OA18327A - Substituted chromene derivatives as selective dual inhibitors of Pi3 delta and gamma protein kinases - Google Patents

Substituted chromene derivatives as selective dual inhibitors of Pi3 delta and gamma protein kinases Download PDF

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OA18327A
OA18327A OA1201600497 OA18327A OA 18327 A OA18327 A OA 18327A OA 1201600497 OA1201600497 OA 1201600497 OA 18327 A OA18327 A OA 18327A
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compound
amino
disease
pyrimidin
fluoro
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OA1201600497
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Swaroop Kumar Venkata Satya VAKKALANKA
Prashant Kashinath Bhavar
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Rhizen Pharmaceuticals Sa
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Abstract

The present invention relates to a selective dual delta (8) and gamma (y) P13K protein kinase modulator (S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-lHpyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl) methane sulfonamide, methods of preparing them, pharmaceutical compositions containing them and methods of treatment, prévention and/or amélioration of PI3K kinase mediated diseases or disorders with them.

Description

The présent invention provides dual delta (δ) and gamma (γ) PI3K protein kinase modulators, methods of preparing them, pharmaceutical compositions containing them and methods of treatment, prévention and/or amelioration of PI3K kinase mediated diseases or disorc ers using them.
BACKGROUND OF THE INVENTION
Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellular lipid kinases that phosphorylate the 3-position hydroxyl group of the inositol ring of phosphoinositide lipids (Pis) generating lipid second messengers. While a and β isoforms of PI3K are ubiquitous in their distribution, expression of δ and y forms of PI3K is restricted to circulating haematogeaous cells and endothélial cells. Unlike PI3Koc or ΡΙ3Κβ, mice lacking expression of ΡΙ3Κδ or PI3Ky do not show any adverse phenotype indicating that targeting of these spécifie isoforms would not resuit in overt toxicity.
Recently, targeted inhibitors of the PI3K pathway hâve been suggested as immunomodulatory agents. This interest stems from the fact that the PI3K pathway serves multiple functions in immune cell signaling, primarily through the génération of phosphatidylinositol (3,4,5)trisphosphate (PIP3), a membrane bound second messenger. PJP3 recruits proteias to the cytoplasmic side of the lipid bilayer, including protein kinases and GTPases, initiating a complex network of downstream signaling cascades important in the régulation of immùne.cell adhesion, migration, and cell-cell communication.
The four class I PI3K isoforms differ significantly in their tissue distribution. PI3Ka and ΡΙ3Κβ are ubiquitous and activated downstream of receptor tyrosine kinases (RTK), whereas ΡΙ3Κδ and PI3Ky are primarily limited to hematopoietic and endothélial cells, and are activated downstream of RTKs, and G protein coupled receptors (GPCR), respectively. Mouse genetic studies hâve revealed that PI3Ka and ΡΙ3Κβ are essential for normal development, whereas Igss of ΡΙ3Κδ and/or ΡΙ3Κγ yields viable offspring with sélective immune déficits.
The expression pattern and functions of ΡΙ3Κδ and ΡΙ3Κγ hâve generated much interest in developing ΡΙ3Κδ/γ inhibitors as active agents for the treatment of many diseascs, including, for example, rheumatoid arthritis, allergies, asthma, chronic obstructive pulmonary diseasc and multiple sclerosis (Hirsch et al., Pharmacol. Ther., 118, 192-205.2008; Marone et al., Biochim. Biophys. Acta., 1784, 159-185, 2008; Rommel et al., Nat. Rev. Immunol., 7, 195-201, 2007; Ruck’e étal., Nat. Rev. Drug Discov., 5, 903-918j2006). Studies using both phaimaeologicand genetic methods hâve shown these two isoforms often demonstrate synergistic interactions with each other (Konrad et al., J. Biol. Chem., 283, 33296-33303, 2008; Laffargue et al., Immunity, 16, 441-451,2002). In mast cells, for example, Ρ13Κδ is essential for degranulation in response to IgE cross-Iinking of Fc-receptors (Ali et al., J. Immunol., 180, 2538-2544, 2008), whiîe ΡΙ3Κγ plays an important rôle in amplifying the response (Laffargue et al., Immunity, 16, 441451, 2002). Similar effects hâve been seen in other cellular functions, including lymphocyte homing and the neutrophil respiratory burst where ΡΙ3Κγ plays a critical rôle and ΡΙ3Κδ amplifies each process. The nonredundant but related rôles of ΡΙ3Κδ and ΡΙ3Κγ hâve made it difficult to détermine which of the two isoforms (alone or in combination) is best targeted in a particular inflammatory disorder.
Studies using mice that lack ΡΙ3Κδ and/or ΡΙ3Κγ or express kinase-dead variants of Ρ13Κδ and ΡΙ3Κγ hâve been valuable tools in understanding their rôles. For example, ΡΙ3Κδ knockout mice demonstrated diminished neutrophil chemotaxis, diminished antibody production (both T cell dépendent and independent) (Jou et al., Mol. Cell.Biol., 22, 8580-8591, 2002), and lowcr numbers of mature B cells (Clayton et al., J. Exp. Med., 196, 753-763, 2002; Jou et al., Mol. Cell.Biol., 22, 8580-8591, 2002), and a decrease in their prolifération in response to anti-IgM (Jou et al., Mol. Cell.Biol., 22, 8580-8591, 2002). This phenotype was replicated in the ΡΙ3Κδ kinase-dead variant and with ΡΙ3Κδ sélective inhibitors along with a decreased number and prolifération of mast cells, and an attenuated allergie response. The ΡΙ3Κγ knockout contained higher numbers of, but less responsive, neutrophils, lower numbers of and less responsivc macrophages, and dendritic cells displayed decreased mast cell degranulation (Laffargue et al., Immunity, 16, 441-451, 2002), a higher ratio of CD4+ to CD8+ T cells, increased thymocyte apoptosis, diminished induction of CXCR3 on activated T cells and decreased cardiac contractility. This latter effect on cardiac tissue was a concem for chronic dosing of patients
-218327 with ΡΙ3Κγ. inhibitors. However, this concem was largely mitigated when the ΡΙ3Κγ kinasedead variant (which better mimics inhibition of the kinase rather than loss of inc protein) showed similar immune cell phenotypes, but importantly had no cardiac defects. The cardiac effect was later shown to be due to scaffolding effects rather than the caiaiytic activity of ΡΙ3Κγ 5 (Olusegon et al., Chemistry & Biology, 1,123-134,2010, including the références cited therein).
The dual ΡΙ3Κδ/ΡΙ3Κγ knockout was viable but exliibitcd serious defects in T celi development and thymocyte survival. The ΡΙ3Κγ knockout/PI3K5 kinase-dead combinaticn produced ·α· similar phenotype suggesting that at least within the immune System, the rôle of PI3K5 is likely only a catalytic one. Interprétation of studies using knockout and kinase-dead rnice can bc ÎO challenging because these models provide only a steady-state picture of the immune system, lack temporal and dose control, and do not permit a fûll understanding of how a dynamic I immune response will react to réversible inhibition. Sélective inhibitors with varying profiles (ΡΙ3Κδ, ΡΙ3Κγ, and ΡΙ3Κδ/γ) are necessary for studies of-leukocyte signaling in order to àssess the relative contributions of each PI3K to immune cell activation (Olusegon et al., supra, 15 including the references cited therein).
Dual inhibition of δ/γ is strongly implicated as an intervention strategy in allergie and nonallergie inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3KÔ and PI3K γ involvement in various cellular processes underlying asthma and chronic obstructive pulmonary disease (COPD) stems from inhibitor studies and gene-targeting 20 approaches (William et.al Chemistry & Biology, 17, 123-134, 2010 and Thompson, et al.
Chemistry & Biology, 17:101-102, 2010). Also, résistance to conventional thérapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K δ/γ pathway. Disruption of ΡΙ3Κδ/γ signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotai rôle played by Ρΐ3Κδ and 25 ΡΙ3Κγ in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well. Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the ΡΙ3Κδ and ΡΙ3Κγ isoforms v/ould be expected to attenuate the progression of immune response encountered in airway inflammation 30 and rheumatoid arthritis (William et.al Chemistry & Biology, 17, 123-134,2010 and Thompson, et al. Chemistry & Biology, 17:101-102,2010)
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Reviews and studies regarding PI3K and related protcin kinase pathways hâve been given by Liu et al., Nature Reviews Drug Discovery, 8. 627-644,2009); Nathan et. aï., Mol Cancer Ther., 8(1), 2009; Marone et al., Biochimica et Biophysica Acta, 1784,159-185, 2008 and Markman et al., Annals of Oncology Advance Access, publis’ned August 2009. Similarly reviews and studies • regarding rôle of ΡΙ3Κδ and ΡΙ3Κγ hâve been given by· William et al.. Chemistry & Bîology, 17,
123-134, 2010 and Timothy et al. J. Med. Chem., 55 (20), 8559-8581, 2012. Ali’ of these literature disclosures are hereby încorporated by reference in iheir entirety. ··
Compounds such as IPI-145 and CAL 130 hâve been reported a; dual inhibitors of Pi3K δ/γ (WO2012/008302 & WO2012/121953 respectively).IPI-145 is under clinical investigation for cancer, asthma and rheumatoid arthritis. IPI-45 has been reported to hâve a maximum tolerated dose (MTD) of 75 mg BID (55th ASH® Annual Meeting. New Orleans-LA, Dec 7-10, 2013). There are no reports of CAL-130 being irivestigated for clinical purposes.
There still remains an unmet need for dual δ/γ PI3K modulators for the treatment of diseases and disorders associated with δ/γ PI3K kinases-mediated events.
Further reference is made herein to International Publication Nos. WO 11/055215 and WO 12/151525 and U.S. Publication Nos. 2011/0118257 and 2012/0289496, each of which is încorporated herein by reference in its entirety.
SUMMARY OF THE INVENTION
The présent invention is directed to sélective dual inhibitors of PI3K delta (δ) and gamma (γ) . protein kinases. These compounds are suitable for use in a pharmaceutical composition for the treatment of PI3K associated diseases, disorders or conditions, e.g., a proliférative disease such as cancer. Inhibition of both ΡΙ3Κδ and ΡΙ3Κγ protein kinases may provide bénéficiai effects in • the treatment of certain diseases and disorders.
The sélective dual inhibitors of the présent invention include N-(5-(4-amino-l-(l-(5-fluoro-3(3-fIuorophenyl)-4-oxo-4H-chromen-2-yl) . ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2methoxyphenyl)methanesulfonamide, pharmaceutically acceptable salts thereof, and prodrugs thereof. For example, the sélective dual inhibitor may be selected from the following compounds, pharmaceutically acceptable salts thereof, and prodrugs thereof:
-418327 (RS)-N-(5-(4-amino-l-(l-(5-f!uoiO-3-(3-tluorophenyl)-4-oxo-4II-chromcn-2-yl) ethyl)lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamid.e (Compound A); and (S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl) ethyl)5 lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyplienyl)methanesulfonamidc (Compound
Al).
In one embodiment, the compound (S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxO 4H-chromen-2-yl)ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2methoxyphenyl)methanesulfonamide or a pharmaceutically acceptable sait thereof is 10 substantially free (e.g., contains less than about 10%, such as less than about 5%, less than about
2.5%, less than about 1%, less than about 0.1% by weight) or is free of (R'.-N-(5-(4-amino-l-(l(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl) ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3yl)-2-methoxyphenyl)methanesulfonamide and pharmaceutically acceptable salts thereof.
In another embodiment, the compound (S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-415 oxo-4H-chromen-2-yl)ethyl)-1 H-pyrazolo[3,4-d]pyrimidin-3-yl)-2methoxyphenyl)methanesulfonamide or a pharmaceutically acceptable sait thereof has an enantiomeric excess of greater than about 90%, such as greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, 20 greater than about 99.5%, greater than about 99.9%, or greater than about 99.99%.
In one preferred embodiment, the présent invention relates to the compound (S)-N-(5-(4-amino1 -( 1 -(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl) ethyl)-1 H-pyrazolo[3,4d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide (Compound Al).
