NZ714465B2 - Dual selective pi3 delta and gamma kinase inhibitors - Google Patents

Abstract

The present invention relates to chromen-4-one derivatives as dual delta (?) and gamma (?) PI3K protein kinase modulators, methods of preparing them, pharmaceutical compositions containing them and methods of treatment, prevention and/or amelioration of Pi3K kinase mediated diseases or disorders with them. h them.

Description

DUAL IVE PI3 DELTA AND GAMMA KINASE INHIBITORS

[01] The present application claims the benefit of Indian Patent Application Nos. 2501/CHE/2013, filed June 7, 2013, and 5567/CHE/2013, filed December 3, 2013 each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[02] The present invention provides dual delta (δ) and gamma (γ) PI3K protein kinase modulators, methods of preparing them, pharmaceutical compositions containing them and methods of treatment, prevention and/or ration of Pi3K kinase mediated diseases or disorders with them.

BACKGROUND OF THE INVENTION

[03] Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellular lipid s that phosphorylate the 3 position hydroxyl group of the inositol ring of phosphoinositide lipids (Pis) generating lipid second messengers. While alpha and beta isoforms are tous in their distribution, expression of delta and gamma is restricted to circulating hematogenous cells and endothelial cells. Unlike PI3K-alpha or beta, mice lacking expression of gamma or delta do not show any adverse phenotype indicating that targeting of these ic isoforms would not result in overt toxicity.

[04] Recently, targeted inhibitors of the phosphoinositidekinase (PI3K) pathway have been suggested as modulatory agents. This interest stems from the fact that the PI3K pathway serves multiple functions in immune cell signaling, primarily through the tion of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a membranebound second messenger .

PIP3 recruits proteins to the cytoplasmic side of the lipid bilayer, including protein kinases and GTPases, initiating a complex network of downstream signaling es important in the regulation of immune cell adhesion, migration, and cell-cell communication.

[05] The four class I PI3K ms differ significantly in their tissue distribution. PI3Ka and ΡΙ3Κβ are ubiquitous and activated ream of receptor tyrosine kinases (RTK), whereas PI3K δ and PI3K γ are primarily limited to hematopoietic and endothelial cells, and are ted downstream of RTKs, and G protein coupled receptors (GPCR), respectively. Mouse genetic studies have revealed that PI3Kα and 1 WO 2014/195888 2014/061954 PI3KB are essential for normal development, whereas loss of PISK 5 andfor PI3K y yields viable offspring with selective immune deficits.

[06] The expression pattern and functions of PI3K 8 and PI3K V have generated much interest in developing PI3K5/y tors as agents for many diseases, including rheumatoid arthritis, allergies, asthma, chronic ctive pulmonary disease and le sclerosis (Hirsch et al., col. Ther., 118, 192—205, 2008; Marone et al., m Biophys. Acta., 1784, 159—185, 2008; Rommel et al., Nat. Rev. Immunol, 7, 191—201, 2007; Ruckle et al., Nat. Rev. Drug Discov., 5, 903—918, 2006). Studies using both pharmacologic and genetic methods have shown these two isoforms often demonstrate synergistic interactions with each other (Konrad et al., J. Biol. Chem, 283, 33296—33303, 2008; gue et al., Immunity, 16, 441—451, 2002). In mast cells, for example, PI3K5 is essential for degranulation in response to IgE cross-linking of Fc-receptors (Ali et al., J.

Immunol, 180, 2538—2544, 2008), but PISKy plays an important role in amplifying the response (Laffargue et al., Immunity, 16, 441—451, 2002). Similar effects have been seen in other cellular functions, including lymphocyte homing and the neutrophil respiratory burst where PI3Ky plays a critical role and PI3K5 amplifies each process. The nonredundant but related roles of PI3K5 and PI3Ky have made it lt to determine which of the two isoforms (alone or in combination) is best targeted in a particular inflammatory disorder. Studies using mice that lack PI3K8 and/or PI3Ky or express kinase—dead variants of PI3K5 and PI3Ky have been valuable tools in understanding their roles. For example, PI3K8 knockout mice demonstrated diminished neutrophil chemotaxis, diminished antibody production (both T cell dependent and independent) (Jou et al., Moi.

Cell.Biol., 22, 591, 2002), and lower numbers of mature B cells (Clayton et al., J.

Exp. Med, 196, 3, 2002; Jou er al., Mal. iol., 22, 8580—8591, 2002), and a decrease in their proliferation in response to anti-IgM (Jou et al., 2002) . This phenotype was replicated in the PI3K5 kinase-dead variant and with PI3K6 ive tors along with decreased numbers of and proliferation of mast cells, and an attenuated allergic response. The P13Ky knockout contained higher numbers of, but less responsive, neutrophils, lower numbers of and less responsive macrophages and dendritic cells displayed decreased mast cell degranulation (Laffargue et al., 2002), a higher ratio of CD4+ to CD8+ T cells), increased yte apoptosis, diminished induction of CXCR3 on activated T cells and decreased c contractility. This latter effect on cardiac tissue was a concern for chronic dosing of patients with PI3K7 inhibitors. However, this concern was largely mitigated when the PI3Ky kinase-dead variant (which better mimics inhibition of the kinase rather than loss of the protein) showed similar immune cell phenotypes, but 2 WO 2014/195888 2014/061954 importantly had no cardiac defects. The cardiac effect was later shown to be due to lding effects rather than the catalytic activity of PI3Ky. The dual PI3K5/PI3Ky knockout was viable but exhibited serious defects in T cell development and thymocyte survival. The PI3Ky knockout/PBKS kinase-dead combination produced a similar phenotype suggesting that at least within the immune system, the role of PI3K8 is likely only a catalytic one. Interpretation of studies using knockout and kinase-dead mice can be challenging because these models provide only a steady-state picture of the immune system, lack temporal and dose control, and do not permit a full understanding of how a dynamic immune response will react to reversible inhibition. Selective inhibitors with varying profiles (PI3K5, PI3Ky, and PI3K5/y) are ary for studies of yte signaling in order to assess the relative contributions of each PI3K to immune cell activation gon et al., try & Biology, 1, 123-134 (2010), including the references cited )

[07] Dual inhibition of 5/y is strongly implicated as an intervention strategy in allergic and lergic inflammation of the airways and other autoimmune diseases.

Scientific evidence for PI3K-8 and y gamma involvement in various cellular processes underlying asthma and COPD stems from tor studies and gene-targeting approaches.

Also, resistance to conventional therapies such as corticosteroids in several COPD ts has been attributed to an up-regulation of the PI3K S/y pathway. Disruption of PI3K- S/y signalling ore es a novel strategy aimed at counteracting the immuno— inflammatory response. Due to the pivotal role played by PI3K- 5 and y 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 ing the 5 and y isoforms would be expected to attenuate the progression of immune se encountered in airway inflammation and rheumatoid arthritis (William et.al Chemistry & Biology, 17, 4, 2010 and Thompson, et al. Chemistry & Biology, 17:101—102, 2010)

[08] Reviews and studies regarding PI3K and related protein kinase pathways have been given by Liu et. al., Nature Reviews Drug Discovery, 8, 627-644, 2009); Nathan T. et. al., Mol Cancer Ther., 8(1), 2009; Marone et, al., Biochimica et Biophysica Acta, 1784, 159-185, 2008 and Markman et. al., Annals of Oncology Advance Access, published August 2009. Similarly s and studies regarding role of PI3K 5 and V have been given by William et.al., Chemistry & Biology, 17, 123-134, 2010 and Timothy et.al. J.

WO 2014/195888 PCT/IB2014/061954 Med. Chem, 55 (20), 8559—8581,2012. All of these literature disclosures are hereby incorporated by reference in their entirety.

[09] Compounds such as 5 and CAL130 have been reported as dual inhibitors of Pi3K 5/7. 5 is under clinical investigation for cancer, asthma and rheumatoid arthiritis. IPI-45 have been ed to have a maximum tolerated dose (MTD) of 75 mg BID (55th ASH® Annula Meeting New Orleans-LA, Dec 7-10, 2013). There are no reports of CAL-130 being investigated for clinical purposes.

[10] There still remains an unmet need for dual 5 y PI3K modulators for the treatment of es and disorders associated with S/y PI3K kinases-mediated events.

[11] Further reference is made herein to International ation Nos. WO 11/055215 and WO 12/151525 and US. Publication Nos. 2011/0118257 and 2012/0289496, each of which is incorporated herein by reference in its entirety.

SUMMARY OF THE INVENTION

[12] The present invention is directed to selective dual inhibitors of PI3K delta and gamma protein kinases. These compounds are suitable for use in a pharmaceutical composition for the treatment of a PI3K associated disease, disorder or condition, e.g., a proliferative disease such as cancer. Inhibition of both PI3K delta and gamma protein kinases may provide cial effects in the treatment of certain diseases and disorders.

[13] The selective dual inhibitors of the t invention include the following compounds, pharmaceutically acceptable salts f, and prodrugs thereof: (RS)—2-(1-(9H-purin-6—ylamino)propy1)-3—(3-fluorophenyl)-4H—chromen—4-one.

(Compound A) (S)-2—(1-(9H—purin-6—ylamino)propyl)—3-(3-fluorophenyl)-4H-chromen-4—one. und A1) (R)-2—(1-(9H—purin-6—ylamino)propyl)—3-(3-fluorophenyl)-4H-chromen-4—one.

(Compound A2)

[14] The chemical structures of the compounds of the t invention are shown below.

WO 2014/195888 PCT/IB2014/061954 (A) (A1) (A2)

[15] In one embodiment, the present invention relates to the compound (S)(1— (9H-purinylamino)propyl)-3—(3-fluorophenyl)-4H-chromenone, or a pharmaceutically acceptable salt thereof.

[16] In one embodiment, the compound (S)-2—(l-(9H-purinylamino)propyl)- 3-(3-fluorophenyl)-4H-chromenone, or a pharmaceutically acceptable salt thereof, is substantially free (e.g., contains less than about 10%, such as less than about 5%, less than about 2.5%, less than about 1%, less than about 0.1% by weight or is free) of (1— (9H-puriny1amino)propyl)-3—(3-fluorophenyl)—4H-chromenone and pharmaceutically acceptable salts f.

[17] In r embodiment, the compound (l-(9H—purin-6— ylamino)propyl)(3—fluorophenyl)-4H—chromenone, or a pharmaceutically acceptable salt thereof, has an enantiomeric excess of greater than about 90%, such as greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, r than about 99%, greater than about 99.5%, greater than about 99.9%, or greater than about 99.99%.

[18] In one preferred embodiment, the present invention relates to the compound (S)(l-(9H-purinylamino)propyl)-3—(3-fluorophenyl)—4H-chromen-4—one (Compound Al).

[19] The invention further provides a ceutical composition comprising one or more compounds of the t invention (such as compound Al) together with a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise one or more of additional active agents (such as anti-cancer agents and the active agents discussed below). In one ment, the pharmaceutical composition includes a therapeutically effective amount of one or more compounds of the present invention.

WO 2014/195888 PCT/IB2014/061954

[20] r embodiment is a method of inhibiting PI3K delta and gamma in a patient by administering to the patient an effective amount of at least one compound of the present invention.

[21] Yet another embodiment is a method of treating, preventing, and/or inhibiting a PI3K protein kinase mediated disease, disorder or condition (such as cancer or other proliferative disease or disorder) in a patient by administering to the patient an effective amount of at least one nd of the present ion.

[22] Yet another embodiment of the present invention is a method for inhibiting PI3K, in particular PI3K delta and gamma kinase in a t by administering to the patient an effective amount of at least one compound of the present invention.

[23] Yet another embodiment of the present ion is a method for treating an inflammatory, autoimmune or proliferative e via tion of a protein kinase (such as P13 delta and gamma kinase) by administering to a patient in need of such treatment an effective amount of at least one compound of the present ion. In one embodiment, the compound of the present invention inhibits both the PI3K delta and gamma protein kinase.

