U.S. patent application number 16/212447 was filed with the patent office on 2019-07-04 for selective dual inhibitors of pi3 delta and gamma protein kinases.
The applicant listed for this patent is Rhizen Pharmaceuticals SA. Invention is credited to Prashant K Bhavar, Swaroop K. Vakkalanka.
Application Number | 20190202836 16/212447 |
Document ID | / |
Family ID | 53761447 |
Filed Date | 2019-07-04 |
View All Diagrams
United States Patent
Application |
20190202836 |
Kind Code |
A1 |
Bhavar; Prashant K ; et
al. |
July 4, 2019 |
SELECTIVE DUAL INHIBITORS OF PI3 DELTA AND GAMMA PROTEIN
KINASES
Abstract
The present invention relates to a selective dual delta
(.delta.) and gamma (.gamma.) PI3K protein kinase modulator
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl) methane
sulfonamide, methods of preparing them, pharmaceutical compositions
containing them and methods of treatment, prevention and/or
amelioration of PI3K kinase mediated diseases or disorders with
them.
Inventors: |
Bhavar; Prashant K;
(Hyderabad, IN) ; Vakkalanka; Swaroop K.; (La
Chaux-de-Fonds, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rhizen Pharmaceuticals SA |
La Chaux-de-Fonds |
|
CH |
|
|
Family ID: |
53761447 |
Appl. No.: |
16/212447 |
Filed: |
December 6, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15480181 |
Apr 5, 2017 |
10179786 |
|
|
16212447 |
|
|
|
|
14752243 |
Jun 26, 2015 |
9708329 |
|
|
15480181 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/08 20180101;
A61P 9/00 20180101; A61P 29/00 20180101; A61P 11/06 20180101; A61P
19/02 20180101; A61P 17/06 20180101; C07C 303/38 20130101; C07D
487/04 20130101; C07F 5/025 20130101; A61P 35/02 20180101; A61P
25/00 20180101; A61P 11/00 20180101; A61K 31/519 20130101; A61P
37/00 20180101; A61P 37/06 20180101; A61P 43/00 20180101; A61P
35/00 20180101; A61K 45/06 20130101; A61P 7/02 20180101; A61P 3/00
20180101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; C07F 5/02 20060101 C07F005/02; A61K 31/519 20060101
A61K031/519; A61K 45/06 20060101 A61K045/06; C07C 303/38 20060101
C07C303/38 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 2014 |
IN |
3144/CHE/2014 |
Claims
1-26. (canceled)
27. A method of inhibiting a catalytic activity of a PI3 .delta.
kinase in a cell comprising contacting the cell with an effective
amount of
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonam-
ide or a pharmaceutically acceptable salt thereof.
28. A method of inhibiting a catalytic activity of a PI3 .gamma.
kinase in a cell comprising contacting the cell with an effective
amount of
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonam-
ide or a pharmaceutically acceptable salt thereof.
29. A method of inhibiting a catalytic activity of a PI3 .delta.
kinase and PI3 .gamma. kinase in a cell comprising contacting the
cell with an effective amount of
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonam-
ide or a pharmaceutically acceptable salt thereof.
30. The method of claim 27, wherein the inhibition takes place in a
subject suffering from a disease, disorder or condition selected
from a proliferative disease, a bone disorder, an inflammatory
disease, an immune disease, a nervous system disease, a metabolic
disease, a respiratory disease, thrombosis, cardiac disease, and
any combination thereof.
31. The method of claim 28, wherein the inhibition takes place in a
subject suffering from a disease, disorder or condition selected
from a proliferative disease, a bone disorder, an inflammatory
disease, an immune disease, a nervous system disease, a metabolic
disease, a respiratory disease, thrombosis, cardiac disease, and
any combination thereof.
32. The method of claim 29, wherein the inhibition takes place in a
subject suffering from a disease, disorder or condition selected
from a proliferative disease, a bone disorder, an inflammatory
disease, an immune disease, a nervous system disease, a metabolic
disease, a respiratory disease, thrombosis, cardiac disease, and
any combination thereof.
33. A method of treating a disease, disorder or condition that
would benefit from inhibiting catalytic activity of a PI3
.delta./.gamma. kinase comprising administering to a subject in
need thereof an effective amount of
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-c-
hromen-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)metha-
nesulfonamide or a pharmaceutically acceptable salt thereof.
34. A method for the treatment of a PI3K associated disease,
disorder or condition comprising administering to a subject in need
thereof an effective amount of
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonam-
ide or a pharmaceutically acceptable salt thereof.
35. The method of claim 34, further comprising administering an
additional active agent selected from anti-cancer agents,
anti-inflammatory agents, immunosuppressive agents, steroids,
non-steroidal anti-inflammatory agents, antihistamines, analgesics,
and any mixture thereof.
36. A compound selected from
(S)-2-(1-(4-amino-3-(4-methoxy-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,
(S)-2-(1-(4-amino-3-(3-amino-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and
pharmaceutically acceptable salts thereof.
Description
[0001] The present application claims the benefit of Indian Patent
Application No. 3144/CHE/2014, filed Jun. 27, 2014 which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides dual delta (.delta.) and
gamma (.gamma.) PI3K protein kinase modulators, methods of
preparing them, pharmaceutical compositions containing them and
methods of treatment, prevention and/or amelioration of PI3K kinase
mediated diseases or disorders using them.
BACKGROUND OF THE INVENTION
[0003] Phosphoinositide-3 kinase (PI3K) belongs to a class of
intracellular lipid kinases that phosphorylate the 3-position
hydroxyl group of the inositol ring of phosphoinositide lipids
(PIs) generating lipid second messengers. While .alpha. and .beta.
isoforms of PI3K are ubiquitous in their distribution, expression
of .delta. and .gamma. forms of PI3K is restricted to circulating
haematogenous cells and endothelial cells. Unlike PI3K.alpha. or
PI3K.beta., mice lacking expression of PI3K.delta. or PI3K.gamma.
do not show any adverse phenotype indicating that targeting of
these specific isoforms would not result in overt toxicity.
[0004] Recently, targeted inhibitors of the PI3K pathway have been
suggested as immunomodulatory agents. This interest stems from the
fact that the PI3K pathway serves multiple functions in immune cell
signaling, primarily through the generation of phosphatidylinositol
(3,4,5)-trisphosphate (PIP3), a membrane bound second messenger.
PIP3 recruits proteins to the cytoplasmic side of the lipid
bilayer, including protein kinases and GTPases, initiating a
complex network of downstream signaling cascades important in the
regulation of immune cell adhesion, migration, and cell-cell
communication.
[0005] The four class I PI3K isoforms differ significantly in their
tissue distribution. PI3K.alpha. and PI3K.beta. are ubiquitous and
activated downstream of receptor tyrosine kinases (RTK), whereas
PI3K.delta. and PI3K.gamma. are primarily limited to hematopoietic
and endothelial cells, and are activated downstream of RTKs, and G
protein coupled receptors (GPCR), respectively. Mouse genetic
studies have revealed that PI3K.alpha. and PI3K.beta. are essential
for normal development, whereas loss of PI3K.delta. and/or
PI3K.gamma. yields viable offspring with selective immune
deficits.
[0006] The expression pattern and functions of PI3K.delta. and
PI3K.gamma. have generated much interest in developing
PI3K.delta./.gamma. inhibitors as active agents for the treatment
of many diseases, including, for example, rheumatoid arthritis,
allergies, asthma, chronic obstructive pulmonary disease and
multiple sclerosis (Hirsch et al., Pharmacol. Ther., 118, 192-205,
2008; Marone et al., Biochim. Biophys. Acta., 1784, 159-185, 2008;
Rommel et al., Nat. Rev. Immunol., 7, 191-201, 2007; Ruckle et al.,
Nat. Rev. Drug Discov., 5, 903-918, 2006). Studies using both
pharmacologic and genetic methods have shown these two isoforms
often demonstrate synergistic interactions with each other (Konrad
et al., J. Biol. Chem., 283, 33296-33303, 2008; Laffargue et al.,
Immunity, 16, 441-451, 2002). In mast cells, for example,
PI3K.delta. is essential for degranulation in response to IgE
cross-linking of Fc-receptors (Ali et al., J. Immunol., 180,
2538-2544, 2008), while PI3K.gamma. plays an important role in
amplifying the response (Laffargue et al., Immunity, 16, 441-451,
2002). Similar effects have been seen in other cellular functions,
including lymphocyte homing and the neutrophil respiratory burst
where PI3K.gamma. plays a critical role and PI3K.delta. amplifies
each process. The nonredundant but related roles of PI3K.delta. and
PI3K.gamma. have made it difficult to determine which of the two
isoforms (alone or in combination) is best targeted in a particular
inflammatory disorder.
[0007] Studies using mice that lack PI3K.delta. and/or PI3K.gamma.
or express kinase-dead variants of PI3K.delta. and PI3K.gamma. have
been valuable tools in understanding their roles. For example,
PI3K.delta. knockout mice demonstrated diminished neutrophil
chemotaxis, diminished antibody production (both T cell dependent
and independent) (Jou et al., Mol. Cell. Biol., 22, 8580-8591,
2002), and lower numbers of mature B cells (Clayton et al., J. Exp.
Med., 196, 753-763, 2002; Jou et al., Mol. Cell. Biol., 22,
8580-8591, 2002), and a decrease in their proliferation in response
to anti-IgM (Jou et al., Mol. Cell. Biol., 22, 8580-8591, 2002).
This phenotype was replicated in the PI3K.delta. kinase-dead
variant and with PI3K.delta. selective inhibitors along with a
decreased number and proliferation of mast cells, and an attenuated
allergic response. The PI3K.gamma. knockout contained higher
numbers of, but less responsive, neutrophils, lower numbers of and
less responsive macrophages, and dendritic cells displayed
decreased mast cell degranulation (Laffargue et al., Immunity, 16,
441-451, 2002), a higher ratio of CD4+ to CD8+ T cells, increased
thymocyte apoptosis, diminished induction of CXCR3 on activated T
cells and decreased cardiac contractility. This latter effect on
cardiac tissue was a concern for chronic dosing of patients with
PI3K.gamma. inhibitors. However, this concern was largely mitigated
when the PI3K.gamma. kinase-dead variant (which better mimics
inhibition of the kinase rather than loss of the protein) showed
similar immune cell phenotypes, but importantly had no cardiac
defects. The cardiac effect was later shown to be due to
scaffolding effects rather than the catalytic activity of
PI3K.gamma. (Olusegon et al., Chemistry & Biology, 1, 123-134,
2010, including the references cited therein). The dual
PI3K.delta./PI3K.gamma. knockout was viable but exhibited serious
defects in T cell development and thymocyte survival. The
PI3K.gamma. knockout/PI3K.delta. kinase-dead combination produced a
similar phenotype suggesting that at least within the immune
system, the role of PI3K.delta. is likely only a catalytic one.
Interpretation of studies using knockout and kinase-dead mice can
be challenging because these models provide only a steady-state
picture of the immune system, lack temporal and dose control, and
do not permit a full understanding of how a dynamic immune response
will react to reversible inhibition. Selective inhibitors with
varying profiles (PI3K.delta., PI3K.gamma., and
PI3K.delta./.gamma.) are necessary for studies of leukocyte
signaling in order to assess the relative contributions of each
PI3K to immune cell activation (Olusegon et al., supra, including
the references cited therein).
[0008] Dual inhibition of .delta./.gamma. is strongly implicated as
an intervention strategy in allergic and non-allergic inflammation
of the airways and other autoimmune diseases. Scientific evidence
for PI3K.delta. and PI3K .gamma. involvement in various cellular
processes underlying asthma and chronic obstructive pulmonary
disease (COPD) stems from inhibitor studies and gene-targeting
approaches (William et. al Chemistry & Biology, 17, 123-134,
2010 and Thompson, et al. Chemistry & Biology, 17:101-102,
2010). Also, resistance to conventional therapies such as
corticosteroids in several COPD patients has been attributed to an
up-regulation of the PI3K .delta./.gamma. pathway. Disruption of
PI3K.delta./.gamma. signaling therefore provides a novel strategy
aimed at counteracting the immuno-inflammatory response. Due to the
pivotal role played by PI3K.delta. and PI3K.gamma. in mediating
inflammatory cell functionality such as leukocyte migration and
activation, and mast cell degranulation, blocking these isoforms
may also be an effective strategy for the treatment of rheumatoid
arthritis as well. Given the established criticality of these
isoforms in immune surveillance, inhibitors specifically targeting
the PI3K.delta. and PI3K.gamma. isoforms would be expected to
attenuate the progression of immune response encountered in airway
inflammation and rheumatoid arthritis (William et. al Chemistry
& Biology, 17, 123-134, 2010 and Thompson, et al. Chemistry
& Biology, 17:101-102, 2010)
[0009] Reviews and studies regarding PI3K and related protein
kinase pathways have been given by Liu et al., Nature Reviews Drug
Discovery, 8, 627-644, 2009); Nathan et. al., Mol Cancer Ther.,
8(1), 2009; Marone et al., Biochimica et Biophysica Acta, 1784,
159-185, 2008 and Markman et al., Annals of Oncology Advance
Access, published August 2009. Similarly reviews and studies
regarding role of PI3K.delta. and PI3K.gamma. have been given by
William et al., Chemistry & Biology, 17, 123-134, 2010 and
Timothy et al. J. Med. Chem., 55 (20), 8559-8581, 2012. All of
these literature disclosures are hereby incorporated by reference
in their entirety.