In another embodiment, the présent invention relates to the compound (S)-N-(5-(4-amino-l-(l25 (5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-1 H-pyrazolo [3,4-d]pyrimidin-3yl)-2-methoxyphenyl)methanesulfonamide or a pharmaceutically acceptable sait thereof.
Another embodiment of the présent invention is (R)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3fluorophenyl)-4-oxo-4H-chromen-2-yl) ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2methoxyphenyl)methanesulfonamide (Compound A2), a pharmaceutically acceptable sait 30 thereof, or prodrug thereof. Compound A2 is an inhibitor of PI3K delta (δ) protein kinase.
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These compounds are suîtable for use in a pharmaceutical composition for the treatmenc of PI3K associated diseases, disorders or conditions, e.g., a proliférative disease such as cancer.
The chemical structures of N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4Hchromen-2-yl) ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-25 methoxyphenÿl)methanesulfonamide, compound Al, and compound A2 arc shown below.
The présent invention further'provides a pharmaceutical composition comprising one or more compounds of the présent invention isuch as compound Al) together with a pharmaceutically 10 acceptable carrier. The pharmaceutical composition may further comprise one or more of addîtional 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 ofthe présent invention.
»*
Another aspect of the présent invention relates to a process for the préparation of N-(5-(415 amino-1 -(1 -(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl) ethyl)-1 H-pyrazolo[3,4d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide:
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The process comprises the steps of:
(a) reacting 5-bromo-2-methoxyaniline
Br
/0 with methane sulphonyl chloride to give N-(5-bromo-2-methoxyphenyl)methanesulfonamide (Intermediate 1): · ·
Intermediate 1;
(b) reacting Intermediate 1 with bis(pinacolato)diboron, for example in the presence of potassium acetate, to giveN-(2-methoxy-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)phenyl)methanesulfonamide (Intermediate 2):
Intermediate 2; and (c) reacting 2-(l-(4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-!-yl)ethyl)-5-fluoro-3 (3-fluorophenyl)-4H-chromen-4-one ·
with intermediate 2 in the presence of a base (such as, for example, sodium carbonate) to give the desired compound N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromcn-2-yl) ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide;
(d) optionally convertingN-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4Hchromen-2-yl) ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide to a pharmaceutically acceptable sait thereof or prodrug thereof.
Yet another embodiment relates to a process for préparation of a compound of formula (Al):
The process comprises the steps of:
(a) subjecting (R)-5-fluoro-3-(3-fluorophenyl)-2-(l -hydroxyethyl)-4H-chromen-4-one:
to a Mitsunobu reaction with 3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-4-amine:
I 12’
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(for example, in the presence of triphenylphosphine and diisopropylazoaicarboxylate) to give (S)-2(l-(4-amino-3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-1-yl)eihyi)-5-fluoiO-3-(3fluorophenyl)-4H-chromen-4-one (Intermediate 3):· .
F
Intermediate 3;
(b) reducing Intermediate 3, for example with a reducing agent such as Raney Ni, to give (S)-2-(l-(4-amino-3-(3-amino-4-methoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-i-yl)ethyl)-5fluoro-3-(3-fluorophenyl)-4H-chromen-4-one (Intermediate 4):
Intermediate 4;
(c) treating Intermediate 4 with methanesulphonyl chloride to give the desired compound of the formula (Al); and (d) optionally converting compound (Al) to a pharmaceutically acceptable sait thereof 15 or prodrug thereof.
-9t · 1
Yet another embodiment are intermediatcs uSelul for preparing the compounds of the présent invention such as (S)-2-(l-(4-amino-3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3.4d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, (S)-2-(l-(4-amino-3(3-amino-4-methoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3fluorophenyl)-4H-chromen-4-one, and salts thereof.
Yet another embodiment of the présent invention is a method of inhibiting PI3KS and ΡΙ3Κγ·ίη a patient comprising administering to the patient an· effective amount of ai least one compound ofthe présent invention.
Yet another embodiment of the présent invention is a method of inhibiting Ρ13Κδ in a patient comprising administering to the patient an effective amount of at least one of (R.)-N-(5-(4amino-1 -( 1 -(5 -fluoro-3 -(3 -fluoropheny l)-4-oxo-4H-chromen-2-yl) ethyl)-1 H-pyrazolo [3,4d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide (compound A2), a pharmaceutically acceptable sait thereof, or a prodrug thereof.
Yet another embodiment of the présent invention is a method of treating, preventing, and/or inhibiting a PI3K protein kinase mediated disease, disorder or condition (such a proliférative disease or disorder, e.g., cancer) in a patient comprising administering to the patient an effective amount of at least one compound of the présent invention.
Yet another embodiment of the présent invention is a method for inhibiting PI3K, in particular ΡΙ3Κδ and ΡΙ3Κγ, in a patient comprising administering to the patient an effective amount of at least one compound of the présent invention.
Yet another embodiment of the présent invention is a method for treating an inflammatory, autoimmune or proliférative disease via modulation of à. protein kinase (such as PI3K5 and ΡΙ3Κγ) comprising administering to a patient in need of such treatment an effective amount of at least one compound of the présent invention. In one embodiment, the compound of the présent invention inhibits both ΡΙ3Κδ and ΡΙ3Κγ. ·
Yet another embodiment of the présent invention is a method for treating an inflammatory, autoimmune or proliférative disease via modulation of a protein kinase (such as ΡΙ3Κδ and ΡΙ3Κγ) by administering to a patient in need of such treatment an effective amount of at least one compound of the présent invention, in combination (simultaneously or sequentially) with at least one other anti-inflammatory, immunomodulator or anti-cancer agent, or a combination
-1018327 thereof. In one embodiment, the compound of the présent invention inhibits both PI3KÔ and ΡΙ3Κγ.
The compounds of the présent invention are useful in the treatment of a variety of cancers, including, but not limited to:
carcinoma, including, but not limited to, that.of the bladder, breast, colon, kidney. liver, lung, including small cell lung cancer, esophagus, gall bladder, ovary, pancréas, stomâch, cervix, thyroid, prostate, and skin, including squamous cell carcinoma;
• hematopoietic tumors of lymphoid lineage, including, but not limited to,-leukernia, acute t lymphocytic leukernia, acute lymphoblastic leukernia, B-cell lymphoma, T-cell lymphoma,
Hodgkin's lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma and Burkett's lymphoma;
hematopoietic tumors of myeloid lineage, including, but not limited to, acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukernia;
tumors of mesenchymal origin, including, but not limited to, fibrosarcoma and 15 rhabdomyosarcoma;
tumors of the central and peripheral nervous system, including, but not limited to, astrocytoma, neuroblastoma, glioma and schwannomas; and other tumors, including, but not limited to, melanoma, seminoma, tcratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and 20 Kaposi's sarcoma.
’ In one embodiment, an effective amount of a compound of the présent invention is administered
-f to treat a leukernia, acute lymphocytic leukernia, acute lymphoblastic leukernia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin’s lymphoma, hairy· cell lymphoma, Burkett's lymphoma, acute and chronic myelogenous leukemias, myelodysplastic 25 syndrome or promyelocytic leukernia.
Due to the key rôle of protein kinases in the régulation of cellular prolifération in general, the compounds of the présent invention may act as réversible cytostatic agents, and may therefore be useful in the treatment of any disease process which features abnormal cellular prolifération,
-11 18327 such as. e.g., benign prostatic hyperplasia, familial adenomatosis polyposis. neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomcrulonephriiis, restenosis following angioplasty or vascular surgery, hypertrophie scar formation, inflammatory bowel disease, transplantation rejection, endotoxic shock, and fungal infections.
The compounds of the présent invention as modulators of apoptosis are useful in the .treatment of cancer (including, but riot limited to, those types mentioned herein above), viral infections (including, but not limited to, herpes virus, poxvirus, Epstein-Barr virus, Sindbis virus· and adenovirus), autoimmune diseases (including, but not limited to, systemie lupus, erythematosus; autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel . 10 disease, and autoimmune diabètes melli-us), neurodegenerative disorders (including, but not limited to, Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophie latéral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebéllar degeneration), myelodysplastic syndromes, aplastic anémia, ischémie injury associated with myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases, haematological discases (including, but not limited to, chronic anémia and aplastic anémia), degenerative diseases of the musculoskeletal System (including, but not limited to, osteoporosis and arthritis) aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases and cancer pain. The compounds of the présent invention are also .· useful in the prévention, inhibition, or suppression of AIDS development in HIV-infected 20 individuals.
The compounds of the présent invention may modulate the level of cellular RNA and DNA . synthesis. The compounds of the présent invention are therefore useful in the treatment of viral infections, including, but not limited to, HIV, human papilloma virus, herpes virus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus.
• *» i The compounds of the présent invention are useful in the chemoprevention of cancer, h · Chemoprevention is defined herein as inhibiting the development of invasive cancer'by either blocking the initiating mutagenic event or by blocking the progression of pre-malignant cells that hâve already suffered an insult or inhibiting tumor relapse. The compounds of the présent. invention are also useful in inhibiting tumor angiogenesis and metastasis. One embodiment of the présent invention is a method of inhibiting tumor angiogenesis or metastasis in a patient in need thereof by administering an effective amount of one or more compounds of the présent invention.
-12' ,ΛΚ·'Γ- .
Another embodiment of the présent invention is a method of treating an immune system-relatcd disease or immune disorder (e.g., an autoimmune disease), a disease or disorder involving inflammation (e.g., asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, glomeruloncphritis, neuroinflammatory diseases, multiple sclerosis, uveitis and disorders of the immune System), cancer or other proliférative disease, a hepatic disease or disorder, a rénal disease or disorder. The method includes administering an effective amount of one or more compounds of the présent invention. ...
• Examples of immune disorders include, but are not limited to, psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, asthma, inflammatory musc'e disease, allergie rhinitis, vaginitis, interstitial eystitis, scleroderma, osteoporosis, eczema, allogeneic or xenogeneic transplantation (organ, bone marrow, stem celis and other cells and tissues) graft rejection, graft-versus-host disease, lupus erythematosus, inflammatory disease, type T diabètes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis), myasthenia gravis, autoimmune haemolytic anémia, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, allergie conjunctivitis and atopie dermatitis.
In one embodiment, the compounds described herein are useful as immunosuppresants to prevent transplant graft rejections, allogeneic or xenogeneic transplantation rejection (organ, bone marrow, stem cells, other cells and tissues), and graft - versus - host disease. In one particular embodiment, transplant graft rejections resuit from tissue or organ transplants, in further embodiments, the graft-versus-host disease results from bone marrow or stem cell transplantation. One embodiment of the présent invention is a method of preventing or decreasing the risk of transplant graft rejection, allogeneic or xenogeneic transplantation rejection (organ, bone marrow, stem cells, other cells and tissues) or graft - versus - host discase comprising administering an effective amount of one or more compounds of the présent invention. «
The compounds of the présent invention are also useful in combination (administered together or sequentially) with known anti-cancer treatments,· such as, for example, radiation therapy or with cytostatic or cytotoxic or anticancer agents, such as, for example, DNA interactive agents, such as cisplatin or doxorubicin; topoisomerase II inhibitors, such as etoposide; topoisomerasc 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
-1318327 agents, such as tamoxifen; thymidilate synthase inhibîtors, such as 5-fluorouracil; and antimetabolites, such as methotrexate, other tyrosine kinase inhibîtors, such as Iressa and OSI-774; angiogenesis inhibîtors; EGF inhibîtors; VEGF inhibîtors; CDK inhibîtors; SRC inhibîtors; cKit inhibîtors; Herl/2 inhibîtors and monoclonal antibodies directcd agair.st growth factor .5 . - receptors such as erbitux (EGF) and herceptin (Her2); BTK inhibitor, such as ibrutinib; and other protein kinase modulators, and any combination thereof.
The compounds of the présent invention are also useful in combination (administered together or sequentially) with one or more steroidal anti-inflammatory drugs. non-steroidai antiinflammatory; drugs (NSAIDs) and immune sélective anti-inflammatory dérivatives (ImSAIDs), 10 and any combination thereof.
The présent invention further provides a pharmaceutical composition comprising one or more compounds of the présent invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise one or more of the active ingrédients identified above, such as other anti-cancer agents.
