U.S. patent application number 17/220806 was filed with the patent office on 2021-12-30 for novel selective pi3k delta inhibitors.
The applicant listed for this patent is Rhizen Pharmaceuticals SA. Invention is credited to Meyyappan Muthuppalaniappan, Dhanapalan Nagarathnam, Swaroop K. Vakkalanka.
Application Number | 20210403475 17/220806 |
Document ID | / |
Family ID | 1000005829978 |
Filed Date | 2021-12-30 |
United States Patent
Application |
20210403475 |
Kind Code |
A1 |
Vakkalanka; Swaroop K. ; et
al. |
December 30, 2021 |
NOVEL SELECTIVE PI3K DELTA INHIBITORS
Abstract
The present invention relates to selective inhibitors of PI3K
delta protein kinases, methods of preparing them, pharmaceutical
compositions containing them and methods of treatment and/or
prevention of kinase mediated diseases or disorders with them.
Inventors: |
Vakkalanka; Swaroop K.;
(Basel, CH) ; Muthuppalaniappan; Meyyappan;
(Hyderabad, IN) ; Nagarathnam; Dhanapalan;
(Bethany, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rhizen Pharmaceuticals SA |
Basel |
|
CH |
|
|
Family ID: |
1000005829978 |
Appl. No.: |
17/220806 |
Filed: |
April 1, 2021 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/519 20130101;
C07D 487/04 20130101; A61K 45/06 20130101; C12N 9/1205 20130101;
C12Y 207/01137 20130101; Y02A 50/30 20180101; A61P 35/00
20180101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61P 35/00 20060101 A61P035/00; A61K 31/519 20060101
A61K031/519; A61K 45/06 20060101 A61K045/06; C12N 9/12 20060101
C12N009/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 4, 2012 |
IN |
2692/CHE/2012 |
Jul 4, 2012 |
IN |
2693/CHE/2012 |
Claims
1.-20. (canceled)
21. A method of inhibiting a catalytic activity of a PI3 .delta.
kinase present in a cell, comprising contacting the cell with an
effective amount of
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3-
,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
or a pharmaceutically acceptable salt thereof.
22. The method of claim 21, wherein the inhibition takes place in a
subject suffering from a disease or disorder which is cancer, bone
disorder, inflammatory disease, immune disease, nervous system
disease, metabolic disease, respiratory disease, thrombosis, or
cardiac disease.
23. The method of claim 21, wherein the inhibition takes place in a
subject suffering from cancer.
24. The method of claim 23, wherein an effective amount of
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate is administered.
25. The method of claim 24, wherein the
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate has an enantiomeric excess of at least
95%.
26. The method of claim 24, wherein the
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate has an enantiomeric excess of at least
98%.
27. A method for the treatment of a PI3K associated disease or
disorder, comprising the step of administering to a subject in need
thereof an effective amount of
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one or a
pharmaceutically acceptable salt thereof.
28. The method of claim 27, further comprising administering
simultaneously or sequentially to a subject in need thereof at
least one other anti-cancer agent, anti-inflammatory agent,
immunosuppressive agent, steroid, non-steroidal anti-inflammatory
agent, antihistamine, analgesic, or a mixture thereof.
29. The method of claim 27, wherein the PI3K associated disease,
disorder or condition is an immune system-related disease, a
disease or disorder involving inflammation, cancer or other
proliferative disease, a hepatic disease or disorder, or a renal
disease or disorder.
30. The method of claim 27, wherein the PI3K associated disease,
disorder or condition is selected from inflammation,
glomerulonephritis, uveitis, hepatic diseases or disorders, renal
diseases or disorders, chronic obstructive pulmonary disease,
rheumatoid arthritis, inflammatory bowel disease, vasculitis,
dermatitis, osteoarthritis, inflammatory muscle disease, allergic
rhinitis, vaginitis, interstitial cystitis, scleroderma,
osteoporosis, eczema, allogeneic or xenogeneic transplantation,
graft rejection, graft-versus-host disease, lupus erythematosus,
pulmonary fibrosis, dermatomyositis, thyroiditis, myasthenia
gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic
relapsing hepatitis, primary biliary cirrhosis, allergic
conjunctivitis, hepatitis, atopic dermatitis, asthma, Sjogren's
syndrome, organ transplant rejection, multiple sclerosis,
Guillain-Barre, autoimmune uveitis, autoimmune hemolytic anemia,
pernicious anemia, autoimmune thrombocytopenia, temporal arteritis,
anti-phospholipid syndrome, vasculitides such as Wegener's
granulomatosis, Behcet's disease, psoriasis, dermatitis
herpetiformis, pemphigus vulgaris, vitiligo, Crohn's disease,
colitis, ulcerative colitis, primary biliary cirrhosis, autoimmune
hepatitis, Type 1 or immune-mediated diabetes mellitus, Grave's
disease. Hash-imoto's thyroiditis, autoimmune oophoritis and
orchitis, autoimmune disorder of the adrenal gland, systemic lupus
erythematosus, polymyositis, dermatomyositis, ankylosing
spondylitis, transplant rejection, skin graft rejection, arthritis,
bone diseases associated with increased bone resorption, ileitis,
Barrett's syndrome, adult respiratory distress syndrome, chronic
obstructive airway disease, corneal dystrophy, trachoma,
onchocerciasis, sympathetic ophthalmitis, endophthalmitis;
gingivitis, periodontitis, tuberculosis, leprosy, uremic
complications, nephrosis, sclerodermatitis, psoriasis, chronic
demyelinating diseases of the nervous system, AIDS-related
neurodegeneration, Alzheimer's disease, infectious meningitis,
encephalomyelitis, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis viral or autoimmune encephalitis;
autoimmune disorders, immune-complex vasculitis, systemic lupus
erythematosus (SLE), cardiomyopathy, ischemic heart disease
hypercholesterolemia, atherosclerosis, preeclampsia, chronic liver
failure, brain and spinal cord trauma, and cancer.
31. The method of claim 27, wherein the PI3K associated disease,
disorder or condition is selected from chronic obstructive
pulmonary disease, asthma, rheumatoid arthritis, chronic
bronchitis, atopic dermatitis, multiple sclerosis, inflammatory
bowel disease, allergic rhinitis, lupus erythematosus and
ulcerative colitis.
32. The method of claim 27, wherein an effective amount of
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate is administered.
33. The method of claim 32, wherein the
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate has an enantiomeric excess of at least
95%.
34. The method of claim 32, wherein the
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate has an enantiomeric excess of at least
98%.
Description
[0001] The present application claims the benefit of Indian Patent
Application Nos. 2692/CHE/2012, filed Jul. 4, 2012, and
2693/CHE/2012, filed Jul. 4, 2012, and U.S. Provisional Application
Nos. 61/691,561, filed Aug. 21, 2012, and 61/691,586, filed Aug.
21, 2012, each of which is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to selective inhibitors of
PI3K delta protein kinases, methods of preparing them,
pharmaceutical compositions containing them and methods of
treatment and/or prevention of kinase mediated diseases or
disorders with them.
BACKGROUND OF THE INVENTION
[0003] Phosphatidylinositol (hereinafter abbreviated as "PI") is
one of a number of phospholipids found in cell membranes. In recent
years it has become clear that PI plays an important role in
intracellular signal transduction. Cell signaling via
3-phosphorylated phosphoinositides has been implicated in a variety
of cellular processes, e.g., malignant transformation, growth
factor signaling, inflammation, and immunity (Rameh et al. (1999)
J. Biol Chem, 274:8347-8350). The enzyme responsible for generating
these phosphorylated signaling products, phosphatidylinositol
3-kinase (also referred to as PI 3-kinase or PI3K), was originally
identified as an activity associated with viral oncoproteins and
growth factor receptor tyrosine kinases that phosphorylate
phosphatidylinositol (PI) and its phosphorylated derivatives at the
3-hydroxyl of the inositol ring (Panayotou et al. (1992) Trends
Cell Biol 2:358-60).
[0004] The phosphoinositide 3-kinases (PI3Ks) are a family of
enzymes that regulate diverse biological functions in every cell
type by generating phosphoinositide second-messenger molecules. As
the activity of these phosphoinositide second messengers is
determined by their phosphorylation state, the kinases and
phosphatises that act to modify these lipids are central to the
correct execution of intracellular signaling events.
Phosphoinositide 3-kinases (PI3K) phosphorylate lipids at the
3-hydroxyl residue of an inositol ring (Whitman et al. (1988)
Nature, 332:664) to generate phosphorylated phospholipids (PIP3s)
which act as second messengers recruiting kinases with lipid
binding domains (including plekstrin homology (PH) regions), such
as Akt and phosphoinositide-dependent kinase-1 (PDK1). Binding of
Akt to membrane PIP3s causes the translocation of Akt to the plasma
membrane, bringing Akt into contact with PDK1, which is responsible
for activating Akt. The tumor-suppressor phosphatase, PTEN,
dephosphorylates PIP3 and therefore acts as a negative regulator of
Akt activation. The PI3-kinases Akt and PDK1 are important in the
regulation of many cellular processes including cell cycle
regulation, proliferation, survival, apoptosis and motility and are
significant components of the molecular mechanisms of diseases such
as cancer, diabetes and immune inflammation (Vivanco et al. (2002)
Nature Rev. Cancer 2:489; Phillips et al. (1998) Cancer 83:41).
[0005] The members of the class I family of PI3Ks are dimers of a
regulatory and a catalytic subunit. The class I family consists of
four isoforms, determined by the 110 kDa catalytic subunits
.alpha., .beta., .gamma. and .delta.. Engelman J A, Nat Rev Genet
2006; 7:606-19; Carnero A, Curr Cancer Drug Targets 2008; 8:187-98;
Vanhaesebroeck B, Trends Biochem Sci 2005; 30:194-204. Class I can
be subdivided into two subclasses: Ia, formed by the combination of
p110 .alpha., .beta., and .delta. and a regulatory subunit (p85,
p55 or p50) and Ib, formed by p110 .gamma. and p101 regulatory
subunits.
[0006] There is considerable evidence indicating that Class Ia PI3K
enzymes contribute to tumourigenesis in a wide variety of human
cancers, either directly or indirectly (Vivanco and Sawyers, Nature
Reviews Cancer, 2002, 2, 489-501; Marone et al., Biochimica et
Biophysica Acta 1784 (2008) 159-185). In particular, the p110 delta
isoform has been implicated in biological functions related to
immune-inflammatory diseases, including signaling from the B-cell
receptor, T cell receptor, FcR signaling of mast cells and
monocyte/macrophage, and osteoclast function/RANKL signaling (Deane
J and Fruman D A (2004) Annu Rev. Immunol. 2004. 22:563-98; Janas
et al., The Journal of Immunology, 2008, 180: 739-746; Marone R et
al., Biochim. Biophy. Acta 2007, 1784:159-185). Deletion of the
PI3K delta gene or selective introduction of a catalytically
inactive mutant of PI3K delta causes a nearly complete ablation of
B cell proliferation and signaling, and impairment of signaling
through T cells as well.