[24] Yet another embodiment of the present invention is a method for treating an inflammatory, autoimmune or proliferative disease via modulation of a protein kinase (such as P13 delta and gamma kinase) by administering to a patient in need of such ent an effective amount of at least one nd of the present invention, in combination (simultaneously or sequentially) with at least one other anti-inflammatory, immunomodulator or anti-cancer agent (or a combination thereof). In one ment, the compound of the present ion inhibits both the PI3K delta and gamma protein kinase.

[25] The compounds of the present invention are useful in the treatment of a variety of cancers, including, but not limited to:

[26] carcinoma, including, but not limited to, that of the bladder, breast, colon, kidney, liver, lung, including small cell lung , esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma;

[27] hematopoietic tumors of lymphoid lineage, including, but not limited to, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B—cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkett's lymphoma;

[28] hematopoietic tumors of myeloid lineage, including, but not limited to, acute and c myelogenous leukemias, myelodysplastic me and promyelocytic leukemia; WO 2014/195888 PCT/IB2014/061954

[29] tumors of mesenchymal origin, including, but not limited to, fibrosarcoma and rhabdomyosarcoma;

[30] tumors of the central and peripheral nervous system, including, but not limited to, ytoma, neuroblastoma, glioma and schwannomas; and

[31] other tumors, including, but not d to, melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma.

[32] In one emobodiment, the compounds of the present invention are administered to treat a leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell ma, Hodgkin's lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, Burkett's lymphoma, acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia.

[33] Due to the key role of protein kinases in the regulation of ar proliferation in general, the protein kinase inhibitors of the present invention could act as reversible cytostatic agents, and may be useful therefore in the treatment of any e process which features abnormal cellular proliferation, e. g., benign prostatic lasia, familial adenomatosis polyposis, neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following lasty or vascular y, hypertrophic scar formation, inflammatory bowel disease, transplantation ion, xic shock, and fungal ions.

[34] The compounds of the t invention as modulators of apoptosis are useful in the treatment of cancer (including but not limited to those types mentioned herein above), viral infections (including, but not limited to, herpevirus, poxvirus, Epstein-Barr Virus, Sindbis virus and adenovirus), autoimmune es (including, but not limited, to systemic lupus, erythematosus, mune ed glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus), neurodegenerative disorders (including, but not limited to, Alzheimer's disease, AIDS— related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration), myelodysplastic syndromes, ic anemia, ischemic injury associated with myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases, hematological diseases (including, but not limited to, chronic anemia and aplastic anemia), degenerative diseases of the musculoskeletal system (including, but not d to, osteoporosis and arthritis) aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases and cancer pain. The compounds of the present invention are 7 WO 2014/195888 PCT/IB2014/061954 also useful in the prevention, tion, or suppression of AIDS development in HIV- infected duals.

[35] The compounds of the t invention can modulate the level of cellular RNA and DNA sis. These agents are therefore useful in the treatment of viral ions, including, but not limited to, HIV, human papilloma virus, herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus.

[36] The compounds of the present invention are useful in the chemoprevention of cancer. Chemoprevention is d as inhibiting the development of invasive cancer by either blocking the initiating mutagenic event or by blocking the progression of pre- malignant cells that have already suffered an insult or inhibiting tumor relapse. The compounds of the present invention are also useful in inhibiting tumor angiogenesis and metastasis. One embodiment of the invention is a method of inhibiting tumor angiogenesis or metastasis in a patient in need thereof by administering an effective amount of one or more compounds of the present invention.

[37] Another embodiment of the present invention is a method of treating an immune system-related disease (e.g., an mune disease), a disease or disorder involving inflammation (e.g., asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, neuroinflammatory diseases, multiple sclerosis, uveitis and disorders of the immune system), cancer or other proliferative disease, a c disease or disorder, a renal disease or disorder. The method includes administering an effective amount of one or more compounds of the present invention.

[38] Examples of immune disorders include, but are not limited to, psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, , inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial is, scleroderma, osteoporosis, , allogeneic or xenogeneic transplantation (organ, bone marrow, stem cells and other cells and tissues) graft rejection, versus-host disease, lupus erythematosus, inflammatory disease, type I diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis), myasthenia gravis, autoimmune hemolytic anemia, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary sis, allergic ctivitis and atopic dermatitis.

[39] In one embodiment, the nds 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 WO 2014/195888 PCT/IB2014/061954 - versus - host disease. In other embodiments, transplant graft rejections result from tissue or organ transplants. In further embodiments, graft-versus-host disease results from bone marrow or stem cell transplantation. One ment is a method of preventing or sing the risk of transplant graft rejection, allogeneic or xenogeneic transplantation rejection (organ, bone marrow, stem cells, other cells and s), or graft - versus - host disease by administering an effective amount of one or more compounds of the present invention.

[40] The compounds of the present invention are also useful in combination (administered er or sequentially) with known anti-cancer treatments, such as radiation therapy or with cytostatic or cytotoxic or anticancer , such as, for e, DNA interactive , such as cisplatin or doxorubicin; topoisomerase II inhibitors, such as etoposide; topoisomerase I tors such as CPT-ll or topotecan; tubulin interacting agents, such as paclitaxel, docetaxel or the epothilones (for e ixabepilone), either naturally occurring or synthetic; al agents, such as tamoxifen; thymidilate synthase inhibitors, such as 5—fluorouracil; and anti—metabolites, such as rexate, other tyrosine kinase tors such as Iressa and OSI-774; angiogenesis inhibitors; EGF inhibitors; VEGF inhibitors; CDK inhibitors; SRC inhibitors; c-Kit inhibitors; Herl/2 inhibitors and monoclonal antibodies ed against growth factor receptors such as erbitux (EGF) and herceptin (Her2) and other protein kinase modulators as well.

[41] The compounds of the present invention are also useful in combination (administered together or sequentially) with one or more steroidal anti-inflammatory drugs, non-steroidal anti-inflammatory drugs (NSAIDs) or immune selective anti— inflammatory derivatives (ImSAIDs).

[42] The invention further provides a pharmaceutical composition comprising one or more compounds of the present invention together with a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise one or more of the active ingredients identified above, such as other ancer agents.

[43] Yet another embodiment is a method of treating leukemia in a patient in need thereof by administering a therapeutically effective amount of a compound of the present invention. For example, the compounds of the present invention are ive for treating chronic lymphocytic ia (CLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) acute myeloid leukemia (AML), multiple a (MM), small lymphocytic lymphoma (SLL), and indolent non-Hodgkin’s lymphoma (I-NHL).

[44] Yet another embodiment is a method of treating leukemia in a patient in need thereof by administering a therapeutically effective amount of a compound of the 9 WO 2014/195888 PCT/IB2014/061954 t invention. For e, the compounds of the present invention are effective for treating autoimmune disorders such as asthma, COPD, rhematoid arthritis, psorias, lupus and experimental mune encephalomyelitis (EAE).

[45] Yet r embodiment is a method of treating allergic rhinitis in a patient in need thereof by administering a therapeutically effective amount of a compound of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS BRIEF DESCRIPTION OF THE DRAWINGS

[46] Figure 1 depicts a bar graph of the neutrophil count in bronchoalveolar lavage fluid (BALF) from animals treated with 0, 0.3, 1, 3, and 10 mgikg of Compound A1 (p0) according to the Lipopolysaccharide d pulmonary neutrophilia model described in Assay 7.

[47] Figure 2 depicts a bar graph of the neutrophil count in neal lavage fluid from animals treated with 0, 1, 3, and 10 mg/kg of Compound A1 (p0) according to the Lipopolysaccharide—induced rat air pouch inflammation model described in Assay 8.

[48] Figure 3 depicts a bar graph of the Lipopolysaccharide-induced plasma TNF-(x concentration in fasted female Wistar rats following administration of 0, 1, 3, and 10 mg/kg of Compound A1 (p0) according to the procedure described in Assay 9.

[49] Figures 4A and 4B depict graphs of enhanced pause (Penh) or PEF/PIF (peak expiratory flow / peak inspiratory flow) ratio, tively, in sensitized male guinea pigs following methacholine challenge and treatment with OVA/SAL or OVA/OVA or 0.3, 1, or 3 mgfkg Compound A1 according to the procedure in Assay 10A.

[50] Figures 4C-4E depict bar graphs of eosinophil count in BALF, total cell count in BALF, and tage eosinophils, respectively, in min-sensitized male guinea pigs and treatment with 0, 0.3, 1, or 3 mg/kg Compound A1 according to the procedure in Assay 10A.

[51] Figures 5A and 5B depict graphs of Penh) or PEF/PIF ratio, respectively, in ovalbumin-sensitized mice following methacholine challenge and treatment with SAL/SAL, L or OVA/OVA or 3 mg/kg Compound A1 according to the procedure in Assay 10B.

[52] Figures SC-SE depict bar graphs of eosinophil count in BALF, total cell count in BALF, and percentage eosinophils, respectively, in ovalbumin-sensitized mice and treated with 0 or 3 mg/kg Compound A1 according to the procedure in Assay 10B. 10 WO 95888 PCT/IB2014/061954

[53] Figures 6A and 6B depict bar graphs of individual histopathological scores for ankle and knee, respectively, in collagen induced arthritis using Lewis rats treated with a control or 15 mg/kg/BID of compound A1 according to the procedure in Assay 11.

[54] Figures 6C and 6D depict bar graphs of summed histopathological scores for ankle and knee, tively, in collagen induced arthritis model using Lewis rats treated with vehicle or 15 mg/kg/BID of compound A1 according to the procedure in Assay 11.

[55] s 7A and 7B depict bar graphs of macrophage and neutrophil cell counts, respectively, in BALF following administration of 0.3, 1, or 3 mg/kg/BID of Compound A1 in male Balb/c mice in a cigarette smoke induced cell ration model as described in Assay 15.

[56] Figure 8 depicts a graph showing the inhibition of AKT phosphorylation in leukemic cell lines (MOLT-4, Jurkat, CCRF-CEM, Hut—78, and HuT-102) by Compound A1 according to the procedure in Assay 3.

[57] Figure 9 depicts a graph showing the inhibition in tage of CD63 positive cells induced by fMLP or anti—FcaRl in human whole blood by Compound A1 according to the procedure in Assay 4.

[58] Figure 10 depicts a graph showing the inhibition of anti-human CD3/CD28— induced cytokines (TNFa, IFNy and IL2) by Compound A1 according to Assay 6D.

DETAILED DESCRIPTION OF THE INVENTION

[59] As used herein the following definitions shall apply unless otherwise ted. Further many of the groups defined herein can be optionally tuted. The g of substituents in the definition is exemplary and is not to be construed to limit the substituents d elsewhere in the specification.

[60] 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 ied, the present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. For the instance, non-limiting example of intermediate mixutures 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 nds described herein n ic double bonds or other s of geometric 11 WO 95888 PCT/IB2014/061954 asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

[61] The term "tautomers" refers to compounds, which are characterized by relatively easy interconversion of ic forms in equilibrium. These isomers are intended to be covered by this invention. "Tautomers" are structurally ct isomers that interconvert by tautomerization. "Tautomerization" is a form of ization and includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. "Prototropic tautomerization" or "proton-shift tautomerization" involves the migration of a proton accompanied by changes in bond order, often the hange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g. in solution), a chemical equilibrium of ers can be d. An example of tautomerization is keto-enol tautomerization. A specific example of keto-enol tautomerization is the interconversion of pentane-2,4-dione and 4-hydroxypent-3—enone tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin—4-ol and pyridin— 4(1H)—one tautomers.