[0010] Compounds such as IPI-145 and CAL130 have been reported as
dual inhibitors of Pi3K .delta./.gamma. (WO2012/008302 &
WO2012/121953 respectively), IPI-145 is under clinical
investigation for cancer, asthma and rheumatoid arthritis. IPI-45
has been reported to have a maximum tolerated dose (MTD) of 75 mg
BID (55th ASH.RTM. Annual Meeting. New Orleans, La., Dec. 7-10,
2013). There are no reports of CAL-130 being investigated for
clinical purposes.
[0011] There still remains an unmet need for dual .delta./.gamma.
PI3K modulators for the treatment of diseases and disorders
associated with .delta./.gamma. PI3K kinases-mediated events.
[0012] Further reference is made herein to International
Publication Nos. WO 11/055215 and WO 12/151525 and U.S. Publication
Nos. 2011/0118257 and 2012/0289496, each of which is incorporated
herein by reference in its entirety.
SUMMARY OF THE INVENTION
[0013] The present invention is directed to selective dual
inhibitors of PI3K delta (.delta.) and gamma (.gamma.) protein
kinases. These compounds are suitable for use in a pharmaceutical
composition for the treatment of PI3K associated diseases,
disorders or conditions, e.g., a proliferative disease such as
cancer. Inhibition of both PI3K.delta. and PI3K.gamma. protein
kinases may provide beneficial effects in the treatment of certain
diseases and disorders.
[0014] The selective dual inhibitors of the present invention
include
N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonami-
de, pharmaceutically acceptable salts thereof, and prodrugs
thereof. For example, the selective dual inhibitor may be selected
from the following compounds, pharmaceutically acceptable salts
thereof, and prodrugs thereof:
[0015]
(RS)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chro-
men-2-yl)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methane-
sulfonamide (Compound A); and
[0016]
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chrom-
en-2-yl)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanes-
ulfonamide (Compound A1).
[0017] In one embodiment, the compound
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonam-
ide or a pharmaceutically acceptable salt thereof is substantially
free (e.g., contains less than about 10%, such as less than about
5%, less than about 2.5%, less than about 1%, less than about 0.1%
by weight) or is free of
(R)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfona-
mide and pharmaceutically acceptable salts thereof.
[0018] In another embodiment, the compound
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonam-
ide or a pharmaceutically acceptable salt thereof has an
enantiomeric excess of greater than about 90%, such as greater than
about 91%, greater than about 92%, greater than about 93%, greater
than about 94%, greater than about 95%, greater than about 96%,
greater than about 97%, greater than about 98%, greater than about
99%, greater than about 99.5%, greater than about 99.9%, or greater
than about 99.99%.
[0019] In one preferred embodiment, the present invention relates
to the compound
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-ch-
romen-2-yl)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonami-
de (Compound A1).
[0020] In another embodiment, the present invention relates to the
compound
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-ch-
romen-2-yl)ethyl)-1H-pyrazolo
[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide or a
pharmaceutically acceptable salt thereof.
[0021] Another embodiment of the present invention is
(R)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfona-
mide (Compound A2), a pharmaceutically acceptable salt thereof, or
prodrug thereof. Compound A2 is an inhibitor of PI3K delta
(.delta.) protein kinase. These compounds are suitable for use in a
pharmaceutical composition for the treatment of PI3K associated
diseases, disorders or conditions, e.g., a proliferative disease
such as cancer.
[0022] The chemical structures of
N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonami-
de, compound A1, and compound A2 are shown below.
##STR00001##
[0023] The present invention further provides a pharmaceutical
composition comprising one or more compounds of the present
invention (such as compound A1) together with a pharmaceutically
acceptable carrier. The pharmaceutical composition may further
comprise one or more of additional active agents (such as
anti-cancer agents and the active agents discussed below). In one
embodiment, the pharmaceutical composition includes a
therapeutically effective amount of one or more compounds of the
present invention.
[0024] Another aspect of the present invention relates to a process
for the preparation of
N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonami-
de:
##STR00002##
The process comprises the steps of:
[0025] (a) reacting 5-bromo-2-methoxyaniline
##STR00003##
with methane sulphonyl chloride to give
N-(5-bromo-2-methoxyphenyl)methanesulfonamide (Intermediate 1):
##STR00004##
[0026] (b) reacting Intermediate 1 with bis(pinacolato)diboron, for
example in the presence of potassium acetate, to give
N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methan-
esulfonamide (Intermediate 2):
##STR00005##
and
[0027] (c) reacting
2-(1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(-
3-fluorophenyl)-4H-chromen-4-one
##STR00006##
with intermediate 2 in the presence of a base (such as, for
example, sodium carbonate) to give the desired compound
N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonami-
de;
[0028] (d) optionally converting
N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonami-
de to a pharmaceutically acceptable salt thereof or prodrug
thereof.
[0029] Yet another embodiment relates to a process for preparation
of a compound of formula (A1):
##STR00007##
The process comprises the steps of:
[0030] (a) subjecting
(R)-5-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one:
##STR00008##
to a Mitsunobu reaction with
3-(4-methoxy-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine:
##STR00009##
(for example, in the presence of triphenylphosphine and
diisopropylazodicarboxylate) to give
(S)-2-(1-(4-amino-3-(4-methoxy-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
(Intermediate 3):
##STR00010##
[0031] (b) reducing Intermediate 3, for example with a reducing
agent such as Raney Ni, to give
(S)-2-(1-(4-amino-3-(3-amino-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
(Intermediate 4):
##STR00011##
[0032] (c) treating Intermediate 4 with methanesulphonyl chloride
to give the desired compound of the formula (A1); and
[0033] (d) optionally converting compound (A1) to a
pharmaceutically acceptable salt thereof or prodrug thereof.
[0034] Yet another embodiment are intermediates useful for
preparing the compounds of the present invention such as
(S)-2-(1-(4-amino-3-(4-methoxy-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,
(S)-2-(1-(4-amino-3-(3-amino-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and
salts thereof.
[0035] Yet another embodiment of the present invention is a method
of inhibiting PI3K.delta. and PI3K.gamma. in a patient comprising
administering to the patient an effective amount of at least one
compound of the present invention.
[0036] Yet another embodiment of the present invention is a method
of inhibiting PI3K.delta. in a patient comprising administering to
the patient an effective amount of at least one of
(R)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-y-
l)
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfona-
mide (compound A2), a pharmaceutically acceptable salt thereof, or
a prodrug thereof.
[0037] Yet another embodiment of the present invention is a method
of treating, preventing, and/or inhibiting a PI3K protein kinase
mediated disease, disorder or condition (such a proliferative
disease or disorder, e.g., cancer) in a patient comprising
administering to the patient an effective amount of at least one
compound of the present invention.
[0038] Yet another embodiment of the present invention is a method
for inhibiting PI3K, in particular PI3K.delta. and PI3K.gamma., in
a patient comprising administering to the patient an effective
amount of at least one compound of the present invention.
[0039] Yet another embodiment of the present invention is a method
for treating an inflammatory, autoimmune or proliferative disease
via modulation of a protein kinase (such as PI3K.delta. and
PI3K.gamma.) comprising administering to a patient in need of such
treatment an effective amount of at least one compound of the
present invention. In one embodiment, the compound of the present
invention inhibits both PI3K.delta. and PI3K.gamma..
[0040] Yet another embodiment of the present invention is a method
for treating an inflammatory, autoimmune or proliferative disease
via modulation of a protein kinase (such as PI3K.delta. and
PI3K.gamma.) by administering to a patient in need of such
treatment an effective amount of at least one compound of the
present invention, in combination (simultaneously or sequentially)
with at least one other anti-inflammatory, immunomodulator or
anti-cancer agent, or a combination thereof. In one embodiment, the
compound of the present invention inhibits both PI3K.delta. and
PI3K.gamma..
[0041] The compounds of the present invention are useful in the
treatment of a variety of cancers, including, but not limited
to:
[0042] carcinoma, including, but not limited to, that of the
bladder, breast, colon, kidney, liver, lung, including small cell
lung cancer, esophagus, gall bladder, ovary, pancreas, stomach,
cervix, thyroid, prostate, and skin, including squamous cell
carcinoma;
[0043] hematopoietic tumors of lymphoid lineage, including, but not
limited to, leukemia, acute lymphocytic leukemia, acute
lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's
lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's
lymphoma;
[0044] hematopoietic tumors of myeloid lineage, including, but not
limited to, acute and chronic myelogenous leukemias,
myelodysplastic syndrome and promyelocytic leukemia;
[0045] tumors of mesenchymal origin, including, but not limited to,
fibrosarcoma and rhabdomyosarcoma;
[0046] tumors of the central and peripheral nervous system,
including, but not limited to, astrocytoma, neuroblastoma, glioma
and schwannomas; and
[0047] other tumors, including, but not limited to, melanoma,
seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum,
keratoctanthoma, thyroid follicular cancer and Kaposi's
sarcoma.
[0048] In one embodiment, an effective amount of a compound of the
present invention is administered to treat a leukemia, acute
lymphocytic leukemia, acute lymphoblastic leukemia, B-cell
lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, hairy cell lymphoma, Burkett's lymphoma, acute and
chronic myelogenous leukemias, myelodysplastic syndrome or
promyelocytic leukemia.
[0049] Due to the key role of protein kinases in the regulation of
cellular proliferation in general, the compounds of the present
invention may act as reversible cytostatic agents, and may
therefore be useful in the treatment of any disease process which
features abnormal cellular proliferation, such as, e.g., benign
prostatic hyperplasia, familial adenomatosis polyposis,
neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis,
psoriasis, glomerulonephritis, restenosis following angioplasty or
vascular surgery, hypertrophic scar formation, inflammatory bowel
disease, transplantation rejection, endotoxic shock, and fungal
infections.
[0050] The compounds of the present invention as modulators of
apoptosis are useful in the treatment of cancer (including, but not
limited to, those types mentioned herein above), viral infections
(including, but not limited to, herpes virus, poxvirus,
Epstein-Barr virus, Sindbis virus and adenovirus), autoimmune
diseases (including, but not limited to, systemic lupus,
erythematosus, autoimmune mediated glomerulonephritis, rheumatoid
arthritis, psoriasis, inflammatory bowel disease, and autoimmune
diabetes mellitus), neurodegenerative disorders (including, but not
limited to, Alzheimer's disease, AIDS-related dementia, Parkinson's
disease, amyotrophic lateral sclerosis, retinitis pigmentosa,
spinal muscular atrophy and cerebellar degeneration),
myelodysplastic syndromes, aplastic anemia, ischemic injury
associated with myocardial infarctions, stroke and reperfusion
injury, arrhythmia, atherosclerosis, toxin-induced or alcohol
related liver diseases, haematological diseases (including, but not
limited to, chronic anemia and aplastic anemia), degenerative
diseases of the musculoskeletal system (including, but not limited
to, osteoporosis and arthritis) aspirin-sensitive rhinosinusitis,
cystic fibrosis, multiple sclerosis, kidney diseases and cancer
pain. The compounds of the present invention are also useful in the
prevention, inhibition, or suppression of AIDS development in
HIV-infected individuals.
[0051] The compounds of the present invention may modulate the
level of cellular RNA and DNA synthesis. The compounds of the
present invention are therefore useful in the treatment of viral
infections, including, but not limited to, HIV, human papilloma
virus, herpes virus, poxvirus, Epstein-Barr virus, Sindbis virus
and adenovirus.
[0052] The compounds of the present invention are useful in the
chemoprevention of cancer. Chemoprevention is defined herein as
inhibiting the development of invasive cancer by either blocking
the initiating mutagenic event or by blocking the progression of
pre-malignant cells that have already suffered an insult or
inhibiting tumor relapse. The compounds of the present invention
are also useful in inhibiting tumor angiogenesis and metastasis.
One embodiment of the present invention is a method of inhibiting
tumor angiogenesis or metastasis in a patient in need thereof by
administering an effective amount of one or more compounds of the
present invention.
[0053] Another embodiment of the present invention is a method of
treating an immune system-related disease or immune disorder (e.g.,
an autoimmune disease), a disease or disorder involving
inflammation (e.g., asthma, chronic obstructive pulmonary disease
(COPD), rheumatoid arthritis, inflammatory bowel disease,
glomerulonephritis, neuroinflammatory diseases, multiple sclerosis,
uveitis and disorders of the immune system), cancer or other
proliferative disease, a hepatic disease or disorder, a renal
disease or disorder. The method includes administering an effective
amount of one or more compounds of the present invention.