Yet another embodiment is a method of treating leukemia in a patient in need thereof comprising administering a therapeutically effective amount of a compound of the présent invention. In one embodiment, the leukemia is selected from chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), acute myeloid leukemia (AML), multiple myeloma (MM), small lymphocytic lymphoma (SLL), and indolent non20 Hodgkin’s lymphoma (I-NHL).
Yet another embodiment of the présent invention is a method of treating an autoimmune disorder in a patient in need thereof comprising administering a therapeutically effective amount of a compound of the présent invention. In one embodiment, the autoimmune disorder is selected from asthma, COPD, rheumatoid arthritis, psoriasis, lupus and experimental 25 autoimmune encephalomyelitis (EAE).
Yet another embodiment of the présent invention is a method of treating allergie rhinitis in a. patient in need thereof comprising administering to the patient a therapeutically effective amount of a compound of the présent invention.
-141 ' • In any of the aforementioned methods, the compound(s) of the présent invention and optional additional active agents can be administered in the form of a pharmaceutical composition as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a bar graph of the neutrophil count in bronchoalveolar lavage fiuid (B A LF) from female Wistar rats treated with 10 mg/kg of Compound Al (po) according to the lipopolysaccharide induced pulmonary neutrophilia model described in Assay 7.
Figure 2 depicts a bar graph of the neutrophil count in peritoneal lavage fiuid from Wistar rats treated with 1, 3, and 10 mg/kg of Compound Al (po) according to the lipopolvsaccharideinduced rat air pouch inflammation model described in Assay 8.
Figures 3A and 3B depict the line and bar graphs of individual clinical scores for hind and fore paws and AUC for clinical score, respectively, in Wistar rats with collagen induced arthritis treated with a control or 10 mg/kg/QD of Compound Al according to the procedure in Assay 11.
Figures 3C and 3D depict line and bar graphs of individual clinical scores for hind and fore paws, respectively, in Wistar rats with collagen induced arthritis treated with vehicle or 10 mg/kg/QD of Compound Al according to the procedure in Assay 11.
Figures 4A and 4B depict the line and bar graphs of volume for hind paws and AUC of paw volume, respectively, in Wistar rats with collagen induced arthritis treated with vehicle or 10 mg/kg/QD of Compound Al according to the procedure in Assay 11.
Figures 4C and 4D depict line and bar graphs of ankle diameter for hind paws and AUC of ankle diameter, respectively, in Wistar rats with collagen induced arthritis treated with vehicle or 10 mg/kg/QD of Compound Al according to the procedure in Assay 11.
Figures 4E to 4G depict bar graphs of histopathological score for inhibition of inflammation, cartilage and pannus, respectively, of ail the hind and fore paws in Wistar rats with collagen induced arthritis treated with vehicle or 10 mg/kg/QD of Compound Al according to the procedure in Assay 11.
-15' ·> I
Figure 4Hdepicts a bar graph of total histopathological score of ali the hind and fore paws in Wistar rats with collagen induced arthritis treated with vehicle or 10 mg/kg/QD of Compound Al according to the procedure in Assay 11.
Figure 5 depicts a bar graph of the percentage incidence of arthritis in Wistar rats with collagen induced arthritis treated with vehicle or 10 mg/kg/QD of Compound Al according to the procedure in Assay 11. · ; «
Figures 6A and 6B depict bar graphs showing the antipsoratic effect of Compound Al (3,10,30 mg/kg) on imiquimod induced psoriasis in Balb/c mice according to the procedure in Assay 13.
ï*
DETAILED DESCRIPTION OF THE INVENTION
As used herein the following définitions shall apply unless otherwise ind.cated. Further many of the groups defined herein can be optionally substituted. The listing of substituents in the définition is exemplary and is not to be construed to limit the substituents defined elsewhere in the spécification.
Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R)- or (S)-. Unless otherwise specified, the présent chemical entities, pharmaceutical compositions and methods are meant to include ail such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. For the instance, non-limiting example of intermediate mixtures include a mixture of R: S or S: R isomers in a ratio of 10:90, 13:87, 17:83, 20:80, or 22:78. Optically active (R)- and (S)isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of géométrie asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z géométrie isomers.
The term tautomers refers to compounds, which are characîerized by relatively easy interconversion of isomeric forms in equilibrium. These isomers are intended to be covered by this invention. Tautomers are structurally distinct isomers that interconvert by tautomerization. Tautomerization is a form of isomerization and includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. Prototropic tautomerization or proton-shift tautomerization involves the migration of a proton
-1618327 accompanied by changes in bond order, ofien the interchange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g. in solution), a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization. A spécifie example of keto-enol tautomerization is the interconversion of pentane-2,4-dione and 45 hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A spécifie example of phenol-keto tautomerization is the interconversicn of pyridin-4-ol and pyridin-4(lH)-one tautomers.......> · ·· · · ·
The term prodrug refers to a compound, which is an inactive precursor of a cor ipound that is converted into its active form in the body by normal metabolic processes. Prodrug design is · 10 .. discussed generally in Hardma, et al. (Eds.), Goodman and Gilman's The Phannacological 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 Sériés, 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 15 through hydrolysis of, for example, an ester or amide linkage, thereby introducing or exposing a functional group on the résultant product. The prodrugs can be designed to react with an .1 endogenous compound to form a water-soluble conjugate that further enhances the pharmacological properties of the compound, for example, increased circulatory half-life. Altematively, prodrugs can be designed to undergo covalent modification on a functional group 20 with, for example, glucuronic acid, sulphate, glutathione, amino acids, or acetate. The resulting conjugate can be inactivated and excreted in the urine, or rendered more potent t han the parent compound. High molecular weight conjugates also can be excreted into the bile, subjected to enzymatic cleavage, and reléased back into the circulation, thereby effectively increasing the biological half-life of the originally administered compound.
The term ester refers to a compound, which is formed by reaction between an acid and an alcohol with élimination of water. An ester can be represented by the general formula RCOOR' (where R is a drug and R’ is a chemical group).
These prodrugs and esters are intended to be covered within the scope of this invention.
Additionally the instant invention also includes the compounds which differ only in the 30 presence of one or more isotopically enriched atoms for example replacement of hydrogen with deuterium or tritium, or the replacement of a carbon by l3C- or l4C-enriched carbon.
-1718327 . The compounds of the présent invention may also contain unnatural proportions of atornic isotopes at one or more of atoms that constitutc such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 • (l25I) or carbon-14 (14C). Ail isotopic variations of the compounds of the présent invention.
-whether radioactive or not, are encompassed within the scope of the présent invention. ·
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 Ν,Ν'.-diacetylethylenediamine, glucamine, triethylamine, choline, hydtoxide, . dicyclohexylamine, metformin, benzylamine, trialkylamine, and thiamine; chiral bases such as 10 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 Mel and (Me^SO.j; nonnatural amino acids such as D-isomers or substituted amino acids; guanidine; and substituted 15 guanidine wherein the substituents are selected from nitro, amino, alkyl, alkenyl, alkynyl, • ammonium or substituted ammonium salts and aluminum salts. Salts may include acid addition salts where appropriate which may be sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides, acétates, tartrates, maleates, citrates, fumarates, succinates, palmoates, methanesulphonates, benzoates, salicylates, benzenesulfonates, ascorbates, glycérophosphates, 20 and ketoglutarates.
When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, ail combinations and subcombinations of ranges and spécifie 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 25 . approximation within experimental variability (or within statistical experimental error), and thus . · ihe number or numerical range may vary from, for example, between 1% and 15% of the· stated number or numerical range. The term comprising (and related terras such as comprise or comprises or having or including) includes those embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, that 30 consist of ’ or consist essentially of ’ the described features.
-1818327
The. following abbreviations and terms hâve the indicated meanings throughout: PI3-K Phosphoinositide 3-kinase; PI = phosphatidylinositol; AIDS = Acquired Immuno Deficiency Syndrome; HIV = Human Immunodeficiency Virus; Mef = Methyl lodidc; ND: Not determined.
Abbreviations used herein hâve their conventional meaning within the chemical and bioiogica!
arts.
The terni cell prolifération refers to a phenomenon by which the cell number has changea as a resuit of division. This term also encompasses cell growth by which the cell morphology has changed (e.g., increased in size) consistent with a proliférative signal.
The terms co-administration, administered in combination with, and their grammatical 10 équivalents, as used herein, encompass administration of two or more agents to an animal so that both agents and/or their métabolites are présent in the animal at the same time. Coadministration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are présent.
The term effective amount or therapeutically effective amount refers to that amount of a compound described herein that is sufficient to effect the intended application including but not limited to disease treatment, as defined below. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and âge cf 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. réduction of platelet adhesion and/or cell migration. The spécifie 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.
As used herein, treatment, treating, or ameliorating are used interchangeably. These terms refers to an approach for obtaining bénéficiai or desired results including but, not limited to, therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant éradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved 30 with the éradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient,
-1918327 notwithstanding that the patient may still be afflicted with the underlying discrder. For prophylactic benefît, 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 hâve been made.
A therapeutic effect, as that term is used herein, encompasses a therapeutic benefît and/or a prophylactic benefît 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.
The term subject or “patient” refers to an animal (e.g., a dog, cat, horse, or pig), such as a · mammal, for example a human. The methods described herein can be useful in both human therapeutics and veterinary applications. In some embodiments, the patient is a mammal. In a preferred embodiment, the patient is human.
Radiation therapy means exposing a patient, using routine .methods and compositions known to the practitioner, to radiation emitters such as alpha-particle emitting radionuclides (e.g., actinium and thorium radionuclides), low linear energy transfer (LET) radiation emitters (i.e. beta emitters), conversion électron emitters (e.g. strontium-89 and samarium- 153-EDTMP), or high-energy radiation, including, without limitation, x-rays, gamma rays, and neutrons.
Signal transduction is a process during which stimulatory cr inhibitory signais are transmitted into and within a cell to elicit an intracellular response. A modulator of a signal transduction pathway refers to a compound which modulâtes the activity of one or more cellular proteins mapped to the same spécifie signal transduction pathway. A modulator may augment (agonist) or suppress (antagonist) the activity of a signalling molécule. ·
The term sélective inhibition or selectively inhibit as applied to a biologically active agent refers to the agent's ability to selectively reduce the target signalling activity as compared to offtarget signalling activity, via direct or indirect interaction with the target.
The term- pharmaceutically acceptable carrier or pharmaceutically acceptable excipient includes, but is not limited to, any and ail solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonie and absorption delaying agents, one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices,
-2018327 colorants/flavouring, carriers, excipients, buffers, stabilizers, solubilizers, and combinations •thereof. Except insofar as any conventional media or agent is incompatible with the active ingrédient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingrédients can also be incorporated into the compositions.
In other embodiments, the compounds of the présent invention selectively inhibit one or more · • members of type I or class I phosphatidylinositol·. 3-kinases (PI3-kinase) with an IC50 value, of about IOO nM or less, about 50 nM or less, about IO 11M or less, about 5 nM or less, about 100 . ·. · .pM or less, about 10 pM or less, or about 1 pM or less as measured in an in vitro kinase assay.
In yet another aspect, an inhibitor that selectively inhibits one or mo.e members of type I PI310 kinases, or an inhibitor that selectively inhibits one or more type I PI3-kinase mediated signalling pathways, altemativeïy can be understood to refer to a compound that exhibits a 50% inhibitory concentration (IC50) with respect to a given type I PI3-kinise, that is at least 10-fold lower, at least 20-fold lower, at least 50-fold lower, at least 100-fold lower, or at least 1000-fold lower than the inhibitor's IC50 with respect to the rest of the other type IPI3 -kinases.
As used herein, the term “dual PI3-kinase δ / γ inhibitor and “dual PI3-kinase δ / γ sélective inhibitor” refers to a compound that inhibits the activity of both the PI3-kinase δ and γ isozyme more effectively than other isozymes of the PI3K. family. A dual PI3-kinase δ / γ inhibitor is therefore more sélective for PI3-kinase δ and γ than conventional PI3K inhibitors such as CAL130, wortmannin and LY294002, which are nonselective PI3K inhibitors.