[0007] There still remains an unmet and dire need for small
molecule kinase modulators in order to regulate and/or modulate
transduction of kinases, particularly PI3K, for the treatment of
diseases and disorders associated with kinase-mediated events.
[0008] International Publication No. WO 2011/055215 and U.S. Patent
Publication No. 2011/0118257 disclose certain 2,3
disubstituted-4H-chromen-4-one as PI3K kinase modulators and are
incorporated herein by reference in their entirety for all
purposes.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to selective inhibitors of
PI3K delta protein kinases. These compounds are suitable for use in
a pharmaceutical composition for the treatment of a PI3K associated
disease, disorder or condition, e.g., a proliferative disease such
as cancer.
[0010] In one embodiment, the PI3K delta inhibitor is
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one (compound-A1)
or a pharmaceutically acceptable salt thereof. In another
embodiment, the PI3K delta inhibitor is
(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one (compound-A2)
or a pharmaceutically acceptable salt thereof.
[0011] In yet another embodiment, the PI3K delta inhibitor is
2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
(compound-B) or a pharmaceutically acceptable salt thereof. The
present invention also includes compound-B, and its
pharmaceutically acceptable salts, in racemic form as well as their
stereoisomers,
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
(compound-B1),
(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
(compound-B2), and pharmaceutically acceptable salts thereof.
[0012] In one embodiment, the PI3K delta inhibitor is
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate. In another embodiment, the PI3K delta
inhibitor is
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]py-
rimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
sulphate. In another embodiment, the PI3K delta inhibitor is
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
hydrochloride. In another embodiment, the PI3K delta inhibitor is
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
benzenesulfonate. In another embodiment, the PI3K delta inhibitor
is
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
maleate. In another embodiment, the PI3K delta inhibitor is
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
camphor sulphonate.
[0013] The chemical structures of compounds A1, A2, B, B1, and B2
are shown below.
##STR00001## ##STR00002##
[0014] In one preferred embodiment, the present invention relates
to the compound
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,-
4-d]pyrimidin-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one
(compound-A1) or a pharmaceutically acceptable salt thereof.
[0015] In another preferred embodiment, the present invention
relates to the compound
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
(compound-B1) or a pharmaceutically acceptable salt thereof.
[0016] The present invention also encompasses prodrugs of these
compounds.
[0017] The invention further provides a pharmaceutical composition
comprising one or more compounds of the present invention (such as
compound A1, A2, B, B1, B2, pharmaceutically acceptable salts
thereof, or mixtures thereof) together with a pharmaceutically
acceptable carrier. The pharmaceutical composition may further
comprise one or more of additional active ingredients, such as
other 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.
[0018] The invention further provides a pharmaceutical composition
comprising compound A1 together with a pharmaceutically acceptable
carrier.
[0019] The invention further provides a pharmaceutical composition
comprising compound B together with a pharmaceutically acceptable
carrier.
[0020] The invention further provides a pharmaceutical composition
comprising compound B1 together with a pharmaceutically acceptable
carrier.
[0021] In one embodiment, the invention provides a pharmaceutical
composition comprising compound A1 or a pharmaceutically acceptable
salt thereof, wherein compound A1 is present in excess of compound
A2
[0022] In a further embodiment, the compound A1 is substantially
free of compound A2.
[0023] In a further embodiment, the compound A1 exists in excess
over compound A2 and has an enantiomeric excess (e.e.) of at least
about 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.
[0024] In another embodiment, the invention provides a
pharmaceutical composition comprising compound B1 or a
pharmaceutically acceptable salt thereof, wherein compound B1 is
present in excess of compound B2
[0025] In a further embodiment, the compound B1 is substantially
free of compound B2.
[0026] In a further embodiment, the compound B1 exists in excess
over compound B2 and has an enantiomeric excess (e.e.) of at least
about 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.
[0027] Another embodiment is a method for preparing the
4-methylbenzenesulfonate (PTSA), sulphate (SA), hydrochloride
(HCl), benzenesulfonate, maleate or camphor sulphonate salt of
compound B or compound B1. The method can include converting
compound B or B1, or a salt of it (other than the desired salt), to
a 4-methylbenzenesulfonate, sulphate, hydrochloride,
benzenesulfonate, maleate or camphor sulphonate salt of compound B
or compound B1.
[0028] Another embodiment is a 4-methylbenzenesulfonate, sulphate,
hydrochloride, benzenesulfonate, maleate or camphor sulphonate salt
of compound B or compound B1 suitable for use in a pharmaceutical
composition for the treatment of a PI3K associated disease,
disorder or condition, e.g., a proliferative disease such as
cancer.
[0029] The invention further provides a pharmaceutical composition
comprising 4-methylbenzenesulfonate, sulphate, hydrochloride,
benzenesulfonate, maleate or camphor sulphonate salt of Compound B
of the present invention together with a pharmaceutically
acceptable carrier. The pharmaceutical composition may further
comprise one or more of additional active ingredients, such as
other 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.
[0030] The invention further provides a pharmaceutical composition
comprising 4-methylbenzenesulfonate, sulphate, hydrochloride,
benzenesulfonate, maleate or camphor sulphonate salt of Compound B
together with a pharmaceutically acceptable carrier.
[0031] In one embodiment, the PTSA salt of compound B or compound
B1 has an enantiomeric excess (e.e.) of at least about 60%, 75%,
80%, 85%, 90%, 95%, 98% or 99%.
[0032] In one embodiment, the SA salt of compound B or compound B1
has an enantiomeric excess (e.e.) of at least about 60%, 75%, 80%,
85%, 90%, 95%, 98% or 99%.
[0033] In one embodiment, the HCl salt of compound B or compound B1
has an enantiomeric excess (e.e.) of at least about 60%, 75%, 80%,
85%, 90%, 95%, 98% or 99%.
[0034] In one embodiment, the benzenesulfonate salt of compound B
or compound B1 has an enantiomeric excess (e.e.) of at least about
60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.
[0035] In one embodiments, the maleate salt of compound B or
compound B1 has an enantiomeric excess (e.e.) of at least about
60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.
[0036] In one embodiment, the camphor sulphonate salt of compound B
or compound B1 has an enantiomeric excess (e.e.) of at least about
60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.
[0037] Another embodiment is a method of inhibiting PI3K delta in a
patient by administering to a patient an effective amount of
compound B or compound B1 of the present invention as a as PTSA
salt.
[0038] Another embodiment is a method of inhibiting PI3K delta in a
patient by administering to a patient an effective amount of at
least one compound of the present invention.
[0039] Yet another embodiment is a method of treating, preventing,
and/or inhibiting a PI3K protein kinase mediated disease, disorder
or condition (such as cancer or other proliferative disease or
disorder) in a patient by administering to the a patient an
effective amount of at least one compound of the present
invention.
[0040] Yet another embodiment is a method of treating a PI3K
associated disease, disorder or condition in a patient by
administering to the patient an effective amount of at least one
compound of the present invention. In one embodiment, the amount of
the compound administered is sufficient to treat a PI3K associated
disease, disorder or condition by inhibition of PI3K delta.
[0041] Yet another embodiment of the present invention is a method
for treating a proliferative disease by 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 amount of
the compound administered is sufficient to treat the proliferative
disease by inhibition of PI3K delta.
[0042] Yet another embodiment of the present invention is a method
for treating a proliferative disease 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-cancer agent. In one
embodiment, the amount of the compound administered is sufficient
to treat (or facilitate treatment of) the proliferative disease by
inhibition of PI3K delta.
[0043] Yet another embodiment is a method of treating a PI3K
associated disease, disorder or condition in a patient, comprising
administering to the patient a pharmaceutical composition
comprising Compound A1, B or B1 or a pharmaceutically acceptable
salt thereof, optionally admixed with at least one pharmaceutically
acceptable excipient. In particular embodiments, the composition
comprises a therapeutically effective amount of a compound of any
of the foregoing embodiments of Compound A1, B or B1 or a
pharmaceutically acceptable salt thereof for the treatment of PI3K
associated disease, disorder or condition.
[0044] Specific embodiments provide a method of treating cancer in
a patient, comprising administering to the patient a pharmaceutical
composition comprising compound A1, B or B1 or a pharmaceutically
acceptable salt thereof, optionally admixed with at least one
pharmaceutically acceptable excipient. In particular embodiments,
the composition comprises of a therapeutically effective amount of
a compound of any of the foregoing embodiments of Compound A1, B or
B1 or a pharmaceutically acceptable salt thereof for the treatment
of cancer in a patient.
[0045] The compounds of the present invention are useful in the
treatment of a variety of cancers, including, but not limited to,
the following: [0046] carcinoma, including that of the bladder,
breast, colon, kidney, liver, lung (including small cell lung
cancer), esophagus, gall bladder, uterus, ovary, testes, larynx,
oral cavity, gastrointestinal tract (e.g., esophagus, stomach,
pancreas), brain, cervix, thyroid, prostate, blood, and skin
(including squamous cell carcinoma); [0047] hematopoietic tumors of
lymphoid lineage, including leukemia, acute lymphocytic leukemia,
acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma,
Hodgkin's lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and
Burkett's lymphoma; [0048] hematopoietic tumors of myeloid lineage,
including acute and chronic myelogenous leukemias, myelodysplastic
syndrome and promyelocytic leukemia; [0049] tumors of mesenchymal
origin, including fibrosarcoma and rhabdomyosarcoma; [0050] tumors
of the central and peripheral nervous system, including
astrocytoma, neuroblastoma, glioma and schwannomas; and [0051]
other tumors, including melanoma, seminoma, teratocarcinoma,
osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid
follicular cancer and Kaposi's sarcoma.
[0052] The compounds of the present invention as modulators of
apoptosis are useful in the treatment, prevention, and inhibition
of cancer (including, but not limited to, those types mentioned
herein above).
[0053] The compounds of the present invention are useful in the
chemoprevention of cancer. Chemoprevention involves inhibiting the
development of invasive cancer by blocking the initiating mutagenic
event, blocking the progression of pre-malignant cells that have
already suffered an insult, or inhibiting tumor relapse. The
compounds are also useful in inhibiting tumor angiogenesis and
metastasis. One embodiment of the invention is a method of
inhibiting tumor angiogenesis or metastasis in a patient by
administering an effective amount of one or more compounds of the
present invention.
[0054] Another embodiment of the present invention is a method of
treating an immune system-related disease (e.g., an autoimmune
disease), a disease or disorder involving inflammation (e.g.,
asthma, chronic obstructive pulmonary disease, 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, or a renal disease or
disorder. The method includes administering an effective amount of
one or more compounds of the present invention.
[0055] Examples of immune disorders which can be treated by the
compounds of the present invention 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, idiopathic
pulmonary fibrosis (IPF) (or cryptogenic fibrosing alveolitis (CFA)
or idiopathic fibrosing interstitial pneumonia), pulmonary
fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g.,
Hashimoto's and autoimmune thyroiditis), myasthenia gravis,
autoimmune hemolytic anemia, multiple sclerosis, cystic fibrosis,
chronic relapsing hepatitis, primary biliary cirrhosis, allergic
conjunctivitis and atopic dermatitis.