[0054] Examples of immune disorders include, but are not limited
to, psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel
disease, dermatitis, osteoarthritis, asthma, inflammatory muscle
disease, allergic rhinitis, vaginitis, interstitial cystitis,
scleroderma, osteoporosis, eczema, allogeneic or xenogeneic
transplantation (organ, bone marrow, stem cells and other cells and
tissues) graft rejection, graft-versus-host disease, lupus
erythematosus, inflammatory disease, type I diabetes, pulmonary
fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g.,
Hashimoto's and autoimmune thyroiditis), myasthenia gravis,
autoimmune haemolytic anemia, multiple sclerosis, cystic fibrosis,
chronic relapsing hepatitis, primary biliary cirrhosis, allergic
conjunctivitis and atopic dermatitis.
[0055] In one embodiment, the compounds described herein are useful
as immunosuppresants to prevent transplant graft rejections,
allogeneic or xenogeneic transplantation rejection (organ, bone
marrow, stem cells, other cells and tissues), and graft-versus-host
disease. In one particular embodiment, transplant graft rejections
result from tissue or organ transplants. In further embodiments,
the graft-versus-host disease results from bone marrow or stem cell
transplantation. One embodiment of the present invention is a
method of preventing or decreasing the risk of transplant graft
rejection, allogeneic or xenogeneic transplantation rejection
(organ, bone marrow, stem cells, other cells and tissues) or
graft-versus-host disease comprising administering an effective
amount of one or more compounds of the present invention.
[0056] The compounds of the present invention are also useful in
combination (administered together or sequentially) with known
anti-cancer treatments, such as, for example, radiation therapy or
with cytostatic or cytotoxic or anticancer agents, such as, for
example, DNA interactive agents, such as cisplatin or doxorubicin;
topoisomerase II inhibitors, such as etoposide; topoisomerase I
inhibitors such as CPT-11 or topotecan; tubulin interacting agents,
such as paclitaxel, docetaxel or the epothilones (for example
ixabepilone), either naturally occurring or synthetic; hormonal
agents, such as tamoxifen; thymidilate synthase inhibitors, such as
5-fluorouracil; and anti-metabolites, such as methotrexate, other
tyrosine kinase inhibitors, such as Iressa and OSI-774;
angiogenesis inhibitors; EGF inhibitors; VEGF inhibitors; CDK
inhibitors; SRC inhibitors; c-Kit inhibitors; Her1/2 inhibitors and
monoclonal antibodies directed against growth factor receptors such
as erbitux (EGF) and herceptin (Her2); BTK inhibitor, such as
ibrutinib; and other protein kinase modulators, and any combination
thereof.
[0057] The compounds of the present invention are also useful in
combination (administered together or sequentially) with one or
more steroidal anti-inflammatory drugs, non-steroidal
anti-inflammatory drugs (NSAIDs) and immune selective
anti-inflammatory derivatives (ImSAIDs), and any combination
thereof.
[0058] The present invention further provides a pharmaceutical
composition comprising one or more compounds of the present
invention and a pharmaceutically acceptable carrier. The
pharmaceutical composition may further comprise one or more of the
active ingredients identified above, such as other anti-cancer
agents.
[0059] Yet another embodiment is a method of treating leukemia in a
patient in need thereof comprising administering a therapeutically
effective amount of a compound of the present invention. In one
embodiment, the leukemia is selected from chronic lymphocytic
leukemia (CLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL),
acute myeloid leukemia (AML), multiple myeloma (MM), small
lymphocytic lymphoma (SLL), and indolent non-Hodgkin's lymphoma
(I-NHL).
[0060] Yet another embodiment of the present invention is a method
of treating an autoimmune disorder in a patient in need thereof
comprising administering a therapeutically effective amount of a
compound of the present invention. In one embodiment, the
autoimmune disorder is selected from asthma, COPD, rheumatoid
arthritis, psoriasis, lupus and experimental autoimmune
encephalomyelitis (EAE).
[0061] Yet another embodiment of the present invention is a method
of treating allergic rhinitis in a patient in need thereof
comprising administering to the patient a therapeutically effective
amount of a compound of the present invention.
[0062] In any of the aforementioned methods, the compound(s) of the
present invention and optional additional active agents can be
administered in the form of a pharmaceutical composition as
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1 depicts a bar graph of the neutrophil count in
bronchoalveolar lavage fluid (BALF) from female Wistar rats treated
with 10 mg/kg of Compound A1 (po) according to the
lipopolysaccharide induced pulmonary neutrophilia model described
in Assay 7.
[0064] FIG. 2 depicts a bar graph of the neutrophil count in
peritoneal lavage fluid from Wistar rats treated with 1, 3, and 10
mg/kg of Compound A1 (po) according to the
lipopolysaccharide-induced rat air pouch inflammation model
described in Assay 8.
[0065] FIGS. 3A and 3B depict the line and bar graphs of individual
clinical scores for hind and fore paws and AUC for clinical score,
respectively, in Wistar rats with collagen induced arthritis
treated with a control or 10 mg/kg/QD of Compound A1 according to
the procedure in Assay 11.
[0066] FIGS. 3C and 3D depict line and bar graphs of individual
clinical scores for hind and fore paws, respectively, in Wistar
rats with collagen induced arthritis treated with vehicle or 10
mg/kg/QD of Compound A1 according to the procedure in Assay 11.
[0067] FIGS. 4A and 4B depict the line and bar graphs of volume for
hind paws and AUC of paw volume, respectively, in Wistar rats with
collagen induced arthritis treated with vehicle or 10 mg/kg/QD of
Compound A1 according to the procedure in Assay 11.
[0068] FIGS. 4C and 4D depict line and bar graphs of ankle diameter
for hind paws and AUC of ankle diameter, respectively, in Wistar
rats with collagen induced arthritis treated with vehicle or 10
mg/kg/QD of Compound A1 according to the procedure in Assay 11.
[0069] FIGS. 4E to 4G depict bar graphs of histopathological score
for inhibition of inflammation, cartilage and pannus, respectively,
of all the hind and fore paws in Wistar rats with collagen induced
arthritis treated with vehicle or 10 mg/kg/QD of Compound A1
according to the procedure in Assay 11.
[0070] FIG. 4H depicts a bar graph of total histopathological score
of all the hind and fore paws in Wistar rats with collagen induced
arthritis treated with vehicle or 10 mg/kg/QD of Compound A1
according to the procedure in Assay 11.
[0071] FIG. 5 depicts a bar graph of the percentage incidence of
arthritis in Wistar rats with collagen induced arthritis treated
with vehicle or 10 mg/kg/QD of Compound A1 according to the
procedure in Assay 11.
[0072] FIGS. 6A and 6B depict bar graphs showing the antipsoratic
effect of Compound A1 (3, 10, 30 mg/kg) on imiquimod induced
psoriasis in Balb/c mice according to the procedure in Assay
13.
DETAILED DESCRIPTION OF THE INVENTION
[0073] As used herein the following definitions shall apply unless
otherwise indicated. Further many of the groups defined herein can
be optionally substituted. The listing of substituents in the
definition is exemplary and is not to be construed to limit the
substituents defined elsewhere in the specification.
[0074] Certain of the compounds described herein contain one or
more asymmetric centers and can thus give rise to enantiomers,
diastereomers, and other stereoisomeric forms that can be defined,
in terms of absolute stereochemistry, as (R)- or (S)-. Unless
otherwise specified, the present chemical entities, pharmaceutical
compositions and methods are meant to include all such possible
isomers, including racemic mixtures, optically pure forms and
intermediate mixtures. For the instance, non-limiting example of
intermediate mixtures include a mixture of R:S or S:R isomers in a
ratio of 10:90, 13:87, 17:83, 20:80, or 22:78. Optically active
(R)- and (S)-isomers can be prepared using chiral synthons or
chiral reagents, or resolved using conventional techniques. When
the compounds described herein contain olefinic double bonds or
other centers of geometric asymmetry, and unless specified
otherwise, it is intended that the compounds include both E and Z
geometric isomers.
[0075] The term "tautomers" refers to compounds, which are
characterized by relatively easy interconversion of isomeric forms
in equilibrium. These isomers are intended to be covered by this
invention. "Tautomers" are structurally distinct isomers that
interconvert by tautomerization. "Tautomerization" is a form of
isomerization and includes prototropic or proton-shift
tautomerization, which is considered a subset of acid-base
chemistry. "Prototropic tautomerization" or "proton-shift
tautomerization" involves the migration of a proton accompanied by
changes in bond order, often the interchange of a single bond with
an adjacent double bond. Where tautomerization is possible (e.g. in
solution), a chemical equilibrium of tautomers can be reached. An
example of tautomerization is keto-enol tautomerization. A specific
example of keto-enol tautomerization is the interconversion of
pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another
example of tautomerization is phenol-keto tautomerization. A
specific example of phenol-keto tautomerization is the
interconversion of pyridin-4-ol and pyridin-4(1H)-one
tautomers.
[0076] The term "prodrug" refers to a compound, which is an
inactive precursor of a compound that is converted into its active
form in the body by normal metabolic processes. Prodrug design is
discussed generally in Hardma, et al. (Eds.), Goodman and Gilman's
The Pharmacological Basis of Therapeutics, 9th ed., pp. 11-16
(1996). A thorough discussion is provided in Higuchi, et al.,
Prodrugs as Novel Delivery Systems, Vol. 14, ASCD Symposium Series,
and in Roche (ed.), Bioreversible Carriers in Drug Design, American
Pharmaceutical Association and Pergamon Press (1987). To
illustrate, prodrugs can be converted into a pharmacologically
active form through hydrolysis of, for example, an ester or amide
linkage, thereby introducing or exposing a functional group on the
resultant product. The prodrugs can be designed to react with an
endogenous compound to form a water-soluble conjugate that further
enhances the pharmacological properties of the compound, for
example, increased circulatory half-life. Alternatively, prodrugs
can be designed to undergo covalent modification on a functional
group with, for example, glucuronic acid, sulphate, glutathione,
amino acids, or acetate. The resulting conjugate can be inactivated
and excreted in the urine, or rendered more potent than the parent
compound. High molecular weight conjugates also can be excreted
into the bile, subjected to enzymatic cleavage, and released back
into the circulation, thereby effectively increasing the biological
half-life of the originally administered compound.
[0077] The term "ester" refers to a compound, which is formed by
reaction between an acid and an alcohol with elimination of water.
An ester can be represented by the general formula RCOOR' (where R
is a drug and R' is a chemical group).
[0078] These prodrugs and esters are intended to be covered within
the scope of this invention.
[0079] Additionally the instant invention also includes the
compounds which differ only in the presence of one or more
isotopically enriched atoms for example replacement of hydrogen
with deuterium or tritium, or the replacement of a carbon by
.sup.13C- or .sup.14C-enriched carbon.
[0080] The compounds of the present invention may also contain
unnatural proportions of atomic isotopes at one or more of atoms
that constitute such compounds. For example, the compounds may be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). All
isotopic variations of the compounds of the present invention,
whether radioactive or not, are encompassed within the scope of the
present invention.
[0081] Pharmaceutically acceptable salts forming part of this
invention include salts derived from inorganic bases such as Li,
Na, K, Ca, Mg, Fe, Cu, Zn, and Mn; salts of organic bases such as
N,N'-diacetylethylenediamine, glucamine, triethylamine, choline,
hydroxide, dicyclohexylamine, metformin, benzylamine,
trialkylamine, and thiamine; chiral bases such as alkylphenylamine,
glycinol, and phenyl glycinol; salts of natural amino acids such as
glycine, alanine, valine, leucine, isoleucine, norleucine,
tyrosine, cystine, cysteine, methionine, proline, hydroxy proline,
histidine, omithine, lysine, arginine, and serine; quaternary
ammonium salts of the compounds of invention with alkyl halides,
alkyl sulphates such as MeI and (Me).sub.2SO.sub.4; non-natural
amino acids such as D-isomers or substituted amino acids;
guanidine; and substituted guanidine wherein the substituents are
selected from nitro, amino, alkyl, alkenyl, alkynyl, ammonium or
substituted ammonium salts and aluminum salts. Salts may include
acid addition salts where appropriate which may be sulphates,
nitrates, phosphates, perchlorates, borates, hydrohalides,
acetates, tartrates, maleates, citrates, fumarates, succinates,
palmoates, methanesulphonates, benzoates, salicylates,
benzenesulfonates, ascorbates, glycerophosphates, and
ketoglutarates.