Inhibition of PI3-kinase δ and γ may be of therapeutic benefit in treatment of various conditions,
e.g., conditions characterized by an inflammatory response including, but not limited to, autoimmune diseases, allergie diseases, and arthritic diseases. Importantly, inhibition of PBkinase δ and γ function does not appear to affect biological functions such as viability and fertility.
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 resuit from infection with pathogenic organisms and viruses, noninfectious means such as trauma or reperfusion following
-21 18327 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 spécifie defence System as well as conditions associated with reactions of the non-specific dcfence 5 System.
The therapeutic methods of the invention include methods for the amelioration of conditions associated with inflammatory cell activation. Inflammatory cell activation refers to the induction by a stimulus (including but not limited to, cytokines, antigens or auto-antibodies) of a proliférative cellular response, the production of soluble mediators (including but not limited r 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 molécules) in inflammatory cells (including but not limited to monocytes, macrophages. T lymphocytes, B lymphocytes, granulocytes r (polymorphonuclear leukocytes including neutrophils, basophils, and eosinophils) mast cells, 15 dendritic cells, Langerhans cells, and endothélial 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, perpétuation, or exacerbation of an inflammatory condition.
Autoimmune disease as used herein refers to any group of disorders in which tissue injury is I associated with humoral or cell-mediated responses to the body's own constituents.
Transplant rejection as used herein refers-to any immune response dirccted against grafted «
tissue (including organs or cells (e.g., bone marrow), characterized by a loss of fonction of the t
grafted and surrounding tissues, pain, swelling, leukocytosis, and thrombocytopenia).
ï
Allergie disease as used herein refers to any symptoms, tissue damage, or loss of tissue fonction resulting from allergy.
i 25 Arthritic disease as used herein refers to any disease that is characterized by inflammatory lésions of the joints attributable to a variety of étiologies.
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 étiologies.
As previously described, the term “dual PI3-kinase δ / γ sélective inhibitor generally refers to a 30 compound that inhibits the activity of the PI3-kinase δ and γ isozyme more effectively than
PA512496/OA/3162740.1
-2218327 other isozymes of the PI3K family. The relative efficacies of compounds as inhibîtors of an enzyme activity (or other biological activity) can be established by determining the concentrations at which each compound inhibits the activity to a predefined extcnt and then comparing the.results. Typically, the preferred détermination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or IC50. IC50 déterminations can be accomplished using conventîonal techniques known in the art. In general, an IC50 can be determined by measuring the activity· of a given eczj’me in the présence üf a· »·' range of concentrations of the inhibitor under studv. The experimentally obtained values of .
Γ enzyme activity then are plotted against the inhibitor concentrations used. The concentration of the inhibitor that shows 50% enzyme activity (as compared to the activity in the absence of any . inhibitor) is taken as the IC50 value. Analogously, other inhibitory concentrations can bc defined through appropriate déterminations of activity. For example, in some settings it can be désirable to establish a 90% inhibitory concentration, i.e., IC90, etc.
• Accordingly, a dual PI3-kinase δ / γ sélective inhibitor altematively can be understood to refer to a compound that exhibits a 50% inhibitory concentration (IC50) with respect to PI3-kinase δ . and γ, that is at least I0-fold lower, at least 20-fold lower, or at least 30-fold lower than the IC50 value with respect to any or ail of the other class I PI3K family members. In an alternative embodiment of the invention, the term dual PI3-kinase δ / γ sélective inhibitor can be understood to refer to a compound that exhibits an IC50 with respect to PI3-kinase δ and γ that is at least 30-fold lower, at least 50-fold lower, at least 100-fold lower, at least 200-fold lower, or at least 500-fold lower than the IC50 with respect to any or ail of the other PI3K class I family , members. A dual PI3-kinase δ / γ sélective inhibitor is typically administered in an amount such that it selectively inhibits both P13-kinase δ and γ activity, as described above.
In certain embodiments, the compounds of the présent invention exhibit PI3-kinase δ and γ inhibition almost equally (~ l:l) or at a maximum ratio of 1:5, i.e., the compound the of the présent invention exhibit almost equal IC50 values for both PI3-kinase δ and γ enzyr.re , or at most a 3 to 8 fold différence between the two.
The methods of the invention may be applied to cell populations in vivo or ex vivo. In vivo means within a living individual, as within an animal or human or in a subject's body. In this context, the methods of the invention may be used therapeutically or prophylactically in an individual. Ex vivo or “in vitro means outside of a living individual. Examples of ex vivo cell populations include in vitro cell cultures and biological samples including but not limited to
-2318327 • fluid or tissue samples obtained from individuals. Such samples may be obtained by methods known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid. urine, and saliva. Exemplary tissue samples include tumors and biopsies thereof. In this context, the invention may be used for a variety of purposes, including therapeutic and experimental purposes. For example, the invention may be used ex vivo or in vitro to déterminé the optimal schedule and/or dosing of administration of a PI3-kinasc δ sélective inhibitor for a given indication, cell type, individual, and other parameters. Information ..jlcaned ficni such ose ηκ y · be used for experimental or diagnostic purposes or in the clinie to set protocols for.in vivo treatment. Other ex vivo uses for which the invention may be suited are described below or will become apparent to those skilled in the art.
The compounds of the présent invention can be prepared by methods known in the art, such as those described in International Publication Nos. WO 2011/055215, WO 2012/151525; and WO 2013/164801, each of which is hereby incorporated by reference in its entirety.
Pharmaceutical Compositions
The présent invention also provides a pharmaceutical composition comprising one or more compounds of the présent invention and one or more pharmaceutically acceptable carriers or excipients. In one embodiment, the pharmaceutical composition includes a therapeutically effective amount of one or more compounds of the présent invention. The pharmaceutical composition may include one or more additional active ingrédients as described herein.
The pharmaceutical carriers and/or excipients may be selected from, for example, diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants, flavourings, buffers, stabilizers, solubilizers, and combinations thereof.
In one embodiment, the pharmaceutical compositions described herein contain from about 0.1 mg to about 1,000 mg, such as from about 1 mg to about 1,000 mg, from about 20 mg to about 800 mg, from about 50 mg to about 600 mg or from about 50 mg to about 600 mg of one or more compounds of the présent invention. In another embodiment, the pharmaceutical compositions described herein contain from about 100 mg to about 400 mg of-one or more compounds of the présent invention.
The pharmaceutical compositions of the présent invention can be administered alone or in combination with one or more other active agents. Where desired, the subject compounds and
-2418327 other agent(s) may be mixed into a préparation or both components may be formulated into separate préparations to use them in combination separately or at the same time.
The compounds and pharmaceutical compositions of the présent 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, intravascularallv. intraperitoneal ly 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 10 comea), sublingually, anally, rectally, vaginally, or by surgical implantation (e.g., embcdded under the splcnic capsule, brain, or in the comea). · :
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, 15 sachets, cachets, gelatins, papers, tablets, suppositories, pellets, pills, troches, and lozenges. The type of packaging will generally dépend on the desired·route of administration. Implantable sustained release formulations are also contemplated, as are transdermal formulations.
Methods of Treatment
The amount of the compound to be administered is dépendent on the mammal being treated, the 20 severity of the disorder or condition, the rate of administration, the disposition of the compound . and the discrétion of the prescribing physician. However, an effective dosage is in the range of . from about 0.001 to about 100 mg/kg body weight per day, preferably from about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to from about 0.05 to about 7 g/day, preferably from about 0.05 to about 2.5 g/day An effective amount of a compound of the invention may be administered in either single or multiple doses (e.g., twice or three times a day).
I · · ,
The compounds of the présent invention may be used in combination with one or more of anticancer agents (e.g., chemotherapeutic agents), therapeutic antibodies, and radiation treatment.
The compounds of the invention may be formulated or administered in conjunction with other : 30 agents that act to relieve the symptoms of inflammatory conditions such as encephalcmyelitis,
-2518327 • · asthma, and. the other diseases described herein. These agents include non-steroîdal anti inflammatory drugs (NSAIDs).
EXAMPLES
- The-examples- and préparations provided below further illusirate and exemplify the comrounds ·.
of thé présent invention and methods of preparing such compounds. It is to be understood that· the scope ofthe présent invention is not limited in any way by the scope of the following · examples and préparations. In the following examples molécules with a single chiral center, · unless otherwise noted, exist as a racemic mixture. Those molecu es with two or more chiral centers, unless otherwise noted, exist as a racemic mixture of diastereomers. Single enantiomers/diastereomers may be obtained by methods known to those skilled in the art.
The intermediates described herein may be prepared by the methods described in International Publication Nos. WO 11/055215 and WO 12/151525, both of which are hereby incorporated by reference.
Intermediate 1: N-(5-bromo-2-methoxyphenyl)mctlianesulfonamidc: To a solution of 5bromo-2-methoxyaniline(1.00 g, 4.94 mmol) in dichloromethane (10 ml), pyridine (0.800 ml, 9.89 mmol) was added and cooled to 0°C. Methane sulphonyl chioride (0.40 ml, 5.19 mmol) was added and stirred for 30 min. The reaction mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulphate and concentrated ur.der reduced pressure. The crude product was chromatographed with ethyl aceta’e : petroleum ether to afford the title compound as a reddish solid (1.20 g, 87%).
Intermediate 2: N-(2-methoxy-5-(4,4,5,5-tetramethyl-13,2-dioxaborolan-2yljphenyl)methanesulfonamide: Potassium acetate (0.841 g, 8.57 mmol) and bis(pinacolato)diboron (1.190 g, 4.71 mmol) were added to a soluti m of intermediate 1 (1.20 g,·
4.28 mmol) in dioxane (17.5 ml) and the solution was degassed for 30 min.[l,l’-· ·
Bis(diphenylphosphino)ferrocene]dichloro palladium(II).CH2C12 (0.104 g, Ό. 128 mmol) was · .· added- undennitrogen atmosphère and heated to 80°C. After 2h the reaction mixture was filtercd through celite and concentrated. The crude product was purified by column chromatography with ethyl acetate : petroleum ether to afford the title compound as a yellow solid (1.00 g,
71%).’H-NMR (δ ppm, CDCI3, 400 MHz): 7. 91 (d, J = 1.2Hz, 1H), 7. 62 (dd, ./= 8.1, 1.2Hz,
1H), 6. 92 (d, J= 8.1Hz, 1H), 6.73 (s, 1H), 3.91 (s, 3H), 2.98 (s, 3H), 1.32 (s, 12H).
40.1
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Intermediate 3: (S)-2-(l-(4-amino-3-(4-methoxy-3-nitrophenyl)-ni-py;razo!o[3,4d]pyrimidin-l-yl)cthyl)-5-fluoro-3-(3-fiuoroj)lienyl)-4H-chromen--,-one: (S)-2-(l-(4-amino3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3fluorophenyl)-4H-chromen-4-one: To a solution of (R)-5-fluoro-3-(3-fluorophenyl)-2-(ihydroxyethyl)-4H-chromen-4-one (0.500 g, 1.64 minol) in nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-4-amine triphenylphesphine (0.649 g, 2.17 mmol) v/eie' (0.564 ’added
THF g, folle
3-(4-mctho?ry-3mtnoi) and the· addition of·· ' diisopropylazodicarboxylate (0.50 ml. 2.47 nimol). ((R)-5-fluoro-3-(3-iluorophenyl)-2-(l‘ hÿdroxyethyl)-4H-chromen-4-one can be prepared as described for Intermediates 23. 25, and 26 ’’ in International Publication No. WO 2012/0151525.). After 4h at room température, the mixture !
’ was concentrated and the residue was purified by column chromatography with ethyl acetate :
petroleum ether to afford the title compound as a brown solid (0.270 g, 29%). ’H-NMR (δ ppm, · : DMSO-d6, 400 MHz): 8.04 (s, 1H), 7.83 (m, 1H), 7.63-7.50 (m, 3H), 7.29 (m, 2H), 7.06 (dt, J = 8.7,2.2Hz, 1H), 6.94 (m, 2H), 6.75 (dd, J = 8.1,2.1Hz, 1H), 5.95 (q, J = 7.0Hz, 1H), 4.98 (s, 15 2H), 3.81 (s, 3H), 1.86 (d, J = 7.0 Hz, 3H).
