response of the
individual patient, the severity of the patient's symptoms, and the
like.
[0068] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical
excipient to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention. When
referring to these preformulation compositions as homogeneous, the
active ingredient is typically dispersed evenly throughout the
composition so that the composition can be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation is then subdivided into unit
dosage forms of the type described above containing from, for
example, about 0.1 to about 1000 mg of the active ingredient of the
present invention.
[0069] The tablets or pills of the present invention can be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0070] The liquid forms in which the compounds and compositions of
the present invention can be incorporated for administration orally
or by injection include aqueous solutions, suitably flavored
syrups, aqueous or oil suspensions, and flavored emulsions with
edible oils such as cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0071] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described supra. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions can be nebulized
by use of inert gases. Nebulized solutions may be breathed directly
from the nebulizing device or the nebulizing device can be attached
to a face masks tent, or intermittent positive pressure breathing
machine. Solution, suspension, or powder compositions can be
administered orally or nasally from devices which deliver the
formulation in an appropriate manner.
[0072] The amount of compound or composition administered to a
patient will vary depending upon what is being administered, the
purpose of the administration, such as prophylaxis or therapy, the
state of the patient, the manner of administration, and the like.
In therapeutic applications, compositions can be administered to a
patient already suffering from a disease in an amount sufficient to
cure or at least partially arrest the symptoms of the disease and
its complications. Effective doses will depend on the disease
condition being treated as well as by the judgment of the attending
clinician depending upon factors such as the severity of the
disease, the age, weight and general condition of the patient, and
the like.
[0073] The compositions administered to a patient can be in the
form of pharmaceutical compositions described above. These
compositions can be sterilized by conventional sterilization
techniques, or may be sterile filtered. Aqueous solutions can be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile aqueous carrier prior to
administration. The pH of the compound preparations typically will
be between 3 and 11, more preferably from 5 to 9 and most
preferably from 7 to 8. It will be understood that use of certain
of the foregoing excipients, carriers, or stabilizers will result
in the formation of pharmaceutical salts.
[0074] The therapeutic dosage of the compounds of the present
invention can vary according to, for example, the particular use
for which the treatment is made, the manner of administration of
the compound, the health and condition of the patient, and the
judgment of the prescribing physician. The proportion or
concentration of a compound of the invention in a pharmaceutical
composition can vary depending upon a number of factors including
dosage, chemical characteristics (e.g., hydrophobicity), and the
route of administration. For example, the compounds of the
invention can be provided in an aqueous physiological buffer
solution containing about 0.1 to about 10% w/v of the compound for
parenteral administration. Some typical dose ranges are from about
1 .mu.g/kg to about 1 g/kg of body weight per day. In some
embodiments, the dose range is from about 0.01 mg/kg to about 100
mg/kg of body weight per day. The dosage is likely to depend on
such variables as the type and extent of progression of the disease
or disorder, the overall health status of the particular patient,
the relative biological efficacy of the compound selected,
formulation of the excipient, and its route of administration.
Effective doses can be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0075] The compositions of the invention can further include one or
more additional pharmaceutical agents such as a chemotherapeutic,
steroid, anti-inflammatory compound, or immunosuppressant, examples
of which are listed hereinabove.
Labeled Compounds and Assay Methods
[0076] Another aspect of the present invention relates to labeled
compounds of the invention (radio-labeled, fluorescent-labeled,
etc.) that would be useful not only in imaging techniques but also
in assays, both in vitro and in vivo, for localizing and
quantitating JAK in tissue samples, including human, and for
identifying JAK ligands by inhibition binding of a labeled
compound. Accordingly, the present invention includes JAK assays
that contain such labeled compounds.