[0056] Yet another embodiment is a method of treating leukemia in a
patient by administering a therapeutically effective amount of a
compound of the present invention. For example, the compounds of
the present invention are effective for treating chronic
lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), acute
myeloid leukemia (AML), multiple myeloma (MM), small lymphocytic
lymphoma (SLL), and indolent non-Hodgkin's lymphoma (I-NHL).
[0057] In the aforementioned methods of treatment, one or more
additional active agents can be administered with the compounds of
the present invention. For example, the compounds of the present
invention are useful in combination (administered together or
sequentially) with known anti-cancer treatments such as
chemotherapy, radiation therapy, biological therapy, bone marrow
transplantation, stem cell transplant or any other anticancer
therapy or with one or more cytostatic, cytotoxic or anticancer
agents or targeted therapy either alone or in combination, such as
but not limited to, for example, DNA interactive agents, such as
fludarabine, cisplatin, chlorambucil, bendamustine or doxorubicin;
alkylating agents, such as cyclophosphamide; 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;
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); CD20 monoclonal antibodies such as rituximab, ublixtumab
(TGR-1101), ofatumumab (HuMax; Intracel), ocrelizumab, veltuzumab,
GA101 (obinutuzumab), AME-133v (LY2469298, Applied Molecular
Evolution), ocaratuzumab (Mentrik Biotech), PRO131921, tositumomab,
hA20 (Immunomedics, Inc.), ibritumomab-tiuxetan, BLX-301 (Biolex
Therapeutics), Reditux (Dr. Reddy's Laboratories), and PR070769
(described in WO2004/056312); other B-cell targeting monoclonal
antibodies such as belimumab, atacicept or fusion proteins such as
blisibimod and BR3-Fe; other monoclonal antibodies such as
alemtuzumab; CHOP (cyclophosphamide, doxorubicin, vincristine,
prednisone); R-CHOP (rituximab-CHOP); hyperCV AD (hyperfractionated
cyclophosphamide, vincristine, doxorubicin, dexamethasone,
methotrexate, cytarabine); R-hyperCV AD (rituximab-hyperCV AD); FCM
(fludarabine, cyclophosphamide, mitoxantrone); R-FCM (rituximab,
fludarabine, cyclophosphamide, mitoxantrone); bortezomib and
rituximab; temsirolimus and rituximab; temsirolimus and
Velcade.RTM.; Iodine-131 tositumomab (Bexxar.RTM.) and CHOP-CVP
(cyclophosphamide, vincristine, prednisone); R-CVP (rituximab-CVP);
ICE (iphosphamide, carboplatin, etoposide); R-ICE (rituximab-ICE);
FCR (fludarabine, cyclophosphamide, rituximab); FR (fludarabine,
rituximab); and D.T. PACE (dexamethasone, thalidomide, cisplatin,
adriamycin, cyclophosphamide, etoposide); and other protein kinase
modulators.
[0058] 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) or immune selective
anti-inflammatory derivatives (ImSAIDs).
BRIEF DESCRIPTION OF THE FIGURES
[0059] FIG. 1 is a bar graph of the percent apoptotic cells after
treatment with compound B1 or Control in primary Multiple Myeloma
patient cells as measured according to Assay 6a.
[0060] FIGS. 2A, 2B, and 2C are bar graphs showing the observed
induction of cytotoxicity (FIG. 2A) and apopotosis (FIG. 2B) in CLL
cells and the corresponding inhibition of PAkt (FIG. 2C), as
measured by Assay 8.
DETAILED DESCRIPTION OF THE INVENTION
[0061] As used herein the following definitions shall apply unless
otherwise indicated.
[0062] 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)-. 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
instance, intermediate mixtures may include a mixture of isomers in
a ratio of about 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 known techniques
[0063] The term "prodrug" refers to a compound, which is an
inactive precursor of a compound, 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, sulfate, 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. Prodrugs of
compounds A1, B, B1 and B2 are intended to be covered within the
scope of this invention.
[0064] Additionally the instant invention also includes 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.
[0065] 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.
[0066] 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; salts of 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; salts
of non-natural amino acids such as D-isomers or substituted amino
acids; salts of guanidine; and salts of 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 are sulphates, nitrates, phosphates,
perchlorates, borates, hydrohalides, acetates, tartrates, maleates,
citrates, fumarates, succinates, palmoates, methanesulphonates,
benzoates, salicylates, benzenesulfonates, ascorbates,
glycerophosphates, and ketoglutarates. In one embodiment, the salt
is 4-methylbenzenesulfonate. In another embodiment, the salt is
sulphate. In yet another embodiment, the salt is hydrochloride. In
yet another embodiment, the salt is benzenesulfonate. In yet
another embodiment, the salt is maleate. In yet another embodiment,
the salt is camphor sulfonate.
[0067] 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.
[0068] The term "comprising" (and related terms such as "comprise"
or "comprises" or "having" or "including") includes, but is not
limited to, 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.
[0069] The following abbreviations and terms have the indicated
meanings throughout: PI3-K=Phosphoinositide 3-kinase;
PI=phosphatidylinositol; DNA-PK=Deoxyribose Nucleic Acid Dependent
Protein Kinase; PTEN=Phosphatase and Tensin homolog deleted on
chromosome Ten; AIDS=Acquired Immuno Deficiency Syndrome; HIV=Human
Immunodeficiency Virus; and MeI=Methyl Iodide.
[0070] Abbreviations used herein have their conventional meaning
within the chemical and biological arts, unless otherwise
indicated.
[0071] 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.
[0072] The term "co-administration," "administered in combination
with," and their grammatical equivalents, as used herein,
encompasses 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.
[0073] 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. 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.
[0074] As used herein, the terms "treatment" and "treating" refer
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.
[0075] 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.
[0076] The term "subject" or "patient" refers to an animal, 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, and in some embodiments,
the patient is human. For veterinary purposes, the term "subject"
and "patient" include, but are not limited to, farm animals
including cows, sheep, pigs, horses, and goats; companion animals
such as dogs and cats; exotic and/or zoo animals; laboratory
animals including mice, rats, rabbits, guinea pigs, and hamsters;
and poultry such as chickens, turkeys, ducks, and geese.
[0077] "Radiation therapy" refers to exposing a patient, using
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.
[0078] "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.
[0079] 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.
[0080] As used herein, the term "PI3-kinase .delta. selective
inhibitor" generally refers to a compound that inhibits the
activity of the PI3-kinase .delta. isozyme more effectively than
other isozymes of the PI3K family (alpha, beta, and gamma). For
instance, the PI3-kinase .delta. selective inhibitor may refer to a
compound that exhibits a 50% inhibitory concentration (IC50) with
respect to the delta type I PI3-kinase that is at least 10-fold, at
least 20-fold, at least 50-fold, at least 100-fold, or lower, than
the inhibitor's IC50 with respect to the rest of the other type 1
PI3-kinases (i.e., alpha, beta, and gamma).
[0081] Inhibition of PI3-kinase .delta. may be of therapeutic
benefit in the 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. function does not
appear to affect biological functions such as viability and
fertility.
[0082] "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.
[0083] The therapeutic methods of the invention include methods for
the treatment 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.
[0084] "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.
[0085] "Transplant rejection" as used herein refers to an 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).
[0086] "Allergic disease" as used herein refers to any symptoms,
tissue damage, or loss of tissue function resulting from
allergy.
[0087] "Arthritic disease" as used herein refers to any disease
that is characterized by inflammatory lesions of the joints
attributable to a variety of etiologies.
[0088] "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.
[0089] The compounds of the present invention can be prepared by
the methods described in International Publication No. WO
2011/055215 and PCT Application No. PCT/IB2013/053544, filed May 3,
2013, both of which are hereby incorporated by reference. Compound
A can be prepared as described in Example 158 of International
Publication No. WO 2011/055215.
[0090] Pharmaceutical Compositions
[0091] The invention 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 a compound of the present
invention. The pharmaceutical composition may include one or more
additional active ingredients as described herein.
[0092] The pharmaceutical carriers and/or excipients may be
selected from diluents, fillers, salts, disintegrants, binders,
lubricants, glidants, wetting agents, controlled release matrices,
colorants, flavorings, buffers, stabilizers, solubilizers, and
combinations thereof.
[0093] 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.
[0094] 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).
[0095] 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.
[0096] 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 about 0.001 to about 100 mg
per kg body weight per day, preferably about 1 to about 35
mg/kg/day, in single or divided doses. For a 70 kg human, this
would amount to about 0.05 to 7 g/day, preferably 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).
[0097] 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.
[0098] 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).
[0099] Preparations of various pharmaceutical compositions are
known in the art. See, e.g., Anderson, Philip O.; Knoben, James E.;
Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth
Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of
Drug Action, Third Edition, Churchill Livingston, N.Y., 1990;
Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition,
McGraw Hill, 2003; Goodman and Gilman, eds., The Pharmacological
Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remingtons
Pharmaceutical Sciences, 20th Ed., Lippincott Williams &
Wilkins., 2000; Martindale, The Extra Pharmacopoeia, Thirty-Second
Edition (The Pharmaceutical Press, London, 1999), all of which are
incorporated by reference herein in their entirety.
[0100] An effective amount of a compound of the invention may be
administered in either single or multiple doses by any of the
accepted modes of administration of agents having similar
utilities, including rectal, buccal, intranasal and transdermal
routes, by intra-arterial injection, intravenously,
intraperitoneally, parenterally, intramuscularly, subcutaneously,
orally, topically, or as an inhalant.
[0101] In one embodiment, Compound A1 or a pharmaceutically
acceptable salt thereof is administered at a dose selected to
produce a concentration of compound in the blood between about 20
to 5,000 ng/mL, and maintaining such concentration during a period
of about 6 to 24 hours following administration. In another
particular embodiment, the dose size and frequency are selected to
achieve a concentration of compound in the blood that is between
about 50 to 2,500 ng/mL and maintain that concentration during a
period of about 6 to 24 hours from the time of administration. In
some embodiments, the dose size and frequency are selected to
achieve a concentration of compound in the blood that is between
about 100 to 1,500 ng/mL following administration. In some
embodiments, the dose size and frequency are selected to achieve a
concentration of compound in the blood that is between about 100 to
750 ng/mL over a period of about 6 to 24 hours from the time of
administration. In further embodiments, the dose size and frequency
is selected to achieve a C.sub.max, plasma level of Compound A1
that is at least about 300 ng/mL and does not exceed about 10,000
ng/mL
[0102] In one embodiment, Compound B1 or a pharmaceutically
acceptable salt thereof is administered at a dose selected to
produce a concentration of compound in the blood between about 20
to 5,000 ng/mL, and maintaining such concentration during a period
of about 6 to 24 hours following administration. In another
particular embodiment, the dose size and frequency are selected to
achieve a concentration of compound in the blood that is between
about 50 to 2,500 ng/mL and maintain that concentration during a
period of about 6 to 24 hours from the time of administration. In
some embodiments, the dose size and frequency are selected to
achieve a concentration of compound in the blood that is between
about 100 to 1,500 ng/mL following administration. In some
embodiments, the dose size and frequency are selected to achieve a
concentration of compound in the blood that is between about 100 to
750 ng/mL over a period of about 6 to 24 hours from the time of
administration. In further embodiments, the dose size and frequency
is selected to achieve a C.sub.max, plasma level of Compound B1
that is at least about 300 ng/mL and does not exceed about 10,000
ng/mL Method of Treatment
[0103] The invention also provides methods of using the compounds
or pharmaceutical compositions of the present invention to treat
disease conditions, including but not limited to diseases
associated with malfunctioning of one or more types of PI3 kinase.