Intermcdiate 4: (S)-2-(l-(4-amino-3-(3-amino-4-methoxyphcnyl)-lH-pyrazolo[3,4d]pyrimidin-l-yl)ethyi)-5-fluoro-3-(3-fluorophenyl)-4H-cliromen-4-onc (S)-2-(l-(4-amino3-(3-amino-4-methoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3fluorophenyl)-4H-chromen-4-one : To a solution of Intermediate 3 (0.260 g, 0.455 mmol) in ’ 20 éthanol (5 ml), Raney Ni (0.130 g) was added and hydrogeneated at 20psi at 50°C for 24h. The reaction mixture was passed through celitepad and concentrated to afford the title compound as v · a brown solid (0.150 g, 60%). Mass : 540.8 (M*).
I ’· · { . Example A
N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromcri-2-yl)ethyl)-lH’ ' pyrazolo[3,4-d]pynmidin-3-yl)-2-methoxyphenyl)mctliàr.esur‘onsrnîde . To, a solution of 2-(l-(4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-flucro-3-(3·. ,·, fluorophenyl)-4H-chromen-4-one (0.200 g, 0.366 mmol) in DME (2.1 ml) and water (0.67 ml),
.. intermediate 2 (0.179 g, 0.550 mmol) and sodium carbonate (0.116 g, 1.10 mmol) were added and the System was degassed for 30 min. (2-(l-(4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidinl-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one can be prepared as described for Intermediates 23, 25, and 26 in International Publication No. WO 2012/0151525).
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Bis(diphenylphosphino) ferrocene]dichloropalladium(n) (0.059 g. 0.075 mmol) was added and kept under microwave irradiation (microwave power 100W, température = 100 °C) for 45 min. The reaction mixture was Celite filtered, concentrated and extracted with ethyl acctate.
The organic layer was dried over sodium sulphate and concentrated under rcduced pressure.
5. The crude prdduct was purified by column chrômatography with methanol: dichloromeîh'ane to afford the title compound as a brown solid (0.080 g, 35%). MP: 216-218 °C. ’l-I-NMR. (δ ppm,
CDClj, 400 MHz): 8.20 (s, 1H), 7.73 (s, 1H), 7.53 (m, 211),-7.31 (i.i-.--7.07-6.73 6H),
- 6.07 (q, J= 6.2 Hz, III), 3.98 (s, 3H), 3.14 (s, 3H), 2.01 (d, J= 6.0Hz, 3H).
Example Al and A2
Method A (S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yJ)ethyl)-lHpyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide and (R)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-lH» * pyrazolo[3,4-d]pyrimîdin-3-yI)-2-methoxyphenyl)methanesulfonamide
• «i : »*· * ?· ; ' ··»·: · -'JiHié two enantiomerically piire isomers were separated by préparative SFC (supercritical fluid) conditions , from N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2yl)ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide (0.500 g) 20 on a CHIRALPAK AS-H column (250 x 30 mm; 5pm) using methanol : CO2 (55:45) as the mobile phase at a flow rate of 80g / min.
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Example Al (S-isomer): Brown solid (0.247 g). Euantiomeric excess: 97.4%. Rétention time: 2.14 min. Mass: 619.1 (M++l). MP: 156-158° C.
Example A2 (R-isomer): Brown solid (0.182 g). Enantiomeric excess: 99.3%. Rétention t: 3.43 min.. Mass: 619.1 (M++l). MP: 168-171° C.
< Method Al ,(S)-N-(5r(4-amino-l-(l-(5-fluoro-3-(3-fluorophcnyl)-4-oxo-4H-cîiromcn-2-yi)cthvî)-2ïîpyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphcnyl)nieÎhanesu!ibnami(îe and (R)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-f uorophenÿl)-4-oxo-4ÎI-chron)en-2-yl)cthy ) lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyJ)mc»hanesuJfo!i2midc
The two enantiomerically pure isomers were separated by préparative SFC (supercritical fluid) conditions from N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2yl)ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl) methanesulfonamide (15.0 g) on a CHIRALPAK AS-H column (250 x 20 mm; 5pm) using methar.ol : CO2 (45:55) as the 15 mobile phase at a fiow rate of 120g / min.
Example Al (S-isomer): Enantiomeric excess: 100 %. Rétention time: 2.21 min. Mass: 6!9.1 (M++l). MP: 175-178° C Spécifie optical rotation (C=l in chloroform, at 25°C) : [a]o := + u 147.16. ' ·
Example A2 (R-isomer): Enantiomeric excess: 99.3%. Rétention t: 3.72 min. Mass: 619.1 20 (M’+l). MP: 154-157° C. Spécifie optical rotation (C=l in chloroform, at 25°C) : [a]o = 159.54.
Method B
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Exampk Λ1 (S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fiuorophenyl)-4-oxo-4H-chiOmen-2-yl)ethyl)-lHpyrazolo[3,4-d]pyrimidin-3-yl)-2-inethoxyphenyl)met’ianesulfonamide
To a solution of Intermediate 4 (0.500 g, 0.923 mmol) in dichloromethane (5 mi) ccoled to 0°C, ' pyridine (0.200 ml, 1.84 mmol) was added and stirrcd for 10 min. Methanesulphonyl chlcride. · · · (0.100 mi, 0.923 mmol) was added stirred for 30 min.' The réaction mixture was quenched Vvith ' '' water, extracted with dichloromethane and dried over sodium sulpiiate. The crude product was ·«. . .. column chjjomatographed with methanol : dichloromethane to afford the title compound as an . · off-white solid (0.240 g, 42%). MP: 211-213°C. ’H-NMR (ô ppm, DMSO-d6,400 MHz): 9.15, 10 (s, 1H), 8.06 (s, 1H), 7.83 (m, 1H), 7.49 (m, 4H), 7.28;(m, 4H), 7.08 (dt, J= 8.6,1.7 Hz, 1H),
6.92 (s, 2H), 5.98 (q, J= 6.9 Hz, 1H), 3.88 (s, 3H), 2.99 (s, 3H). 1.88 (d, J= 7.0 Hz, 3H).. . Enantiomeric excess: 85.4% as determined by HPLC on a chiralpak AS-3R coiumn, enriched in the fast eluting isomer (rétention time = 7.46 min.).
Metabolic Stability
Metabolic stability studies were conducted on Compounds A, Al, and A2 as well as Example 128 of WO 2012/151525 using mouse, rat, dog, monkey, and human liver microsomes. The protocol for the studies with mouse, rat, and human liver microsomes (ail from BD Gentest, USA) is provided below. 0.4 mg protein was preincubated with 2mM NADPH (cofactor) in phosphate buffer (pH~7.4) for 15 minutes at 37° C and then added with ΙμΜ test item and incubated further for 60 minutes in triplicate. The reaction mixture was terminated with methanol containing an internai standard and centrifuged further to analyze the test item remaining ïn the supematant by LC-MS/MS. The percent parent compound remaining was . calculated in comparison with similar samples terminated at 0 minutes. The results are provided in Table 1 below.
. The metabolic stability data for Compound Al indicates that it exhibits a superior pharmacokinetic profile.
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Table l
Compound Metaholie stability in liver nricrosomes
Mouse Rat Dog Monkey Human
Example 128 of WO 2012/151525 85.0 73.3 ' ND ND 70.4.
Compound A 96 91 • 64.3 ?? 3 • 69.7
Compound Al 85.9 94.2 83.5 73.S 95.7
Compound A2 68.9 79.5 52.3. 1.9 66.2
ND- Not Determined
Protein Binding
Below is provided the procedure for measuring plasma protein binding (using an equilibrium dialysis method). 745pL of plasma was transferred into a 2ml micro centrifuge tube. To that 5pL of Compound Al (150μΜ) was added. Samples were mixed in the table top vortexer for 2 minutes. 50pL plasma (n=2) was transferred in a pre-labeled l.5mL micro centrifuge tube treated as 0 hour sample.
The remaining 650pL plasma sample were incubated for 30 minutes at 37°C in a water bath.
After 30 minute incubation, 50pL plasma (n=2) was removed in a pre-labelled 1.5mL micro centrifuge tube treated as 0.5 hour sample. 200pL of the plasma sample (n=2) was transferred into the sample chamber which was indicated by the red ring. The red insert was placed into the base plate and 350pL of buffer was transferred into the buffer chamber. Plates were incubated. at 37°C at approximately 100 RPM on an orbital shaker or 20 RPM on an υρ-and-down shaker . 15, ... - , fo,r 4 hours. 50pL of post dialysis-sample from' the buffer and the plasma chambsrs were transferred into a pre-labelled micro centrifuged tube. '50pL of plasma was adècd to.the buffer samples and an equal volume of buffer (KH2PO4 Buffer pH 7.4) to the collecte·:! plasma samples. 150pL of methanol containing internai standard (Tolbutamide 250ng/ml) was added· to precipitate the protein and release compound. Samples were vortexed for 3 minutes in a table.
top vortexer and centrifuge for 5 minutes at 14,000 RPM. Supematant was subjected to LCMS/MS analysis.
The plasma protein binding data for Compound Al is provided in Table 2 below:
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Table 2
Protein Binding (%)
Mouse Rat Dog Monkcy Human
97.61 99.04 95.85 94.71 97.24
Pharmacokinetics * The oral bioavailability of Compound Al (free base) was evaluated in rats and micc. The 5 protocol for the pharmacokiuetics studies in rat is provided below.
Ail animais were fasted ovemight (12 hours) before dosing and continued till 4.0 hours after administration of test item. Test item formulations were prepared in 2% Tween 80 and’99% media (0.5% Methyl cellulose, 4000cPs, pH 2.2). The blood samples (150 pL from each animal) were collected from the orbital sinus, and placed into a micro centrifuge tube containing 10 disodium EDTA as an anticoagulant. Blood samples were centrifuged immediately with a speed of 1000g for 10 min at 4° C and separated plasma samples were frozen at below -80°C and stored until analysis. The concentrations of test item in ail formulations were analyzed by HPLC. The plasma concentrations of test item in ail samples were analyzed by LC-MS/MS. Pharmacokinetic parameters (Cmax, AUCo.t, Tmax, and t·/,) were estimated by using WinNonlin 15 . software. Results are provided in Table 3 for Compound A, Al, and Example 128 of WO 2012/151525 in rats and Compound Al in mice.
; · ..· ·?? ' · ;
J ·.··;·; r
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Table 3
Compound Units Ex. 128 ofWO 2012/151525 Compound A Compound Al Compound At
Anima! . Rat- Mice
Route Oral . Oral Oral Oral J
Dose mg/kg 10 iO 10 • 10
N 2 n 2. 4 3
Cmax μΜ 0.68 1.02 11.38 3.78
AUCo-t μΜ-hr 2.01 7.95 97.76 7.49
Tmax Hr 0.83 2.67 1.83 0.50
t’/2 Hr 1.56 4.52 2.45 1.45
Compounds A and Al showed superior pharmacokinetic profiles compared to Example 128 of WO 2012/151525. For instance, Compound A showed a ~1.5 fold incrcase in Cniax, -4 fold increase in AUCo-t, and - 2.8 fold increase in t·/, as compared to Example 128 of WO 2012/151525. Compound Al showed a -16 fold increase in Cm3x, 48 fold incrcase in AUCo-t, and -1.6 fold increase in t>/2 as compared to Example 128 of WO 2012/151525.
Biological Assays
The pharmacological properties of the compounds described herein may be eonfirmed by a number of pharmacological assays, as exemplified below,
Assay 1: Fluorescent Détermination of PI3 Kinase Enzvme Activity
Phosphoinositide 3 kinases (PI3K) belong to a class of lipid kinases that play a critical rôle in the régulation of several key cellular processes. The PI3K are capable of phosphorylating the 315 hydroxy position of phosphoinositols thereby generatïng second messengers involved in downstream signalling events. The homogenous time resolved fluorescence (HTRF) assay allows détection of 3,4,5-triphosphate (PIP3) formed as a resuit of phosphorylation of phosphotidylinositol 4,5-biphosphate (PIP2) by PI3K isoforms such as α, β, γ or δ.