[0082] When ranges are used herein for physical properties, such as
molecular weight, or chemical properties, such as chemical
formulae, all combinations and subcombinations of ranges and
specific embodiments therein are intended to be included. The term
"about" when referring to a number or a numerical range means that
the number or numerical range referred to is an approximation
within experimental variability (or within statistical experimental
error), and thus the number or numerical range may vary from, for
example, between 1% and 15% of the stated number or numerical
range. The term "comprising" (and related terms such as "comprise"
or "comprises" or "having" or "including") includes those
embodiments, for example, an embodiment of any composition of
matter, composition, method, or process, or the like, that "consist
of" or "consist essentially of" the described features.
[0083] The following abbreviations and terms have the indicated
meanings throughout: PI3-K=Phosphoinositide 3-kinase;
PI=phosphatidylinositol; AIDS=Acquired Immuno Deficiency Syndrome;
HIV=Human Immunodeficiency Virus; MeI=Methyl Iodide; ND: Not
determined.
[0084] Abbreviations used herein have their conventional meaning
within the chemical and biological arts.
[0085] The term "cell proliferation" refers to a phenomenon by
which the cell number has changed as a result of division. This
term also encompasses cell growth by which the cell morphology has
changed (e.g., increased in size) consistent with a proliferative
signal.
[0086] The terms "co-administration," "administered in combination
with," and their grammatical equivalents, as used herein, encompass
administration of two or more agents to an animal so that both
agents and/or their metabolites are present in the animal at the
same time. Co-administration includes simultaneous administration
in separate compositions, administration at different times in
separate compositions, or administration in a composition in which
both agents are present.
[0087] The term "effective amount" or "therapeutically effective
amount" refers to that amount of a compound described herein that
is sufficient to effect the intended application including but not
limited to disease treatment, as defined below. The therapeutically
effective amount may vary depending upon the intended application
(in vitro or in vivo), or the subject and disease condition being
treated, e.g., the weight and age of the subject, the severity of
the disease condition, the manner of administration and the like,
which can readily be determined by one of ordinary skill in the
art. The term also applies to a dose that will induce a particular
response in target cells, e.g. reduction of platelet adhesion
and/or cell migration. The specific dose will vary depending on the
particular compounds chosen, the dosing regimen to be followed,
whether it is administered in combination with other compounds,
timing of administration, the tissue to which it is administered,
and the physical delivery system in which it is carried.
[0088] As used herein, "treatment," "treating," or "ameliorating"
are used interchangeably. These terms refers to an approach for
obtaining beneficial or desired results including but, not limited
to, therapeutic benefit and/or a prophylactic benefit. By
therapeutic benefit is meant eradication or amelioration of the
underlying disorder being treated. Also, a therapeutic benefit is
achieved with the eradication or amelioration of one or more of the
physiological symptoms associated with the underlying disorder such
that an improvement is observed in the patient, notwithstanding
that the patient may still be afflicted with the underlying
disorder. For prophylactic benefit, the compositions may be
administered to a patient at risk of developing a particular
disease, or to a patient reporting one or more of the physiological
symptoms of a disease, even though a diagnosis of this disease may
not have been made.
[0089] A "therapeutic effect," as that term is used herein,
encompasses a therapeutic benefit and/or a prophylactic benefit as
described above. A prophylactic effect includes delaying or
eliminating the appearance of a disease or condition, delaying or
eliminating the onset of symptoms of a disease or condition,
slowing, halting, or reversing the progression of a disease or
condition, or any combination thereof.
[0090] The term "subject" or "patient" refers to an animal (e.g., a
dog, cat, horse, or pig), such as a mammal, for example a human.
The methods described herein can be useful in both human
therapeutics and veterinary applications. In some embodiments, the
patient is a mammal. In a preferred embodiment, the patient is
human.
[0091] "Radiation therapy" means exposing a patient, using routine
methods and compositions known to the practitioner, to radiation
emitters such as alpha-particle emitting radionuclides (e.g.,
actinium and thorium radionuclides), low linear energy transfer
(LET) radiation emitters (i.e. beta emitters), conversion electron
emitters (e.g. strontium-89 and samarium-153-EDTMP), or high-energy
radiation, including, without limitation, x-rays, gamma rays, and
neutrons.
[0092] "Signal transduction" is a process during which stimulatory
or inhibitory signals are transmitted into and within a cell to
elicit an intracellular response. A modulator of a signal
transduction pathway refers to a compound which modulates the
activity of one or more cellular proteins mapped to the same
specific signal transduction pathway. A modulator may augment
(agonist) or suppress (antagonist) the activity of a signaling
molecule.
[0093] The term "selective inhibition" or "selectively inhibit" as
applied to a biologically active agent refers to the agent's
ability to selectively reduce the target signaling activity as
compared to off-target signaling activity, via direct or indirect
interaction with the target.
[0094] The term "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable excipient" includes, but is not
limited to, any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, one or more suitable diluents, fillers, salts,
disintegrants, binders, lubricants, glidants, wetting agents,
controlled release matrices, colorants/flavouring, carriers,
excipients, buffers, stabilizers, solubilizers, and combinations
thereof. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions of the invention is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
[0095] In other embodiments, the compounds of the present invention
selectively inhibit one or more members of type I or class I
phosphatidylinositol 3-kinases (PI3-kinase) with an IC.sub.50 value
of about 100 nM or less, about 50 nM or less, about 10 nM or less,
about 5 nM or less, about 100 pM or less, about 10 pM or less, or
about 1 pM or less as measured in an in vitro kinase assay.
[0096] In yet another aspect, an inhibitor that selectively
inhibits one or more members of type I PI3-kinases, or an inhibitor
that selectively inhibits one or more type I PI3-kinase mediated
signaling pathways, alternatively can be understood to refer to a
compound that exhibits a 50% inhibitory concentration (IC.sub.50)
with respect to a given type I PI3-kinase, that is at least 10-fold
lower, at least 20-fold lower, at least 50-fold lower, at least
100-fold lower, or at least 1000-fold lower than the inhibitor's
IC.sub.50 with respect to the rest of the other type I
PI3-kinases.
[0097] As used herein, the term "dual PI3-kinase .delta./.gamma.
inhibitor" and "dual PI3-kinase .delta./.gamma. selective
inhibitor" refers to a compound that inhibits the activity of both
the PI3-kinase .delta. and .gamma. isozyme more effectively than
other isozymes of the PI3K family. A dual PI3-kinase
.delta./.gamma. inhibitor is therefore more selective for
PI3-kinase .delta. and .gamma. than conventional PI3K inhibitors
such as CAL-130, wortmannin and LY294002, which are nonselective
PI3K inhibitors.
[0098] Inhibition of PI3-kinase .delta. and .gamma. may be of
therapeutic benefit in treatment of various conditions, e.g.,
conditions characterized by an inflammatory response including, but
not limited to, autoimmune diseases, allergic diseases, and
arthritic diseases. Importantly, inhibition of PI3-kinase .delta.
and .gamma. function does not appear to affect biological functions
such as viability and fertility.
[0099] "Inflammatory response" as used herein is characterized by
redness, heat, swelling and pain (i.e., inflammation) and typically
involves tissue injury or destruction. An inflammatory response is
usually a localized, protective response elicited by injury or
destruction of tissues, which serves to destroy, dilute or wall off
(sequester) both the injurious agent and the injured tissue.
Inflammatory responses are notably associated with the influx of
leukocytes and/or leukocyte (e.g., neutrophil) chemotaxis.
Inflammatory responses may result from infection with pathogenic
organisms and viruses, noninfectious means such as trauma or
reperfusion following myocardial infarction or stroke, immune
responses to foreign antigens, and autoimmune diseases.
Inflammatory responses amenable to treatment with the methods and
compounds according to the invention encompass conditions
associated with reactions of the specific defense system as well as
conditions associated with reactions of the non-specific defense
system.
[0100] The therapeutic methods of the invention include methods for
the amelioration of conditions associated with inflammatory cell
activation. "Inflammatory cell activation" refers to the induction
by a stimulus (including but not limited to, cytokines, antigens or
auto-antibodies) of a proliferative cellular response, the
production of soluble mediators (including but not limited to
cytokines, oxygen radicals, enzymes, prostanoids, or vasoactive
amines), or cell surface expression of new or increased numbers of
mediators (including, but not limited to, major histocompatibility
antigens or cell adhesion molecules) in inflammatory cells
(including but not limited to monocytes, macrophages, T
lymphocytes, B lymphocytes, granulocytes (polymorphonuclear
leukocytes including neutrophils, basophils, and eosinophils) mast
cells, dendritic cells, Langerhans cells, and endothelial cells).
It will be appreciated by persons skilled in the art that the
activation of one or a combination of these phenotypes in these
cells can contribute to the initiation, perpetuation, or
exacerbation of an inflammatory condition.
[0101] "Autoimmune disease" as used herein refers to any group of
disorders in which tissue injury is associated with humoral or
cell-mediated responses to the body's own constituents.
[0102] "Transplant rejection" as used herein refers-to any immune
response directed against grafted tissue (including organs or cells
(e.g., bone marrow), characterized by a loss of function of the
grafted and surrounding tissues, pain, swelling, leukocytosis, and
thrombocytopenia).
[0103] "Allergic disease" as used herein refers to any symptoms,
tissue damage, or loss of tissue function resulting from
allergy.
[0104] "Arthritic disease" as used herein refers to any disease
that is characterized by inflammatory lesions of the joints
attributable to a variety of etiologies.
[0105] "Dermatitis" as used herein refers to any of a large family
of diseases of the skin that are characterized by inflammation of
the skin attributable to a variety of etiologies.
[0106] As previously described, the term "dual PI3-kinase
.delta./.gamma. selective inhibitor" generally refers to a compound
that inhibits the activity of the PI3-kinase .delta. and .gamma.
isozyme more effectively than other isozymes of the PI3K family.
The relative efficacies of compounds as inhibitors of an enzyme
activity (or other biological activity) can be established by
determining the concentrations at which each compound inhibits the
activity to a predefined extent and then comparing the results.
Typically, the preferred determination is the concentration that
inhibits 50% of the activity in a biochemical assay, i.e., the 50%
inhibitory concentration or "IC.sub.50". IC.sub.50 determinations
can be accomplished using conventional techniques known in the art.
In general, an IC.sub.50 can be determined by measuring the
activity of a given enzyme in the presence of a range of
concentrations of the inhibitor under study. The experimentally
obtained values of enzyme activity then are plotted against the
inhibitor concentrations used. The concentration of the inhibitor
that shows 50% enzyme activity (as compared to the activity in the
absence of any inhibitor) is taken as the IC.sub.50 value.
Analogously, other inhibitory concentrations can be defined through
appropriate determinations of activity. For example, in some
settings it can be desirable to establish a 90% inhibitory
concentration, i.e., IC.sub.90, etc.
[0107] Accordingly, a dual PI3-kinase .delta./.gamma. selective
inhibitor alternatively can be understood to refer to a compound
that exhibits a 50% inhibitory concentration (IC.sub.50) with
respect to PI3-kinase .delta. and .gamma., that is at least 10-fold
lower, at least 20-fold lower, or at least 30-fold lower than the
IC.sub.50 value with respect to any or all of the other class I
PI3K family members. In an alternative embodiment of the invention,
the term dual PI3-kinase .delta./.gamma. selective inhibitor can be
understood to refer to a compound that exhibits an IC.sub.50 with
respect to PI3-kinase .delta. and .gamma. that is at least 30-fold
lower, at least 50-fold lower, at least 100-fold lower, at least
200-fold lower, or at least 500-fold lower than the IC.sub.50 with
respect to any or all of the other PI3K class I family members. A
dual PI3-kinase .delta./.gamma. selective inhibitor is typically
administered in an amount such that it selectively inhibits both
PI3-kinase .delta. and .gamma. activity, as described above.
[0108] In certain embodiments, the compounds of the present
invention exhibit PI3-kinase .delta. and .gamma. inhibition almost
equally (.about.1:1) or at a maximum ratio of 1:5, i.e., the
compound the of the present invention exhibit almost equal
IC.sub.50 values for both PI3-kinase .delta. and .gamma. enzyme, or
at most a 3 to 8 fold difference between the two.
[0109] The methods of the invention may be applied to cell
populations in vivo or ex vivo. "In vivo" means within a living
individual, as within an animal or human or in a subject's body. In
this context, the methods of the invention may be used
therapeutically or prophylactically in an individual. "Ex vivo" or
"in vitro" means outside of a living individual. Examples of ex
vivo cell populations include in vitro cell cultures and biological
samples including but not limited to fluid or tissue samples
obtained from individuals. Such samples may be obtained by methods
known in the art. Exemplary biological fluid samples include blood,
cerebrospinal fluid, urine, and saliva. Exemplary tissue samples
include tumors and biopsies thereof. In this context, the invention
may be used for a variety of purposes, including therapeutic and
experimental purposes. For example, the invention may be used ex
vivo or in vitro to determine the optimal schedule and/or dosing of
administration of a PI3-kinase .delta. selective inhibitor for a
given indication, cell type, individual, and other parameters.