A detailed description of conditions and disorders mediated by PI3
.delta. kinase activity is set forth in WO 2001/81346, US
2005/043239, WO 2010/123931, WO 2010/111432 and WO 2010/057048, all
of which are incorporated herein by reference in their entireties
for all purposes.
[0104] The treatment methods provided herein comprise administering
to the subject a therapeutically effective amount of a compound of
the invention. In one embodiment, the present invention provides a
method of treating an inflammation disorder, including autoimmune
diseases in a mammal. The method comprises administering to said
mammal a therapeutically effective amount of a compound of the
present invention.
[0105] The disorders, diseases, or conditions treatable with a
compound provided herein, include, but are not limited to, [0106]
inflammatory or allergic diseases, including systemic anaphylaxis
and hypersensitivity disorders, atopic dermatitis, urticaria, drug
allergies, insect sting allergies, food allergies (including celiac
disease and the like), anaphylaxis, serum sickness, drug reactions,
insect venom allergies, hypersensitivity pneumonitis, angioedema,
erythema multiforme, Stevens-Johnson syndrome, atopic
keratoconjunctivitis, venereal keratoconjunctivitis, giant
papillary conjunctivitis, and mastocytosis; [0107] inflammatory
bowel diseases, including Crohn's disease, ulcerative colitis,
ileitis, enteritis, and necrotizing enterocolitis; [0108]
vasculitis, and Behcet's syndrome; [0109] psoriasis and
inflammatory dermatoses, including dermatitis, eczema, allergic
contact dermatitis, viral cutaneous pathologies including those
derived from human papillomavirus, HIV or RLV infection, bacterial,
flugal, and other parasital cutaneous pathologies, and cutaneous
lupus erythematosus; [0110] asthma and respiratory allergic
diseases, including allergic asthma, exercise induced asthma,
allergic rhinitis, otitis media, hypersensitivity lung diseases,
chronic obstructive pulmonary disease and other respiratory
problems; [0111] autoimmune diseases and inflammatory conditions,
including but are not limited to, lupus erythematosus, systemic
lupus erythematosus (SLE), multiple sclerosis, polyarthritis,
primary biliary cirrhosis, psoriasis, rheumatoid arthritis,
psoriatic arthritis, gouty arthritis, spondylitis, reactive
arthritis, chronic or acute glomerulonephritis, lupus nephritis,
Reiter's syndrome, Takayasu's arteritis, temporal arteritis (also
known as "giant cell arteritis"), autoimmune pulmonary
inflammation, autoimmune thyroiditis, autoimmune inflammatory eye
disease, vitiligo, and vulvodynia. Other disorders include
bone-resorption disorders and thromobsis; [0112] cancers of the
breast, skin, prostate, cervix, uterus, ovary, testes, bladder,
lung, liver, larynx, oral cavity, colon and gastrointestinal tract
(e.g., esophagus, stomach, pancreas), brain, thyroid, blood, and
lymphatic system; and [0113] pulmonary or respiratory conditions
including but not limited to asthma, chronic bronchitis, allergic
rhinitis, adult respiratory distress syndrome (ARDS), severe acute
respiratory syndrome (SARS), chronic pulmonary inflammatory
diseases (e.g., chronic obstructive pulmonary disease), silicosis,
pulmonary sarcoidosis, pleurisy, alveolitis, vasculitis, pneumonia,
bronchiectasis, hereditary emphysema, and pulmonary oxygen
toxicity.
[0114] In certain embodiments, the cancer or cancers treatable with
the methods provided herein includes, but is or are not limited to,
[0115] leukemias, including, but not limited to, acute leukemia,
acute lymphocytic leukemia, acute myelocytic leukemias such as
myeloblasts, promyelocyte, myelomonocytic, monocytic,
erythroleukemia leukemias and myelodysplastic syndrome or a symptom
thereof (such as anemia, thrombocytopenia, neutropenia, bicytopenia
or pancytopenia), refractory anemia (RA), RA with ringed
sideroblasts (RARS), RA with excess blasts (RAEB), RAEB in
transformation (RAEB-T), preleukemia, and chronic myelomonocytic
leukemia (CMML); [0116] chronic leukemias, including, but not
limited to, chronic myelocytic (granulocytic) leukemia, chronic
lymphocytic leukemia, and hairy cell leukemia; [0117] polycythemia
vera; [0118] lymphomas, including, but not limited to, Hodgkin's
disease and non-Hodgkin's disease; [0119] multiple myelomas,
including, but not limited to, smoldering multiple myeloma,
nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,
solitary plasmacytoma, and extramedullary plasmacytoma; [0120]
Waldenstrom's macroglobulinemia; [0121] monoclonal gammopathy of
undetermined significance; [0122] benign monoclonal gammopathy;
[0123] heavy chain disease; [0124] bone and connective tissue
sarcomas, including, but not limited to, bone sarcoma,
osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell
tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma,
soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma,
Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,
metastatic cancers, neurilemmoma, rhabdomyosarcoma, and synovial
sarcoma; [0125] brain tumors, including, but not limited to,
glioma, astrocytoma, brain stem glioma, ependymoma,
oligodendroglioma, nonglial tumor, acoustic neurinoma,
craniopharyngioma, medulloblastoma, meningioma, pineocytoma,
pineoblastoma, and primary brain lymphoma; [0126] breast cancer,
including, but not limited to, adenocarcinoma, lobular (small cell)
carcinoma, intraductal carcinoma, medullary breast cancer, mucinous
breast cancer, tubular breast cancer, papillary breast cancer,
primary cancers, Paget's disease, and inflammatory breast cancer;
[0127] adrenal cancer, including, but not limited to,
pheochromocytom and adrenocortical carcinoma; [0128] thyroid
cancer, including, but not limited to, papillary or follicular
thyroid cancer, medullary thyroid cancer, and anaplastic thyroid
cancer; [0129] pancreatic cancer, including, but not limited to,
insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting
tumor, and carcinoid or islet cell tumor; [0130] pituitary cancer,
including, but limited to, Cushing's disease, prolactin-secreting
tumor, acromegaly, and diabetes insipidus; [0131] eye cancer,
including, but not limited, to ocular melanoma such as iris
melanoma, choroidal melanoma, and cilliary body melanoma, and
retinoblastoma; [0132] vaginal cancer, including, but not limited
to, squamous cell carcinoma, adenocarcinoma, and melanoma; [0133]
vulvar cancer, including, but not limited to, squamous cell
carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,
and Paget's disease; [0134] cervical cancers, including, but not
limited to, squamous cell carcinoma, and adenocarcinoma; [0135]
uterine cancer, including, but not limited to, endometrial
carcinoma and uterine sarcoma; [0136] ovarian cancer, including,
but not limited to, ovarian epithelial carcinoma, borderline tumor,
germ cell tumor, and stromal tumor; [0137] esophageal cancer,
including, but not limited to, squamous cancer, adenocarcinoma,
adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous
carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma,
and oat cell (small cell) carcinoma; [0138] stomach cancer,
including, but not limited to, adenocarcinoma, fungating
(polypoid), ulcerating, superficial spreading, diffusely spreading,
malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma;
[0139] colon cancer; [0140] rectal cancer; [0141] liver cancer,
including, but not limited to, hepatocellular carcinoma and
hepatoblastoma; [0142] gallbladder cancer, including, but not
limited to, adenocarcinoma; [0143] cholangiocarcinomas, including,
but not limited to, pappillary, nodular, and diffuse; [0144] lung
cancer, including, but not limited to, non-small cell lung cancer,
squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,
large-cell carcinoma, and small-cell lung cancer; [0145] testicular
cancer, including, but not limited to, germinal tumor, seminoma,
anaplastic, classic (typical), spermatocytic, nonseminoma,
embryonal carcinoma, teratoma carcinoma, and choriocarcinoma
(yolk-sac tumor); [0146] prostate cancer, including, but not
limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma;
[0147] penal cancer; [0148] oral cancer, including, but not limited
to, squamous cell carcinoma; [0149] basal cancer; [0150] salivary
gland cancer, including, but not limited to, adenocarcinoma,
mucoepidermoid carcinoma, and adenoidcystic carcinoma; [0151]
pharynx cancer, including, but not limited to, squamous cell cancer
and verrucous; [0152] skin cancer, including, but not limited to,
basal cell carcinoma, squamous cell carcinoma and melanoma,
superficial spreading melanoma, nodular melanoma, lentigo malignant
melanoma, and acral lentiginous melanoma; [0153] kidney cancer,
including, but not limited to, renal cell cancer, adenocarcinoma,
[0154] hypernephroma, fibrosarcoma, and transitional cell cancer
(renal pelvis and/or uterer); [0155] Wilms' tumor; [0156] bladder
cancer, including, but not limited to, transitional cell carcinoma,
squamous cell cancer, adenocarcinoma, and carcinosarcoma; and other
cancer, including, not limited to, myxosarcoma, osteogenic sarcoma,
endotheliosarcoma, lymphangio-endotheliosarcoma, mesothelioma,
synovioma, hemangioblastoma, epithelial carcinoma,
cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, and papillary
adenocarcinomas See Fishman et al., 1985, Medicine, 2d Ed., J.B.
Lippincott Co., Philadelphia and Murphy et al., 1997, Informed
Decisions: The Complete Book of Cancer Diagnosis, Treatment, and
Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States
of America.
[0157] It will be appreciated that the treatment methods of the
invention are useful in the fields of human medicine and veterinary
medicine. Thus, the individual to be treated may be a mammal,
preferably human, or other animals. For veterinary purposes,
individuals include but are not limited to farm animals including
cows, sheep, pigs, horses, and goats; companion animals such as
dogs and cats; exotic and/or zoo animals; laboratory animals
including mice, rats, rabbits, guinea pigs, and hamsters; and
poultry such as chickens, turkeys, ducks, and geese.
[0158] The invention also relates to a method of treating a
hyperproliferative disorder in a subject that comprises
administering to said mammal a therapeutically effective amount of
a compound of the present invention or a pharmaceutically
acceptable salt thereof. In some embodiments, said method relates
to the treatment of cancer such as acute myeloid leukemia, thymus,
brain, lung, squamous cell, skin, eye, retinoblastoma, intraocular
melanoma, oral cavity and oropharyngeal, bladder, gastric, stomach,
pancreatic, bladder, breast, cervical, head, neck, renal, kidney,
liver, ovarian, prostate, colorectal, esophageal, testicular,
gynecological, thyroid, CNS, PNS, AIDS-related (e.g. Lymphoma and
Kaposi's Sarcoma) or viral-induced cancer. In some embodiments,
said method relates to the treatment of a non-cancerous
hyperproliferative disorder such as benign hyperplasia of the skin
(e. g., psoriasis), restenosis, or prostate (e.g., benign prostatic
hypertrophy (BPH)).