[62] The term "prodrug" refers to a compound, which is an inactive precursor of a compound that is converted into its active form in the body by normal metabolic processes. g design is sed generally in Hardma, et al. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed., pp. 11-16 (1996). A thorough discussion is provided in Higuchi, et al., Prodrugs as Novel Delivery Systems, Vol. 14, ASCD Symposium , and in Roche (ed.), Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987). To illustrate, prodrugs can be converted into a pharmacologically active form through hydrolysis of, for example, an ester or amide linkage, y introducing or exposing a functional group on the resultant product. The gs can be designed to react with an nous compound to form a water-soluble conjugate that further enhances the pharmacological properties of the compound, for example, increased circulatory half-life. Alternatively, gs can be designed to undergo covalent modification on a functional group with, for e, glucuronic acid, sulfate, glutathione, amino acids, or e. The resulting conjugate can be inactivated and excreted in the urine, or rendered more potent than the parent compound. High molecular weight conjugates also can be excreted into the bile, subjected to enzymatic cleavage, and released back into the circulation, thereby effectively increasing the biological half-life of the originally administered compound. 12 WO 2014/195888 PCT/IB2014/061954

[63] The term "ester" refers to a compound, which is formed by reaction between an acid and an l with elimination of water. An ester can be ented by the general formula RCOOR' (where R is a drug and R’ is a chemical group).

[64] These prodrugs and esters are intended to be covered within the scope of this invention.

[65] Additionally the instant invention also includes the compounds which differ only in the presence of one or more isotopically enriched atoms for example replacement of hydrogen with deuterium or tritium, or the ement of a carbon by 13C— or 14C- enriched carbon.

[66] The compounds of the present invention may also contain unnatural proportions of atomic es at one or more of atoms that constitute such compounds.

For e, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine—125 (1251) or carbon-l4 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[67] Pharmaceutically acceptable salts forming part of this invention e salts derived from inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Zn, and Mn; salts of organic bases such as N,N'-diacetylethylenediamine, glucamine, ylamine, choline, hydroxide, dicyclohexylamine, metformin, benzylamine, trialkylamine, and thiamine; chiral bases such as henylamine, 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 s, alkyl tes such as Mel and (Me)ZSO4; non-natural amino acids such as D— isomers or substituted amino acids; ine; and substituted guanidine n the substituents are selected from nitro, amino, alkyl, alkenyl, l, ammonium or tuted ammonium salts and aluminum salts. Salts may include acid addition salts where appropriate which may be sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides, es, tartrates, maleates, citrates, fumarates, succinates, palmoates, methanesulphonates, benzoates, salicylates, benzenesulfonates, ascorbates, glycerophosphates, and ketoglutarates.

[68] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as al formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included.

The term "about" when referring to a number or a numerical range means that the number 13 WO 2014/195888 PCT/IB2014/061954 or numerical range referred to is an approximation within mental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range. The term "comprising" (and d terms such as ise" or "comprises" or "having" or "including") includes those embodiments, for example, an embodiment of any composition of matter, composition, , or process, or the like, that "consist of” or "consist essentially of” the described features.

[69] The ing abbreviations and terms have the indicated meanings throughout: PI3-K = Phosphoinositide 3-kinase; PI = phosphatidylinositol; AIDS = Acquired Immuno Deficiency Syndrome; HIV = Human Immunodeficiency Virus; MeI = Methyl Iodide; ND: Not determined.

[70] Abbreviations used herein have their conventional meaning within the al and biological arts.

[71] The term "cell proliferation" refers to a phenomenon by which the cell number has changed as a result of division. This term also encompasses cell growth by which the cell morphology has changed (e.g., increased in size) consistent with a proliferative signal.

[72] The terms "co-administration, II II administered in ation with," and their grammatical equivalents, as used herein, encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. Co-administration includes simultaneous administration in separate compositions, stration at different times in separate compositions, or administration in a composition in which both agents are present.

[73] The term tive amount" or "therapeutically effective amount" refers to that amount of a compound bed herein that is ient to effect the intended application including but not limited to disease treatment, as defined below. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e. g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can y 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. ion of et adhesion andi'or cell migration. The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried. 14 WO 2014/195888 PCT/IB2014/061954

[74] As used herein, "treatment," ing," or "ameliorating" are used interchangeably. These terms refers to an approach for obtaining beneficial or d results including but, not d to, therapeutic benefit and/or a lactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is ed with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of ping a particular disease, or to a patient ing one or more of the physiological symptoms of a disease, even though a diagnosis of this e may not have been made.

[75] A "therapeutic effect," as that term is used herein, encompasses a therapeutic benefit andfor a lactic benefit as described above. A prophylactic effect includes delaying or eliminating the ance 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.

[76] The term "subject" or “patient” refers to an animal (e. g., a dog, cat, horse, or pig), such as a , for example a human. The methods described herein can be useful in both human therapeutics and veterinary applications. In some embodiments, the patient is a mammal, and in some embodiments, the patient is human.

[77] "Radiation y" means exposing a patient, using routine methods and compositions known to the practitioner, to radiation emitters such as alpha—particle emitting uclides (e.g., actinium and thorium radionuclides), low linear energy transfer (LET) radiation emitters (i.e. beta emitters), conversion electron emitters (e.g. strontium-89 and samarium- lS3—EDTMP), or high-energy radiation, including, without limitation, x-rays, gamma rays, and neutrons.

[78] l transduction" is a process during which stimulatory or inhibitory signals are itted into and within a cell to elicit an intracellular response. A tor of a signal transduction pathway refers to a compound which modulates the activity of one or more cellular proteins mapped to the same specific signal transduction pathway. A modulator may augment (agonist) or suppress (antagonist) the activity of a signaling molecule.

[79] The term "selective inhibition" or "selectively inhibit" as applied to a biologically active agent refers to the agent's ability to selectively reduce the target 15 WO 2014/195888 PCT/IB2014/061954 signaling activity as compared to off—target signaling activity, via direct or indirect interaction with the target.

[80] The term "pharmaceutically able carrier" or "pharmaceutically acceptable excipient" includes, but is not limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying , one or more suitable diluents, s, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants/flavoring, carriers, excipients, buffers, stabilizers, solubilizers, and combinations thereof. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic itions of the invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[81] In certain embodiments, one or more of the compounds described herein bind specifically to a P13 kinase or a protein kinase selected from the group consisting of mTor, DNA-dependent n kinase (Pubmed protein accession number (PPAN) AAA79184), AbI tyrosine kinase (CAA52387), Bcr-Abl, hemopoietic cell kinase (PPAN CA119695), Src (PPAN CAA24495), vascular endothelial growth factor receptor 2 (PPAN 19), epidermal growth factor or (PPAN AG43241), EPH receptor B4 (PPAN EAL23820), stem cell factor receptor (PPAN AAF22141), Tyrosine-protein kinase receptor TIE—2 (PPAN Q02858), lated tyrosine kinase 3 (PPAN l 10), platelet-derived growth factor receptor alpha (PPAN NP_990080), RET (PPAN CAA73l3l), and any other related n kinases, as well as any onal mutants thereof.

[82] In other embodiments, the 1C50 of a compound described herein for pi 10a, pi lOB, pi 107, or pi 105 is less than about 1 uM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than 1 nM or less than about 0.5nM. In some embodiments, the 1C50 of a compound described herein for mTor is less than about 1 uM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than 1 nM or less than about 0.5nM. In some other embodiments, one or more of the compounds described herein exhibit dual binding specificity and are capable of inhibiting a PI3 kinase (e.g., a class I P13 kinase) as well as a protein kinase (e.g., mTor) with an 1C50 value less than about 1 uM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than 1 nM or less than about 0.5 nM.

[83] In additional embodiments, the compounds of the present invention exhibit one or more functional characteristics disclosed . For e, one or more of the compounds described herein bind specifically to a PI3 kinase. In some embodiments, the 16 WO 2014/195888 PCT/IB2014/061954 IC50 of a nd described herein for pi 10a, pi 10B, pi 10y, or pi 105 is less than about 1 uM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than about 1 nM, less than about 0.5nM, less than about 100pM, or less than about 50 pM.

[84] In other embodiments, the compounds of the present invention selectively inhibit one or more members of type I or class I phosphatidylinositol 3-kinases (P13- ) with an IC50 value of about 100 nM or less, about 50 nM or less, about 10 nM or less, about 5 nM or less, about 100 pM or less, about 10 pM or less, or about 1 pM or less as measured in an in vitro kinase assay.

[85] In yet r , an tor that selectively inhibits one or more members of type I PI3—kinases, or an tor that selectively inhibits one or more type I PI3—kinase mediated signaling pathways, alternatively can be understood to refer to a compound that exhibits a 50% inhibitory concentration (IC50) with respect to a given type I PI3—kinase, that is at least 10—fold lower, at least 20-fold lower, at least 50-fold lower, at least 100-fold lower, at least lOOO-fold lower than the inhibitor's IC50 with respect to the rest of the other type I P13 -kinases.

[86] As used herein, the term “dual PI3-kinase 5 / y inhibitor" and “dual PI3— kinase 5 / y selective inhibitor” refers to a compound that inhibits the activity of both the PI3-kinase 5 and y isozyme more effectively than other isozymes of the PI3K family. A dual PI3-kinase 5 / y inhibitor is therefore more selective for PI3-kinase 5 and y than conventional PI3K tors such as CAL-130, wortmannin and LY294002, which are nonselective PI3K inhibitors.

[87] Inhibition of PI3—kinase 5 and 7 may be of therapeutic benefit in treatment of various conditions, e. g., conditions characterized by an inflammatory response including, but not limited to, autoimmune es, ic diseases, and arthritic diseases.

Importantly, inhibition of PI3-kinase 8 and y function does not appear to affect ical functions such as viability and fertility.

[88] "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, tive response elicited by injury or destruction of tissues, which serves to destroy, dilute or wall off (sequester) both the injurious agent and the injured tissue. Inflammatory responses are notably associated with the influx of leukocytes and/or leukocyte (e.g., neutrophil) chemotaxis. Inflammatory responses may result from infection with pathogenic organisms and Viruses, noninfectious means such as trauma or reperfusion following myocardial infarction or stroke, immune responses to foreign ns, and autoimmune diseases. Inflammatory responses 17 WO 2014/195888 PCT/IB2014/061954 amenable to treatment with the methods and compounds according to the invention encompass conditions associated with reactions of the specific defense system as well as conditions associated with reactions of the non-specific defense system.

[89] The therapeutic methods of the invention include methods for the amelioration of conditions associated with atory cell activation. "Inflammatory cell activation" refers to the induction by a stimulus (including but not d to, cytokines, antigens or auto-antibodies) of a proliferative cellular response, the production of soluble mediators (including but not limited to cytokines, oxygen radicals, enzymes, prostanoids, or vasoactive ), or cell surface expression of new or increased numbers of mediators (including but not limited to, major histocompatibility antigens or cell adhesion molecules) in inflammatory cells (including but not limited to monocytes, macrophages, T lymphocytes, B lymphocytes, ocytes (polymorphonuclear leukocytes including neutrophils, ils, and eosinophils) mast cells, dendritic cells, Langerhans cells, and endothelial cells). It will be appreciated by persons d in the art that the activation of one or a combination of these phenotypes in these cells can contribute to the initiation, perpetuation, or exacerbation of an inflammatory ion.

[90] "Autoimmune disease" as used herein refers to any group of disorders in which tissue injury is associated with humoral or cell-mediated responses to the body's own constituents.

[91] "Transplant rejection" as used herein refers-to any immune response directed against d tissue (including organs or cells (e. g., bone marrow), characterized by a loss of function of the grafted and surrounding tissues, pain, swelling, leukocytosis, and thrombocytopenia).

[92] "Allergic disease" as used herein refers to any symptoms, tissue damage, or loss of tissue function resulting from y.

[93] "Arthritic disease" as used herein refers to any disease that is characterized by inflammatory lesions of the joints attributable to a variety of etiologies.

[94] "Dermatitis" as used herein refers to any of a large family of diseases of the skin that are characterized by inflammation of the skin attributable to a variety of etiologies.