[0077] The present invention further includes isotopically-labeled
compounds of the invention. An "isotopically" or "radio-labeled"
compound is a compound of the invention where one or more atoms are
replaced or substituted by an atom having an atomic mass or mass
number different from the atomic mass or mass number typically
found in nature (i.e., naturally occurring). Suitable radionuclides
that may be incorporated in compounds of the present invention
include but are not limited to .sup.2H (also written as D for
deuterium), .sup.3H (also written as T for tritium), .sup.11C,
.sup.13C, .sup.14N, .sup.15N, .sup.15O, .sup.17O, .sup.18O,
.sup.18F, .sup.35S, .sup.36Cl, .sup.82Br, .sup.75Br, .sup.76Br,
.sup.77Br, .sup.123I, .sup.124I, .sup.125I and .sup.131I. The
radionuclide that is incorporated in the instant radio-labeled
compounds will depend on the specific application of that
radio-labeled compound. For example, for in vitro metalloprotease
labeling and competition assays, compounds that incorporate
.sup.3H, .sup.14C, .sup.82Br, .sup.125I, .sup.131I, .sup.35S or
will generally be most useful. For radio-imaging applications
.sup.11C, .sup.18F, .sup.125I, .sup.123I, .sup.124I, .sup.131I,
.sup.75Br, .sup.76Br or .sup.77Br will generally be most
useful.
[0078] It is understood that a "radio-labeled" or "labeled
compound" is a compound that has incorporated at least one
radionuclide. In some embodiments the radionuclide is selected from
the group consisting of .sup.3H, .sup.14C, .sup.125I, .sup.35S and
.sup.82Br.
[0079] The present invention can further include synthetic methods
for incorporating radio-isotopes into compounds of the invention.
Synthetic methods for incorporating radio-isotopes into organic
compounds are well known in the art, and a person of ordinary skill
in the art will readily recognize the methods applicable for the
compounds of invention.
[0080] A labeled compound of the invention can be used in a
screening assay to identify/evaluate compounds. For example, a
newly synthesized or identified compound (i.e., test compound)
which is labeled can be evaluated for its ability to bind a JAK by
monitoring its concentration variation when contacting with the
JAK, through tracking of the labeling. For example, a test compound
(labeled) can be evaluated for its ability to reduce binding of
another compound which is known to bind to a JAK (i.e., standard
compound). Accordingly, the ability of a test compound to compete
with the standard compound for binding to the JAK directly
correlates to its binding affinity. Conversely, in some other
screening assays, the standard compound is labeled and test
compounds are unlabeled. Accordingly, the concentration of the
labeled standard compound is monitored in order to evaluate the
competition between the standard compound and the test compound,
and the relative binding affinity of the test compound is thus
ascertained.
Kits
[0081] The present invention also includes pharmaceutical kits
useful, for example, in the treatment or prevention of
JAK-associated diseases or disorders, such as cancer, which include
one or more containers containing a pharmaceutical composition
comprising a therapeutically effective amount of a compound of the
invention. Such kits can further include, if desired, one or more
of various conventional pharmaceutical kit components, such as, for
example, containers with one or more pharmaceutically acceptable
carriers, additional containers, etc., as will be readily apparent
to those skilled in the art. Instructions, either as inserts or as
labels, indicating quantities of the components to be administered,
guidelines for administration, and/or guidelines for mixing the
components, can also be included in the kit.
[0082] The invention will be described in greater detail by way of
specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of noncritical parameters which can be changed or modified
to yield essentially the same results.
EXAMPLES
Example 1
3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-
-pyrazol-1-yl]propanenitrile and
3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1-
H-pyrazol-1-yl]propanenitrile
##STR00008##
[0083] Step 1. 6-bromo-2-oxabicyclo[2.2. 1]heptan-3-one
##STR00009##
[0085] Bromotrimethysilane (3.1 mL, 0.023 mol) was added dropwise
to a solution of dimethyl sulfoxide (1.6 mL, 0.023 mol) in
chloroform (38.0 mL) in a round-bottom flask at 0.degree. C. The
resulting mixture was stirred at 0.degree. C. for 2 hours. To the
reaction mixture was added dropwise a solution of
cyclopent-3-ene-1-carboxylic acid (2.00 g, 0.0178 mol) in
chloroform (12 mL) over a period of 15 minutes and the reaction
mixture was stirred at 0.degree. C. for 10 minutes.