EXAMPLES
[0159] The examples and preparations provided below further
illustrate and exemplify the methods of preparing compounds of the
invention. 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. Single enantiomers may be obtained by methods known to
those skilled in the art.
[0160] Unless otherwise mentioned, work-up refers to distribution
of the reaction mixture between the aqueous and organic phases
indicated within parentheses, separation and drying over
Na.sub.2SO.sub.4 of the organic layer and evaporating the solvent
to give a residue. Unless otherwise stated, purification implies
column chromatography using silica gel as the stationary phase and
a mixture of petroleum ether (boiling at 60-80.degree. C.) and
ethyl acetate or dichloromethane and methanol of suitable polarity
as the mobile phases. RT refers to ambient temperature
(25-28.degree. C.).
Intermediate 1
##STR00003##
[0162] Intermediate 1:
6-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one: To
a solution of
2-(1-bromoethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one (15.0
g, 40.84 mmol) in DMSO (150 ml), n-butanol (7.5 ml) was added and
heated to 120.degree. C. for 3 h. The reaction mixture was cooled
to RT, quenched with water 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 ethyl acetate:petroleum ether to afford the
title compound as an off-white solid (7.90 g, 64%). .sup.1H-NMR
(.delta. ppm, CDCl.sub.3, 400 MHz): 7.85 (dd, J=8.1, 3 Hz, 1H),
7.54 (dd, J=9.2, 4.2 Hz, 1H), 7.47-7.37 (m, 2H), 7.15-6.98 (m, 3H),
4.74 (quintet, J=6.8 Hz, 1H), 2.23 (d, J=7.4 Hz, 1H), 1.54 (d,
J=6.6 Hz, 3H).
Intermediate 2
##STR00004##
[0164] Intermediate 2:
2-acetyl-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one: DMSO (5.60
ml, 79.14 mmol) was added to dichloromethane (40 ml) cooled to
-78.degree. C., followed by oxalyl chloride (3.40 ml, 39.57 mmol).
After 10 min. intermediate 1 (6.00 g, 19.78 mmol) in
dichloromethane (54 ml) was added dropwise and stirred for 20 min.
Triethylamine (12 ml) was added and stirred for 1 h. The reaction
mixture was quenched with water and extracted with dichloromethane.
The organic layer was dried over sodium sulphate and concentrated
under reduced pressure. The crude product was purified by column
chromatography with ethyl acetate:petroleum ether to afford the
title compound as a yellow solid (4.2 g, 71%) which was used as
such in the next step.
Intermediate 3
##STR00005##
[0166] Intermediate 3:
(S)-6-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one:
To intermediate 2 (2.00 g, 6.66 mmol), R-Alpine borane (0.5M in
THF, 20 ml) was added and heated to 60.degree. C. for 20 h. The
reaction mixture quenched with aq. 2N HCl, 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 ethyl acetate:petroleum ether to
afford the title compound as an off-white solid (1.51 g, 75%).
Enantiomeric excess: 94.2%, enriched in the fast eluting isomer
(retention time: 8.78 min.) as determined by HPLC on a chiralpak
AD-H column.
Intermediate 4
##STR00006##
[0168] Intermediate 4:
(R)-1-(6-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl
4-chlorobenzoate: To a solution of intermediate 3 (1.45 g, 4.78
mmol) in THF (15 ml), 4-chlorobenzoic acid (0.748 g, 4.78 mmol) and
triphenylphosphine (1.88 g, 7.17 mmol) were added and heated to
45.degree. C. followed by diisopropylazodicarboxylate (1.4 ml, 7.17
mmol). After 1 h, the reaction mixture was concentrated and the
residue was purified by column chromatography with ethyl
acetate:petroleum ether to afford the title compound as an
off-white solid (1.81 g, 86%) which was used without purification
in the next step.
[0169] Intermediate 5
[0170] Method A
##STR00007##
[0171] Intermediate 5:
(R)-6-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one:
To intermediate 4 (1.75 g, 3.96 mmol) in methanol (17 ml) cooled to
10.degree. C., potassium carbonate (0.273 g, 1.98 mmol) was added
and stirred for 30 min. The reaction mixture was concentrated,
acidified with 2N HCl solution, extracted with ethyl acetate, dried
over sodium sulphate and concentrated under reduced pressure. The
crude product was purified by column chromatography with ethyl
acetate:petroleum ether to afford the title compound as a yellow
solid (1.05 g, 87%). Enantiomeric excess: 93.6%, enriched in the
late eluting isomer (retention time: 11.12 min.) as determined by
HPLC on a chiralpak AD-H column.
[0172] Method B:
[0173] Step-1:
(R)-2-(1-(benzyloxy)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
To 1-(5-fluoro-2-hydroxyphenyl)-2-(3-fluorophenyl)ethanone (11.00
g, 44.31 mmol) in dichloromethane, HATU (33.7 g, 88.63 mmol) and
R-(+)2-benzyloxypropionic acid (9.58 g, 53.17 mmol) were added and
stirred for 10 min. Triethylamine (66.7 ml, 0.47 mol) was added
dropwise and stirred at RT for 24 h. The reaction mixture was
quenched with water, extracted with dichloromethane, dried over
sodium sulphate and concentrated under reduced pressure. The crude
product was purified by column chromatography with ethyl
acetate:petroleum ether to afford the title compound as a yellow
solid (10.5 g, 60%). .sup.1H-NMR (8 ppm, CDCl.sub.3, 400 MHz): 7.85
(dd, J=8.1.3 Hz, 1H), 7.58 (dd, J=9.1, 4.1 Hz, 1H), 7.47-7.39 (m,
1H), 7.39-7.34 (m, 1H), 7.28-7.20 (m, 3H), 7.20-7.14 (m, 2H),
7.16-7.07 (m, 1H), 6.99-6.89 (m, 2H), 4.50-4.31 (m, 3H), 1.56 (d,
J=6.4 Hz, 3H).
[0174] Step-2:
(R)-2-(1-(benzyloxy)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
(10.5 g, 26.69 mmol) in dichloromethane (110 ml) cooled to
0.degree. C., aluminium chloride (5.35 g, 40.03 mmol) was added
portionwise and stirred at RT for 6 h. The reaction mixture was
quenched with 2N HCl solution, extracted with dichloromethane,
dried over sodium sulphate and concentrated under reduced pressure.
The crude product was purified by column chromatography with ethyl
acetate:petroleum ether to afford the desired intermediate as a
yellow solid (6.1 g, 76%). Enantiomeric excess: 97.7%, enriched in
the late eluting isomer (retention time: 11.12 min.) as determined
by HPLC on a chiralpak AD-H column.
Intermediate 6
##STR00008##
[0176] Intermediate 6:
(R)-2-(1-(benzyloxy)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one: To
2-(3-fluorophenyl)-1-(2-hydroxyphenyl)ethanone (10.0 g, 43.43 mmol)
in dichloromethane (75 ml), HATU (33.0 g, 86.86 mmol) and
R-(+)2-benzyloxypropionic acid (9.39 g, 52.12 mmol) were added and
stirred for 10 min. Triethylamine (65.4 ml, 0.469 mol) was added
dropwise and stirred at RT for 24 h. The reaction mixture was
quenched with water, extracted with dichloromethane, dried over
sodium sulphate and concentrated under reduced pressure. The crude
product was purified by column chromatography with ethyl
acetate:petroleum ether to afford the title compound as a off-white
solid (9.0 g, 55%). .sup.1H-NMR (8 ppm, CDCl.sub.3, 400 MHz): 8.23
(dd, J=7.9, 1.2 Hz, 1H), 7.74-7.70 (m, 1H), 7.58 (d, J=8.3 Hz, 1H),
7.43 (t, J=7.2 Hz, 1H), 7.37 (q, J=7.2 Hz, 1H), 7.29-7.15 (m, 5H),
7.09 (dt, J=8.6, 1.7 Hz, 1H), 7.00-6.90 (m, 2H), 4.51-4.35 (m, 3H),
1.57 (d, J=6.4 Hz, 3H).
Intermediate 7
##STR00009##
[0178] Intermediate 7:
(R)-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one: To
intermediate 6 (5.0 g, 13.35 mmol) in dichloromethane (50 ml)
cooled to -78.degree. C., boron tribromide (1M in dichloromethane,
36.5 ml, 0.145 mmol) was added dropwise and stirred for 1 h. The
reaction mixture was quenched with 2N HCl solution, extracted with
dichloromethane, dried over sodium sulphate and concentrated under
reduced pressure. The crude product was purified by column
chromatography with ethyl acetate:petroleum ether to afford
intermediate II as an off-white solid (3.05 g, 80%). .sup.1H-NMR
(.delta. ppm, CDCl.sub.3, 400 MHz): 8.24 (dd, J=7.9, 1.5 Hz, 1H),
7.73 (m, 1H), 7.54 (d, J=8.1 Hz, 1H), 7.44 (m, 2H), 7.13-7.01 (m,
3H), 4.71 (q, J=6.6 Hz, 1H), 1.56 (d, J=6.5 Hz, 3H). Mass: 284.9
(M.sup.+). Purity: 99.73%. [.alpha.].sup.25.sub.D-0.605 (c=1,
CHCl.sub.3). Enantiomeric excess: 95.2%, enriched in the late
eluting isomer (retention time: 10.19 min.) as determined by HPLC
on a chiralpak AD-H column.
Intermediate 7a and 7b
##STR00010##
[0180] Intermediate 7a and 7b:
(S)-2-(1-bromoethyl)-3-(3-fluorophenyl)-4H-chromen-4-one and
(R)-2-(1-bromoethyl)-3-(3-fluorophenyl)-4H-chromen-4-one: The two
enantiomerically pure isomers were separated by preparative SFC
conditions from
2-(1-bromoethyl)-3-(3-fluorophenyl)-4H-chromen-4-one (10 g) using
CO.sub.2:MeOH and analysed on a XBridge C18 column (50.times.4.6
mm; 3.5 m) using water (10 mM ammonium bicarbonate):acetonitrile
(gradient: 5%-95% acetonitrile in 1.2 min.) as the mobile phase at
a flow rate of 2.0 ml/min.
[0181] Intermediate 7a: Off-white solid (3.80 g). e.e. 100%. Rt:
1.79 min. Mass: 348.9 (M.sup.++1).
[0182] Intermediate 7b: Off-white solid (3.8 g). e.e. 100%. Rt:
1.79 min. Mass: 348.9 (M.sup.++1).
Intermediate 8
##STR00011##
[0184] Intermediate 8:
3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one: To a
solution of 2-(1-bromoethyl)-3-(3-fluorophenyl)-4H-chromen-4-one
(30 g, 86.51 mmol) in DMSO (300 ml), n-butanol (15 ml) was added
and heated to 120.degree. C. for 3 h., The reaction mixture was
cooled to RT, quenched with water 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 ethyl acetate:petroleum ether to afford the
title compound as a off-white solid (16 g, 64%) which was used as
such in next step.