[95] As previously bed, the term “dual PI3-kinase 5 / y selective inhibitor" generally refers to a compound that ts the activity of the PI3—kinase 5 and y e more ively than other isozymes of the PI3K family. The relative efficacies of compounds as inhibitors of an enzyme activity (or other ical activity) can be ished by determining the trations at which each compound inhibits the activity 18 WO 2014/195888 PCT/IB2014/061954 to a predefined extent and then ing the results. Typically, the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or "IC50". IC50 determinations can be accomplished using conventional techniques known in the art. In general, an IC50 can be determined by ing the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study. The mentally obtained values of enzyme activity then are plotted against the tor 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 be defined through appropriate determinations of ty. For example, in some settings it can be desirable to establish a 90% inhibitory concentration, i.e., ICgo, etc.

[96] Accordingly, a dual PI3-kinase 6 / y ive inhibitor alternatively can be tood to refer to a compound that exhibits a 50% inhibitory concentration (IC50) with respect to PI3—kinase 5 and y, that is at least 10-fold lower, at least 20—fold lower, or at least 30-fold lower than the IC50 value with respect to any or all of the other class I PI3K family members. In an alternative embodiment of the invention, the term dual PI3—kinase 8 / y selective inhibitor can be tood to refer to a compound that exhibits an IC50 with respect to PI3—kinase 5 and y that is at least 30—fold lower, at least 50—fold lower, at least lOO-fold lower, at least ZOO-fold lower, or at least 500-fold lower than the IC50 with respect to any or all of the other PI3K class I family members. A dual PI3-kinase 5 / 7 selective inhibitor is typically administered in an amount such that it selectively inhibits both PI3-kinase 8 and 7 activity, as bed above.

[97] In certain embodiments, the compounds of the present invention exhibit PI3-kinase 5 and y inhibition almost equally (~ 1:1) or at a maximum ratio of 1:5, i.e., the nd the of the present invention exhibit almost equal IC50 values for both P13— kinase 5 and y enzyme or at most a 3 to 8 fold difference between the two. ,

[98] The methods of the ion 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 eutically or prophylactically in an individual. "Ex vivo" or “in vitro” means outside of a living individual. Examples of ex vivo cell populations include in vitro cell cultures and biological samples including but not limited to fluid or tissue samples obtained from individuals. Such samples may be obtained by methods known in the art. Exemplary biological fluid s include blood, cerebrospinal fluid, urine, and saliva. Exemplary tissue samples include tumors and es f. In this context, the invention may be 19 WO 2014/195888 2014/061954 used for a variety of purposes, including therapeutic and experimental purposes. For example, the ion may be used ex vivo or in vitro to ine the optimal schedule and/or dosing of administration of a PI3—kinase 5 selective inhibitor for a given indication, cell type, individual, and other parameters. Information gleaned from such use may be used for experimental or diagnostic purposes or in the clinic to set protocols for in vivo treatment. Other ex vivo uses for which the invention may be suited are described below or will become apparent to those skilled in the art.

[99] The compounds of the present ion can be prepared by methods known in the art, such as those described in International Publication Nos. WO 2011/055215, WO 51525, and WO 2013/164801, all of which are hereby incorporated by reference.

Pharmaceutical Compositions

[100] The invention provides a pharmaceutical composition sing one or more compounds of the present invention and one or more pharmaceutically acceptable carriers or excipients. In one embodiment, the pharmaceutical composition includes a therapeutically effective amount of a compound of the present invention. The pharmaceutical composition may include one or more additional active ingredients as described herein.

[101] The pharmaceutical carriers and/or excipients may be selected from diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release es, colorants, ngs, buffers, stabilizers, solubilizers, and combinations thereof.

[102] In one embodiment, the pharmaceutical compositons bed herein contain from about 0.1 mg to about 1,000 mg, such as from about 1 mg to about 1,000 mg or from about 20 mg to about 800 mg or 50 mg to about 600 mg or 50 mg to about 600 mg of one or more compounds of the present invention. 100 mg to about 400 mg of one or more compounds of the present invention.

[103] The pharmaceutical compositions of the t invention can be administered alone or in combination with one or more other active . Where desired, the subject compounds and other agent(s) may be mixed into a preparation or both components may be formulated into te preparations to use them in combination separately or at the same time.

[104] The compounds and pharmaceutical compositions of the present invention can be administered by any route that enables delivery of the compounds to the site of 20 WO 2014/195888 PCT/IB2014/061954 action, such as orally, asally, topically (e.g., transdermally), intraduodenally, parenterally (including intravenously, intraarterially, intramuscularally, intravascularally, intraperitoneally or by injection or infusion), intradermally, by intramammary, intrathecally, intraocularly, retrobulbarly, intrapulmonary (e. g., aerosolized drugs) or subcutaneously (including depot stration for long term release e.g., embedded-under the-splenic capsule, brain, or in the cornea), sublingually, anally, rectally, vaginally, or by surgical implantation (e.g., embedded under the splenic capsule, brain, or in the cornea).

[105] The itions can be administered in solid, olid, liquid or gaseous form, or may be in dried powder, such as lyophilized form. The pharmaceutical compositions can be packaged in forms ient for delivery, including, for example, solid dosage forms such as es, sachets, cachets, gelatins, papers, tablets, suppositories, pellets, pills, troches, and lozenges. The type of ing will generally depend on the desired route of administration. Implantable sustained release formulations are also contemplated, as are transdermal formulations.

Methods of Treatment

[106] The amount of the compound to be administered is dependent on the mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the ibing physician. However, an ive dosage is in the range of about 0.001 to about 100 mg/kg body weight per day, preferably about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, ably about 0.05 to about 2.5 gfday An effective amount of a compound of the invention may be administered in either single or le doses (e. g., twice or three times a day).

[107] The compounds of the present invention may be used in combination with one or more of anti-cancer agents (e. g., chemotherapeutic agents), therapeutic dies, and radiation treatment.

[108] The compounds of the invention may be formulated or administered in conjunction with other agents that act to relieve the symptoms of inflammatory conditions such as encephalomyelitis, asthma, and the other diseases described herein. These agents include non-steroidal anti-inflammatory drugs (NSAIDs).

EXAMPLES

[109] The examples and preparations ed below further illustrate and exemplify the compounds of the present invention and methods of preparing such compounds. It is to be understood that the scope of the present invention is not limited in 21 WO 2014/195888 PCT/IB2014/061954 any way by the scope of the following examples and preparations. In the following examples molecules with a single chiral center, unless otherwise noted, exist as a racemic mixture. Those molecules with two or more chiral s, 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.

[110] As used herein, superscript 1 refers to International Publication No. W0 11/055215 and superscript 2 refers to International Publication No. W0 12/151525. These references describe how various intermediates are prepared.

Intermediates Intermediate 1: 3-(3-flu0rophenyl)(1-hydr0xypropyl)-4H-chromenone: To a solution of 2—(1-bromopropy1)(3-fluorophenyl)-4H-chromenone1 (8.80 g, 24.36 mmol ) in DMSO (85 ml), n—butanol (5 ml) was added and heated to 120° C for 3h. The reaction mixture was cooled to room temperature (RT), quenched with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated under d pressure. The crude product was purified by column chromatography with ethyl acetate: petroleum ether to afford the title compound as a yellow solid (2.10 g, 29 %) which was used without further purification in next step.

Intermediate 2: 3-(3-flu0r0phenyl)pr0pi0nyl-4H-chr0men0ne: DMSO (1.90 ml, 26.82 mmol) was added to dichloromethane (70 ml) and cooled to -78°C. Oxalyl chloride (1.14 ml, 13.41 mmol) was then added. After 10 minutes, intermediate 1 (2.00 g, 6.70 mmol) in dichloromethane (20 ml) was added dropwise and stirred for 20 min. ylamine (7 ml) was added and stirred for 1h. The reaction mixture was quenched with water and extracted with dichloromethane. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude t was purified by column chromatography with ethyl acetate: eum ether to afford the title compound as a yellow liquid (1.20 g, 60%) which was used as such in next step.

Intermediate 3: (+)/(—)—3-(3-flu0rophenyl)(1-hydroxypropyl)-4H-chr0men0ne : To a on of intermediate 2 (0.600 g, 2.02 mmol) in DMF (7.65 ml) under nitrogen purging, formic acid : lamine 5 : 2 azeotrope (1.80 ml) was added followed by [(S,S)tethTstenRuCl] (3.0 mg). The reaction mixture was heated at 80°C for 1.5 hours under continuous en g. The reaction mixture was quenched with water, extected with ethyl acetate, dried over sodium sulphate and concentrated. The crude t was purified by column chromatography with ethyl e: petroleum ether to afford the title compound as a yellow solid (0.450 g, 74%). Mass: 299.0 (M+). 22 WO 2014/195888 PCT/IB2014/061954 Enantiomeric excess: 78%, enriched in the late eluting isomer (retention time: 9.72 min.) as determined by HPLC on a chiralpak AD—H column.

Intermediate 4: (+)/(-)(3-flu0r0phenyl)(1-hydr0xypr0pyl)-4H-chr0men0ne : The title nd was obtained as yellow solid (0.500 g, 83%) by using a procedure similar to the one described for intermediate 3, using intermediate 2 (0.600 g, 2.02 mmol), DMF (7.65 ml), formic acid : trietylamine 5 : 2 ope (1.80 ml) and [(R,R)tethTstenRuCl] (3.0 mg). Mass: 298.9 (M+). Enantiomeric excess: 74.8%, enriched in the fast eluting isomer (retention time: 8.52 min.) as determined by HPLC on a chiralpak AD-H column.

Intermediate 5: (3-flu0r0phenyl)(1-hydr0xypr0pyl)-4H-chr0men0ne: Step 1 : (R)(1-(benzyloxy)propyl)(3-fluorophenyl)-4H-chromenone: To 2-(3- fluorophenyl)(2-hydroxyphenyl)ethanone (2.15 g, 9.36 mmol ), in dichloromethane ( 20 ml), HATU (4.27 g, 11.23 mmol), R-(+)2-benzyloxybutyric acid (2.00 g, 10.29 mmol) were added and stirred for 10min, then triethylamine (14.0 ml, 101.1 mmol) was added dropwise and stirred at RT for 24h. The reaction mixture was quenched with water, extracted with romethane, dried over sodium sulphate and concentrated under reduced pressure. The crude product was ed by column chromatography with ethyl acetate: petroleum ether to afford the title compound as yellow solid (1.65 g, 45%). 1H- NMR (8 ppm, CDC13, 400 MHz): 8.24 (dd, J = 79,15 Hz, 1H), 7.74 (dt, J = 7.1,1.7 Hz, 1H), 7.58 (dd, J: 8304 Hz, 1H), .06 (m, 10H), 4.51 (d, J: 7.8 Hz, 1H), 4.34 (d, J = 7.8 Hz, 1H), 4.25 (dd, J = 7862 Hz, 1H), 2.17-1.90 (m, 2H), 0.95 (t, J = 7.5 Hz, 3H).

Mass: 389.0 (M+).