Information gleaned from such use may be used for experimental or
diagnostic purposes or in the clinic to set protocols for in vivo
treatment. Other ex vivo uses for which the invention may be suited
are described below or will become apparent to those skilled in the
art.
[0110] The compounds of the present invention can be prepared by
methods known in the art, such as those described in International
Publication Nos. WO 2011/055215, WO 2012/151525, and WO
2013/164801, each of which is hereby incorporated by reference in
its entirety.
Pharmaceutical Compositions
[0111] The present invention also provides a pharmaceutical
composition comprising one or more compounds of the present
invention and one or more pharmaceutically acceptable carriers or
excipients. In one embodiment, the pharmaceutical composition
includes a therapeutically effective amount of one or more
compounds of the present invention. The pharmaceutical composition
may include one or more additional active ingredients as described
herein.
[0112] The pharmaceutical carriers and/or excipients may be
selected from, for example, diluents, fillers, salts,
disintegrants, binders, lubricants, glidants, wetting agents,
controlled release matrices, colorants, flavourings, buffers,
stabilizers, solubilizers, and combinations thereof.
[0113] In one embodiment, the pharmaceutical compositions described
herein contain from about 0.1 mg to about 1,000 mg, such as from
about 1 mg to about 1,000 mg, from about 20 mg to about 800 mg,
from about 50 mg to about 600 mg or from about 50 mg to about 600
mg of one or more compounds of the present invention. In another
embodiment, the pharmaceutical compositions described herein
contain from about 100 mg to about 400 mg of one or more compounds
of the present invention.
[0114] The pharmaceutical compositions of the present invention can
be administered alone or in combination with one or more other
active agents. Where desired, the subject compounds and other
agent(s) may be mixed into a preparation or both components may be
formulated into separate preparations to use them in combination
separately or at the same time.
[0115] The compounds and pharmaceutical compositions of the present
invention can be administered by any route that enables delivery of
the compounds to the site of action, such as orally, intranasally,
topically (e.g., transdermally), intraduodenally, parenterally
(including intravenously, intraarterially, intramuscularally,
intravascularally, intraperitoneally or by injection or infusion),
intradermally, by intramammary, intrathecally, intraocularly,
retrobulbarly, intrapulmonary (e.g., aerosolized drugs) or
subcutaneously (including depot administration for long term
release e.g., embedded-under the-splenic capsule, brain, or in the
cornea), sublingually, anally, rectally, vaginally, or by surgical
implantation (e.g., embedded under the splenic capsule, brain, or
in the cornea).
[0116] The compositions can be administered in solid, semi-solid,
liquid or gaseous form, or may be in dried powder, such as
lyophilized form. The pharmaceutical compositions can be packaged
in forms convenient for delivery, including, for example, solid
dosage forms such as capsules, sachets, cachets, gelatins, papers,
tablets, suppositories, pellets, pills, troches, and lozenges. The
type of packaging will generally depend on the desired route of
administration. Implantable sustained release formulations are also
contemplated, as are transdermal formulations.
Methods of Treatment
[0117] The amount of the compound to be administered is dependent
on the mammal being treated, the severity of the disorder or
condition, the rate of administration, the disposition of the
compound and the discretion of the prescribing physician. However,
an effective dosage is in the range of from about 0.001 to about
100 mg/kg body weight per day, preferably from about 1 to about 35
mg/kg/day, in single or divided doses. For a 70 kg human, this
would amount to from about 0.05 to about 7 g/day, preferably from
about 0.05 to about 2.5 g/day An effective amount of a compound of
the invention may be administered in either single or multiple
doses (e.g., twice or three times a day).
[0118] The compounds of the present invention may be used in
combination with one or more of anti-cancer agents (e.g.,
chemotherapeutic agents), therapeutic antibodies, and radiation
treatment.
[0119] The compounds of the invention may be formulated or
administered in conjunction with other agents that act to relieve
the symptoms of inflammatory conditions such as encephalomyelitis,
asthma, and the other diseases described herein. These agents
include non-steroidal anti-inflammatory drugs (NSAIDs).
EXAMPLES
[0120] The examples and preparations provided below further
illustrate and exemplify the compounds of the present invention and
methods of preparing such compounds. It is to be understood that
the scope of the present invention is not limited in any way by the
scope of the following examples and preparations. In the following
examples molecules with a single chiral center, unless otherwise
noted, exist as a racemic mixture. Those molecules with two or more
chiral centers, unless otherwise noted, exist as a racemic mixture
of diastereomers. Single enantiomers/diastereomers may be obtained
by methods known to those skilled in the art.
[0121] The intermediates described herein may be prepared by the
methods described in International Publication Nos. WO 11/055215
and WO 12/151525, both of which are hereby incorporated by
reference.
[0122] Intermediate 1:
N-(5-bromo-2-methoxyphenyl)methanesulfonamide: To a solution of
5-bromo-2-methoxyaniline (1.00 g, 4.94 mmol) in dichloromethane (10
ml), pyridine (0.800 ml, 9.89 mmol) was added and cooled to
0.degree. C. Methane sulphonyl chloride (0.40 ml, 5.19 mmol) was
added and stirred for 30 min. The reaction mixture was quenched
with water, extracted with ethyl acetate, dried over anhydrous
sodium sulphate and concentrated under reduced pressure. The crude
product was chromatographed with ethyl acetate:petroleum ether to
afford the title compound as a reddish solid (1.20 g, 87%).
[0123] Intermediate 2:
N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methan-
esulfonamide: Potassium acetate (0.841 g, 8.57 mmol) and
bis(pinacolato)diboron (1.190 g, 4.71 mmol) were added to a
solution of intermediate 1 (1.20 g, 4.28 mmol) in dioxane (17.5 ml)
and the solution was degassed for 30 min.
[1,1'-Bis(diphenylphosphino)ferrocene]dichloro
palladium(II).CH.sub.2Cl.sub.2 (0.104 g, 0.128 mmol) was added
under nitrogen atmosphere and heated to 80.degree. C. After 2 h the
reaction mixture was filtered through celite and concentrated. The
crude product was purified by column chromatography with ethyl
acetate:petroleum ether to afford the title compound as a yellow
solid (1.00 g, 71%). .sup.1H-NMR (.delta. ppm, CDCl.sub.3, 400
MHz): 7. 91 (d, J=1.2 Hz, 1H), 7. 62 (dd, J=8.1, 1.2 Hz, 1H), 6. 92
(d, J=8.1 Hz, 1H), 6.73 (s, 1H), 3.91 (s, 3H), 2.98 (s, 3H), 1.32
(s, 12H).
[0124] Intermediate 3:
(S)-2-(1-(4-amino-3-(4-methoxy-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one:
(S)-2-(1-(4-amino-3-(4-methoxy-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one: To a
solution of
(R)-5-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one
(0.500 g, 1.64 mmol) in THF (5 ml),
3-(4-methoxy-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(0.564 g, 1.97 mmol) and triphenylphosphine (0.649 g, 2.47 mmol)
were added followed by the addition of diisopropylazodicarboxylate
(0.50 ml, 2.47 mmol).
((R)-5-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-o-
ne can be prepared as described for Intermediates 23, 25, and 26 in
International Publication No. WO 2012/0151525.). After 4 h at room
temperature, the mixture was concentrated and the residue was
purified by column chromatography with ethyl acetate:petroleum
ether to afford the title compound as a brown solid (0.270 g, 29%).
.sup.1H-NMR (.delta. ppm, DMSO-d6, 400 MHz): 8.04 (s, 1H), 7.83 (m,
1H), 7.63-7.50 (m, 3H), 7.29 (m, 2H), 7.06 (dt, J=8.7,2.2 Hz, 1H),
6.94 (m, 2H), 6.75 (dd, J=8.1,2.1 Hz, 1H), 5.95 (q, J=7.0 Hz, 1H),
4.98 (s, 2H), 3.81 (s, 3H), 1.86 (d, J=7.0 Hz, 3H).
[0125] Intermediate 4:
(S)-2-(1-(4-amino-3-(3-amino-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one:
(S)-2-(1-(4-amino-3-(3-amino-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one: To a
solution of Intermediate 3 (0.260 g, 0.455 mmol) in ethanol (5 ml),
Raney Ni (0.130 g) was added and hydrogenated at 20 psi at
50.degree. C. for 24 h. The reaction mixture was passed through
celitepad and concentrated to afford the title compound as a brown
solid (0.150 g, 60%). Mass: 540.8 (M.sup.+).
Example A
N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethy-
l)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide
[0126] To a solution of
2-(1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-fluoro-3-(-
3-fluorophenyl)-4H-chromen-4-one (0.200 g, 0.366 mmol) in DME (2.1
ml) and water (0.67 ml), intermediate 2 (0.179 g, 0.550 mmol) and
sodium carbonate (0.116 g, 1.10 mmol) were added and the system was
degassed for 30 min.
(2-(1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-f-
luoro-3-(3-fluorophenyl)-4H-chromen-4-one can be prepared as
described for Intermediates 23, 25, and 26 in International
Publication No. WO 2012/0151525). Bis(diphenylphosphino)
ferrocene]dichloropalladium(II) (0.059 g, 0.075 mmol) was added and
kept under microwave irradiation (microwave power=100 W,
temperature=100.degree. C.) for 45 min. The reaction mixture was
Celite filtered, concentrated and extracted with ethyl acetate. The
organic layer was dried over sodium sulphate and concentrated under
reduced pressure. The crude product was purified by column
chromatography with methanol: dichloromethane to afford the title
compound as a brown solid (0.080 g, 35%). MP: 216-218.degree. C.
.sup.1H-NMR (.delta. ppm, CDCl.sub.3, 400 MHz): 8.20 (s, 1H), 7.73
(s, 1H), 7.53 (m, 2H), 7.31 (m, 2H), 7.07-6.73 (m, 6H), 6.07 (q,
J=6.2 Hz, 1H), 3.98 (s, 3H), 3.14 (s, 3H), 2.01 (d, J=6.0 Hz,
3H).
Example A1 and A2
Method A
[0127]
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chrom-
en-2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesu-
lfonamide
and
(R)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen--
2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfo-
namide
##STR00012##
[0129] The two enantiomerically pure isomers were separated by
preparative SFC (supercritical fluid) conditions from
N-(5-(4-amino-1-(145-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethy-
l)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide
(0.500 g) on a CHIRALPAK AS-H column (250.times.30 mm; 5 .mu.m)
using methanol: CO.sub.2 (55:45) as the mobile phase at a flow rate
of 80 g/min.
[0130] Example A1 (S-isomer): Brown solid (0.247 g). Enantiomeric
excess: 97.4%. Retention time: 2.14 min. Mass: 619.1 (M.sup.++1).
MP: 156-158.degree. C.
[0131] Example A2 (R-isomer): Brown solid (0.182 g). Enantiomeric
excess: 99.3%. Retention t: 3.43 min. Mass: 619.1 (M.sup.++1). MP:
168-171.degree. C.
Method A1
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl-
)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonami-
de
and
(R)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen--
2-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfo-
namide
##STR00013##
[0133] The two enantiomerically pure isomers were separated by
preparative SFC (supercritical fluid) conditions from
N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)eth-
yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)
methanesulfonamide (15.0 g) on a CHIRALPAK AS-H column
(250.times.20 mm; 5 .mu.m) using methanol: CO.sub.2 (45:55) as the
mobile phase at a flow rate of 120 g/min.
[0134] Example A1 (S-isomer): Enantiomeric excess: 100%. Retention
time: 2.21 min. Mass: 619.1 (M.sup.++1). MP: 175-178.degree. C.
Specific optical rotation (C=1 in chloroform, at 25.degree. C.):
[.alpha.].sub.D=+147.16.
[0135] Example A2 (R-isomer): Enantiomeric excess: 99.3%. Retention
t: 3.72 min. Mass: 619.1 (M.sup.++1). MP: 154-157.degree. C.
Specific optical rotation (C=1 in chloroform, at 25.degree. C.):
[.alpha.].sub.D=-159.54.
Method B
Example A1
(S)--N-(5-(4-amino-1-(1-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl-
)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonami-
de
[0136] To a solution of Intermediate 4 (0.500 g, 0.923 mmol) in
dichloromethane (5 ml) cooled to 0.degree. C., pyridine (0.200 ml,
1.84 mmol) was added and stirred for 10 min. Methanesulphonyl
chloride (0.100 ml, 0.923 mmol) was added stirred for 30 min. The
reaction mixture was quenched with water, extracted with
dichloromethane and dried over sodium sulphate. The crude product
was column chromatographed with methanol:dichloromethane to afford
the title compound as an off-white solid (0.240 g, 42%). MP:
211-213.degree. C. .sup.1H-NMR (.delta. ppm, DMSO-d6, 400 MHz):
9.15 (s, 1H), 8.06 (s, 1H), 7.83 (m, 1H), 7.49 (m, 4H), 7.28 (m,
4H), 7.08 (dt, J=8.6,1.7 Hz, 1H), 6.92 (s, 2H), 5.98 (q, J=6.9 Hz,
1H), 3.88 (s, 3H), 2.99 (s, 3H), 1.88 (d, J=7.0 Hz, 3H).