Intermediate 9
##STR00012##
[0186] Intermediate 9:
2-acetyl-3-(3-fluorophenyl)-4H-chromen-4-one: DMSO (16.0 ml, 227
mmol) was added to dichloromethane (200 ml) cooled to -78.degree.
C., followed by oxalyl chloride (9.80 ml, 113.5 mmol). After 10
min. intermediate 8 (16.2 g, 56.79 mmol) in dichloromethane (54 ml)
was added dropwise and stirred for 20 min. Triethylamine (32 ml)
was added and stirred for 1 h. The reaction mixture was quenched
with water and extracted with dichloromethane. The organic layer
was dried over sodium sulphate and concentrated under reduced
pressure. The crude product was purified by column chromatography
with ethyl acetate:petroleum ether to afford the title compound as
a yellow solid (8.2 g, 51%). .sup.1H-NMR (8 ppm, CDCl.sub.3, 400
MHz): 8.26 (dd, J=8.0, 1.5 Hz, 1H), 7.79 (m, 1H), 7.58 (d, J=8.3
Hz, 1H), 7.50 (dt, J=8.0, 0.8 Hz, 1H), 7.41 (m, 1H), 7.15 (m, 1H),
7.01 (m, 2H), 2.37 (s, 3H).
Intermediate 10
##STR00013##
[0188] Intermediate 10:
(S)-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one: To
intermediate 8 (1.00 g, 3.53 mmol) in THF (2 ml), R-Alpine borane
(0.5M in THF, 10 ml) was added and heated to 60.degree. C. for 20
h. The reaction mixture quenched with aq. 2N HCl, 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 ethyl acetate:petroleum
ether to afford the title compound as a off-white solid (0.400 g,
40%). Enantiomeric excess: 94.8%, enriched in the fast eluting
isomer (retention time: 8.71 min.) as determined by HPLC on a
chiralpak AD-H column.
Intermediate 11
[0189] Intermediate 11: 4-bromo-2-fluoro-1-isopropoxybenzene: To a
solution of 4-bromo-2-fluorophenol (10 g, 52.35 mmol) in THF (100
ml), isopropyl alcohol (4.8 ml, 62.62 mmol) and triphenylphosphine
(20.6 g, 78.52 mmol) were added and heated to 45.degree. C.
followed by diisopropylazodicarboxylate (15.4 ml, 78.52 mmol). The
mixture was refluxed for 1 h, concentrated and the residue was
purified by column chromatography with ethyl acetate:petroleum
ether to afford the title compound as a colourless liquid (13.1 g,
99%) which was used without purification in the next step.
Intermediate 12
[0190] Intermediate 12:
2-(3-fluoro-4-isopropoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane:
Potassium acetate (10.52 g, 107.2 mmol) and bis(pinacolato)diboron
(15 g, 58.96 mmol) were added to a solution of intermediate 11
(10.52 g, 107.2 mmol) in dioxane (125 ml), and the solution was
degassed for 30 min. [1,1'-Bis(diphenylphosphino)ferrocene]dichloro
palladium(II).CH.sub.2Cl.sub.2 (4.4 g, 5.36 mmol) was added under
nitrogen atmosphere and heated to 80.degree. C. After 12 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
oil (13.9 g, 99%) which was used without purification in the next
step.
Intermediate 13
[0191] Intermediate 13:
3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine:
To a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (11.0
g, 42.14 mmol) in DMF 110 ml), ethanol (55 ml) and water (55 ml),
intermediate 12 (23.4 g, 84.28 mmol) and sodium carbonate (13.3 g,
126.42 mmol) were added and degassed for 30 min.
Tetrakis(triphenylphosphine)palladium(0) (2.4 g, 2.10 mmol) was
added under nitrogen atmosphere and heated to 80.degree. C. After
12 h, the reaction mixture was filtered though celite, concentrated
and extracted with ethyl acetate. The organic layer was dried over
sodium sulphate and concentrated under reduced pressure. The crude
product was triturated with diethyl ether, filtered and dried under
vacuum to afford the title compound as light brown solid (3.2 g,
26% yield) which is used as such for the next step.
Example A
2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin--
1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one
[0192] The title compound is prepared as described in Example 158
of International Publication No. WO 2011/055215.
Example A1
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one
[0193] To a solution of intermediate 13 (3.35 g, 11.60 mmol) in THF
(2.0 ml), intermediate 7 (3.00 g, 10.55 mmol) and
triphenylphosphine (5.57 g, 15.82 mmol) were added and stirred at
RT for 5 min. Diisopropylazodicarboxylate (3.2 ml, 15.82 mmol) was
added heated to 45.degree. C. After 2 h, the reaction mixture was
quenched with water 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 ethyl acetate:petroleum ether to afford the title compound as
off-white solid (2.79 g, 48%). MP: 200-203.degree. C. Mass: 554.3
(M.sup.++1). Enantiomeric excess: 94.0% as determined by HPLC on a
chiralpak AD-H column, enriched in the fast eluting isomer
(retention time=12.63 min).
Example A2
Method 1
(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one
[0194] To intermediate 13 (0.039 g, 0.143 mmol), cesium hydroxide
(0.013 g, 0.074 mmol) in ethanol was added and refluxed for 30 min.
The solvent was concentrated and the residue was dissolved in DMF
(0.5 ml). Intermediate 7a (0.050 g, 0.143 mmol) was added and
stirred at room temperature for 4 h. The reaction mixture was
diluted with water 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
off-white solid (0.025 g, 231%). MP. 205-207.degree. C. Mass: 554.3
(M.sup.++1). Enantiomeric excess: 74.0% as determined by HPLC on a
chiralpak AD-H column, enriched in the late eluting isomer
(retention time=14.77 min.).
Method 2
(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl)-3-(3-fluorophenyl)-4H-chromen-4-one
[0195] To a solution of intermediate 13 (0.143 g, 0.527 mmol) in
THF (7.5 ml), intermediate 10 (0.150 g, 0.527 mmol) and
triphenylphosphine (0.200 g, 0.791 mmol) were added and stirred at
RT for 5 min. Diisopropylazodicarboxylate (0.15 ml, 0.791 mmol) was
added heated to 45.degree. C. After 3 h, the reaction mixture was
quenched with water 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 ethyl acetate:petroleum ether to afford the title compound as
off-white solid (0.035 g, 12%). MP: 204-206.degree. C. Mass: 554.3
(M.sup.++1). Enantiomeric excess: 98.8% as determined by HPLC on a
chiralpak AD-H column, enriched in the late eluting isomer
(retention time=14.77 min).
Example B
2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin--
1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
[0196] To a solution of intermediate 13 (0.080 g, 0.293 mmol) in
DMF (2 ml), potassium carbonate (0.081 g, 0.587 mmol) was added and
stirred at RT for 10 min. To this mixture intermediate
2-(1-bromoethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
(0.215 g, 0.587 mmol) was added and stirred for 12 h. The reaction
mixture was diluted with water 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 pale yellow solid (0.045 g, 270%). MP.
175-177.degree. C. .sup.1H-NMR (.delta. ppm, DMSO-D.sub.6, 400
MHz): .delta. 8.20 (s, 1H), 7.85 (dd, J=8-1, 3.0 Hz, 1H), 7.48-7.33
(m, 5H), 7.14 (t, J=8.3 Hz, 1H), 7.02 (m, 2H), 6.90 (m, 1H), 6.10
(q, J=7.1 Hz, 1H), 5.42 (s, 2H), 4.64 (quintet, J=6.0 Hz, 1H), 1.99
(d, J=7.1 Hz, 3H), 1.42 (d, J=6.1 Hz, 6H).
Example B1
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
[0197] To a solution of intermediate 13 (0.134 g, 0.494 mmol) in
THF (2.0 ml), intermediate 5 (0.150 g, 0.494 mmol) and
triphenylphosphine (0.194 g, 0.741 mml) were added and stirred at
RT for 5 min. Diisopropylazodicarboxylate (0.15 ml, 0.749 mmol) was
added heated to 45.degree. C. After 2 h, the reaction mixture was
quenched with water 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 ethyl acetate:petroleum ether to afford the title compound as
an off-white solid (0.049 g, 20%). MP: 139-142.degree. C. Mass:
571.7 (M.sup.+). .sup.1H-NMR (.delta. ppm, CDCl.sub.3 400 MHz):
8.24 (s, 1H), 7.85 (dd, J=8.2, 3.1 Hz, 1H), 7.50-7.29 (m, 5H), 7.14
(t, J=8.4 Hz, 1H), 7.02 (m, 2H), 6.92 (d, J=8.4 Hz, 1H), 6.11 (q,
J=7.1 Hz, 1H), 5.40 (s, 2H), 4.66 (quintet, J=6.1 Hz, 1H), 2.00 (d,
J=7.1 Hz, 3H), 1.42 (d, J=6.1 Hz, 6H). Enantiomeric excess: 89.8%
as determined by HPLC on a chiralpak AD-H column, enriched in the
fast eluting isomer (retention time=10.64 min.).
Example B2
(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
[0198] To a solution of intermediate 13 (0.284 g, 0.989 mmol) in
THF (5.0 ml), intermediate 3 (0.250 g, 0.824 mmol) and
tris(4-methoxy)phenylphosphine (0.435 g, 1.23 mml) were added and
stirred at RT for 5 min. Diisopropylazodicarboxylate (0.25 ml, 1.23
mmol) was added stirred at room temperature. After 12 h, the
reaction mixture was quenched with water 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 ethyl acetate:petroleum ether to
afford the title compound as an off-white solid (0.105 g, 22%). MP:
145-148.degree. C. Mass: 571.7 (M.sup.+). .sup.1H-NMR (.delta. ppm,
CDCl.sub.3, 400 MHz): 8.23 (s, 1H), 7.85 (dd, J=8.1, 3.0 Hz, 1H),
7.50-7.29 (m, 5H), 7.14 (t, J=8.4 Hz, 1H), 7.02 (m, 2H), 6.92 (d,
J=8.4 Hz, 1H), 6.10 (q, J=7.1 Hz, 1H), 5.42 (s, 2H), 4.64 (quintet,
J=6.1 Hz, 1H), 1.99 (d, J=7.2 Hz, 3H), 1.42 (d, J=6.0 Hz, 6H).
Enantiomeric excess: 95.4% as determined by HPLC on a chiralpak
AD-H column, enriched in the late eluting isomer (retention
time=14.83 min.).