N,N-diisopropylethylamine (4.0 mL, 0.023 mol) was then added and
the resulting mixture was stirred at 0.degree. C. After 10 minutes,
the mixture was heated to reflux for 16 hours. The reaction mixture
was diluted with chloroform, washed with water, brine, dried
(MgSO.sub.4), and stripped in vacuo. The residue was purified by
chromatography on silica gel using 30% EtOAc/hexanes as eluent to
give the product. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 4.88
(brs, 1H), 4.34 (m, 1H), 2.90 (m, 1H), 2.66 (m, 1H), 2.31 (m, 1H),
1.93 (m, 1H), 1.83 (m, 1H).
Step 2. 2-oxabicyclo[2.2.1]heptan-3-one
##STR00010##
[0087] Tris(trimethylsilyl)silane (4.7 mL, 15 mmol) was added
dropwise to a solution of 6-bromo-2-oxabicyclo[2.2.1]heptan-3-one
(1.96 g, 10.3 mmol) and 2,2'-azo-bis-isobutyronitrile (0.2 g, 1
mmol) in toluene (100 mL) in a round-bottom flask and the resulting
mixture was stirred at 80.degree. C. for 5 hours. The reaction
mixture was concentrated by rotoary evaporation and the residue was
diluted with ethyl acetate, washed with saturated NH.sub.4Cl, dried
(MgSO.sub.4), and stripped in vacuo. The residue was purified by
chromatography on silica gel using 100% hexanes, grading to 25%
EtOAc/hexanes then 33% EtOAc/hexanes as eluents to give the
product. .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 4.93 (m, 1H),
2.91 (m, 1H), 2.19 (m, 1H), 1.60-1.99 (m, 5H).
Step 3. (2E)- and
(2Z)-3-[(1S,3R)-3-hydroxycyclopentyl]acrylonitrile and (2E)- and
(2Z)-3-[(1R,3S)-3-hydroxycyclopentyl]acrylonitrile
##STR00011##
[0089] 1.00 M of diisobutylaluminum hydride in toluene (8.0 mL) was
added dropwise to a solution of 2-oxabicyclo[2.2.1]heptan-3-one
(600 mg, 5 mmol) in methylene chloride (20 mL) in a round-bottom
flask at -78.degree. C. The resulting mixture was stirred at
-78.degree. C. for 45 minutes. The reaction mixture was treated
with saturated Rochelle's salt solution. After stirring for 15
minutes, the reaction mixture was extracted with ethyl acetate and
the combined organic extracts were washed with water, saturated
NaCl, dried (MgSO.sub.4), and stripped in vacuo. The crude product
was used in the next reaction without further purification.
[0090] A solution of the crude 2-oxabicyclo[2.2.1]heptan-3-ol (400
mg, 4 mmol) and (triphenylphosphoranylidene)acetonitrile (1.2 g,
3.8 mmol) in toluene (12 mL) in a round-bottom flask was heated at
80.degree. C. for 2 hours. The reaction mixture was then purified
by chromatography on silica gel using 40% EtOAc/hexanes to give the
racemic products. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 6.78
(dd, 1H), 5.30 (d, 1H), 5.20 (m, 1H), 2.67 (m, 1H), 2.20 (m, 1H),
1.40-1.90 (m, 6H).
Step 4.
3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy-
]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
and
3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]me-
thyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
##STR00012##
[0092] 1,8-Diazabicyclo[5.4.0]undec-7-ene (0.54 mL, 3.6 mmol) was
added to a solution of a mixture of (2E)- and
(2Z)-3-[(1S,3R)-3-hydroxycyclopentyl]acrylonitrile and (2E)- and
(2Z)-3-[(1R,3S)-3-hydroxycyclopentyl]acrylonitrile (0.250 g, 1.82
mmol) and
4-(1H-pyrazol-4-yl)-7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-
-d]pyrimidine (0.57 g, 1.8 mmol) in acetonitrile (5 mL) in a
round-bottom flask. The resulting mixture was stirred at 25.degree.
C. for 2 days at which time LCMS analysis showed .about.80% of the
starting materials had been consumed. The reaction mixture was
purified by chromatography on silica gel using 1:1 EtOAc/hexanes to
give the product. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.90
(d, 1H), 8.39 (m, 2H), 7.46 (m, 1H), 6.86 (m 1H), 5.73 (s, 2H),
4.52 (m, 2H), 3.59 (m, 2H), 3.2 (m, 1H), 3.02 (m, 1H), 2.78 (m,
1H), 2.3 (m, 1H), 1.30-1.90 (m, 6H), 0.99 (m, 2H), 0.08 (s, 9H).
