EA045057B1 - TREATMENT OF B-CELL MALIGNANTS USING A COMBINATION OF JAK AND PI3K INHIBITORS - Google Patents

Description

This application claims priority to application No. 61/976815, filed April 8, 2014, which is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to methods of treating B cell malignancies using a combination of JAK1 and/or JAK2 inhibitors and PI3Kδ inhibitors.

State of the art

The B cell receptor (BCR) is present on both normal and most malignant B cells. BCR engagement provides important survival signals, and interruption of the BCR signal can lead to B cell death. Studies using siRNA to inhibit BCR expression have demonstrated that constitutive BCR signaling is critical for the survival and proliferation of human B-cell lymphomas. The primary role of the BCR signal in these cells is to activate spleen tyrosine kinase (Syk), which in turn leads to several downstream events that promote cell survival, including activation of Bruton's tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and AKT . It has been demonstrated that many B-cell malignancies, including diffuse large B-cell lymphomas (DLBCL), are particularly dependent on BCR survival signals, as evidenced by their sensitivity to genetic and pharmacological inhibition of BCR signal components in vitro. It has been demonstrated that DLBCL cells use PI3K, which enhances the anti-apoptotic NF-κB and survival signal, and inhibition of the PI3K/AKT pathway is enhanced by NFκB inhibition when killing DLBCL cell lines in vitro.

Impaired activation of JAKs through the production of cytokines and growth factors is also associated with increased proliferation and survival of malignant cells in many tumor types. JAKs activate many downstream pathways involved in malignant cell proliferation and survival, including STATs, a group of important latent transcription factors. Clinically significant, serum levels of IL-10 and IL-6, which signal through JAK, were found to be elevated in patients with DLBCL compared to normal control subjects (Gupta et al, 2012). Additionally, patients with high serum IL-10 levels showed shorter event-free survival (Gupta et al, 2012). Within the JAK family of kinases, JAK1 has been demonstrated to interact with JAK2, JAK3, and TYK2 and play a dominant role in mediating the signal of many inflammatory cytokines, including IL-6, IL-10, and interferon.

In DLBCL, activation of the JAK signal occurs through both autocrine and paracrine mechanisms. In tumor cells, the BCR signal leads to increased production of IL-6 and IL-10 through activation of the NF-kB pathway (Lam et al, 2008). A subset of DLBCL variants are characterized as having high levels of expression of STAT3, IL-6, and/or IL-10, and JAK inhibition in these DLBCL cell lines has been demonstrated to be cytotoxic and enhanced by NF-κB inhibitors. In addition to activating the JAK/STAT pathway through autocrine pathways, the stromal compartment may also provide a source of these cytokines in a paracrine manner (Hodge et al, 2005).

For these reasons, there is a need to develop new therapies that can be used to treat B-cell malignancies such as DLBCL. This invention addresses this and other needs.

Description of the figures

Fig. 1A shows Western blot analysis for the presence of IL6 and IL10 for various DLBCL cell lines.

Fig. 1B shows Western blotting for actin and p-Stat3 for Pfeiffer cells treated with IL6 or IL10.

Fig. 2A shows the % inhibition in the cell proliferation assay for Pfeiffer cells as a function of the concentration of compound 28 with backbone (DMSO), DMSO+IL10, and DMSO+IL10+ruxolitinib.

Fig. 2B shows the % inhibition in the cell proliferation assay for Pfeiffer cells as a function of the concentration of Compound 28 with vehicle (DMSO), DMSO+IL10, and DMSO+IL10+Compound 7.

Fig. 3 shows the % inhibition in the cell proliferation assay for HBL-1 cells as a function of the concentration of compound 28 with backbone (DMSO), DMSO+IL10, and DMSO+IL10+ruxolitinib.

Fig. 4 shows Western blot analysis of Pfeiffer cells after treatment with vehicle (DMSO), ruxolitinib, compound 28, or compound 28 and ruxolitinib with or without IL10.

Fig. 5 shows a Western blot of Pfeiffer cells after treatment with vehicle (DMSO), compound 7, compound 28, or compound 28 and compound 7 with or without IL10.

Fig. 6 shows the % inhibition in the cell proliferation assay for Pfeiffer cells as a function of the concentration of compound 28 with base (DMSO), DMSO+IL10, and DMSO+IL10+compound 16.