4-Methylbenzenesulfonate salt of Compound B1
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate
##STR00014##
[0200]
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d-
]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
4-methylbenzenesulfonate: To
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one (22.7
g, 39.69 mmol) in isopropanol (600 ml), p-toluenesulphonic acid
(8.30 g, 43.66 mmol) was added and refluxed for 1 h. The reaction
mixture was concentrated, co-distilled with petroleum ether and
dried. To the residue water (300 ml) was added and stirred for 30
min. The solid was filtered, washed with petroleum ether and dried
under vacuum to afford the title compound as off-white solid (28.2
g, 95%). MP: 138-141.degree. C. .sup.1H-NMR (.delta. ppm,
CDCl.sub.3, 400 MHz): 8.11 (s, 1H), 7.85 (dd, J=8.0, 3.0 Hz, 1H),
7.80 (d, J=8.2 Hz, 2H), 7.51 (dd, J=9.3, 4.3 Hz, 1H), 7.45 (dd,
J=7.5, 3.1 Hz, 1H), 7.42-7.31 (m, 3H), 7.29 (m, 2H), 7.22 (d, J=8.0
Hz, 2H), 7.16 (t, J=8.3 Hz, 1H), 7.08 (dt, J=8.5, 2.5 Hz, 1H), 6.97
(br s, 1H), 6.88 (br s, 1H), 6.11 (q, J=7.2 Hz, 1H), 4.67 (quintet,
J=6.0 Hz, 1H), 2.36 (s, 3H), 2.03 (d, J=7.1 Hz, 3H), 1.43 (d, J=6.0
Hz, 6H). Mass: 572.4 (M.sup.++1-PTSA). Enantiomeric excess: 93.4%
as determined by HPLC on a chiralpak AD-H column, enriched in the
fast eluting isomer (retention time=12.35 min.)
Sulphate Salt of Compound B1
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
sulfate
##STR00015##
[0202]
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d-
]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one
sulphate: To
(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrim-
idin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one (15.0
g, 26.24 mmol) in isopropanol (600 ml) was cooled to 0.degree. C.
To this Sulphuric acid (2.83 g, 28.86 mmol) was added and stirred
at room temperature for 24 h. The reaction mass was filtered and
washed with petroleum ether and dried under vacuum. To the solid,
water (150 ml) was added and stirred for 30 min. The solid was
filtered, washed with petroleum ether and dried under vacuum to
afford the title compound as off-white solid (13.5 g, 76%). MP:
125-127.degree. C. .sup.1H-NMR (.delta. ppm, CDCl.sub.3, 400 MHz):
8.11 (s, 1H), 7.85 (dd, J=8.0, 3.0 Hz, 1H), 7.51 (dd, J=9.2, 4.2
Hz, 1H), 7.45-7.31 (m, 3H), 7.29 (m, 1H), 7.15 (t, J=8.3 Hz, 1H),
7.08 (dt, J=8.5, 2.4 Hz, 1H), 6.96 (br s, 1H), 6.88 (br s, 1H),
6.09 (q, J=7.1 Hz, 1H), 4.676 (quintet, J=6.1 Hz, 1H), 2.01 (d,
J=7.1 Hz, 3H), 1.42 (d, J=6.1 Hz, 6H). Mass: 572.2
(M.sup.++1-H.sub.2SO.sub.4). Enantiomeric excess: 89.6% as
determined by HPLC on a chiralpak AD-H column, enriched in the fast
eluting isomer (retention time=12.08 min.)
[0203] Various other acid addition salts of compound B1 were
prepared as provided in Table 1.
TABLE-US-00001 TABLE 1 Melting Point Acid Method of preparation
(.degree. C.) Hydrochloric Compound B1 (1 eq.) dissolved in THF,
130-132 acid excess HCl/Et.sub.2O was added, the clear solution
obtained was evaporated completely. The residue obtained was washed
with water. p- Compound B1 (1 eq.) dissolved in 138-141.degree. C.
Toluenesulfonic isopropyl alcohol (IPA), refluxed for acid 30 min.,
acid (1.1 eq.) in IPA was added, the clear solution obtained
wasevaporated completely. The residue obtained was washed with
water. Benzenesulphonic Compound B1 (1 eq.) dissolved in IPA,
170-172 acid refluxed for 30 min., acid(1.1 eq.) in IPA was added,
the clear solution not obtained, the residue was evaporated
completely and was washed with water. Maleic acid Compound B1 (1
eq.) dissolved in IPA, 107-109 refluxed for 30 min., acid (1.1 eq.)
in IPA was added, the clear solution not obtained, the residue was
evaporated completely and was washed with water. Camphor sulfonic
Compound B1 (1 eq.) dissolved in IPA, 120-121 acid refluxed for 30
min., acid (1.1 eq.) in IPA was added, the clear solution not
obtained, the residue was evaporated completely and was washed with
water. Sulphuric acid Compound B1 (1 eq.) dissolved in IPA, 125-127
refluxed for 30 min., acid(1.1 eq.) in IPA was added, the clear
solution obtained was evaporated completely. The residue obtained
was washed with water.
Metabolic Stability
[0204] Metabolic stability studies were conducted using mouse, rat,
and human liver microsomes. The protocol for the studies with
mouse, rat, and human liver microsomes (all from BD Gentest, USA)
is provided below. In brief, 0.4 mg protein was preincubated with 2
mM NADPH (cofactor) in phosphate buffer (pH-7.4) for 15 min 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 the tables below.
[0205] The data below surprisingly show that compound A1 of the
present invention has significantly greater metabolic stability in
human liver microsomes over its enantiomer A2 and racemic compound
A. For instance, the compound of Example A1 has an almost 5 fold
greater metabolic stability in human liver microsomes than that of
Example A2. Due to their enhanced metabolic stability, the
presently claimed compounds have a superior pharmacokinetic
profile.
TABLE-US-00002 Comparative data for Compound A and its individual
isomers A1 & A2 Metabolic stability in liver microsomes Example
Mouse Rat Human A 32.6 43.1 38.4 A1 46.1 35.8 54.5 A2 34.7 44.2
11.4
[0206] Similarly, the data below surprisingly show that compound B1
of the present invention has significantly greater metabolic
stability in human liver microsomes over its enantiomer B2 and
racemic compound B. For instance, the compound of Example B1 has an
almost 3 fold greater metabolic stability in human liver microsomes
than that of Example B2. Due to their enhanced metabolic stability,
the presently claimed compounds have a superior pharmacokinetic
profile.
TABLE-US-00003 Comparative data for Compound B and its individual
isomers B1 & B2 Metabolic stability in liver microsomes Example
Mouse Rat Human B 54.9 51.4 46.9 B1 36.6 23.1 74.9 B2 54.1 48.6
27.5
Pharmacokinetics
[0207] The oral bioavailability of compound B1 (free base) and its
PTSA salt were evaluated in rats. The protocol for the
pharmacokinetics studies in rat is provided below.
[0208] 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, 4000 cPs, 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 formulation were
analyzed by HPLC. The plasma concentrations of test item in all
samples were analyzed by LC-MS/MS. Pharmacokinetic parameters viz.
C.sub.max, AUC.sub.0-t, T.sub.max, and t1/2 were estimated by using
WinNonlin software.
[0209] The PTSA salt of compound of Example B1 exhibited a
C.sub.max about twice that, and an area under the curve (AUC) of
nearly three times that, of the free base of compound B1.
[0210] Similarly the oral bioavailability of compound B1 (free
base) and its PTSA salt were evaluated in Dogs. The PTSA salt of
compound B1 exhibited a C.sub.max more than twice that, and an area
under the curve (AUC) of about four times that, of the free base of
compound B1.
Biological Assay
Assay 1: Fluorescent Determination of PI3K Enzyme Activity
[0211] The homogenous time resolved fluorescence (HTRF) assay
allows detection of 3,4,5-triphosphate (PIP3) formed as a result of
phosphorylation of phosphotidylinositol 4,5-biphosphate (PIP2) by
PI3K isoforms such as .alpha., .beta., .gamma. or .delta..
[0212] 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. Briefly, 0.5 .mu.l of
40.times. inhibitor (in 100% DMSO) or 100% DMSO were added to each
well of a 384-well black 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
and incubated for 10 min. After the initial incubation, 5
.mu.l/well of 400 .mu.M ATP was added and incubated for an
additional 30 minutes. Reaction was terminated by adding 5
.mu.l/well stop solution (Millipore, Billerica, Mass.). Five
microliters of detection mix (Millipore, Billerica, Mass.) were
then added to each well and was incubated for 6-18 h 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 620 nm with an integration time of 400
.mu.sec.
[0213] The results are shown below.
Comparative data for Compound A and its individual isomers A1 &
A2
TABLE-US-00004 % Inhibition @ 1 .mu.M Example Pi3K delta IC.sub.50
(nM) Pi3K .alpha. Pi3K .beta. Pi3K .gamma. A 37.32 2.63 9.95 55.85
A1 13.83 8.91 47.87 80.60 A2 >10 .mu.M 0.95 38.74 66.3
TABLE-US-00005 Selectivity profile of Compound A1 IC.sub.50 (nM)
Fold-Selectivity Assay PI3K.delta. PI3K.alpha. PI3K.beta.
PI3K.gamma. Enzyme 13.83 >1000 >54 >9
[0214] Comparative Data for Compound B and its Individual Isomers
B1 & B2
TABLE-US-00006 % Inhibition @ 1 .mu.M Example Pi3K .delta.
IC.sub.50 (nM) Pi3K .alpha. Pi3K .beta. Pi3K .gamma. B 24.89 37.90
18 18.3 B1 22.33 19.13 44.88 47.21 B2 1447 25.29 52.01 68.10
TABLE-US-00007 Selectivity profile of Compound B1 IC.sub.50 (nM)
Fold-Selectivity Assay PI3K.delta. PI3K.alpha. PI3K.beta.
PI3K.gamma. Enzyme 22.23 >10000 >50 >48
Assay 2: In Vitro Cell Proliferation Assay in Leukemic Cell
Lines
[0215] 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 compound at a
concentration range of 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 h (prior to the addition of the test
compound) and 48 h after the addition of test compound. Absorbance
was read on a Fluostar Optima (BMG Labtech, Germany) at a wave
length of 450 nm. Data were analysed using GraphPad Prism and
percent inhibition due to the test compound compared to the control
was calculated accordingly.
[0216] Results: While a slight dose-dependent reduction in cell
viability was observed, the compounds did not display any apparent
cytotoxicity over the 72 h incubation period.
Assay 3: Inhibition of AKT Phosphorylation in Leukemic Cell
Lines:
[0217] Inhibition of AKT phosphorylation in leukemic cell lines:
THP-1, HL-60, MOLT-4, RPMI-8226, or DLBCL cells were incubated with
desired concentrations of compound for 48 hours. Cells were lysed
and pAKT was determined by Western Blotting. Bands were quantified
using ImageJ and normalized to actin.
[0218] Results: Compound A1 and Compound B1 when tested at 1 .mu.M
exhibited 50 to 90% inhibition.