Enantiomeric excess: 85.4% as determined by HPLC on a chiralpak
AS-3R column, enriched in the fast eluting isomer (retention
time=7.46 min.).
Metabolic Stability
[0137] Metabolic stability studies were conducted on Compounds A,
A1, and A2 as well as Example 128 of WO 2012/151525 using mouse,
rat, dog, monkey, and human liver microsomes. The protocol for the
studies with mouse, rat, and human liver microsomes (all from BD
Gentest, USA) is provided below. 0.4 mg protein was preincubated
with 2 mM NADPH (cofactor) in phosphate buffer (pH-7.4) for 15
minutes at 37.degree. C. and then added with 1 .mu.M test item and
incubated further for 60 minutes in triplicate. The reaction
mixture was terminated with methanol containing an internal
standard and centrifuged further to analyze the test item remaining
in the supernatant by LC-MS/MS. The percent parent compound
remaining was calculated in comparison with similar samples
terminated at 0 minutes. The results are provided in Table 1
below.
[0138] The metabolic stability data for Compound A1 indicates that
it exhibits a superior pharmacokinetic profile.
TABLE-US-00001 TABLE 1 Metabolic stability in liver microsomes
Compound Mouse Rat Dog Monkey Human Example 128 of 85.0 73.3 ND ND
70.4 WO 2012/151525 Compound A 96 91 64.3 42.3 69.7 Compound A1
85.9 94.2 83.5 78.8 95.7 Compound A2 68.9 79.5 52.3 1.9 60.2
ND--Not Determined
Protein Binding
[0139] Below is provided the procedure for measuring plasma protein
binding (using an equilibrium dialysis method). 745 .mu.L of plasma
was transferred into a 2 ml micro centrifuge tube. To that 5 .mu.L
of Compound A1 (150 .mu.M) was added. Samples were mixed in the
table top vortexer for 2 minutes. 50 .mu.L plasma (n=2) was
transferred in a pre-labeled 1.5 mL micro centrifuge tube treated
as 0 hour sample.
[0140] The remaining 650 .mu.L plasma sample were incubated for 30
minutes at 37.degree. C. in a water bath. After 30 minute
incubation, 50 .mu.L plasma (n=2) was removed in a pre-labelled 1.5
mL micro centrifuge tube treated as 0.5 hour sample. 200 .mu.L of
the plasma sample (n=2) was transferred into the sample chamber
which was indicated by the red ring. The red insert was placed into
the base plate and 350 .mu.L of buffer was transferred into the
buffer chamber. Plates were incubated at 37.degree. C. at
approximately 100 RPM on an orbital shaker or 20 RPM on an
up-and-down shaker for 4 hours. 50 .mu.L of post dialysis-sample
from the buffer and the plasma chambers were transferred into a
pre-labelled micro centrifuged tube. 50 .mu.L of plasma was added
to the buffer samples and an equal volume of buffer
(KH.sub.2PO.sub.4 Buffer pH 7.4) to the collected plasma samples.
150 .mu.L of methanol containing internal standard (Tolbutamide 250
ng/ml) was added to precipitate the protein and release compound.
Samples were vortexed for 3 minutes in a table top vortexer and
centrifuge for 5 minutes at 14,000 RPM. Supernatant was subjected
to LC-MS/MS analysis.
[0141] The plasma protein binding data for Compound A1 is provided
in Table 2 below:
TABLE-US-00002 TABLE 2 Protein Binding (%) Mouse Rat Dog Monkey
Human 97.61 99.04 95.85 94.71 97.24
Pharmacokinetics
[0142] The oral bioavailability of Compound A1 (free base) was
evaluated in rats and mice. The protocol for the pharmacokinetics
studies in rat is provided below.
[0143] All animals were fasted overnight (12 hours) before dosing
and continued till 4.0 hours after administration of test item.
Test item formulations were prepared in 1% Tween 80 and 99% media
(0.5% Methyl cellulose, 4000cPs, pH 2.2). The blood samples (150
.mu.L from each animal) were collected from the orbital sinus, and
placed into a micro centrifuge tube containing disodium EDTA as an
anticoagulant. Blood samples were centrifuged immediately with a
speed of 1000 g for 10 min at 4.degree. C. and separated plasma
samples were frozen at below -80.degree. C. and stored until
analysis. The concentrations of test item in all formulations were
analyzed by HPLC. The plasma concentrations of test item in all
samples were analyzed by LC-MS/MS. Pharmacokinetic parameters
(C.sub.max, AUC.sub.0-t, T.sub.max, and t.sup.1/2) were estimated
by using WinNonlin software. Results are provided in Table 3 for
Compound A, A1, and Example 128 of WO 2012/151525 in rats and
Compound A1 in mice.
TABLE-US-00003 TABLE 3 Ex. 128 of WO Compound Compound Units
2012/151525 Compound A Compound A1 A1 Animal Rat Mice Route Oral
Oral Oral Oral Dose mg/kg 10 10 10 10 N 2 2 4 3 C.sub.max .mu.M
0.68 1.02 11.38 3.78 AUC.sub.0-t .mu.M hr 2.01 7.95 97.76 7.49
T.sub.max Hr 0.83 2.67 1.83 0.50 t.sub.1/2 Hr 1.56 4.52 2.45
1.45
[0144] Compounds A and A1 showed superior pharmacokinetic profiles
compared to Example 128 of WO 2012/151525. For instance, Compound A
showed a .about.1.5 fold increase in C.sub.max, .about.4 fold
increase in AUC.sub.0-t, and .about.2.8 fold increase in t.sub.1/2
as compared to Example 128 of WO 2012/151525. Compound A1 showed a
.about.16 fold increase in C.sub.max, 48 fold increase in
AUC.sub.0-t, and .about.1.6 fold increase in t.sub.1/2 as compared
to Example 128 of WO 2012/151525.
Biological Assays
[0145] The pharmacological properties of the compounds described
herein may be confirmed by a number of pharmacological assays, as
exemplified below.
Assay 1: Fluorescent Determination of PI3 Kinase Enzyme
Activity
[0146] Phosphoinositide 3 kinases (PI3K) belong to a class of lipid
kinases that play a critical role in the regulation of several key
cellular processes. The PI3K are capable of phosphorylating the
3-hydroxy position of phosphoinositols thereby generating second
messengers involved in downstream signaling events. The homogenous
time resolved fluorescence (HTRF) assay allows detection of
3,4,5-triphosphate (PIPS) formed as a result of phosphorylation of
phosphotidylinositol 4,5-biphosphate (PIP2) by PI3K isoforms such
as .alpha., .beta., .gamma. or .delta..
[0147] PI3K isoform activity for .alpha., .beta., .gamma. or
.delta. was determined using a PI3K human HTRF.TM. Assay Kit
(Millipore, Billerica, Mass.) with modifications. All incubations
were carried out at room temperature. 0.5 .mu.l of 40.times.
inhibitor (in 100% DMSO) or 100% DMSO were added to each well of a
384-well white plate (Greiner Bio-One, Monroe, N.C.) containing
14.5 .mu.l 1.times. reaction buffer/PIP2 (10 mM MgCl.sub.2, 5 mM
DTT, 1.38 .mu.M PIP2) mix with or without enzyme, followed by 5
.mu.l/well of 400 .mu.M ATP and incubated for an additional 30
minutes. The reaction was terminated by adding 5 .mu.l/well stop
solution (Millipore, Billerica, Mass.). 5 .mu.l of detection mix
(Millipore, Billerica, Mass.) was then added to each well and
incubated for 6-18 hours in the dark. HRTF ratio was measured on a
microplate reader (BMG Labtech., Germany) at an excitation
wavelength of 337 nm and emission wavelengths of 665 and 615 nm
with an integration time of 400 msec counting delay of 50 msec. The
results for Compounds A, A1 and A2 are shown in Table 4 below.
Comparative data for Compound A1 and Example 128 of WO2012/151525
are provided in Table 5 below.
TABLE-US-00004 TABLE 4 IC.sub.50 (nM) Compound Pi3K.delta.
Pi3K.alpha. Pi3K.beta. Pi3K.gamma. A 102.8 ND ND 82.94 A1 30.46
>10000 1359 48.72 A2 92.95 ND ND >10 .mu.M ND: Not
Determined
TABLE-US-00005 TABLE 5 Selectivity profile Assay IC.sub.50 (nM)
Fold-Selectivity Compound PI3K.delta. PI3K.gamma. PI3K.alpha.
PI3K.beta. Example 128 of 76.01 70.70 NC (38.29*) NC (51.04*) WO
2012/151525 Compound A 102.8 82.94 ND ND Compound A1 30.46 48.72
>329 >45 (46.8*) (23.02**) (IC.sub.50 = 1359 nM) Compound A2
92.95 >10000 ND ND *% inhibition @ 1 .mu.M; **% inhibition @ 10
uM; NC--Not Calculated and ND: Not Determined
Assay 2: In Vitro Cell Proliferation Assay in Leukemic Cell
Lines
[0148] Growth inhibition assays were carried out using 10% FBS
supplemented media. Cells were seeded at a concentration of
5000-20,000 cells/well in a 96-well plate. Test compounds at a
concentration ranging from 0.01 to 10000 nM were added after 24
hours. Growth was assessed using the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
dye reduction test at 0 hour (prior to the addition of the test
compound) and 72 hours after the addition of test compound.
Absorbance was read on a Fluostar Optima (BMG Labtech, Germany) at
a wavelength of 450 nm. Data were analysed using GraphPad Prism and
percent inhibition due to the test compound compared to the control
was calculated accordingly.
[0149] Compound A1 caused a reduction in T-lymphoma (MOLT-4,
Jurkat, CCRF-CEM, Hut-78 and HuT-102) cell viability with GI.sub.50
values ranging from 2.5-12.8 .mu.M for the dose range tested.
Additionally, compound A1 did not display any apparent cytotoxicity
over the 72 hour incubation period.
Assay 3: Inhibition of AKT Phosphorylation in Leukemic Cell
Lines
[0150] MOLT-4, Jurkat, CCRF-CEM, and Hut-78 cells were incubated
with desired concentrations of compound for 48 hours. Cells were
lysed and pAKT determined by Western Blotting. Bands were
quantified using ImageJ and normalized to actin.
[0151] Compound A1 caused a reduction in pAKT expression in
T-lymphoma (MOLT-4, Jurkat, CCRF-CEM and Hut-78) cell lines with
EC.sub.50 values ranging from 0.02-1.6 .mu.M for the dose range
tested.
Assay 4: Inhibition of PI3K .delta. and .gamma. Signaling in
Basophils from Human Whole Blood
[0152] PI3K .delta. and .gamma. signaling in basophils manifested
by an alteration of anti-Fc.epsilon.R1 or fMLP induced CD63
expression is a useful pharmacodynamic marker determined using the
Flow2CAST.RTM. kit (Buhlmann Laboratories, Switzerland). The test
procedure involves the following steps: [0153] Mix the
anti-coagulated blood sample by inverting the venipuncture tube
several times; [0154] Prepare fresh and pyrogen-free 3.5 ml
polypropylene or polystyrene tubes suitable for Flow Cytometry
measurements; [0155] Add 49 .mu.l of patient's whole blood to each
tube; [0156] Add 1 .mu.l of 10% DMSO (background) or test compound
(10% DMSO) to the assigned tubes and mix gently. Incubate at room
temperature for 15 minutes; [0157] Pipet 50 .mu.l of the
Stimulation buffer (background) or anti-Fc.epsilon.RI Ab or fMLP to
each tube; [0158] Add 100 .mu.l of Stimulation Buffer to each tube;
[0159] Mix gently. Add 20 .mu.l Staining Reagent (1:1 mix of
FITC-CD63 and PE-CCR3) to each tube; [0160] Mix gently, cover the
tubes and incubate for 15 minutes at 37.degree. C. in a water bath.
(using an incubator will take about 10 minutes longer incubation
time due to less efficient heat transfer); [0161] Add 2 ml
pre-warmed (18-28.degree. C.) Lysing Reagent to each tube, mix
gently; [0162] Incubate for 5-10 minutes at 18-28.degree. C.;
[0163] Centrifuge the tubes for 5 minutes at 500.times.g; [0164]
Decant the supernatant by using blotting paper; [0165] Resuspend
the cell pellet with 300-800 .mu.l of Wash Buffer; and [0166]
Vortex gently and acquire the data on the flow cytometer within the
same day.
[0167] Percent CD63 positive cells within the gated basophil
population were determined in different treatment groups and
normalized to vehicle control.
[0168] Compound A1 exhibited an EC.sub.50 of <30 nM for
Fc.epsilon.R1(PI3K 6) and an IC.sub.50 of <70 nM for fMLP (PI3K
.gamma.)(n=1).