Step 2: (R)-3—(3-fluorophenyl)-2—(1-hydroxypropyl)-4H-chromen-4—one : To (R)-2—(l- (benzyloxy)propyl)(3-fluorophenyl)-4H-chromenone (1.50 g, 3.86 mmol) in dichloromethane (15 ml) cooled to 0°C and aluminium chloride (1.00 g, 7.72 mmol) was added portion wise and stirred at RT for 6h. The reaction mixture was quenched with 2N HCl on, ted with dichloromethane, dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography with ethyl acetate: petroleum ether to afford the title compound as yellow solid (0.552 g, 48%).‘1H-NMR (5 ppm, CDClg, 400 MHz): ' 8.24 (dd, J: 8.0,1.6 Hz, 1H), 7.72 (m, = 84,05 Hz, 1H), 7.44 (m, 2H), 7.12-7.01(m,3H), 4.49 , 1H), 7.52 (dd, J (t, J = 7.0 Hz, 1H), 1.94 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H). Mass: (299.0(M+). Purity: 96.93%. [a]25D —14.73 (c = 1, CHCl3). Enantiomeric : 85.92%, enriched in the fast eluting isomer (retention time: 8.57 min.) as determined by HPLC on a chiralpak AS-3R column. 23 WO 2014/195888 PCT/IB2014/061954 Compound A (RS)- 2-(1-(9H-purinylamino)propyl)(3-fluorophenyl)-4H-chromenone To a solution of intermediate 1 (2.50 g, 8.41 mmol) in THF (25 ml), tert-butyl 9-trityl-9H- 6-ylcarbamate (4.81 g, 10.09 mmol) and triphenylphosphine (3.31 g, 12.62 mmol) were added and stirred at RT for 5 min. ropylazodicarboxylate (2.5 ml, 12.62 mmol) was added and stirred at RT for 2h. The reaction mixture was concentrated and column chromatographed with ethyl acetate : petroleum ether to afford a yellow coloured intermediate. To the intermediate, romethane (65 ml) and trifluoroacetic acid (7.9 ml) were added and the resulting mixture was stirred at RT for 12 h. The reaction mixture was then basified with aqueous sodium bicarbonate solution, extracted with dichloromethane and dried over sodium sulphate. The crude product was purified by column chromatography with ol: dichloromethane to afford the title compound as pale—brown solid (1.05 g, 30 %). MP: 148-150°C. Mass: 415.6 (M+).

Compound A1 (S)(1-(9H-purinylamino)propyl)(3-fluorophenyl)-4H-chromenone Method A: To a solution of intermediate 3 (0.250 g, 0.838 mmol) in THF (5ml), tert— butyl 9-trityl-9H-purin—6-ylcarbamate (0.479 g, 1.00 mmol) and triphenylphosphine (0.329 g, 1.25 mmol) were added and the resulting e was stirred at RT for 5 min.

Diisopropylazodicarboxylate (0.25 ml, 1.25 mmol) was then added and d at RT for 12 h. The reaction mixture was concentrated and column chromatographed with ethyl acetate: pet.ether to afford the yellow coloured intermediate. To the intermediate in dichloromethane (6 ml), roacetic acid (1.2 ml) was added d at RT for 12 h. The reaction mixture was basified with aqueous sodium bicarbonate solution, extracted with dichloromethane and dried over sodium sulphate. The crude product was purified by column chromatography with ol: dichloromethane to afford the title compound as an off-white solid (0.015 g, 4 %). MP: 137-140°C. 1H-NMR (5 ppm, DMSO'dfi, 400 MHz): 12.94 (s, 1H), 8.12-8.10 (m, 4H), 7.84-7.80 (m, 1H), 7.61 (d, J: 8.3 Hz, 1H), 7.50- 7.41 (m, 2H), 7.28-7.18 (m, 3H), 5.20-5.06 (m, 1H), 2.10-1.90 (m, 2H), 0.84 (t, J: 3.7 Hz, 3H). Enantiomeric excess: 77.4% as determined by HPLC on a chiralpak AD-H column, enriched in the fast eluting isomer (retention time = 7.90 min). 24 WO 2014/195888 PCT/IB2014/061954 Method B : To a solution of intermediate 5 (2.60 g, 8.68 mmol) in THF (52 ml), tert— butyl 9-trityl-9H-purinylcarbamate (4.96 g, 10.42 mmol) and triphenylphosphine (2.76 g, 13.03 mmol) were added and the resulting mixture was stirred at RT for 5 min.

Diisopropylazodicarboxylate (0.25 ml, 1.25 mmol) was then added and stirred at RT for 12 h. The reaction mixture was concentrated and column chromatographed with ethyl acetate: petroleum ether to afford the yellow coloured intermediate. To the intermediate in dichloromethane (55 ml), trifluoroacetic acid (14.2 ml) was added and stirred at RT for 12 h. The reaction mixture was basified with aqueous sodium bicarbonate on, extracted with dichloromethane and dried over sodium sulphate. The crude product was purified by column chromatography with methanol: dichloromethane to afford the title compound as pale—yellow solid (1.00 g, 27 %). MP: 168—170°C. Mass: 416.5(M++1) Enantiomeric : 86.5% as determined by HPLC on a pak AD-H column, enriched in the fast eluting isomer (retention time = 7.90 min.).

Method C : The title compound was separated by preparative SFC conditions from Compound A (1.090 g) on a CHIRALPAK AY-H column (250 x 30 mm; Sum) using methanol : C02 (35:65) as the mobile phase at a flow rate of 80 g / min. Off-white solid (0.378 g). e.e. 100%. Rt: 2.37 min. Mass: 416.1(M++1). MP: 149—152°C.

Compound A2 (R)(1-(9H-purinylamino)propyl)(3-fluorophenyl)-4H-chromenone Method A: The title compound was obtained as an off-white solid (0.015 g, 4 %) by using a procedure similar to the one bed for compound A1 (Method A) using utyl 9— trityl-9H-purinylcarbamate (0.479 g, 1.00 mmol), intermediate 4 (0.250 g, 0.838 mmol), triphenylphosphine (0.329 g, 1.25 mmol), THF (5 ml) and ropylazodicarboxylate (0.25 ml, 1.25 mmol), followed by the cleavage of the intermediate with trifluoroacetic acid (1.2 ml) and dichloromethane (6 ml). MP: 139-141°C. Mass: 415.6 (M+).

Enantiomeric excess: 81.6% as determined by HPLC on a chiralpak AD-H , enriched in the late eluting isomer (retention time = 10.81 min.) Method B : The title compound was separated by preparative SFC conditions from nd A (1.090 g) on a CHIRALPAK AY-H column (250 x 30 mm; Sum) using methanol : C02 (35:65) as the mobile phase at a flow rate of 80 g / min. ite solid (0.434 g). e.e. 98%. Rt: 3.71 min. Mass: 416.1(M++1). MP: 162—164°C. 25 WO 2014/195888 PCT/IB2014/061954 BIOLOGICAL ASSAYS

[111] The pharmacological properties of the compounds described herein may be confirmed by a number of pharmacological assays. The cological assays which have been carried out with the compounds according to the invention and/or their pharmaceutically acceptable salts are exemplified below Assay 1: Fluorescent Determination of P13 Kinase Enzyme Activity

[112] Phosphoinositide 3 kinases (PI3K) belong to a class of lipid kinases that play a critical role in the regulation of several key cellular processes. The PI3K are e of phosphorylating the oxy position of phosphoinositols thereby generating second gers involved in downstream signalling events. The homogenous time resolved fluorescence (HTRF) assay allows detection of 3,4,5—triphosphate (PIP3) formed as a result of orylation of phosphotidylinositol 4,5-biphosphate (PIP2) by PI3K isoforms such as a, [3, y or 8.

[113] PI3K m activity for 0t, [5, y or 5 was determined using a PI3K human HTRFTM Assay Kit (Millipore, Billerica, MA) with modifications. All incubations were carried out at room temperature. Briefly, 0.5 ul 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) ning 14.5 ul 1X on buffer /PIP2 (10 mM MgClz, 5 mM DTT, 1.38 uM PIP2) mix with or without enzyme, followed by 5 ul/well of 400 uM ATP and incubated for an additional 30 minutes. Reaction was terminated by adding 5 ul/well stop solution pore, Billerica, MA). 5 pl of detection mix (Millipore, Billerica, MA) were then added to each well and was incubated for 6-18 hours in the dark. HRTF ratio was measured on a microplate reader (BMG Labtech., y) at an excitation wavelength of 337 nm and emission wavelengths of 665 and 615 nm with an integration time of 400 msec counting delay of 50 msec. The results for Compounds A1 and A2 are shown in Table 1 below. Comparative data for Compound A1 and Example 47 of WO 11/055215 are provided in Table 2.

TABLE 1 IC50 (nM) Compound ----Pi3K5 Pi3K 0c Pi3K [5 Pi3K y A1 >4000 24.05 A2 ND >10 uM WO 95888 PCT/IB2014/061954 TABLE 2 PI3K 8 PI3K y % Inhibition at 1 mm Example 47 of 105.9 25.54 ND Compound A1 23.85 _ 24.05 Assay 2: In Vitro Cell Proliferation Assay in Leukemic Cell Lines

[114] 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 tration range from 0.01 to 10000 nM were added after 24 h.

Growth was assessed using the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide (MTT) dye reduction test at 0 h (prior to the addition of the test compound) and 72 h after the addition of test compound. Absorbance was read on a Fluostar Optima (BMG Labtech, y) at a wave length of 450 nm. Data were analysed using GraphPad Prism and percent tion due to the test nd compared to the control was calculated accordingly.

[115] Compound A1 caused a reduction in T-lymphoma (MOLT-4, Jurkat, CCRF—CEM, Hut-78 & HuT-102) cell ity with G150 values ranging from 1—5 uM for the dose range tested. Additionally, the compound did not display any apparent cytotoxicity over the 72-h incubation period up to 10 uM.

Assay 3: Inhibition of AKT phosphorylation in leukemic cell lines

[116] MOLT-4, , CCRF—CEM, Hut-78, HUT-102, $624 and HH cells were incubated with desired concentrations of compound for 48 h. Cells were lysed and pAKT determined by Western Blotting. Bands were quantified using ImageJ and normalized to actin.

[117] Compound A1 caused a reduction in pAKT expression in T-lymphoma 4, Jurkat, CCRF—CEM, Hut-78 & HuT—102) cell lines with EC50 values ranging from 0.5-2 [1M for the dose range tested. The results are shown in Figure 8.

Assay 4: Inhibition of PI3K 6 and y signalling in basophils from Human Whole Blood 27 WO 95888 PCT/IB2014/061954

[118] PI3K 5 and y signalling in basophils manifested by an tion of anti— FcaRl or fMLP induced CD63 expression is a useful pharmacodynamic marker determined using the AST® kit (Buhlmann Laboratories, Switzerland). Briefly, it involves the following steps: 0 Mix the anti-coagulated blood sample by inverting the venipuncture tube several times; 0 Prepare fresh and pyrogen—free 3.5 ml polypropylene or polystyrene tubes suitable for Flow Cytometry measurements; 0 Add 49 ul of patient’s whole blood to each tube; 0 Add 1 ul of 10% DMSO (background) or compound (10% DMSO) to the assigned tubes and mix gently. Incubate at room temperature for 15 minutes; 0 Pipet 50 ul of the ation buffer (background) or anti- FcaRI Ab or or fMLP to each tube; 0 Add 100 pl of Stimulation Buffer to each tube; 0 Mix gently. Add 20 ul Staining Reagent (1:1 mix of FITC-CD63 and PE— CCR3) to each tube; 0 Mix gently, cover the tubes and incubate for 15 minutes at 37°C in a water bath. (using an incubator will take about 10 minutes longer incubation time due to less efficient heat transfer); 0 Add 2 ml pre—warmed (18-280C) Lysing Reagent to each tube, mix gently; 0 Incubate for 5 -10 minutes at 18-280C; 0 Centrifuge the tubes for 5 minutes at 500 x g; 0 Decant the supernatant by using blotting paper; 0 Resuspend the cell pellet with 300—800 ul of Wash ; and 0 Vortex gently and acquire the data on the flow cytometer within the same day.

[119] Percent CD63 positive cells within the gated il tion were determined in different treatment groups and normalized to e control.

[120] Compound A1 exhibited a EC50 of < 40 nM for chR1(PI3K 5) and a IC50 of < 40 nM for fMLP ( PI3K y) ( n=10). The results are shown in Figure 9.

Assay 4A: Cellular Activity Demonstrating ivity of Compound A1 towards PI3K Delta and PI3K Gamma Isoform Assay 4A1: Anti-IgM induced B-Cell Proliferation (for PI3K6 selectivity) 28 WO 2014/195888 PCT/IB2014/061954

[121] The ive of this study was to assess the inhibitory potential of Compound Al on gM induced human B-cell proliferation.