Assay 4: Inhibition of PI3K6 Signalling in Basophils from Human
Whole Blood
[0219] PI3K8 signalling in basophils manifested by an alteration of
anti-Fc.epsilon.R1 induced CD63 expression is a useful
pharmacodynamic marker determined using the Flow2CAST.RTM. kit
(Buhlmann Laboratories, Switzerland). Briefly, it involves the
following steps: [0220] Mix the anti-coagulated blood sample by
inverting the venipuncture tube several times [0221] Prepare fresh
and pyrogen-free 3.5 ml polypropylene or polystyrene tubes suitable
for Flow Cytometry measurements [0222] Add 49 .mu.l of patient's
whole blood to each tube. [0223] Add 1 .mu.l of 10% DMSO
(background) or compound (10% DMSO) to the assigned tubes and mix
gently. Incubate at room temperature for 15 min [0224] Pipette 50
.mu.l of the Stimulation buffer (background) or anti-Fc.epsilon.RI
Ab to each tube [0225] Add 100 .mu.l of Stimulation Buffer to each
tube [0226] Mix gently. Add 20 .mu.l Staining Reagent (1:1 mix of
FITC-CD63 and PE-CCR3) to each tube [0227] 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) [0228] Add 2 ml
pre-warmed (18-28.degree. C.) Lysing Reagent to each tube, mix
gently [0229] Incubate for 5-10 minutes at 18-28.degree. C. [0230]
Centrifuge the tubes for 5 minutes at 500.times.g [0231] Decant the
supernatant by using blotting paper [0232] Resuspend the cell
pellet with 300-800 .mu.l of Wash Buffer [0233] Vortex gently and
acquire the data on the flow cytometer within the same day. [0234]
Percent CD63 positive cells within the gated basophil population
are to be determined in different treatment groups and normalized
to vehicle control.
[0235] Results: Compound A1 and Compound B1 inhibited anti-FcER1
mediated CD63 expression in human whole blood basophils with EC50s
of .ltoreq.100 nM respectively.
Assay 4a: Cell Based Compound Specificity Towards Inhibition of
PI3K .delta., .alpha., .beta. or .gamma. Isoforms
[0236] Compound specificity towards PI3K.delta. was determined in
an IgM-induced B cell proliferation assay. B-cells isolated from
blood of healthy subjects were seeded in a 96-well tissue culture
plate and incubated with desired concentrations of compound for 30
min. Cells were stimulated with 5 .mu.g/ml purified goat anti-human
IgM. Growth was assessed using the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
dye reduction test. For selectivity against PI3K .alpha., .beta.,
or .gamma. isoforms, NIH-3T3 or RAW macrophages were seeded in a
6-well tissue culture plate and incubated overnight. Complete
medium was replaced with serum-free media the following day and
compound at the desired concentrations were added. After 15
minutes, 20 ng/ml PDGF, 5 .mu.M LPA, or 50 ng/ml c5a was added and
incubated for an additional 10 minutes. Cells were lysed and AKT
phosphorylation was determined by Western Blotting. Intensity of
the bands was determined using ImageJ 1.42q (NIH, USA) and
normalized to Actin (loading control).
TABLE-US-00008 EC.sub.50 (nM) Fold-Selectivity Assay PI3K.delta.
PI3K.alpha. PI3K.beta. PI3K.gamma. Selectivity profile of Compound
A1 Cell-based <50 nM >1000 >30 >8 Selectivity profile
of Compound B1 Cell-based <30 nM >10000 >34 >17
Assay 5: Inhibition of Apoptosis in Leukemic Cell Lines
[0237] Apoptosis in leukemic cells was determined using an in situ
Caspase 3 kit (Millipore, US) as outlined below: [0238] Seed
leukemic cells at a density of 1.times.10.sup.6 cells/well in a 6
well plate [0239] Add test compound/DMSO at desired concentrations
[0240] Incubate the plate for 24 hrs at 37.degree. C. in 5%
CO.sub.2 incubator [0241] Collect cells in a 2 ml centrifuge tube
[0242] Add 1.6 .mu.L of freshly prepared 5.times.FLICA reagent and
mix cells by slightly flicking the tubes [0243] Incubate tubes for
1 hour at 37.degree. C. under 5% CO.sub.2 [0244] Add 2 ml of
1.times. wash buffer to each tube and mix [0245] Centrifuge cells
at <400.times.g for 5 minutes at room temperature. [0246]
Carefully remove and discard supernatant, and gently vortex cell
pellet to disrupt any cell-to-cell clumping. [0247] Resuspend cell
pellet in 300 ul of 1.times. wash buffer [0248] Place 100 .mu.L of
each cell suspension into each of two wells of a black microtiter
plate. Avoid creation of bubbles. [0249] Read absorbance of each
microwell using an excitation wavelength of 490 nm and an emission
wavelength of 520 nm [0250] Percent increase in caspase-3 activity
manifested by an increase in fluorescence compared to the control
blank is to be calculated.
[0251] Results: Compound A1 and Compound B1 dose-dependently
induced Caspase-3 activity in the cell lines tested.
Assay 6: Screening for Anticancer Activity in Human Primary
Leukemic Cells
[0252] 6-I: Flow cytometry analysis of apoptotic induction in AML
patient bone marrow leukemic cells upon compound treatment using
Annexin V and 7-AAD staining: Mononuclear cells were extracted by
the Ficoll method and seeded in plates. The cells were treated by
different compounds for 48 hrs before they were analyzed by flow
cytometry. After washing with PBS, 1.times.10.sup.5 cells were
stained by Annexin V-APC and 7-AAD. Annexin V positive staining
measures total apoptotic cells, including early and late apoptotic
cells. For Annexin V positive cells, 7-AAD negative signal reflects
early apoptotic cells.
[0253] 6-II: pAKT analysis of AML patient bone marrow sample using
pAKT ELISA kit: Mononuclear cells were extracted by the Ficoll
method and seeded in plates. The cells were treated by different
compounds for 48 hrs before they were analyzed by pAKT ELISA kit
following the product protocol. Briefly, 1.times.10.sup.6 cells
were transferred into an ELISA kit well and lyzed with 10 .mu.L
5.times. Cell Lysis Mix (phospho-AKT 1/2/3 (Ser473) InstantOne.TM.
ELISA Kit, eBioscience, 85-86042). The cells were then incubated
with 50 .mu.l antibody cocktail for 1 hr at room temp. on a
microplate shaker (.about.300 rpm). After incubating with detection
reagent, the result was measured using a SpectraMAX Plus microplate
spectrophotometer set at 450 nm.
[0254] 6-III: Cell proliferation analysis of AML patient bone
marrow sample using MTS assay: Mononuclear cells were extracted by
the Ficoll method and seeded in plates. The cells were treated by
different compounds for 48 hrs and 72 hrs before they were analyzed
by MTS assay following product instruction. Briefly, 20 .mu.L of
the MTS solution was added into each well containing the 100 .mu.L
cell suspension, followed by incubation for 4 hours at 37.degree.
C., in 95% humidity with presence of 5% CO.sub.2. The absorbance of
490 nm (A490) was read using SpectraMAX Plus microplate
spectrophotometer.
[0255] Results: Treatment with compound A1 and compound B1 caused a
dose dependent reduction in proliferation and AKT phosphorylation
with a concomitant increase in the number of apoptotic cells.
TABLE-US-00009 Compound Results A1 >50% inhibition of PAKT @ 0.3
.mu.M; ~1.5 fold increase in appotosis @ 3 .mu.M and Dose dependent
reduction in cell viability. B1 >50% inhibition of PAKT @ 0.3
.mu.M; ~1.5 fold increase in appotosis @ 3 .mu.M and Dose dependent
reduction in cell viability.
Assay 6a: Screening for Anticancer Activity in Human Multiple
Myeloma Cells
[0256] Samples were taken from two patients with newly diagnosed
stage II IgG Kappa and stage III IgG Lambd restricted disease. This
screening was performed by inducing apoptosis using doses and times
determined from the MTT assay. 1-5.times.10.sup.5 cells were
collected by centrifugation. The cells were re-suspended in 500
.mu.l of 1.times. Binding Buffer. 5 .mu.l of Annexin V-FITC and 5
.mu.l of propidium iodide were added. The cells were incubated at
room temperature for 5 minutes in the dark.
[0257] Quantification by flow cytometry: Annexin V-FITC binding was
analyzed by flow cytometry (Ex=488 nm; Em=530 nm) using FITC signal
detector (usually FL1) and PI staining using a phycoerythrin
emission signal detector (usually FL2). The results are shown below
and in FIG. 1.
TABLE-US-00010 B1 >75% inhibition of PAKT @ 3.0 .mu.M; ~1.5 fold
increase in apoptosis @ 3 .mu.M and Dose dependent reduction in
cell viability.
Assay 7: Screening for Anticancer Activity in Various Leukemic Cell
Line
[0258] Proliferation of immortalized leukemic cells representative
of various indications was determined by a MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
assay. Cells were incubated with Compound B1 for different
time-periods (72-96 h) based on their doubling time.
TABLE-US-00011 Cell Line Disease Cell Type Organ TOLEDO Diffuse
large cell lymphoma/ B lymphocyte Peripheral Non-Hodgkin's B-cell
Blood lymphoma U266B1 Myeloma, Plasmacytoma B lymphocyte Peripheral
(CD40-) Blood MOLT-4 ALL T lymphoblast Peripheral Blood Jurkat
Acute T-cell leukemia T lymphocyte Peripheral Blood THP-1 Acute
Monocytic Leukemia Monocyte Peripheral Blood MM-1R immunoglobulin A
lambda B lymphoblast Peripheral myeloma Blood DLBCL Large Cell
Lymphoma B lymphoblast Ascites Fluid MM-1S immunoglobulin A lambda
B lymphoblast Peripheral myeloma Blood U937 Histiocytic Lymphoma
Monocyte Pleural Effusion Raji Burkitt's Lymphoma B lymphoblast
Maxilla CCRF- ALL T lymphoblast Peripheral CEM Blood HL-60 AML
Promyeloblast Peripheral Blood
[0259] Results: Overall, a 50% growth inhibition for the majority
of B, T, and monocytic cell lines was achieved at a concentration
between 0.5-7.5 .mu.M of Compound B1. The data demonstrated the
ability of Compound B1 to inhibit leukemic cell proliferation
albeit with different potencies based on the cell type.
Assay 8: Screening for Anticancer Activity in Human CLL Cells
[0260] Primary CLL cells were incubated with serial dilutions of
test compound (Compound B1) for 48 hours and tested for apoptosis
by activated caspase-3 and 7AAD staining measured by flow
cytometry. After 72 hours of incubation, CLL cells were evaluated
for cytotoxicity using the colorimetric MTS reagent. Phosphorylated
Akt (5473) was measured by flow cytometry after one hour of
incubation of test compound and ten minutes of incubation with
anti-IgM or anti-IgD. Akt phosphorylation was quantified by median
fluorescent intensity (MFI). Of the seven CLL patient samples used
for experiments, five had mutated IGHV, five had 13q deletion or
normal cytogenetics determined by fluorescent in situ
hybridization, three were ZAP-70 negative, and seven were CD38
negative. IgM expression ranged between 13% and 90%, whereas IgD
expression was uniformly elevated. The test compound significantly
induced apoptosis (caspase-3+/7AAD+) and cytotoxicity in a
dose-dependent manner in concentrations between 0.1 and 25.6 .mu.M.
Incubation with anti-surface immunoglobulin significantly induced
Akt phosphorylation compared to media alone while the addition of
test compound significantly abrogated this effect and returned Akt
phosphorylation to baseline.
[0261] Results: The test compound induces cytotoxicity and
apoptosis in CLL cells, via inhibition of pAKT. The results are
also shown in FIGS. 2A, 2B, and 2C.
[0262] 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.
[0263] All publications, patents and patent applications cited in
this application are herein incorporated by reference to the same
extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated herein by reference.
* * * * *