LC/MS: 453 (M+H).sup.+.
Step 5.
3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin--
4-yl)-1H-pyrazol-1-yl]propanenitrile and
3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1-
H-pyrazol-1-yl]propanenitrile
[0093] Lithium tetrafluoroborate (0.176 g, 1.88 mmol) was added to
a solution of
3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-
-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1-pyrazol-1-yl]propanenitrile and
3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-
-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
(85.0 mg, 0.188 mmol) in acetonitrile (1.5 mL) and water (0.135 mL)
in a vial. The resulting mixture was heated at 85.degree. C. for 26
hours. After the reaction mixture was allowed to cool to 25.degree.
C., ethylenediamine (63 .mu.L, 0.94 mmol) was added and the
resulting mixture was stirred at 25.degree. C. for 3 hours. The
reaction mixture was purified by prep. LC to give the product as
the trifluoroacetic acid salt. This was dissolved in methanol and
Amberlyst 26 was added. The resulting mixture was stirred for 10
minutes, filtered, and concentrated. The residue was purified by
chiral chromatography to give 4 major peaks and 4 minor peaks.
(Column: ChiralPak IA, 4.6.times.250 mm, 5 micron particle. Mobile
phase: 30% Ethanol in hexanes. Flow Rate: 0.8 ml/min-analytical;
Column: ChiralPak IA, 20.times.250 mm, 5 micron particle. Mobile
Phase: 30% Ethanol in hexanes. Flow rate: 12 ml/min-preparative)
The minor peaks were attributed to the trifluoroacetate esters
which are very mobile and are cleaved on standing in methanol to
the corresponding alcohols.
[0094] Major Peak 1 [Retention time: 18.56 minutes]: .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 8.66 (brs, 1H), 8.64 (s, 1H), 8.38
(s, 1H), 7.51 (m, 1H), 6.97 (m, 1H), 4.57 (m, 1H), 4.20 (m, 1H),
3.16 (m, 2H), 2.65 (m, 1H), 1.64-2.00 (m, 5H), 1.28 (m, 1H). LC/MS:
323 (M+H).sup.+.
[0095] Major Peak 2 [Retention time: 25.88 minutes]: .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 8.66 (brs, 1H), 8.64 (s, 1H), 8.38
(s, 1H), 7.50 (m, 1H), 6.96 (m, 1H), 4.60 (m, 1H), 4.30 (m, 1H),
3.18 (m, 2H), 2.61 (m, 1H), 2.23 (m, 1H), 1.40-1.80 (m, 5H). LC/MS:
323 (M+H).sup.+.
[0096] Major Peak 3 [Retention time: 39.84 minutes]: .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 8.66 (brs, 1H), 8.64 (s, 1H), 8.38
(s, 1H), 7.50 (m, 1H), 6.96 (m, 1H), 4.60 (m, 1H), 4.30 (m, 1H),
3.18 (m, 2H), 2.61 (m, 1H), 2.23 (m, 1H), 1.40-1.80 (m, 5H). LC/MS:
323 (M+H).sup.+.
[0097] Major Peak 4 [Retention time: 51.48 minutes]: .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 8.66 (brs, 1H), 8.64 (s, 1H), 8.38
(s, 1H), 7.51 (m, 1H), 6.97 (m, 1H), 4.57 (m, 1H), 4.20 (m, 1H),
3.16 (m, 2H), 2.65 (m, 1H), 1.64-2.00 (m, 5H), 1.28 (m, 1H). LC/MS:
323 (M+H).sup.+.