Plating and treatment Isolated B-cells were re-suspended to 1.0 X 106 cells per ml. 100 ul of cell suspension was added to each well of a 96-well plate. Triplicates were ined. 50 ul of drug dilution was added and mixed well. A DMSO blank and inducer blank were maintained.

Treated plate was incubated for 30 min at 37°C, 5% C02 and then 50 ul of 4X inducer was added and mixed by ing.

Plate was ted at 37°C, 5% C02 for 72 h.

Media was aspirated and 150 pl of DMSO was added to dissolve the formazan crystals.

Absorbance was read at A560 and A640 nm.

[122] The data demonstrates the inhibitory potential of Compound Al on PI3K8 mediated induction of human B-cell proliferation. See, e.g., Backer er al. Journal of Immunology, 134: 3532-3538, 1985.

Assay 4A2: LPA induced AktS473 Phosphorylation in 3T3 Fibroblasts (for PI3KB Selectivity)

[123] The objective of this study was to determine the effect of Compound Al on PI3KB kinase mediated LPA induced AktS473 phosphorylation in 3T3 fibroblasts. 3T3 cells were treated with desired concentrations of the test compound for 15 min. 1 ml of 2X LPA was added such that the final concentration was 5 uM and incubated for 5 min.

Media was discarded and washed with 1 ml of ld 1X PBS. 250 pl of cell lysis buffer was added and incubated on ice for 30 min.

Samples were centrifuged and supernatant was at —80°C until is.

Samples were analyzed by Western Blotting using pAKT (S473) as the primary and anti-rabbit IgG-HRP as a secondary antibody.

Intensity of the bands was determined using ImageJ 1.42q (NIH, USA) and normalized to Actin (loading control). Data was d using GraphPad Prism (Version 5.02). 29 WO 2014/195888 PCT/IB2014/061954

[124] The results demonstrate the selectivity of nd A1 over the beta isoform of PI3K. See erque et al., J. Biol. Chem. 278, 39830-39838, 2003.

Assay 4A3: c5a Induced AktS473 Phosphorylation in RAW 264.7 Macrophages (for PI3K7 Selectivity)

[125] The objective of this study was to determine the effect of Compound Al on PI3Ky kinase mediated c5a induced AktS473 phosphorylation in RAW 264.7 macrophages. 0 RAW 264.7 cells were treated with desired concentrations of the test nd for 15 min. 1 ml of 2X c5a was added such that the final concentration was 50 ng/ml and incubated for 15 min. 0 Media was ded and washed with 1 ml of ice—cold 1X PBS. 0 250 pl of cell lysis buffer was added and incubated on ice for 30 min. 0 Samples were centrifuged and supernatant was stored at -80°C until analysis 0 Samples were analyzed by Western Blotting using pAKT (S473) as the primary and abbit IgG-HRP as a secondary antibody. 0 ity of the bands was determined using ImageJ 1.42q (NIH, USA) and normalized to Actin (loading control). Data was plotted using GraphPad Prism on 5.02).

[126] Inhibition of pAktS473, a downstream marker of PI3K8 signaling suggests a role 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 Selectivity)

[127] The objective of this study was to determine the effect of Compound Al on PI3KOL kinase mediated AktS473 phosphorylation in PDGF induced 3T3 fibroblasts. 0 3T3 cells were treated with desired trations of the test compound for 15 min. 1 ml of 2X PDGF was added such that the final concentration was 20 ng/ml and incubated for 10 min. 0 Media was discarded and washed with 1 ml of ice-cold 1X PBS. 0 250 pl of cell lysis buffer was added and incubated on ice for 30 min. 0 Samples were centrifuged and supernatant was collected and stored at -80°C until analysis. 30 WO 95888 PCT/IB2014/061954 0 Samples were ed by Western Blotting using pAKT (S473) as the primary and anti-rabbit IgG-HRP as a secondary antibody. 0 Intensity of the bands was determined using Image] 1.42q (NIH, USA) and normalized to Actin (loading control). Data was plotted using GraphPad Prism (Version 5.02).

[128] No inhibition was observed at 10 uM of Compound Al, demonstrating the selectivity of Compound A1 over the alpha isoform of PI3K. See Albuquerque er al., J.

Biol. Chem. 278, 39830-39838, 2003.

[129] The Table below summarizes the results from Assyas 4.

CELLULAR ACTIVITY DEMONSTRATING SELECTIVITY OF COMPOUND Al TOWARDS PI3K DELTA AND PI3K GAMMA M Cellular IC50 PI3K alpha (PDGF induced pAKT in 3T3 fibroblasts) >10000 nM Cellular IC50 PI3K beta (LPA induced pAKT in 3T3 lasts) 2067 nM Cellular IC50 PI3K delta (anti-IgM induced human B-cell proliferation 38.1 nM Cellular IC50 PI3K gamma (05a induced pAKT in RAW macrophages) 223 nM Assay 5: tion of Apoptosis in Leukemic Cell Lines

[130] Apoptosis in leukemic cells was determined using an in-situ Caspase 3 kit (Millipore, US) as outlined below: 0 Seed leukemic cells - at a density of 1X106 cells/well in a 6 well plate 0 Add test compound/DMSO at desired concentrations 0 Incubate the plate for 24 hrs at 37°C in 5% C02 incubator 0 Collect cells in a 2ml fuge tube 0 Add 1.6 uL of y prepared 5X FLICA reagent and mix cells by slightly flicking the tubes 0 Incubate tubes for 1 hour at 37°C under 5% C02 0 Add 2 ml of lX wash buffer to each tube and mix 0 Centrifuge cells at <400 x g for 5 minutes at room temperature. 0 Carefully remove and discard supernatant, and gently vortex cell pellet to disrupt any cell-to-cell clumping. 0 Resuspend cell pellet in 300ul of lX wash buffer 0 Place 100 uL of each cell suspension into each of two wells of a black microtiter plate. Avoid creation of bubbles. 0 Read ance of each microwell using an excitation wavelength of 490 nm and an emission wavelength of 520 nm 31 WO 2014/195888 PCT/IB2014/061954 0 Percent increase in caspase-3 activity manifested by an increase in cence compared to the control blank is to be calculated.

[131] Compound Al caused a dose-dependent induction in caspase-3 activity in T-lymphoma (MOLT-4, Jurkat, CCRF-CEM, Hut-78 & HuT-102) cell lines.

Assay 6: Screening for pAKT Inhibition in Human Primary CTCL Cells

[132] Flow cytometry analysis of pAKT inhibition: Purified malignant T cells were isolated from donors and cultured overnight in RPMI/1% BSA. Cells were incubated with the test nd for 1.5 hr with a cytokine mix added for the final 30 minutes. The composition of the cytokine mix was 20 ng/ml 1L2 + 5 ng/ml 1L7 + 10 ng/ml IL15 + 10% FBS. AKT phosphorylation was determined by flow cytometry.

[133] Treatment with Compound A1 caused a dose dependent reduction AKT orylation with EC50 ranging from 40-300 nM (% inhibition data).

Assay 6A: Screening for Anti-Cancer Activity in Human CLL Cells

[134] Primary CLL cells were enriched using Rosette-Sep B cells from Stem Cell Technology, generally giving purity of > 97% of B cells/CLL cells. Cells were seeded at 2.5 x 105 per well in a 96 well with either serum free medium (SFM) or SFM + 10% heat- inactivated fetal bovine serum (volume 100 microliters) in the presence of desired concentrations of the test compound and ed for 3 days at 370 C in a carbon dioxide incubator. Cytotoxicity was determined using the MTS assay.

[135] Compound A1 induces cytotoxicity in CLL cells with a median ECSO of < 100 nM in serum-free and < 700 nM in 10% FBS media.

Assay 6B: Screening for Anticancer Activity in Patient Derived B-Lymphoma Cells

[136] Primary cells from lymphoid tumors were exposed to the test compound [Compound Al] to assess the induction of cell death. Cells were derived from diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), splenic al zone lymphoma (SMZL), extranodal al zone ma (EMZL), or chronic lymphocytic leukemia (CLL, no.=l). Cells were d with desired concentrations of the compound and apoptosis (Annexin V/PI) was ed by flow-cytometry after a 48-h incubation .

[137] Almost all the primary cells d from B-cell lymphomas underwent an increase in cell death when exposed to the test compound (Compound A1) at a 32 WO 2014/195888 PCT/IB2014/061954 concentration of 4 uM. The phenomenon appeared more evident among primary cells derived from small cell lymphomas (MZL, MCL, and CLL).

Assay 6C: Inhibition of AKT orylation in B-Lymphoma Cell Lines

[138] LY-l, LY-10, Daudi, JEKO, REC and MAVER cells were incubated with desired concentrations of [compound Al] for 48 hours. Cells were lysed and pAKT was determined by Western Blotting. Bands were quantified using ImageJ and normalized to actin.

[139] Compound A1 exhibited an EC50 of 20 — 200 nM across the B-lymphoma cell lines tested.

Assay 6D: Cytokine Assay in Anti-Human CD3 and CD28 Vo-Stimulated Primary T- Cells

[140] The ive of this study was to assess the inhibitory potential of Compound Al on cytokines produced by anti-human CD3/CD28 co-stimulated primary T- cells.

Plating and Treatment 0 Plates were coated with 50 ul of anti-human CD3 at a concentration of 100 ng/ml either overnight at 4°C or for 2 h at 37°C. After incubation, plates were washed twice with sterile PBS to remove unbound antibody. 0 Isolated human T-cells were re—suspended to 0.625 x 106 cells per ml in 1.5ml tubes and 1 ul of drug dilution ) was added. A DMSO blank and un- induced blank were maintained. 0 Cells were incubated with compound at room temperature for 30 min and then added to uman CD3 coated wells of a 96-well plate, 240 pl each. Anti- human CD28 was added immediately, 10 ul (25X) per well. 0 Plate was incubated at 37°C, 5% C02 for 24 h. 0 Plates were centrifuged at 4000 rpm at room temperature, supernatant was collected and stored at —20°C. 0 Cytokines were determined using a commercial ELISA kit with absorbance read at 450 and 570 nm.

[141] IC50 values were ated from eight independent ments.

Compound A1 inhibited anti-human 28 induced T cell cytokines with an IC50 of 33 WO 2014/195888 PCT/IB2014/061954 24, 9.54 and 20.6 nM for TNFOL, IFNy and IL2 respectively. The results are shown in Figure 10.

Assay 7: lysaccharide Induced ary Neutrophilia in Female Wistar Rat Model

[142] An exaggerated recruitment and subsequent activation of neutrophil is likely to be important for the development and course of several inflammatory diseases in the airways and lungs, such as severe asthma, chronic ctive pulmonary e, cystic fibrosis, and acute atory distress syndrome. The mechanisms by which neutrophil bute to these diseases may e the release of proteolytic enzymes, such as phil elastase, and free oxygen radicals. When released, these compounds can cause bronchoconstriction, bronchial hyperreactivity, hyper-secretion, epithelial damage, and tissue remodelling in the airways.

[143] After the quarantine period, fasted animals were randomized and divided into various groups depending on their body s. The test compound [Compound Al] was prepared as a suspension in a e consisting of 0.5% methylcellulose in which Tween 80 as a suspending agent. The compound or vehicle was administered by oral gavage in a volume of lOmL/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 h after LPS instillation, animals were exsanguinated under hesia, and then a was cannulated and the lungs were lavaged with 5-ml aliquots of heparinised PBS (1 unit/ml) four times through tracheal cannula (total volume 20 ml). Bronchoalveolar lavage (BAL) fluid was been stored at 2-8 C’C until assayed for total cell and differential leukocyte count. Bronchioalveolar fluid was centrifuged (500xg for 10 min) and the resulting cell pellet was resuspended in 0.5 ml of heparinised saline.