-33· .-ζγ,Α. '
PI3K isoform activity for α, β, γ or δ was determined using a PI3K human HTRF™ Ztssay Kit (Millipore, Billerica, MA) with modifications. Ail incubations were C3nied out at room température. 0.5 μΐ of 40X inhibitor (in 100% DMSO) or 100% DMSO were added to each · well of a 384-well white plate (Greiner Bio-One, Monroe, NC) containing 14.5 μΐ IX réaction buffer / PIP2 (10 mM MgCh, 5 mM DTT. 1.38 μΜ PIP2) mix with or without enzyme, followed by 5 μΐ/well of 400 μΜ ATP and incubated for an additional 30 minutes. The reaction ‘was termiriated by adding 5 μΐ/well stop solution (Millipore, Billerica, MA}.· 5 μ! of détection · <· i mix (Millipore, Billerica, MA) was then added to cach.well and incubated for 6-18 houn in the dark. HRTF ratio was measured on a microplate reader (BMG Labtech., Germany; at an excitation wavelength of 337 nm and émission wavelengths of 565 and 615 nm vnth an intégration time of 400 msec counting delay of 50 msec. The results for Compounds A, Al and ;A2 are shown in Table 4 below. Comparative data for Compound Al and Example 128 of.
i <WO2012/151525 are provided in Table 5 below.
Table 4
Compound IC50 («M)
Pi3KÔ Pi3Ka Ρί3Κβ Ρί3Κγ
A 102.8 ND ND 82.94
Al 30.46 >10000 1359 48.72
A2 92.95 ND ND >10 μΜ
ND: N ot Determined
Table 5
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Sclcctivity profile
Assay IC50 (nM) FolJ-Sclcctivity
Compound ΡΙ3Κδ PI3Ky PI3Ka PJ3Kp
Example 128 ofWO 2012/151525 76.01 . 70,70 NC (38.29*) NC (51.04*)
Compound A 102.8 82.94· ND , ND
Compound Al 30.46 48.72'·. >329 (23.02**) >45 (46.8*) ;(IC50= 1359 nM)
Compound A2 92.95 >10009 ND ND
* % inhibition @ ΙμΜ ; ** % inhibition @ 10 uîv Determined ; NC-Not Calcul ated and ND: Not
Assay 2: In Vitro Cell Prolifération Assay in Leukemic Cell Lines
Growth inhibition assays were carried out using 10% FBS supplemented media. Cells were seeded at a concentration of 5000 - 20,000 cells/well in a 96-well plate. Test compounds at a concentration ranging from 0.01 to 10000 nM were added after 24 hours. GroXvth was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylletrazolium bromide (MTT) dye réduction · test at 0 hour (prior to the addition of the test compound) and 72 hours after the addition of test · compound. Absorbance was read on a Fluostar · Optima (BMG Labtech, Germany) at a wavelength of 450 nm. Data were analysed using GraphPad Prism and percent inhibition due to the test compound compared to the control was calculated accordingly.
Compound Al caused a réduction in T-lymphoma (MOLT-4, Jurkat, CCRF-CEM, Hut-78 and HuT-102) cell viability with GI50 values ranging from 2.5-12.8 μΜ for the dose range tested. 1 Additionally, compound Al did not display any apparent cytotoxicity over the 72 hour incubation period. ·<
Assay 3: Inhibition of AKT Phosphorylation in Leukemic Cell Lines
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MOLT-4, Jurkat, CCRF-CEM, and Hur-78 cells were incubated with dcsired concentrations of compound for 48 hours. Cells were lysed and ρΛΚ,Τ determined by Western Blotting. Bar.ds· were quantified using ImageJ and normalized to actin.
<
Compound Al caused a réduction in pAKT expression in T-lyrnphoma (MOLT-4. Jurkat, CCRF-CEM and Hut-78) cell lines with EC50 values ranging from 0.02-1.6 μΜ for the dose range tested.
Assay 4: Inhibition of PI3K δ and γ Signalling in Basophils from Hnman Whole Bloo.! . ·. : ··
PI3K δ and γ signalling in basophils manifested by an alteration of anti-FceRl or fMLP inducedCD63 expression is a useful pharmacodynamie marker determined using the Flow2CAST®’ kit .. (Buhlmann Laboratories, Switzerland). The test procedure involves the following steps:
Mix the anti-coagulated blood sample by inverting the venipuncture tube several times;
Préparé fresh and pyrogen-free 3.5 ml polypropylene or polystyrène tubes suitable for Flow Cytometry measurements;
Add 49 μΐ of patient’s whole blood to each tube;
Add l μΐ of 10% DMSO (background) or test compound (10% DMSO) to the assigned tubes and mix gently. Incubate at room température for 15 minutes;
Pipet 50 μΐ of the Stimulation buffer (background) or anti- FccRI Ab or fMLP to each tube;
Add 100 μΐ of Stimulation Buffer to each tube;
Mix gently. Add 20 μΐ Staining Reagent (1:1 mix of FITC-CD63 and PE-CCR3) to each tube; i
Mix gently, cover the tubes and incubate for 15 minutes at 37°C in a water batb. (using an incubator will take about 10 minutes longer incubation time due to less efficient beat transfer);.
Add 2 ml pre-warmed (18-28° C) Lysing Reagent to each tube, mix gently; >
Incubate for 5 -10 minutes at 18-28° C;
Centrifuge the tubes for 5 minutes at 500 x g ;
Decant the supematant by using blotting paper;
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Vortex gently and acquire the data on the flow cytometcr within the same day.
Percent CD63 positive cells within the gated basophil population were determined in different .treatment groups and normalized to vehiclc control. .
. ·.·· ’t
Compound Al exhibited an EC50 of <30 nM for ’EccRl(PI3K δ) and an IC50 of <70 nM for fMLP ( ΡΙ3Κγ)( n=l).
» · . . 4
Assay 4A: Cellular Activity Demonstrating Selectivity of Compound Al towards PI3K.Deitaand
PI3K Gamma Isoforms .. ·
Assay 4A1: Anti-lgM inauced B-Cell Prolifération (for PI?Κδ Selectivity)
The objective of this study was to assess the inhibitory potential of Compound Al on anti-IgM induced human B-cell prolifération.
Plating and Treatment
Isolated B-cells were re-suspended to 1.0 x 10e cells per ml. 100 μΙ of cell suspension was added to each well of a 96-well plate. Triplicates were maintained.
μΙ of drug dilution was added and mixed well. A DMSO blank and inducer blank were maintained.
The treated plate was incubated for 30 minutes at 37° C, 5% CO2 and then 50·μΙ of 4X inducer was added and mixed by pipetting.
The plate was incubated at 37e C, 5% CO2 for 72 hours.
' ‘ Media was aspirated and 150 μΙ of DMSO was added to dissolve the formazan ’crystals.
Absorbance was read at A56o and A64o nm.
The data demonstrates the inhibitory potential of Compound Al on ΡΙ3Κδ mediated induction of human B-cell prolifération. See, e.g., Baeker et al., Journal of Immunology, 134: 3532-3538, 1985.
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Assay 4A2: LPA Induced AktS473 Phosphorvlclion in 3T3 Fibroblasts (for PI3KB Selectiv<ty)
The objective of this study was to détermine the effect of Compound AJ on ΡΙ3Κβ kinase mediated LPA induced AktS473 phosphorylation in 3T3 fibroblasts.
3T3 cells were treated with desired concentrationsof the test compound for 15 minutes. 1 ml of2X LPA was added such th<t the final concentration was 5 μΜ and incubated for 5 minutes.
Media was discarded and washed with 1 ml of ice-cold IX P35.!
250 μΙ of cell lysis buffer was added and incubated on ice for 30 minutes.
> Samples were centrifuged and supernatant was maintained at-80°.Cuntil analysis.
• 10 Samples were analyzed by Westarn Blotting using pAKT (S473) as the primar·/ and anti-rabbit IgG-HRP as a secondary antibody.
Intensity ofthe bands was determined using ImageJ 1.42q (NIH, USA) and normalized to Actin (loading control). Data was plotted using GraphPad Prism (Version 5.02).
The results demonstrate the selectivity of Compound Al over the beta isoform of PI3K. See 15 Albuquerque et al., J. Biol. Chem., 278, 39830-39838, 2003.
Assay 4A3: c5a Induced AktS473 Phosphorylation in RAW 264.7 Macrophages (for PI3Ky Selectivity)
The objective of this study was to détermine the effect of Compound Al on ΡΙ3Κγ kinase mediated c5a induced AktS473 phosphorylation in RAW 264.7 macrophages;
RAW 264.7 cells were treated with desired concentrations ofthe test compound for 15 minutes. 1 ml of2Xc5a was added such that the final concentration was 50 r:g/ml and ·· incubated for 15 minutes.
Media was discarded and washed with 1 ml of ice-cold IX PBS.
250 μΙ of cell lysis buffer was added and incubated on ice for 30 minutes.
Samples were centrifuged and supernatant was stored at -80° C until analysis
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IgG-HRP as a secondary antibody.
Intensity ofthe bands was determined using ImageJ 1.42q (NIH, USA) and normalized to Actin (loading control). Data was plotted using GraphPad Prism (Version 5.02).
» · -Inhibition of pAktS473, a downstream marker of ΡΙ3Κγ signaîling suggcsts r. roie for
Compound Al in the oncogenic pathways regulated by Akt in c5a induced RAW 264.7 cells. See To et al., Am. J. Respir. Crit. Care Med., 182, 897-904, 2010.
Assay 4A4: PDGF Induced Akt Phosphorylation in 3T3 cells (for PI3K a Selectiv'ty)
The objective of this study was to détermine the effect of Compound Al on PI3Ka kinase mediated AktS473 phosphorylation in PDGF induced 3T3 fibroblasts.
3T3 cells were treated with desired concentrations ofthe test compound for 15 minutes. 1 ml of 2X PDGF was added such that the final concentration was 20 ng/ml and incubated for 10 minutes.
Media was discarded and washed with 1 ml of ice-cold IX PBS.
250 μΙ of cell lysis buffer was added and incubated on ice for 30 minutes.
Samples were centrifuged and supernatant was collected and stored at -80’ C until analysis.
Samples were analyzed by Western Blotting using pAKT (S473) as the primary and anti-rabbit «
IgG-HRP as a secondary antibody.
Intensity ofthe bands was determined using ImageJ 1.42q (NIH, USA) and normalized to Actin « « (loading control). Data was plotted using GraphPad Prism (Version 5.02).
• ** I . *
No inhibition was observed at 10 μΜ of Compound Al, demonstrating the selectivity of Compound Al over the alpha isoform of PI3K. See Albuquerque et al., J. Biol. Chem. 278, 39830-39838,2003.
Table 6 below summarizes the results from Assays 4A1-4A4.
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Table 6
CELLULAR ACTIVITY DEMONSTRATING SELECTIVITY OF COMFOUND Al TOWARDS PI3K δ AND PI3K γ ISOFORMS
Cellular ICJ0 PI3K alpha (PDGF induced pAKT in 3T3 fibroblasts) >10000 nM
Cellular IC<0 PI3K beta (LPA induced pAKT in 3T3 ùbroblasts) 1324 nM
Cellular PI3K delta (anti-IgM induced human B-cell prolifération) 11.03 nM
Cejlular ICJO PI3K gamma (c5a induced pAKT in RAW macrophages) 51.73 nM
Assay 5: Inhibition of Apoptosis in Leukemic Cell Lines · ;
Apoptosis in leukemic cells was determined using an in silu Caspase 3 kit (Millipore, US) as 5 outlined below:
Seed leukemic cells at a density of 1X106 cells/well in a 6 well plate
Add test compound/DMSO at desired concentrations
Incubate the plate for 24 hours at 37° C in 5% CO2 incubator
Collect cells in a 2ml centrifuge tube
Add 1.6 pL of freshly prepared 5X FLICA reagent and mix cells by slightly flicking the tubes
Incubate tubes for 1 hour at 37° C under 5% CO2
Add 2 ml of IX wash buffer to each tube and mix
Centrifuge cells at <400 x g for 5 minutes atroorn température. f • I *“ i . ·
Carefully remove and discard supernatant, and gently vortex cell pcllet to disrupt any cell-to·;
cell dumping.