Example 2
3-[(1S,3S)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-
-pyrazol-1-yl]propanenitrile trifluoroacetic acid salt and
3-[(1R,3R)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1-
H-pyrazol-1-yl]propanenitrile trifluoroacetic acid salt
##STR00013##
[0098] Step 1:
(1S,3S)-3-{2-cyano-1-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,-
3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]ethyl}cyclopentyl benzoate and
(1R,3R)-3-{2-cyano-1-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,-
3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]ethyl}cyclopentyl benzoate
[0099] Diisopropyl azodicarboxylate (0.38 mL, 1.9 mmol) was added
to a solution of
3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-
-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
and
3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-
-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
(0.51 g, 1.9 mmol) in tetrahydrofuran (5.3 mL) in a round-bottom
flask at 0.degree. C. The resulting mixture was stirred for 10
minutes and benzoic acid (0.24 g, 1.9 mmol) was added. The reaction
mixture was stirred at 0.degree. C. for 2 hours at which time TLC
analysis showed no starting material. The reaction mixture was
diluted with ethyl acetate, washed with sat. NaHCO.sub.3, water,
saturated NaCl, dried (MgSO.sub.4), and stripped in vacuo. The
residue was chromatographed on silica gel using 20% EtOAc/hexanes
to give the product. .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.
8.91 (d, 1H), 8.39 (m, 2H), 8.08 (m, 2H), 7.75 (m, 1H), 7.61 (m,
1H), 7.48 (m, 2H), 7.46 (m, 1H), 6.87 (m 1H), 5.74 (s, 2H),
5.40-5.50 (m, 1H), 4.40 (m, 1H), 3.60 (m, 2H), 3.25 (m, 1H), 3.07
(m, 1H), 2.27 (m, 2H), 1.30-1.90 (m, 6H), 0.99 (m, 2H), 0.08 (s,
9H). LC/MS: 557 (M+H).sup.+.
Step 2:
3-[(1S,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy-
]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
and
3-[(1R,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]me-
thyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
[0100] Lithium hydroxide (22.7 mg, 0.000948 mol) was added to a
solution of
(1S,3S)-3-{2-cyano-1-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo-
[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]ethyl}cyclopentyl benzoate
and
(1R,3R)-3-{2-cyano-1-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,-
3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]ethyl}cyclopentyl benzoate (440
mg, 0.00079 mol) dissolved in a mixture of 1,4-dioxane (10.0 mL,
0.128 mol), methanol (4.0 mL, 0.099 mol), and water (4.0 mL, 0.22
mol) in a round-bottom flask. The resulting mixture was stirred for
20 hours at which time LCMS analysis showed no starting material.
The reaction mixture was extracted with ethyl acetate and the
organic extracts were washed with sat. NaHCO.sub.3, water,
saturated NaCl, dried (MgSO.sub.4), and stripped in vacuo. The
residue was used in the next reaction without further purification.
LC/MS: 453 (M+H).sup.+.
Step 3:
3-[(1S,3S)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin--
4-yl)-1H-pyrazol-1-yl]propanenitrile trifluoroacetic acid salt and
3-[(1R,3R)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1-
H-pyrazol-1-yl]propanenitrile trifluoroacetic acid salt
[0101] The mixture of
3-[(1S,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-
-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
and
3-[(1R,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-
-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
was deprotected under the same conditions described in Example 1,
Step 5. The mixture was separated using chiral LC and further
purified by LC to give the isomers as the trifluoroacetate salts.
Column: ChiralPak IA, 4.6.times.250 mm, 5 micron particle. Mobile
phase: 30% Ethanol in hexanes. Flow Rate: 0.8 ml/min-analytical;
Column: ChiralPak IA, 20.times.250 mm, 5 micron particle.
MobilePhase: 30% Ethanol in hexanes. Flow rate: 12 ml/min
preparative).
[0102] Pk1 [Retention time: 16.98 minutes]: .sup.1H (500 MHz,
DMSO-d.sub.6): .delta. 8.11 (brs, 1H), 8.07 (brs, 1H), 7.70 (s,
1H), 7.03 (d, 1H), 6.46 (m, 1H), 3.80 (m, 1H), 3.43 (m, 1H), 2.20
(m, 2H), 2.08 (m, 1H), 1.29 (m, 1H), 1.20 (m, 1H), 0.60-0.90 (m,
4H). LC/MS: 323 (M+H).sup.+.