Assay 4A: Cellular Activity Demonstrating Selectivity of Compound
A1 towards PI3K Delta and PI3K Gamma Isoforms
Assay 4A1: Anti-IgM Induced B-Cell Proliferation (For PI3K.delta.
Selectivity)
[0169] The objective of this study was to assess the inhibitory
potential of Compound A1 on anti-IgM induced human B-cell
proliferation.
Plating and Treatment
[0170] Isolated B-cells were re-suspended to 1.0.times.10.sup.6
cells per ml. 100 .mu.l of cell suspension was added to each well
of a 96-well plate. Triplicates were maintained.
[0171] 50 .mu.l of drug dilution was added and mixed well. A DMSO
blank and inducer blank were maintained.
[0172] The treated plate was incubated for 30 minutes at 37.degree.
C., 5% CO.sub.2 and then 50 .mu.l of 4.times. inducer was added and
mixed by pipetting.
[0173] The plate was incubated at 37.degree. C., 5% CO.sub.2 for 72
hours.
[0174] Media was aspirated and 150 .mu.l of DMSO was added to
dissolve the formazan crystals.
[0175] Absorbance was read at A560 and A640 nm.
[0176] The data demonstrates the inhibitory potential of Compound
A1 on PI3K.delta. mediated induction of human B-cell proliferation.
See, e.g., Baeker et al., Journal of Immunology, 134: 3532-3538,
1985.
Assay 4A2: LPA Induced AktS473 Phosphorylation in 3T3 Fibroblasts
(for PI3K.beta. Selectivity)
[0177] The objective of this study was to determine the effect of
Compound A1 on PI3K.beta. kinase mediated LPA induced AktS473
phosphorylation in 3T3 fibroblasts.
[0178] 3T3 cells were treated with desired concentrations of the
test compound for 15 minutes. 1 ml of 2.times. LPA was added such
that the final concentration was 5 .mu.M and incubated for 5
minutes.
[0179] Media was discarded and washed with 1 ml of ice-cold
1.times. PBS.
[0180] 250 .mu.l of cell lysis buffer was added and incubated on
ice for 30 minutes.
[0181] Samples were centrifuged and supernatant was maintained at
-80.degree. C. until analysis.
[0182] Samples were analyzed by Western Blotting using pAKT (S473)
as the primary and anti-rabbit IgG-HRP as a secondary antibody.
[0183] Intensity of the bands was determined using ImageJ 1.42q
(NIH, USA) and normalized to Actin (loading control). Data was
plotted using GraphPad Prism (Version 5.02).
[0184] The results demonstrate the selectivity of Compound A1 over
the beta isoform of PI3K. See Albuquerque et al., J. Biol. Chem.,
278, 39830-39838, 2003.
Assay 4A3: c5a Induced AktS473 Phosphorylation in RAW 264.7
Macrophages (For PI3K.gamma. Selectivity)
[0185] The objective of this study was to determine the effect of
Compound A1 on PI3K.gamma. kinase mediated c5a induced AktS473
phosphorylation in RAW 264.7 macrophages.
[0186] RAW 264.7 cells were treated with desired concentrations of
the test compound for 15 minutes. 1 ml of 2.times. c5a was added
such that the final concentration was 50 ng/ml and incubated for 15
minutes.
[0187] Media was discarded and washed with 1 ml of ice-cold
1.times. PBS.
[0188] 250 .mu.l of cell lysis buffer was added and incubated on
ice for 30 minutes.
[0189] Samples were centrifuged and supernatant was stored at
-80.degree. C. until analysis
[0190] Samples were analyzed by Western Blotting using pAKT (S473)
as the primary and anti-rabbit IgG-HRP as a secondary antibody.
[0191] Intensity of the bands was determined using ImageJ 1.42q
(NIH, USA) and normalized to Actin (loading control). Data was
plotted using GraphPad Prism (Version 5.02).
[0192] Inhibition of pAktS473, a downstream marker of PI3K.gamma.
signaling suggests a role for Compound A1 in the oncogenic pathways
regulated by Akt in c5a induced RAW 264.7 cells. See To et al., Am.
J. Respir. Crit. Care Med., 182, 897-904, 2010.
Assay 4A4: PDGF Induced Akt Phosphorylation in 3T3 cells (For PI3K
a Selectivity)
[0193] The objective of this study was to determine the effect of
Compound A1 on PI3K.alpha. kinase mediated AktS473 phosphorylation
in PDGF induced 3T3 fibroblasts. [0194] 3T3 cells were treated with
desired concentrations of the test compound for 15 minutes. 1 ml of
2.times. PDGF was added such that the final concentration was 20
ng/ml and incubated for 10 minutes. [0195] Media was discarded and
washed with 1 ml of ice-cold 1.times. PBS. [0196] 250 .mu.l of cell
lysis buffer was added and incubated on ice for 30 minutes. [0197]
Samples were centrifuged and supernatant was collected and stored
at -80.degree. C. until analysis. [0198] Samples were analyzed by
Western Blotting using pAKT (S473) as the primary and anti-rabbit
IgG-HRP as a secondary antibody. [0199] Intensity of the bands was
determined using ImageJ 1.42q (NIH, USA) and normalized to Actin
(loading control). Data was plotted using GraphPad Prism (Version
5.02).
[0200] No inhibition was observed at 10 .mu.M of Compound A1,
demonstrating the selectivity of Compound A1 over the alpha isoform
of PI3K. See Albuquerque et al., J. Biol. Chem. 278, 39830-39838,
2003.
[0201] Table 6 below summarizes the results from Assays
4A1-4A4.
TABLE-US-00006 TABLE 6 CELLULAR ACTIVITY DEMONSTRATING SELECTIVITY
OF COMPOUND A1 TOWARDS PI3K .delta. AND PI3K .gamma. ISOFORMS
Cellular IC.sub.50 PI3K alpha (PDGF induced pAKT in 3T3 >10000
nM fibroblasts) Cellular IC.sub.50 PI3K beta (LPA induced pAKT in
3T3 1324 nM fibroblasts) Cellular IC.sub.50 PI3K delta (anti-IgM
induced human B-cell 11.03 nM proliferation) Cellular IC.sub.50
PI3K gamma (c5a induced pAKT in RAW 51.73 nM macrophages)
Assay 5: Inhibition of Apoptosis in Leukemic Cell Lines
[0202] Apoptosis in leukemic cells was determined using an in situ
Caspase 3 kit (Millipore, US) as outlined below:
[0203] Seed leukemic cells at a density of 1.times.10.sup.6
cells/well in a 6 well plate
[0204] Add test compound/DMSO at desired concentrations
[0205] Incubate the plate for 24 hours at 37.degree. C. in 5%
CO.sub.2 incubator
[0206] Collect cells in a 2 ml centrifuge tube
[0207] Add 1.6 .mu.L of freshly prepared 5.times. FLICA reagent and
mix cells by slightly flicking the tubes
[0208] Incubate tubes for 1 hour at 37.degree. C. under 5%
CO.sub.2
[0209] Add 2 ml of 1.times. wash buffer to each tube and mix
[0210] Centrifuge cells at <400.times.g for 5 minutes at room
temperature.
[0211] Carefully remove and discard supernatant, and gently vortex
cell pellet to disrupt any cell-to-cell clumping.
[0212] Re-suspend cell pellet in 300 ul of 1.times. wash buffer
[0213] Place 100 .mu.L of each cell suspension into each of two
wells of a black microtiter plate. Avoid creation of bubbles.
[0214] Read absorbance of each microwell using an excitation
wavelength of 490 nm and an emission wavelength of 520 nm.
[0215] Percent increase in caspase-3 activity manifested by an
increase in fluorescence compared to the control blank is to be
calculated.
Assay 6A: Cytokine Assay in Human PBMC
[0216] The objective of this study was to assess the inhibitory
potential of Compound A1 on antigen-induced cytokine release in
human PBMC
Plating and Treatment
[0217] Heparinized human whole blood was diluted 1:1 with PBS, over
laid on leukocyte separation medium and centrifuged at 400 g for 40
minutes. [0218] Buffy layer was removed and washed with PBS [0219]
0.15*10.sup.6 of PBMCs were plated in 100 .mu.l per well in RPMI
media and incubated for 2 h. [0220] 50 .mu.l of 3.times. of the
compound dilution in media was added and incubated for 15 min.
[0221] TNF.alpha.--induced with 50 .mu.l of LPS in RPMI such that
final concentration was 1 .mu.g/ml. Supernatant was collected at 6
hours. [0222] IL-2-induced with 50 .mu.l of PHA in RPMI such that
final concentration was 20 .mu.g/ml. Supernatant was collected at
24 hours. [0223] IL-4-induced with 50 .mu.l of PHA in RPMI such
that final concentration was 20 .mu.g/ml. Supernatant was collected
at 48 hours. [0224] ELISA was performed used kits from eBioscience.
[0225] EC.sub.50 was calculated using GraphPad Prism 5.
[0226] EC.sub.50 values were calculated from 2-3 independent
experiments. Compound A1 inhibited antigen-induced TNF.alpha.,
IL-2, and IL-4 with an EC.sub.50 of 7.1, 9.5, and 3.5 nM,
respectively.
Assay 6B: Inhibition of LPS Induced CD19 or CD45R in Human or Mouse
Whole Blood
[0227] The effect of Compound A1 on modulating B-cell receptor
(BCR)-activated proliferation of human or mouse B-lymphocytes was
determined. CD19 is a protein present on B cells from the earliest
recognizable B-lineage cells during development to B-cell blasts
but is however lost on maturation to plasma cells. LPS is an
endotoxin and a major component of environmental microbes with a
potent mitogenic activity on B-cells via the BCR signaling
pathway.
[0228] Diluted human whole blood was treated with DMSO or desired
concentrations of Compound A1. Samples were induced with LPS 15
minutes after addition of compound and incubated for 72 hours at
37.degree. C. and 5% CO.sub.2. Cells positive for CD45 and CD19
were determined by flow cytometry and data are expressed as
percentage CD19 positive cells in the total population. Treatment
with Compound A1 resulted in a dose-dependent inhibition of
LPS-induced human whole blood B-cell proliferation (EC.sub.50=117.7
nM) manifested by a reduction in CD19 expression.
[0229] Similar to CD19, CD45R (B220) is expressed on mouse
B-lymphocytes throughout their development from early pro-B stages
onwards and is down-regulated upon terminal differentiation to
plasma cells. Briefly, diluted mouse whole blood was treated with
of DMSO or desired concentrations of Compound A1. Samples were
induced with LPS 15 minutes after compound addition, and incubated
for 72 hours at 37.degree. C. and 5% CO.sub.2. Cells positive for
CD45 and CD45R were determined by flow cytometry. Data are
expressed as percentage CD45R positive cells in the total
population. Consistent with CD19+ cell proliferation data,
treatment with Compound A1 resulted in a dose-dependent inhibition
of LPS-induced mouse whole blood B-cell proliferation
(EC.sub.50=128.2 nM) manifested by a reduction in CD45R
expression.
Assay 6C: Inhibition of AKT Phosphorylation in Isolated Mouse
Splenocytes
[0230] The PI3K pathway is regulated downstream by AKT, a
serine-threonine kinase that modulates several oncogenic processes
such as cell proliferation, growth, and survival. Because the
spleen is a repertoire for vast quantities of B- and T-lymphocytes,
inhibition of LPS-induced AKT phosphorylation was determined ex
vivo using isolated mouse splenocytes. Cells were plated and
incubated with a desired concentration of Compound A1 for 15
minutes followed by induction with LPS (20 .mu.g/mL) for 30
minutes. Following induction, cells were lysed and pAKT was
determined by ELISA using pAKT.sup.S473 capture/detection antibody
pair and anti-mouse-HRP secondary antibody. Blank subtracted
absorbance values were obtained to calculate percent inhibition of
pAKT in test samples. Compound A1 caused a dose-dependent reduction
(EC.sub.50=347.4 nM) in phosphorylation of the downstream marker,
AKT, at low concentrations thereby elucidating the signaling
pathway
Assay 7: Lipopolysaccharide Induced Pulmonary Neutrophilia in
Female Wistar Rat Model
[0231] An exaggerated recruitment and subsequent activation of
neutrophil is likely to be important for the development and course
of several inflammatory diseases in the airways and lungs, such as
severe asthma, chronic obstructive pulmonary disease, cystic
fibrosis, and acute respiratory distress syndrome. The mechanisms
by which neutrophil contributes to these diseases may involve the
release of proteolytic enzymes, such as neutrophil elastase, and
free oxygen radicals. When released, these compounds can cause
bronchoconstriction, bronchial hyperreactivity, hyper-secretion,
epithelial damage, and tissue remodeling in the airways.
[0232] After the quarantine period, fasted animals were randomized
and divided into various groups depending on their body weights.