Re-suspend cell pellet in 300ul of IX wash buffer.
Place 100 pL of each cell suspension into each of two wells of a black microtiter plate. Avoid création of bubbles.
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Read absorbance of each microwell using an excitation waveîength of49G nm and an émission wavelength of 520 nm.
Percent increase in caspase-3 activity manifested by an increase in fluorescence compared to the control blank is to be calculated.
· · . Assay 6A: Cytokine Assav in human PBMC
The objective of this study was to assess the inhibitory potential of Compound Λ1 on antigeninduced cytokine release in human PBMC
Platiug and Treatment t * ·
Heparinized human whole blood was diluted 1:1 with PBS, over laid on leukocyte séparation 10 medium and centrifuged at 400 g for 40 minutes.
Buffy layer was removed and washed with PBS
0.15* 106 of PBMCs were plated in 100 μΐ per well in RPMI media and incubated for 2h.
μΐ of 3X of the compound dilution in media was added and incubated for 15 min.
TNFa - induced with 50 μΐ of LPS in RPMI such that final concentration was lpg/ml. 15 Supematant was collected at 6 hours.
IL-2 - induced with 50 μΐ of PHA in RPMI such that final concentration was 20 pg/ml. Supematant was collected at 24 hours.
IL-4 - induced with 50 μΐ of PHA in RPMI such that final concentration was 20 pg/ml. Supematant was collected at 48 hours.
1- :20- · ELISA was-performed used kits from eBioscience.
EC50 was calculated using GraphPad Prism 5.
EC50 values were calculated from 2-3 independent experiments. Compound Al inhibitcd antigen-induced TNFa, IL-2, and IL-4 with an EC50 of 7.1,9.5, and 3.5 nM, respectively.
Assay 6B: Inhibition of LPS Induced CD 19 or CD45R in Human or Mouse Whole Blood
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The effect of Compound Al on modulating B-ccll reccptor (BCR)-activated prolifération of. human or mouse B-lymphocytes was determined. CD19 is a protein présent on B cells from the earliest recognizable B-lineage cells during development to B-cell blasts but is however lost on maturation to plasma cells. LPS is an endotoxin and a major component of environmental microbes with a potent mitogenic activity on B-cells via the BCR signaling pathway.
Diluted human whole bîood was treated with DMSO or desired concentrations oi* Compound’ Al. Samples were induced with LPS 15 minutes after addition of compound and incubated for 72 hours at 37° C and 5% CO2. Cells positive for CD45 and CD19 were determined by -tlow ·· cytometry and data are expressed as percentage CD19 positive cells in the total population: Treatment with Compound Al resulted in a dose-dependent inhibition of LPS-induced human’ whole blood B-cell prolifération (EC50 =117.7 nM) manifested by a réduction in CD19 expression.
Similar to CD19, CD45R (B220) is expressed on mouse B-lymphocytes throughout their development from early pro-B stages onwards and is down-regulated upon terminal différentiation to plasma cells. Briefly, diluted mouse whole blood was treated with of DMSO or desired concentrations of Compound Al. Samples were induced with LPS 15 minutes after compound addition, and incubated for 72 hours at 37°C and 5% CO2. Cells positive for CD45 and CD45R were determined by flow cytometry. Data are expressed as percentage CD45R positive cells in the total population. Consistent with CD19+ cell prolifération data, treatment. with Compound Al resulted in a dose-dependent inhibition of LPS-induced mouse whole blood B-cell prolifération (EC50 = 128.2 nM) manifested by a réduction in CD45R expression.
Assay 6C: Inhibition of AKT Phosphorylation in Isolated Mouse Splénocytes
The PI3K pathway is regulated downstream by AKT, a serine-threonine kinase tirât modulâtes. several oncogenic processes such as cell prolifération, growth, and survival. Because the spleen is a répertoire for vast quantifies of B- and T- lymphocytes, inhibition of LPS-induced AKT · phosphorylation was determined ex vivo using isolated mouse splénocytes. Cells were plated · and incubated with a desired concentration of Compound Al for 15 minutes followed by induction with LPS (20 pg/rnL) for 30 minutes. Following induction, cells were lysed and · pAKT was determined by ELISA using pAKTs473 capture/detection antibody pair and antimouse-HRP secondary antibody. Blank subtracted absorbance values were obtained te calculate percent inhibition of pAKT in test samples. Compound Al caused a dose-dependent réduction
-4218327 (EC50 347.4 nM) in phosphorylation of the Jownstream marker.
ART, at low concentrations » thereby elucidating the signaling pathway
Assay 7: Lipopolysaccharide Induced Pulmonarv Neutrcphilia in Femalc Wistar Rat Model
Ah exaggerated recruitment and subséquent activation of neutrophii is likely to the development and course of several inflammatory diseases in the airwaÿs and lungs, such· as severe asthma, chronic obstructive pulmonary diseuse, cystic fibrosis/and acuta respiratory • · * ·» , .
distress syndrome. The mechanisms by which neutrophii contributes .0 these diseases may involve the release of proteolytic enzymes, such as neutrophii elastase, and free oxygen radicals.· . When released, these compounds can cause bronchoconstriction, bro· '.chiai hypcrreactivity, hyper-secretion, épithélial damage, and tissue remodelling in the airways.·
After the quarantine period, fasted animais were randomized and divided into various groups · depending on their body weights. The test compound (Compound Al) was prepared as a suspension in a vehicle consisting of 0.5% methylcellulose in which Tween 80 as a suspending agent. The compound or vehicle was administered by oral gavage at a volume of 10 mL/kg.
Female Wistar rats were anaesthetized with ketamine and LPS solution was administered intratracheally one hour after compound administration at a dose of 1 mg/kg. 6 hours after LPS instillation, animais were exsanguinated under anaesthesia, and then the trachea was cannulated and the lungs were lavaged with 5 ml aliquots of heparinised PBS (1 unit ml) four times through a trachéal cannula (total volume 20 mL). Bronchoalveolar lavage (BAL) fluid was stored at 2-8 °C until assayed for total cell and differential leukocyte count. Bronchioaiveolar fluid was centrifuged (500*g for 10 minutes) and the resulting cell pellet was resuspended in 0.5 ml of heparinised saline. The total numbers of white blood cells were delernined in BAL fluid or blood by using a blood cell counter and was adjusted to l*106 cell/ml. Differential cell count was calculated manually. One hundred microliters of the cell suspension was centrifuged using a Cytospin 3 to préparé a cell smear. The cell smear was stained with a >lood staining solution for différentiation and slides were microscopically observed to identify eosinephii according to their morphological characteristics. The number of each cell type among 300 white blood cells in the cell smear was determined and expressed as a percentage. The number of eosinophil in each BÀLf or blood was calculated.
-4318327
Compound Λ1 showed a réduction of neutrophil infiltration into the lungs with an inhibition of · 65.29% at 10 mg/kg compared to the controi group, suggesting a therapeutic rôle in inflammatory disorders. The results are shown in Figure 1.
Assay 8: Lipopolysaccharide-Induced Rat Air Pouch Model of Inflammation . * · »
F FemaleAVistar rats (175-200 g) were acclimatized for scven days prior to the start of the . experiment. Animais were randomiy distributed to varions groups based' on their body wcights. ·.· · ••-Animais were anaesthetised with ether and subcutaneous air pouches were made by injecting 20 . .ml of stérile air under the skin in the intra-scapular area (day 0) and maintained with a second r 10 ml injection of sterile-filtered air on day 4. On day 6, oral treatment was commenced 1 hour prior to induction of inflammation by s.c. injection of LPS solution on day 6. A volume of 5 ml of LPS solution dissolved in stérile saline (100pg/kg) was injected into each pouch. Samples of pouch fluid were taken at 6 h after administration of LPS by flushing the pouch with 5 ml of stérile saline and withdrawing 4 ml of fluid. The number of leukocytes présent in pouch fluid was determined microscopically using a haemocytometer. Differential cell content was determined by microscopie examination of fluid smears stained with Diff-Quik.
t
Compound Al caused a dose-dependent réduction of neutrophil migration into the rat air pouch with an ED5oof2.65 mg/kg suggesting a therapeutic rôle in rheumatoidarthritis. The results are shown in Figure 2.
Assay 9: Ovalbumin Induced Pulmonary Eosinophilia in Male Guinea Pigs
20’ After the quarantine period, 0.3 ml of blood samples are collected from orbital vein by retro1 · · orbital plexus method from each individual animal and analysed on a cell analyser (ADVIA
2120, Siemens). Based on their total cell count, guinea pigs are randomized and divided into 1 ’* Various groups. Ear pinna is marked with an indelible marking pen for identification. On day 0, ‘ i ; · · ·· wêightS are recorded and animais are sensitized with 50,ug of ovalbumin (OVA) and 10 mg of ·.
25·' ' ’i-'àium solution (1 ml) intraperitoneallÿ. On day Ί and day 14, the above sensitization protocol is ’ · répeated. Animais are observed for any signs of illness or reaction to the sensitization up to day ' '19 and recorded if any. On day 19, 20, and 21, after the treatment with test compound by oral gavage, 30 minutes later animais are exposed to 0.5 % w/v, 0.5% and 1% ovalbumin challenge respectively. Control and sham group animais are treated with 0.5% w/v methyl cellulose (vehicle). Sham control groups are sensitized with 10 mg of alum on day 0, 7 and 14 and exposed to saline solution (SAL) with the same nebulization rate on day 19. 20 and 21. Twenty
-44r/.5x. .. ‘1 hours after the last OVA challenge, airway hvperresponsiveness is measured by ’.vhole body plethysmograph against cumulative doses of methacholine challenge (75, 100, 125 and 150 pg/ml). After measuring the airway response, blood samples and BAL fluid are collected. Samples are analysed for total cell couni by using a neubuear chamber under microscope and 5 differential leukocyte count is done manually.
Assay 10: Murine Asthma Model · After the quarantine period, based on their body weights, mice were randomized and dividcd into four groups (n=7), Tails were marked with an indelible marking pen for identification. On day 0, weights were recorded and animais were sensitized with lOOpg of ovalbumin and .10 mg .10 of alum solution (0.2 mL) intraperitoneally. On day 7 and day 14, the above sensitization protocol was repeated. Animais were obsçrved for any signs of illness or reaction ;to the sensitization up to day 24 and recorded if any. On day 24, 25, and 25, after the treatment with test compound by oral gavage, 30 minutes later animais were exposed to 10 % w/v ovalbumin challenge. Control and sham group animais were treated with 0.5% w/v methyl cellulose 15 (vehicle). Sham control groups were sensitized with 10 mg of alum on day 0, 7 and 14 and exposed to saline solution with the same nebulization rate on day 24, 25 and 26. Forty eight hours after the last OVA challenge, airway hyperresponsiveness was measured by whole body plethysmograph against cumulative doses of methacholine challenge (2.5, 10, 50 and 100 mg/ml). After measuring the airway response, blood samples and BAL fluid were collected.
Samples were analysed for total cell count by using a neubuear chamber under microscope and differential leukocyte count was done manually.
. Assay 11 : Collagen Induced Arthritis (CIA) in Wistar Rats
Female wistar rats were acclimatized for seven days prior to the start of the experiment and were randomly distributed to various groups based on their body weights. On day 0, animais 25 were treated by intradermal injection of 5C0 pg of bovine collagen type II emulsifiect with complété Freund’s adjuvant (IFA) containing MTB (4 mg/mL) delivered at the base of the tail. On day 7 after primary immunization, animais were treated by booster injection of 300 pg Cil in incomplète Freund’s adjuvant by intradermal injection at the base of the tail. Onset of arthritis in ankle joints usually became visually apparent between days 12 and 14. Animais were treated 30 with test compound or vehicle (orally administered) from the day after onset of arthritis and the treatment continued for the next 9 consecutive days. Arthritis Scores were taken by visual
-4518327 . ' examination for signs of joint inflammation regularly throughov.t the study period. . Measurements of body weights, paw volumes, and paw thickness were taken on days 0, 1,3, 5.