[0103] Pk2 [Retention time: 18.68 minutes]: .sup.1H (500 MHz,
CD.sub.3OD): .delta. 8.91 (s, 1H), 8.087 (s, 1H), 8.51 (s, 1H),
7.84 (d, 1H), 7.28 (m, 1H), 4.60 (m, 1H), 4.34 (m, 1H), 3.20 (m,
2H), 2.91 (m, 1H), 1.92 (m, 2H), 1.60 (m, 3H), 1.35 (m, 1H). LC/MS:
323 (M+H).sup.+.
[0104] Pk3 and Pk4 eluted together (23.13 minutes).
Example 3
3-[(1S)-3-oxocyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazo-
l-1-yl]propanenitrile trifluoroacetate salt and
3-[(1R)-3-oxocyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyraz-
ol-1-yl]propanenitrile trifluoroacetic acid salt
##STR00014##
[0105] Step 1:
3-[(1S)-3-oxocyclopentyl-3-[4-(7-[2-(trimethylsilyl)ethoxymethyl-7H-pyrro-
lo[2,3-d]pyrimidin-4-yl)-]H-pyrazol-1-yl]propanenitrile and
3-[(]R)-3-oxocyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
[0106] Dimethyl sulfoxide (0.340 mL, 4.79 mmol) was added to a
solution of oxalyl chloride (0.20 mL, 2.4 mmol) in methylene
chloride (25 mL) -78.degree. C. in a round-bottom flask. The
resulting mixture was stirred at -78.degree. C. for 15 minutes and
a solution of
3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-
-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
and
3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-
-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile
(0.84 g, 1.8 mmol) in methylene chloride (17 mL) was added
dropwise. The resulting mixture was stirred at -78.degree. C. for
60 minutes and triethylamine (0.722 mL, 5.18 mmol) was added. After
stirring at -78.degree. C. for 60 minutes, the reaction mixture was
warmed to 0.degree. C. and stirred for 1 hour. The reaction mixture
was diluted with ethyl acetate, washed with water, saturated NaCl,
dried (MgSO.sub.4), and stripped in vacuo. The residue was purified
by chromatography on silica gel using 40% EtOAc/hexanes to give the
product. .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.91 (m, 1H),
8.40 (d, 1H), 8.38 (s, 1H), 7.47 (m, 1H), 6.85 (t, 1H), 5.74 (s,
2H), 4.51 (m, 1H), 3.60 (t, 2H), 3.00-3.30 (m, 3H), 1.50-2.70 (m,
6H), 0.98 (t, 2H), 0.00 (s, 9H). LC/MS: 451 (M+H).sup.+.
Step 2:
3-[(1S)-3-oxocyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1-
H-pyrazol-1-yl]propanenitrile trifluoroacetate salt and
3-[(1R)-3-oxocyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyraz-
ol-1-yl]propanenitrile trifluoroacetic acid salt
[0107] The mixture of
3-[(1S)-3-oxocyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile and
3-[(1R)-3-oxocyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile was
deprotected under conditions similar to Example 1, Step 5, to give
the two diastereomeric ketones which were separated by chiral
chromatography and purified by LC to give the diastereomers and
enantiomers as the trifluoroacetate salts. Column: ChiralPak IA,
4.6.times.250 mm, 5 micron particle. Mobile phase: 30% Ethanol in
hexanes. Flow Rate: 0.8 ml/min-analytical; Column: ChiralPak IA,
20.times.250 mm, 5 micron particle. MobilePhase: 30% Ethanol in
hexanes. Flow rate: 12 ml/min preparative).
[0108] Pk1 [Retention time: 11.82 minutes].
[0109] Pk2 [Retention time: 13.94 minutes]: .sup.1H (500 MHz,
CDCl.sub.3): .delta. 10.07 (brs, 1H), 8.79 (brs, 1H), 8.27 (s, 1H),
8.25 (s, 1H), 7.32 (d, 1H), 6.71 (m, 1H), 4.40 (m, 1H), 3.12 (m,
1H), 2.97 (m, 2H), 2.00-2.32 (m, 5H), 1.61 (m, 1H). LC/MS: 321
(M+H).sup.+.