The test compound (Compound A1) was prepared as a suspension in a
vehicle consisting of 0.5% methylcellulose in which Tween 80 as a
suspending agent. The compound or vehicle was administered by oral
gavage at a volume of 10 mL/kg. Female Wistar rats were
anaesthetized with ketamine and LPS solution was administered
intratracheally one hour after compound administration at a dose of
1 mg/kg. 6 hours after LPS instillation, animals were exsanguinated
under anaesthesia, and then the trachea was cannulated and the
lungs were lavaged with 5 ml aliquots of heparinised PBS (1
unit/ml) four times through a tracheal cannula (total volume 20
mL). Bronchoalveolar lavage (BAL) fluid was stored at 2-8.degree.
C. until assayed for total cell and differential leukocyte count.
Bronchioalveolar fluid was centrifuged (500.times.g for 10 minutes)
and the resulting cell pellet was resuspended in 0.5 ml of
heparinised saline. The total numbers of white blood cells were
determined in BAL fluid or blood by using a blood cell counter and
was adjusted to 1.times.10.sup.6 cell/ml. Differential cell count
was calculated manually. One hundred microliters of the cell
suspension was centrifuged using a Cytospin 3 to prepare a cell
smear. The cell smear was stained with a blood staining solution
for differentiation and slides were microscopically observed to
identify eosinophil according to their morphological
characteristics. The number of each cell type among 300 white blood
cells in the cell smear was determined and expressed as a
percentage. The number of eosinophil in each BALf or blood was
calculated.
[0233] Compound A1 showed a reduction of neutrophil infiltration
into the lungs with an inhibition of 65.29% at 10 mg/kg compared to
the control group, suggesting a therapeutic role in inflammatory
disorders. The results are shown in FIG. 1.
Assay 8: Lipopolysaccharide-Induced Rat Air Pouch Model of
Inflammation
[0234] Female Wistar rats (175-200 g) were acclimatized for seven
days prior to the start of the experiment. Animals were randomly
distributed to various groups based on their body weights. Animals
were anaesthetised with ether and subcutaneous air pouches were
made by injecting 20 ml of sterile air under the skin in the
intra-scapular area (day 0) and maintained with a second 10 ml
injection of sterile-filtered air on day 4. On day 6, oral
treatment was commenced 1 hour prior to induction of inflammation
by s.c. injection of LPS solution on day 6. A volume of 5 ml of LPS
solution dissolved in sterile saline (100 .mu.g/kg) was injected
into each pouch. Samples of pouch fluid were taken at 6 h after
administration of LPS by flushing the pouch with 5 ml of sterile
saline and withdrawing 4 ml of fluid. The number of leukocytes
present in pouch fluid was determined microscopically using a
haemocytometer. Differential cell content was determined by
microscopic examination of fluid smears stained with Diff-Quik.
[0235] Compound A1 caused a dose-dependent reduction of neutrophil
migration into the rat air pouch with an ED.sub.50 of 2.65 mg/kg
suggesting a therapeutic role in rheumatoid arthritis. The results
are shown in FIG. 2.
Assay 9: Ovalbumin Induced Pulmonary Eosinophilia in Male Guinea
Pigs
[0236] After the quarantine period, 0.3 ml of blood samples are
collected from orbital vein by retro-orbital plexus method from
each individual animal and analysed on a cell analyser (ADVIA 2120,
Siemens). Based on their total cell count, guinea pigs are
randomized and divided into various groups. Ear pinna is marked
with an indelible marking pen for identification. On day 0, weights
are recorded and animals are sensitized with 50 .mu.g of ovalbumin
(OVA) and 10 mg of alum solution (1 ml) intraperitoneally. On day 7
and day 14, the above sensitization protocol is repeated. Animals
are observed for any signs of illness or reaction to the
sensitization up to day 19 and recorded if any. On day 19, 20, and
21, after the treatment with test compound by oral gavage, 30
minutes later animals are exposed to 0.5% w/v, 0.5% and 1%
ovalbumin challenge respectively. Control and sham group animals
are treated with 0.5% w/v methyl cellulose (vehicle). Sham control
groups are sensitized with 10 mg of alum on day 0, 7 and 14 and
exposed to saline solution (SAL) with the same nebulization rate on
day 19. 20 and 21. Twenty hours after the last OVA challenge,
airway hyperresponsiveness is measured by whole body plethysmograph
against cumulative doses of methacholine challenge (75, 100, 125
and 150 .mu.g/ml). After measuring the airway response, blood
samples and BAL fluid are collected. Samples are analysed for total
cell count by using a neubuear chamber under microscope and
differential leukocyte count is done manually.
Assay 10: Murine Asthma Model
[0237] After the quarantine period, based on their body weights,
mice were randomized and divided into four groups (n=7). Tails were
marked with an indelible marking pen for identification. On day 0,
weights were recorded and animals were sensitized with 100 .mu.g of
ovalbumin and 10 mg of alum solution (0.2 mL)
intraperitoneally.
[0238] On day 7 and day 14, the above sensitization protocol was
repeated. Animals were observed for any signs of illness or
reaction to the sensitization up to day 24 and recorded if any. On
day 24, 25, and 26, after the treatment with test compound by oral
gavage, 30 minutes later animals were exposed to 10% w/v ovalbumin
challenge.
[0239] Control and sham group animals were treated with 0.5% w/v
methyl cellulose (vehicle). Sham control groups were sensitized
with 10 mg of alum on day 0, 7 and 14 and exposed to saline
solution with the same nebulization rate on day 24, 25 and 26.
[0240] Forty eight hours after the last OVA challenge, airway
hyperresponsiveness was measured by whole body plethysmograph
against cumulative doses of methacholine challenge (2.5, 10, 50 and
100 mg/ml). After measuring the airway response, blood samples and
BAL fluid were collected. Samples were analysed for total cell
count by using a neubuear chamber under microscope and differential
leukocyte count was done manually.
Assay 11: Collagen Induced Arthritis (CIA) in Wistar Rats
[0241] Female wistar rats were acclimatized for seven days prior to
the start of the experiment and were randomly distributed to
various groups based on their body weights. On day 0, animals were
treated by intradermal injection of 500 .mu.g of bovine collagen
type II emulsified with complete Freund's adjuvant (IFA) containing
MTB (4 mg/mL) delivered at the base of the tail. On day 7 after
primary immunization, animals were treated by booster injection of
300 .mu.g CII in incomplete Freund's adjuvant by intradermal
injection at the base of the tail. Onset of arthritis in ankle
joints usually became visually apparent between days 12 and 14.
Animals were treated with test compound or vehicle (orally
administered) from the day after onset of arthritis and the
treatment continued for the next 9 consecutive days. Arthritis
Scores were taken by visual examination for signs of joint
inflammation regularly throughout the study period. Measurements of
body weights, paw volumes, and paw thickness were taken on days 0,
1, 3, 5, 7, 9, and 10. After the ten day treatment, at the end of
the study, blood was withdrawn at necropsy and processed to serum
or plasma and all joints were taken and both fore paw and hind paws
were fixed in 10% formalin for histopathology analysis after taking
a small piece of tissue from each joint and stored at -80.degree.
C. for cytokine analysis in tissue homogenate. Clinical Scoring
Criteria for Fore and Hind Paws: 0=normal; 1=one hind or fore paw
joint affected or minimal diffuse erythema and swelling; 2=two hind
or fore paw joints affected or mild diffuse erythema and swelling;
3=three hind or fore paw joints affected or moderate diffuse
erythema and swelling; 4=marked diffuse erythema and swelling, or
=four digit joints affected; 5=severe diffuse erythema and severe
swelling entire paw, unable to flex digits.
[0242] Compound A1 dosed therapeutically in the rat CIA model
demonstrates significant efficacy in the reduction of the clinical
score (FIGS. 3A and 3B) observed in both prophylactic paws (FIG.
3C) and therapeutic paws (FIG. 3D).
[0243] Compound A1 dosed therapeutically in the rat CIA model
demonstrates significant efficacy in reducing the average paw
volumes of both the hind paws (FIGS. 4A and 4B) and in ankle
diameter (FIGS. 4C and 4D).
[0244] Histological analysis: Compound A1 dosed therapeutically in
the rat CIA model demonstrates significant efficacy in inhibition
of inflammation (58.3%, see FIG. 4A), cartilage (46.51%, see FIG.
4B) and pannus (49.18%, see FIG. 4C) observed by histopathology of
all the hind and fore paws.
[0245] The incidence and progression of arthritis was significantly
reduced in treatment group compared to control group animals (FIG.
5).
Assay 12: Acute Cigarette Smoke Induced Cell Infiltration in Male
Balb/c Mice
[0246] Animals (male Balb/c mice) are to be acclimatized for seven
days prior to the start of the experiment. Animals are then to be
randomly distributed to various groups based on their body weights.
On day 1, the mice are to be administered test compound or vehicle
by oral/intranasal route and after 1 hour, the test compound
administered animals are to be placed in a whole body exposure box.
On day 1 and day 2, mice are exposed to the mainstream smoke of 6
cigarettes, of 8 cigarettes on day 3, and of 10 cigarettes on day
4. Exposure to the smoke of each cigarette will last for 10
minutes. The cigarettes are to be completely burned in the first
two minutes, followed by an air flow with animal ventilator and the
next 20 minutes will be exposure with fresh room air. After every
second cigarette, an additional break of 20 minutes with exposure
to fresh room air is to be conducted. Control animals are to be
exposed to room air chamber. From day 1 to day 4, animals will be
administered the test compound either by oral or intranasal route.
On day 5, 24 hours after the last cigarette smoke (CS) exposure,
animals will be exsanguinated under anaesthesia, and the trachea
will be cannulated and the lungs lavaged with 0.5-ml aliquots of
heparinised PBS (1 unit/ml) four times through tracheal cannula
(total volume 2 ml). Bronchioalveolar (BAL) collected is to be
stored at 2-8.degree. C. until assayed for total cell and
differential leukocyte count. BAL fluid is to be centrifuged
(500.times.g for 10 min) and the resulting cell pellet is
resuspended in 0.5 ml of heparinised saline. The total number of
white blood cells is to be determined in BAL fluid and blood using
a blood cell counter and adjusted to 1.times.10.sup.6 cell/ml.
Differential cell count is calculated manually. Forty microliters
of the cell suspension is centrifuged using Cytospin 3 to prepare a
cell smear. The cell smear is stained with a blood staining
solution for differentiation and microscopically observed to
identify eosinophil according to their morphological
characteristics. The number of each cell type among 300 white blood
cells in the cell smear are to be determined and expressed as a
percentage, and the number of neutrophils and macrophages in each
BAL fluid are to be calculated.
Assay 13: Imiquimod Induced Plaque Psoriasis in Balb/c Mice
Model
[0247] Imiquimod (IMQ) is a ligand for TLR7 and TLR8, originally
used for the treatment of non-melanoma skin cancers. The topical
application of IMQ on the shaved back skin of the mouse induces a
psoriasis-like skin condition exhibiting most of the human
psoriasis pathology characteristic features including acanthosis,
parakeratosis, and infiltration of immune cells and involvement of
the IL23/IL17/IL22 pathway. Animals (male Balb/c mice) were
acclimatized for seven days prior to the start of the experiment.
Animals were randomly distributed to various groups based on their
body weights. On day 0, the back skin of the mice was shaved by
topical application of hair removal cream. On day 1, mice were
administered the test compound or vehicle by the oral route and
after 1 hour the mice that received the test compound received a
topical application of 62.5 mg of commercially available IMQ cream
(5%; Beselna Cream; Mochida Pharmaceuticals, Tokyo, Japan) on the
shaved back skin. The mice were treated with topical application of
imiquimod for the next 5 consecutive days, one hour after test
compound or vehicle administration. Animals were allowed to dry for
one hour before returning to their cages after topical application
on every day. Four hours after the final application of IMQ cream,
the mice were killed and skin samples were obtained. Back skin
thickness was measured using dial thickness gauge. After measuring
skin thickness, skin samples were fixed in 10% neutral buffered
formalin solution and embedded in paraffin. Deparaffinised sections
were stained with hematoxylin-eosin (HE). Epidermal thickness was
quantified by averaging the values of five independent fields per
section. To score the severity of inflammation of the back skin, an
objective scoring system was used based on the human clinical
Psoriasis Area and Severity Index (PASI). Erythema, scaling, and
thickening were scored independently on a scale from 0 to 4:
0=none; 1=slight; 2=moderate; 3=marked; and 4=very marked.
[0248] As shown in FIGS. 6A and 6B, Compound A1 reduced back skin
thickness, erythema, and scaling (as shown by the histopathological
score) compared to the control group animals.
[0249] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as described above. It is intended that the appended
claims define the scope of the invention and that methods and
structures within the scope of these claims and their equivalents
be covered thereby.
[0250] All publications and patent and/or patent applications cited
in this application are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated
herein by reference.
* * * * *