7, 9, and 10. After the ten day treatment, at the end of the study, blood was withdrawn at necropsy and processed to sérum or plasma and ail joints were taken and both fore paw and hind paws were fixed in 10% formalin for histopathology analysis after taking a small pièce-of iissuc ·· from each joint and stored at -80°C for cytokine analysis in tissue homogenate. Clinical Scoring- . Criteria for Fore and Hind Paws: 0 -- normal;· [ ~ or.C: hind or fore pav jbint nr’ccfti. or miuÎîn.s· <' · . diffuse erythema and swelling: 2 = two hind. or -ore paw joints affected or mild diffuse ;
erythema and swelling; 3 - three hind or fore paw jomts affected or moderato diffuse erythema· . and swelling; 4 = marked diffuse erythema and swelling. or = four digit joints affected;. 5 = : severe diffuse erythema and severe swelling entire paw, unable to flex digits.
Compound Al dosed therapeutically in the rat CIA model demonstrates significant efficacy in the réduction of the clinical score (Figures 3A and 3B) observed in both prophylactic paws (Figure 3C) and therapeutic paws (Figure 3D).
Compound Al dosed therapeutically in the rat CIA model demonstrates significant efficacy in · reducing the average paw volumes of both the hind paws (Figures 4A and 4B) and in ankle : diameter (Figures 4C and 4D).
Histological analysis: Compound Al dosed therapeutically in the rat CIA model demonstrates : significant efficacy in inhibition of inflammation (58.3 %, see figure 4A), cartilage (46.51%, see figure 4B) and pannus (49.18%, see figure 4C) observed by histopathology of ail the hind.and fore paws.
The incidence and progression of arthritis was significantly reduced in treatment group compared to control group animais (Figure 5).
Assay 12: Acute Cigarette Smoke Induced Cell. Infiltration in Male Balb/c Mice ·. · . 3 '
». · . · i.
Animais (male Balb/c mice) are to be acclimatizec for seven days prior îo the start of the· i experiment. Animais are then to be randomly distributed to various groups based on their body weights. On day 1, the mice are to be administered test compound or vehiclo by oral/intrailasal route and after 1 hour, the test compound administered animais are to be placed in a whole body exposure box. On day 1 and day 2, mice are exposed to the mainstream smoke of 6 cigarettes, of
8 cigarettes on day 3, and of 10 cigarettes on day 4. Exposure to the smoke of each cigarette will last for 10 minutes. The cigarettes are to be completely bumed in the first two minutes, followed
P'··’.?
-4618327 •5 by an air· flow with animal ventilator and ihc next 20 minutes will be exposure with fresh ïeom air. After every second cigarette, an additional break of 20 minutes with exposure to îresh room air is to be conducted. Control animais are to be exposed to room air chamber. From day J to • day 4, animais will be administered the test compound either by oral or intvanarm route. On day . . 5, 24 hours-after the last cigarate smoke (CS) exposure, animais will b? exsa.agu.inated under. . anaesthesia, and the trachea will be cannulated and the lungs lavaged· wifr. O.D-ml 'allqucts of heparinised PBS (1 unit/ml) four times -through: trachée· caimJô ·-tcm'. 2· ml;:-· ·’
Bronchioalveolar (BAL) collcctcd is to be stored at 2-S °C until assayed -for tôt·»! cell and . . .. differential leukocyte count. BAL fluid is to be centrifnged (500*g for 10 min) and tîte resulting ..
• cell pellet is resuspended in 0.5 ml of heparinised· saline. The total number'of whitc blood cells is to be determined in BAL fiuid and blood using a blood cell couiiter and adjusted to l*106 cell/ml. Differential cell count is calculated manually. Forty microliters of the cell suspension is centrifuged using Cytospin 3 to préparé a cell smear. The cell smear is stained with a blood staining solution for différentiation and microscopically obser/ed to identify eosinophil according to their morphological characteristics. The number of each cell type among 300 white blood cells in the cell smear are to be determined and expressed as a percentage, and the number of neutrophils and macrophages in each BAL fluid are to be calculated.
.1’
Assay 13: Imiquimod Induced Plaque Psoriasis in Balb/c Mice Model
I·
Imiquimod (IMQ) is a ligand for TLR7 and TLR8, originally used for the treatment of nonmelanoma skin cancers. The topical application of IMQ on the shaved back skin of the mouse induces a psoriasis-like skin condition exhibiting most of the human psoriasis pathologv characteristic features including acanthosis, parakeratosis, and infiltration of immune cells and involvement of the IL23 / IL 17 / IL22 pathway. Animais (male Balb/c micc) were acclimatized for seven days prior to the start of the experiment. Animais were randomly distributed to various groups based on their body weights. On day 0, the back skin of the mice was shaved by topical application of hair removal cream. On day 1, mice were a Jministered the.· test compound or ' vehiclè by the oral route and after 1 hour the mice that received the test compound received a ' topical application of 62.5 mg of commercially available IMQ cream (5%; Bcselna Cream;
' Mochida Pharmaceuticals, Tokyo, Japon) on the shaved back skin. The nrice were !;:safed with· · 30 topical application of imiquimod for the next 5 consecutive days, one hour after test compound or vehicle administration. Animais were allowed to dry for one hour before retuming to their cages after topical application on every day. Four hours after the final application of IMQ
-4718327 cream, the mice were killed and skin samples were obtained. Back skin thickness was measured using dial thickness gauge. After measuring skin thickness, skin samples were fixed in 10% neutrai buffered formalin solution and embedded in paraffin. Deparaffinised sections were . . stained· with hematoxylin-eosin (IIE). Epidermal thickness was quantified by averaging the •5 : : '·.·values of five independent fields per section. To score the severity ofinflammation ofthe back · ' skin, an objective scoring System was used based on the human clinical Psoriasis Area and
Severity Index (PASI). Erythema, scaling, and’thickenir.g were scc.vd indopendently sn a-ccrùc*·-. ·*>·
... from 0 to 4: 0 = none; 1 = slight; 2 = moderate; 3 rnarked; and 4 - very marked.
As shown in Figures 6Λ and 6B, Compound Al reduced back skin thickness, erythema, and 10 scaling (as shown by the histopathological score) comparcd to the control group animais.
Although the invention herein has been described with rcference to p; xticuiar embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the présent invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be 15 devised without departing from the spirit and scope of the présent 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 équivalents be covered thereby.
Ail publications and patent and/or patent applications cited in this application are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated herein by reference..

Claims (15)

1. A compound selected from (RS)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chrornen-
2-yl)ethyl)-lH. pyrazolo [3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)me{hanesulfonamide;
5 (8)-Ν-(5-(4-3ΐηίηο-1-(1-(5-ί1·.ιθΓθ-3-(3-ί1ηοΓορ1ΐ2^1)-4-οχο-4Η-οΗΓθπ·ιοή-2^1)εΙΙη'1)-1Ι-!- .
pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide;
(R)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-lHpyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyI)methanesulfonamide;
and pharmaceutically acceptable salts thereof.d •4 1 10 2. A compound according to claim 1 selected from (S)-N-(5-(4-amino-l-(l-(5-fluoro-3(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-2methoxyphenyl) methanesulfonamide and pharmaceutically acceptable salts thereof.
3. The compound according to claim 2, wherein the compound is snbstantially free of (R)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl)-lH-
15 pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)methanesulfonamide and pharmaceutically ; acceptable sait thereof.
4. The compound of claim 3, wherein the compound has an enantioireric excess greater than about 95%.
5. A pharmaceutical composition comprising a compound accord'ng to any one of 20 daims 1-4 and at least one pharmaceutically acceptable carrier.. .
6. Use of a compound of any one of daims 1-4, in the manufacture of a médicament for the treatment of a disease, disorder cr condition that would benefit from inhibiting catalytic activity of a PI3 δ/γ kinase.
7. The use of compound of any one of daims 1-4, in the manufacture of a médicament 25 ' for the treatment of a PI3K associated disease, disorder or condition.
8. The use of compound of any one of daims 1-4, wherein further comprising administering an additional active agent selected from anti-cancer agents, anti-inflammatory agents,
-4918327 immunosuppressive agents, steroids, non-steroidal anti-inflammatory agents, antihistamines, analgésies, and mixtures thereof.
9. , The use of claim 6, wherein the disease, disorder or condition that would benefit from inhibiting catalytic activity of a PI3 ό/γ kinase is selected from the carcinoma of bladder,
5 breast, colon, kidney, liver, lung, small cell lung cancer, esophagus, gali bladder, ovary, pancréas, stonlach, cervix. thyroid, prostate, and skin; including squamous oeil carcinoma; leukernia. acute iymphocytic leukernia, acute lymphoblastic leukernia, B~cel! lymphoma, T-cell lymphoma, . Hodgkin's lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma and Burkett's lymphoma; acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocyticleukemia;
10 fîbrosarcoma and rhabdomyosarcoma; astrocytoma, neuroblastoma, glioma and. sch\ ‘annomas; melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoina . pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma in a patient in need thereof.
10. The use of claim 7, wherein the PI3K associated disease, disorder or condition is an immune system-related disease, a disease or disorder involving inflammation, cancer or other
15 proliférative disease, a hepatic disease or disorder, or a rénal disease or disorder.
11. The use of claim 6 or 7, wherein the disease, disorder or condition is selected from leukernia, acute Iymphocytic leukernia, acute lymphoblastic leukernia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma, Burkett's lymphoma, acute and chronic myelogenous leukemias, myelodysplastic syndrome or promyelocytic
20 ïeukemia in a patient in need thereof.
12. The use of claim 6 or 7, wherein the disease, disorder or condition is selected from asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflainma'ory bowcl disease, glomerulonephritis, neuroinflammatory diseases, multiple sclerosis, uveitis, psorias, lupus, psteoarthritis, allergie rhinitis, lupus erythematosus and disorders of the immune System.’
25
13. ’ A compound selected from (S)-2-(l-(4-amino-3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5flüoro-3-(3-fluorophenyl)-4H-chromen-4-one, (S)-2-(l-(4-amino-3-(3-amino-4-methoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,
-50F > · · ·' and salts thereof.
14. A process for the préparation of compound of formula (I) of claim 1
5 comprising the steps of:
(a) reacting 5-bromo-2-methoxyaniline with methane sulphonyl chloride (b)
Br
10 to give N-(5-bromo-2-methoxyphenyl)methanesulfonamide (Intermediate 1)
Intermediate 1;
15 (c) reacting Intermediate 1 with bis(pinacolato)diboron to give N-(2-methoxy-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanesulfonamide (Intermediate 2)
-51 ./31 ... '
Intermediate 2; and (d) reacting 2-(l-(4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(35 fluorophenyl)-4H-chromen-4-one with Intermediate 2 in the presence of a suitable base to give a compound of formula (I).
15. A process for the préparation of compound of formula (Al) of claim 2 (Al) comprising the steps of:
(a) reacting (R)-5-fluoro-3-(3-fluorophenyl)-2-(l-hydroxyethyl)-4H-chromen-4-one
-5218327 with 3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrirnidin-4 -amine under Mitsunobu conditions using triphenylphosphine and diisopropylazodicarboxylate to give (S)2-(l-(4-amino-3-(4-methoxy-3-nitrophenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3(3-fluorophenyl)-4H-chromen-4-one (Intermediate 3)
10 Intermediate 3;
(b) reducing intermediate 3 to give (S)-2-(l -(4-amino-3-(3-amino-4-methoxyphenyl)-lHpyrazolo[3,4-d]pyrimidin-l-yl)ethyl)-5-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one (Intermediate
4)
-5318327 (c) reacting Intermediate 4 with methanesulphonyl chloride to give a compound of the formula·
5 (Al).
-5418327
ABSTRACT
The présent invention relates to a sélective dual delta (δ) and gamma (γ) PI3K protein kinase modulator (S)-N-(5-(4-amino-l-(l-(5-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yI)elhyl)-lrIpyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl) méthane sulfonamide, methods of pieparir.g 5 them, pharmaceutical compositions containîng them and methods of treatment, prévention and/or amelioration of PI3K kinase mediated diseases or disorders xvith them.
OA1201600497 2014-06-27 2015-06-26 Substituted chromene derivatives as selective dual inhibitors of Pi3 delta and gamma protein kinases OA18327A (en)

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