The total numbers of white blood cells were determined in BAL fluid or blood by using a blood cell counter and was adjusted to 1x106 cell/ml. Differential cell count was calculated ly. One hundred microliters of the cell suspension was centrifuged using cytospin 3 to prepare a cell smear. The cell smear was stained with a blood staining solution for differentiation and slides were microscopically observed to identify eosinophil according to their morphological characteristics. The number of each cell type among 300 white blood cells in the cell smear was determined and expressed as a percentage. The number of eosinophil in each BALf or blood was calculated.

[144] Compound Al showed a dose-dependent reduction of neutrophil infiltration into the lungs with an ED50 of 6.5 mg/kg suggesting a therapeutic role in inflammatory disorders. The results are shown in Figure l. 34 WO 2014/195888 PCT/IB2014/061954 Assay 8: Lipopolysaccharide-Induced Rat Air Pouch Model of Inflammation

[145] Leukocyte recruitment and the formation of pro—inflammatory mediators, including different cytokines, are the hallmark of an atory response. The air-pouch model was originally ped as a facsimile um for the study of inflammatory processes that occur in rheumatoid arthritis (RA). The model allows the differential quantification of leukocyte species that accumulate in the air-pouch wall (tissue) as well as those that transmigrate into the air-pouch cavity (lavage), and it allows the characterization of the chemokines and adhesion molecules responsible for diapedesis induced by a variety of inflammatory stimuli.

[146] Female Wistar rats (175—200 g) were acclimatized for seven days prior to the start of the experiment. Animals were randomly distributed to various groups based on their body weights. Animals were anaesthetised with ether and subcutaneous air pouches were made by injecting 20 ml of sterile air under the skin in the scapular area (day 0) and maintained with a second lO-ml ion of sterile—filtered air on day 4. On day 6, oral treatment was commenced l h prior to induction of inflammation by so. injection of LPS solution on day 6. A volume of 5-m1 of LPS solution dissolved in sterile saline (lOOug/kg) was injected into each pouch. Samples of pouch fluid were taken at 6 h after administration of LPS by flushing the pouch with 5 ml of sterile saline and withdrawing 4 ml of fluid. The numbers of ytes present in pouch fluid was determined microscopically using a haemocytometer. Differential cell content was determined by microscopic examination of fluid smears stained with Diff-Quik.

[147] Compound A1 caused a dose-dependent reduction of neutrophil migration into the rat air pouch with an ED50 of 2.6 mg/kg ting a therapeutic role in rheumatoid arthritis. The results are shown in Figure 2.

Assay 9: Lipopolysaccharide Induced TNF-oc Production

[148] Fasted female Wistar rats were randomized into different groups ing on their body weights. Test compound (Compound Al) was prepared as a suspension in a vehicle ting of 0.5% methylcellulose. The compound or vehicle was administered by oral gavage in a volume of lOmL/kg. LPS solution was administered intraperitoneally one hour after nd stration at a dose of 0.3 mg/kg. Blood was collected in serum separator tubes Via cardiac re ninety minutes after LPS ion. Serum was separated and stored at —20 CC and will be analysed for TNFa by ELISA. 35 WO 2014/195888 PCT/IB2014/061954

[149] Compound A1 reduced plasma TNFa concentrations suggesting a therapeutic role in inflammatory disorders (percent inhibition observed at 1, 3 and 10 mg/kg was 5%, 15%, and 40%, respectively). The results are shown in Figure 3.

Assay 10A: min Induced Pulmonary Eosinophilia in Male Guinea Pigs

[150] Airway inflammation and hyper-responsiveness (AHR) are hallmarks and distinguishing features of bronchial asthma. Provocation of pre-sensitized mice with the same allergen induces airway inflammation with preferential philic infiltration and, as a consequence, AHR. Pulmonary eosinophilia and airway lling in conjunction with altered neural l of airway tone and airway epithelial mation may contribute to AHR in asthma.

[151] After the quarantine period, 0.3 mL of blood samples was collected from orbital vein by retro-orbital plexus method from each individual animal and ed on a cell analyser (ADVIA 2120, Siemens). Based on their total cell count, guinea pigs were randomized and divided into various groups. Ear pinna was marked with an indelible marking pen for identification. On day 0, weights were recorded and animals were sensitized with 50ug of min (OVA) and 10 mg of alum solution (1 mL) intraperitoneally. On day 7 and day 14, the above sensitization protocol was repeated.

Animals were ed 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 mins later animals were exposed to 0.5 % w/v, 0.5% and 1% Ovalbumin challenge respectively. Control & sham group animals were treated with 0.5% w/v methyl ose (vehicle). Sham control groups were sensitized with 10 mg of alum on day 0, 7 & 14 and exposed to saline solution (SAL) with the same nebulization rate on day 19. 20 and 21. Twenty hours after last OVA challenge, airway hyperresponsiveness was measured by whole body plethysmograph against cumulative doses of methacholine challenge (75, 100, 125 & 150 ug/ml), after measuring the airway response, blood samples and BAL fluid was collected. Samples were analysed for total cell count by using neubuear r under microscope and differential leukocyte count was done manually.

[152] As depicted in the Figures 4A and 4B, nd A1 caused a icant dose dependent reduction in airway hyperresponsiveness against methacholine challenge of sensitized Guinea pigs.

[153] As depicted in the Figures 4C-4E, Compound A1 caused a icant dose dependent reduction in eosinophil infiltration into the bronchoalveolar lavage fluid of sensitized Guinea pigs. 36 WO 2014/195888 PCT/IB2014/061954 Assay 10B: Murine Asthma Model

[154] Airway inflammation and hyper—responsiveness (AHR) are hallmarks and distinguishing features of bronchial asthma. Provocation of pre-sensitized mice with the same allergen induces airway inflammation with preferential philic infiltration and, as a consequence, AHR. Pulmonary eosinophilia and airway remodelling in conjunction with altered neural control of airway tone and airway lial desquamation may contribute to AHR in asthma. After the quarantine period, based on their body weights, mice were randomized and divided into four groups (n=7). Tails were marked with an indelible g pen for identification. On day 0, s were recorded and animals were sensitized with IOOug of Ovalbumin and 10 mg of alum solution (0.2 mL) intraperitoneally. On day 7 and day 14, the above sensitization protocol was repeated. s were observed for any signs of illness or reaction to the sensitization up to day 24 and recorded if any. On day 24, 25, and 26, after the ent with test compound by oral gavage, 30 mins later animals were exposed to 10 % w/v min challengeControl and sham group animals were treated with 0.5% w/v methyl ose (vehicle). Sham control groups were sensitized with 10 mg of alum on day 0, 7 & l4 and exposed to saline solution with the same nebulization rate on day 24, 25 & 26. Forty eight hours after 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 was collected.

Samples were analysed for total cell count by using neubuear chamber under microscope and differential leukocyte count was done manually.As depicted in the Figures 5A and 5B, Compound A1 at a dose of 3 mg/kg caused a significant reduction in airway hyperresponsiveness against holine nge of ovalbumin sensitized mice.

[159] As depicted in the Figures 5C-5E, Compound A1 at a dose of 3 mg/kg caused a significant inhibition of eosinophil infiltration into the oalveolar lavage fluid of ovalbumin sensitized mice.

Assay 11: Collagen Induced Arthritis in Lewis Rats

[160] Female wistar rats were acclimatized for seven days prior to the start of the ment and were randomly distributed to various groups based on their body weights.

On day 0, s were treated by intradermal injection of 500 pg of bovine collagen type II emulsified with complete Freund’s adjuvant (IFA) containing MTB (4 mg/mL) delivered at the base of the tail. On day 7 after primary immunization, animals were treated 37 WO 2014/195888 2014/061954 by r injection of 300 ug C11 in incomplete Freund's adjuvant by intradermal injection at the base of the tail. Onset of arthritis in ankle joints usually became visually apparent between days 12 and 14. Animals were treated with test compound or vehicle (orally administered) from the day after onset of arthritis until end of the experiment (day 28) as a therapeutic group. Arthritis Scores were taken by visually examination for signs of joint inflammation regularly throughout the study period. Body weights and paw volumes, paw thickness has been taken on day 0, 3, 7, 10, 12, 14, 17 21, 24 and 28. On d28, at the end of the study, blood has been withdrawn at sy and processed to serum or plasma and all joints were taken and both fore paw and hind paws were fixed in 10% formalin for histopathology analysis after taking the small piece of tissue from each joint and stored at — 80°C for cytokine analysis in tissue homogenate. Clinical Scoring Criteria for Fore and Hind Paws: 0 = normal; 1 = one hind or fore paw joint affected or minimal diffuse erythema and ng; 2 = two hind or fore paw joints affected or mild diffuse erythema and ng; 3 = three hind or fore paw joints affected or moderate diffuse erythema and swelling; 4 = marked diffuse erythema and swelling, or = four digit joints affected; 5 = severe diffuse erythema and severe swelling entire paw, unable to flex digits.

[161] Compound Al dosed therapeutically in the rat CIA model demonstrates significant efficacy in reduction of knee and as well as ankle swelling.

[162] ogical analysis of joints at study end demonstrates complete structural preservation at 15 mg/kg Compound Al. For comparison, a vehicle-dosed animal shows synovial (S) inflammation and significant ce of bone resorption, pannus formation, and cartilage degradation and, as ed in Figures 6A-6D, Compound Al shows significant reduction in individual and summed histopathological scores for both knee and ankle.

Assay 12: Acute CSE Induced Cell ation in Male Balb/c Mice

[163] Animals (male Balb/c mice) are to be acclimatized for seven days prior to the start of the experiment. Animals are to be randomly distributed to various groups based on their body weights. On day 1, mice are to be administered the test compound or e by oral/intranasal route and after 1 hr the test compound stration the animals are to be anaesthetised with ether and cigarette smoke extract is to be administered by asal route in volume of SOul/mouse and repeated the CSE exposure to animals daily after the test compound administration for four days (dl to d4). On day 5, 24 hours after last CSE exposure animals are to be exsanguinated under anesthesia, and the trachea is to be cannulated and the lungs are lavaged with 0.5-ml aliquots of heparinised PBS (1 unit/ml) 38 WO 2014/195888 PCT/IB2014/061954 four times through tracheal cannula (total volume 2 m1). BAL stored at 2-8 0C until assayed for total cell and differential leukocyte count. Bronchioalveolar fluid is to be centrifuged (500xg for 10 min) and the resulting cell pellet has to be resuspended in 0.5 ml of heparinised saline. The total number of white blood cells is to be determined in BAL fluid and blood using a blood cell counter and adjusted to 1x106 cell/ml. Differential cell count is to be calculated manually. Forty microliters of the cell sion is to be centrifuged using cytospin 3 to prepare a cell smear. The cell smear is to be d with a blood staining solution for differentiation and microscopically has to be observed to fy 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 to be sed as a percentage, and the number of neutrophils and macrophages in each BALf are to be calculated.

Assay 13: Sub-chronic CSE d Cell Infiltration in Male Balb/c Mice

[164] Animals (male Balb/c mice) are to be acclimatized for seven days prior to the start of the experiment. Animals are to be randomly distributed to various groups based on their body weights. On day 1, animals are to be anaesthetised with ether and cigarette smoke extract is to be stered by intranasal route in volume of SOul/mouse and repeated the CSE exposure to animals daily for eight days (dl to d8). On day 9, mice are to be administered by test compound or vehicle by oral/intranasal route and after 1 hr test compound administration animals are to be anaesthetised with ether and cigarette smoke extract is to be administered by asal route in volume of 50ul/mouse and animals are to be d to CSE daily after the test compound administration for next three days (d9 to d1 1), on day 12, twenty four hours after last CSE re animals are to be exsanguinated under anesthesia, and the trachea is to be ated and the lungs are to be lavaged with 0.5-ml aliquots of heparinised PBS (1 unit/n11) four times h tracheal cannula (total volume 2 ml). BAL stored at 2-8 °C until assayed for total cell and differential leukocyte count. Bronchioalveolar fluid was centrifuged (SOOXg for 10 min) and the resulting cell pellet is to be resuspended in 0.5 ml of heparinised saline. The total numbers of white blood cells are to be determined in BAL fluid and blood using a blood cell counter and adjusted to l><106 cell/ml. Differential cell count was calculated ly.