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Fig. 7 shows Annexin-V staining of Pfeiffer cells treated with Compound ± Compound 16 demonstrating synergistic induction of apoptosis by combination therapy.

Fig. 8 shows Western blot analysis of Pfeiffer cells following treatment with compound 28 ± compound 16 demonstrating effects on STAT3 and pAKT.

Brief description of the invention

This application provides a method of treating a B cell malignancy in patients in need thereof, comprising administering to the patient: (a) a JAK1 and/or JAK2 inhibitor; and (b) a PI3Kδ inhibitor.

This application also provides a method of treating a disease selected from diffuse large B cell lymphoma, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, villous cell leukemia, mantle cell lymphoma, small cell lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, extra anodal lymphomas from marginal zone cells, Hodgkin's lymphoma, Burkitt's lymphoma, Waldenström's macroglobulinemia, prolymphocytic leukemia, acute lymphoblastic leukemia, myelofibrosis, mucosal lymphatic tissue (MALT) lymphoma, mediastinal (thymic) large B-cell lymphoma, lymphomatoid granulomatosis, lymphomas s of the marginal zone of the spleen, primary effusion lymphoma, intravascular large B-cell lymphoma, plasma cell leukemia, extramedullary plasmacytoma, smoldering myeloma (also known as silent myeloma), monoclonal gammopathy of unknown origin (MGUS), activated B-cell-like (ABC), diffuse large B-cell lymphoma (ABC) -DLBCL) and germinal center B-cell lymphoma (GCB)-diffuse large B-cell lymphoma (GCB-DLBCL) in patients who require it, comprising administering to the patient: (a) a JAK1 and/or JAK2 inhibitor; and (b) a PI3Kδ inhibitor.

In some embodiments of the methods, the JAK1 and/or JAK2 inhibitor is selected from:

3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile;

3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol1 -yl] propanenitrile;

3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl )1H-pyrazol-1-yl]propanenitrile;

4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1yl)carbonyl] -3-fluorobenzonitrile;

4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1yl)carbonyl] -3-fluorobenzonitrile;

{1-{ 1-[3-Fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl} -3-[4-(7H-pyrrolo[2,3 -d]pyrimidin-4yl)-1H-pyrazole -1 -yl]azetidin-3-yl}acetonitrile;

4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[ 4fluoro-2-(trifluoromethyl)phenyl]piperidine-1-carboxamide;

[3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1 H-pyrazol-1 -yl] -1-(1-{ [2-(trifluoromethyl)pyrimidin-4yl] carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile;

[trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1 -yl]-3 -(4-{[2-(trifluoromethyl)pyrimidin4-yl ]carbonyl}piperazin-1-yl)cyclobutyl]acetonitrile;

{trans-3-(4-{[4-[(3-hydroxyazetidin-1-yl)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1yl)-1-[4-(7H -pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;

{trans-3-(4-{[4-{[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2yl]oxy}piperidin-1-yl)- 1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;

{trans-3-(4-{[4-{[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2yl]oxy}piperidin-1-yl)- 1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;

4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy} piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-1 H- pyrazol-1-yl]butanenitrile;

5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-Nisopropylpyrazin-2 -carboxamide;

4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2.5 difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;

5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-Nisopropylpyrazin-2 -carboxamide;

{1-(cis-4-{[6-(2-hydroxyethyl)-2-(trifluoromethyl)pyrimidin-4-yl]oxy}cyclohexyl)-3-[4-(7Hpyrrolo[2,3-d]pyrimidin- 4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;

{1-(cis-4-{[4-[(ethylamino)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7Hpyrrolo[2,3-d]pyrimidine -4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;

- 2 045057 {1-(cis-4-{[4-(1-hydroxy-1-methylethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4(7H-pyrrolo[ 2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;

{1-(cis-4-{[4-{[(3R)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;

{1 -(cis-4- {[4-{[(3 S)-3-hydroxypyrrolidin-1 -yl]methyl} -6-(trifluoromethyl)pyridin-2-yl]oxy} cyclohexyl)-3-[4 -(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;

{trans-3 -(4-{[4-({[(1 S)-2-rugpoKcu-1 -methylethyl]amino}methyl)-6-(trifluoromethyl)pyridin-2yl]oxy}piperidin-1-yl) -1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;

{trans-3-(4-{[4-({[(2R)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;

{trans-3-(4-{[4-({[(2S)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;

{trans-3-(4-{[4-(2-hydroxyethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7Hpyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;

((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2Hpyran- 2-yl)acetonitrile;

4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1Ή-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N- [(1S)2,2,2-trifluoro-1-methylethyl]benzamide; and pharmaceutically acceptable salts of any of the above compounds.