[0110] Pk3 [Retention time: 17.61 minutes]: .sup.1H (500 MHz,
CDCl.sub.3): .delta. 10.70 (brs, 1H), 8.83 (brs, 1H), 8.34 (s, 1H),
8.30 (s, 1H), 7.35 (d, 1H), 6.73 (m, 1H), 4.37 (m, 1H), 3.10 (m,
1H), 2.90 (m, 2H), 2.51 (m, 1H), 2.27 (m, 1H), 2.15 (m, 1H), 1.91
(m, 1H), 1.84 (m, 1H), 1.60 (m, 1H). LC/MS: 321 (M+H).sup.+.
[0111] Pk4 [Retention time: 20.31 minutes].
Example A
TABLE-US-00002 [0112] TABLE 2 JAK 1 JAK 2 JAK 3 Fraction Human IC50
IC50 IC50 Unbound (% Intrinsic CL Compound (nM) (nM) (nM) human
serum) (L/h/kg) Compound 1 <10 <10 <10 <5 0.68
Metabolite 1 2.5-12 0.7-2.5 8.3-45 26-35 <0.50 Metabolite 2 3-15
2-2.8 17-30 5-27 <0.50 Metabolite 3 2.7-12 2.1-5.9 11-41 14-56
<0.57
[0113] Metabolites 1, 2, and 3 were isolated from rat or dog urine
after administration of Compound 1 in connection with
pharmacokinetic and toxicokinetic studies. Activity data for
Metabolites 1, 2, and 3, along with free fraction and intrinsic
clearance data, was compared with that for the parent compound,
Compound 1. JAK activity assays, free fraction assays, and
intrinsic clearance assays are described below. Data points were
obtained for some individual stereoisomers of Metabolites 1, 2, and
3, and the numerical range provided above reflects the highest and
lowest values obtained for all the stereoisomers tested. As can be
seen in Table 1, the metabolites are potent inhibitors of JAK1,
JAK2, and JAK3, like Compound 1. However, the free fractions
obtained for the metabolites are unexpectedly higher and the
intrinsic clearance desirably lower than for Compound 1.
In Vitro JAK Kinase Assay
[0114] Compounds herein were tested for inhibitory activity of JAK
targets according to the following in vitro assay described in Park
et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic
domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and
Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed
using baculovirus in insect cells and purified. The catalytic
activity of JAK1, JAK2 or JAK3 was assayed by measuring the
phosphorylation of a biotinylated peptide. The phosphorylated
peptide was detected by homogenous time resolved fluorescence
(HTRF). IC.sub.50s of compounds were measured for each kinase in
the reactions that contain the enzyme, ATP and 500 nM peptide in 50
mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL
(0.01%) BSA. The ATP concentration in the reactions was 90 .mu.M
for Jak1, 30 .mu.M for Jak2 and 3 .mu.M for Jak3. Reactions were
carried out at room temperature for 1 hr and then stopped with 20
.mu.L 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer
(Perkin Elmer, Boston, Mass.). Binding to the Europium labeled
antibody took place for 40 minutes and HTRF signal was measured on
a Fusion plate reader (Perkin Elmer, Boston, Mass.). Compounds
having an IC.sub.50 of 10 .mu.M or less for any of the
above-mentioned JAK targets were considered active.
Free Fraction Assay
[0115] The protein binding of a test compound was determined by
equilibrium dialysis using a Dianorm system from Harvard Apparatus
(Holliston, Mass.). The dialysis was performed at 37.degree. C. for
2 hrs in human serum. The metabolites were incubated at 3 .mu.M,
and Compound 1 at 3 and 10 .mu.M. The compound concentrations in
serum and buffer post-dialysis were determined by LC/MS/MS
analysis. Free fraction is defined as the ratio of the buffer
versus serum concentration.
Intrinsic Clearance Assay
[0116] Intrinsic clearance was determined by incubating 1 .mu.M of
test compound in human mixed gender liver microsomes (0.5 mg/mL
protein) at 37.degree. C. in the presence of 1 mM NADPH. The
disappearance of the test compound was monitored by LC/MS at 0, 5,
10, 20 and 30 min. The slope of decline in compound concentration
was used to calculate the human intrinsic clearance by employing
standard methods reported in the literature.
[0117] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference cited
in the present application is incorporated herein by reference in
its entirety.
* * * * *