Forty microliters of the cell suspension is to be centrifuged using cytospin 3 to prepare a cell smear. The cell smear is to be stained with a blood staining solution for differentiation and microscopically observed to identify eosinophil according to their morphological characteristics. The number of each cell type among 300 white blood cells in the cell 39 WO 2014/195888 2014/061954 smear has to be determined and expressed as a percentage, and the number of neutrophils and macrophages in each BALf are to be calculated.

Assay 14: Reversal of Corticosteroid Insentivity in Cigarette Smoke Extract Induced Pulmonary Inflammation (COPD) Model

[165] Female Balb/c mice are to be acclimatized for seven days prior to the start of the experiment. Animals are then to be randomly distributed to various groups based on their body weights. On day 1, s are to be anaesthetised with ether and cigarette smoke extract is to be administered by intranasal route in volume of SOul/mouse and animals are to be exposed to CSE daily for next five days (dl to d6). On day 7, mice are to be administered by dexamethasone at 10 mg/kg by oral gavage and 60 mins later, mice are to be stered with CSE by asal route and it has to be repeated for next four days (d7 to dll). From day 9 to dayll, s are to be administered by test compound or vehicle by oral/intranasal route and 30 mins after dexamethasone administration and 30 mins later animals are to be anaesthetised with ether and cigarette smoke extract is to be administered by intranasal route in volume of 50ul/mouse and animals are to be exposed to CSE daily after the test compound administration for next two days (i.e. d9 to dll), on d12, twenty four hours after last CSE exposure animals are to be exsanguinated under anesthesia, and the trachea is to be cannulated and the lungs are to be lavaged with 0.5-ml ts of heparinised PBS (1 unit/n11) four times through tracheal cannula (total volume 2 ml). BAL has to be stored at 2-8 0C until assayed for total cell and differential leukocyte count. Bronchioalveolar fluid is to be centrifuged (SOOXg for 10 min) and the ing cell pellet has to be resuspended in 0.5 ml of heparinised saline. The total number of white blood cells is to be determined in BAL fluid and blood using a blood cell counter and adjusted to 1x106 cell/ml. Differential cell count is to be calculated manually. Forty microliters of the cell suspension is to be centrifuged using cytospin 3 to prepare a cell smear. The cell smear is to be stained with a blood staining solution for differentiation and microscopically has to be observed to identify eosinophil according to their morphological characteristics. The number of each cell type among 300 white blood cells in the cell smear are to be determined and will be expressed as a percentage, and the number of phils and macrophages in each BAL fluid are to be calculated.

Assay 15: Acute Cigarette Smoke Induced Cell Infiltration in Male Balb/c Mice

[166] Animals (male Balb/c mice) are to be atized for seven days prior to the start of the experiment. Animals are then to be randomly buted to various groups 40 WO 2014/195888 PCT/IB2014/061954 based on their body s. On day 1, mice is to be administered test compound or vehicle by oral/intranasal route and after 1 hr test compound administration animals are to be placed in whole body exposure box. On day 1 and d2 mice are d to the mainstream smoke of 6 cigarettes and of 8 ttes on day 3, and of 10 cigarettes on day 4. Exposure to the smoke of each cigarette lasts for 10 min (cigarette are to be completely burned in the first two minutes and followed by an air flow with animal ventilator and next 20 min exposure with fresh room air. After every second cigarette an onal break of 20 min with exposure to fresh room air is to be conducted. Control animals are to be exposed to room air chamber. From day 1 to d4 animals are administered by test compound either oral or intranasal route. On day 5, 24 hours after last cigarate smoke (CS) exposure animals are exsanguinated under anesthesia, and the trachea is to be cannulated and the lungs are d with 0.5-ml aliquots of heparinised PBS (1 unit/n11) four times through tracheal cannula (total volume 2 ml). Bronchioalveolar (BAL) collected is to be stored at 2-8 C’C until assayed for total cell and differential leukocyte count. BAL fluid is to be centrifuged (SOOXg for 10 min) and the resulting cell pellet is resuspended in 0.5 ml of heparinised saline. The total number of white blood cells is to be ined in BAL fluid and blood using a blood cell counter and adjusted to 1x106 cell/ml. Differential cell count is calculated manually. Forty microliters of the cell suspension is centrifuged using cytospin 3 to prepare a cell smear. The cell smear is stained with a blood staining solution for differentiation and microscopically observed to identify eosinophil according to their morphological characteristics. The number of each cell type among 300 white blood cells in the cell smear are to be ined and expressed as a percentage, and the number of phils and macrophages in each BAL fluid are to be ated.

Results: As depicted in s 7A and 7B, Compound Al reduced macrophages and neutrophil infiltration into BALF thereby indicating a therapeutic role in chronic ctive ary diseases Assay 16: Ovalbumin-Induced Nasal phil and Neutrophil Accumulation in Mice

[167] Animals (mice) are to be acclimatized for seven days prior to the start of the experiment. Animals are then to be randomly distributed to various groups based on their body weights. Animals are to be immunized with OVA (40 ug/kg i.p.) on day 1 and 5. In order to elicit local inflammatory responses in the nose, mice are to be repeatedly challenged intra-nasally (IOuL/per nostril) on days 12-19 with OVA (3% OVA in saline).

On day 19 non-fasted mice are to be dosed intra—nasally (lOuL/nostril) with either vehicle 41 WO 2014/195888 PCT/IB2014/061954 or test compound 2 hours before to the start of the final OVA challenge. Two hrs later, each animal is to be received a final intranasal OVA (3%) challenge). After a further 8 hr, each animal is to be anaesthetized and nasal lavage is to be carried out by ling 1 ml of PBS into the posterior nares via a rostrally implanted tracheal cannula extending to a position that is approximately 1 mm before the posterior nares. This procedure has to be ed to give a yield of approximately 2 ml of lavage fluid. Total cell numbers in the nasal lavage fluid samples are to be measured using a haemocytometer. Cytospin smears of the nasal lavage fluid samples are to be prepared by centrifugation at 1200 rpm for 2 min at RT and d using a Diff-Quik stain system (Dade Behring) for differential cell counts. Cells are to be counted using oil immersion microscopy.

Assay 17: P01y-l:C-Induced Cell Accumulation in Mice

[168] Specific pathogen-free AIJ mice , 5 weeks old) are to be acclimatized for seven days prior to the start of the experiment. Animals are then to be randomly distributed to various groups based on their body weights. Animals are to be administered with poly (l:C)—LMW 1C; 1 mg/mL, 40 uL) intranasally twice daily for 3 days under anaesthesia with 3% isoflurane. Animals are to be treated with test compound by intra— nasally (35 uL of on in 50% DMSO/PBS) 2hr before each poly-l:C treatment.

Twenty four hr after the last poly-l:C challenge, animals are to be anesthetized, the trachea has to be cannulated and BALF is to be collected. The trations of alveolar macrophages and phils in BALF are to be determined by using a blood cell counter 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 prepare a cell smear.

The cell smear is stained with a blood staining on for differentiation and microscopically ed to fy 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.

[169] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these ments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as described above. It is intended that the appended claims define the 42 W0 95888 PCT/IB2014/061954 scope of the invention and that methods and ures within the scope of these claims and their equivalents be covered thereby. ceutical Composition of Compound Al Example I

[170] The es described below containing 5 or 10 mg of Compound A1 are prepared.

Ingredients Comound A1 4; u: Microcr stalline cellulose (Avicel PH102) — H drox r0 0 l cellulose l LF) Purified Water Low substituted hydroxyl-oroyl cellulose (L-HPC; LH-l l) Talc Colloidal silicon dioxide (Aerosil-200) .09 wt» Magnesium stearate Example 11

[171] The capsules described below containing 25, 50, or 100 mg of Compound A1 are prepared. —‘&-

[172] All publications and patent and/or patent applications cited in this application are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated herein by reference.

Claims (21)

WHAT IS CLAIMED IS:

1. A compound selected from 2-(1-(9H-purinylamino)propyl)(3-fluorophenyl)-4H- chromenone and pharmaceutically acceptable salts thereof.

2. The compound according to claim 1, n the compound is ed from (S)(l- (9H-purinylamino)propyl)(3- fluorophenyl)-4H-chromenone and ceutically acceptable salts thereof.

3. The compound according to claim 2, wherein the nd is free of (l-(9H- purinylamino)propyl)(3-fluorophenyl)-4H-chromenone, and pharmaceutical acceptable salts thereof.

4. The compound of claim 2, wherein the compound has an enantiomeric excess greater than 95%.

5. The compound according to claim 1, wherein the compound is (S)(l-(9H-purin ylamino)propyl)(3-fluorophenyl)-4H-chromenone.

6. The compound of claim 5, wherein the compound is free of (R)(l-(9H-purin ylamino)propyl)(3-fluorophenyl)-4H-chromenone.

7. The nd of claim 5, wherein the compound has an enantiomeric excess greater than 95%.

8. A pharmaceutical composition comprising a compound according to any one of claims 1-7 and at least one pharmaceutically acceptable carrier.

9. Use of a compound according to any one of claims 1-7 in the cture of a medicament for ting a catalytic activity of a PI3 δ kinase present in a cell.

10. Use of a compound according to any one of claims 1-7 in the manufacture of a medicament for inhibiting a catalytic activity of a PI3 γ kinase present in a cell.

11. Use of a compound according to any one of claims 1-7 in the manufacture of a medicament for inhibiting a catalytic ty of a PI3 δ kinase and PI3 γ kinase present in a cell.

12. The use of any one of claims 9-11, wherein the inhibition takes place in a subject suffering from a disease, disorder or condition selected from cancer, a bone disorder, an 44 inflammatory disease, an immune disease, a nervous system e, a metabolic disease, a respiratory disease, thrombosis, and cardiac disease.

13. Use of a compound according to any one of claims 1-7 in the manufacture of a medicament for treating leukemia.

14. Use of a compound according to any one of claims 1-7 in the manufacture of a medicament for treating asthma or chronic obstructive pulmonary disease.

15. Use of a compound according to any one of claims 1-7 in the manufacture of a ment for treating rheumatoid arthritis, psoriasis, lupus or experimental mune encephalomyelitis (EAE).

16. Use of a compound according to any one of claims 1-7 in the manufacture of a medicament for treating chronic lymphocytic ia (CLL), non-Hodgkin lymphoma (NHL), n lymphoma (HL) acute myeloid leukemia (AML), multiple myeloma (MM), small lymphocytic ma (SLL), or indolent non-Hodgkin's ma (I-NHL) disease.

17. Use of a compound of any one of claims 1-7 in the cture of a medicament for the treatment of a disease, disorder or condition that would benefit from inhibiting catalytic activity of a PI3 δ/γ kinase.

18. Use of a compound according to any one of claims 1-7 in the manufacture of a medicament for the treatment of a PI3K associated e, disorder or condition.

19. The use of claim 18, wherein the ment is to be administered with an additional active agent selected from anti-cancer agents, anti-inflammatory agents, immunosuppressive agents, steroids, non-steroidal anti-inflammatory agents, antihistamines, analgesics, and mixtures thereof.

20. The use of claim 18, wherein the PI3K associated e, disorder or condition is an immune system-related disease, a disease or disorder involving inflammation, cancer or other proliferative disease, a hepatic disease or er, or a renal disease or disorder.

21. The use of claim 18, wherein the PI3K associated disease, disorder or condition is selected from leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, n's ma, non-Hodgkins lymphoma, hairy cell lymphoma, Burkett's lymphoma, acute and chronic myelogenous leukemias, myelodysplastic 45 syndrome and locytic leukemia, psoriasis, rheumatoid arthritis, osteoarthritis, asthma, COPD, allergic rhinitis and lupus erythematosus. 46