In some embodiments of the methods, the PI3Kδ inhibitor is selected from:

7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one;

(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one;

4-[1 -(4-amino-3 -methyl-1 H-pyrazolo[3,4-d]pyrimidin-1 -yl)ethyl]-6-chloro-2-{1 -[(2S)-2- hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile;

4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-[1-(2-hydroxyethyl)azetidin3-yl ]-3-methoxybenzonitrile;

5-{3-[1 -(4-amino-3-methyl-1 H-pyrazolo[3,4-d]pyrimidin-1 -yl)ethyl]-6-cyano-2-ethoxy-5-methylphenyl} N ,N-dimethylpyridine-2-carboxamide;

4-{3-[1 -(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1 -yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin- 2-she; And

N-{1-[5-chloro-8-(3-fluorophenyl)cinnolin-7-yl]ethyl}-9H-purine-6-amine;

4-chloro-3'-fluoro-3-methyl-6-[1-(9H-purin-6-ylamino)ethyl]biphenyl-2-carbonitrile; and pharmaceutically acceptable salts of any of the above compounds.

This application also provides a JAK1 and/or JAK2 inhibitor for use in combination with a PI3Kδ inhibitor for the treatment of B cell malignancy or any of the diseases specified herein.

This application also provides the use of a JAK1 and/or JAK2 inhibitor and a PI3Kδ inhibitor for the preparation of a medicament for the treatment of B cell malignancy or any of the diseases specified herein.

Detailed description of the invention

This application provides, among other things, a method of treating a disease selected from diffuse large B-cell lymphoma, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, villous cell leukemia, mantle cell lymphoma, small cell lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, extranodal marginal zone cell lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma, Waldenström's macroglobulinemia, prolymphocytic leukemia, acute lymphoblastic leukemia, myelofibrosis, mucosal lymphatic tissue (MALT) lymphoma, mediastinal (thymic) large B-cell lymphoma, lymphomatoid granulomatosis, marginal zone lymphoma spleen, primary effusion lymphoma, intravascular large B-cell lymphoma, plasma cell leukemia, extramedullary plasmacytoma, smoldering myeloma (also known as silent myeloma), monoclonal gammopathy of unknown origin (MGUS), activated B-cell-like (ABC) diffuse large B-cell lymphoma (ABC-DLBCL) and germinal center B-cell lymphoma (GCB)-diffuse large B-cell lymphoma (GCB-DLBCL) in patients in need, which involves administering to the patient: (a) a JAK1 and/or JAK2 inhibitor; and (b) a PI3Kδ inhibitor.

In some embodiments, the non-Hodgkin's lymphoma is non-Hodgkin's lymphoma (NHL) in the form of relapsed or refractory NHL or relapsed follicular NHL.

In some embodiments, the disease is diffuse large B cell lymphoma (DLBCL).

In some embodiments, the disease is activated B-cell-like (ABC) diffuse large B-cell lymphoma (ABC-DLBCL) or germinal center B-cell lymphoma (GCB)-diffuse large B-cell lymphoma (GCB-DLBCL).

In some embodiments, the JAK1 and/or JAK2 inhibitor and the PI3Kδ inhibitor are administered simultaneously.

In some embodiments, the JAK1 and/or JAK2 inhibitor and the PI3Kδ inhibitor are administered sequentially.

In some embodiments, the JAK1 and/or JAK2 inhibitor is selective for JAK1 and JAK1 over JAK3 and TYK2. In some embodiments, the JAK1 and/or JAK2 inhibitor is selective for JAK1 over JAK2, JAK3, and TYK2. For example, some of our disclosed compounds or pharmaceutically acceptable salts thereof inhibit JAK1 preferentially over one or more of JAK2, JAK3 and TYK2. In some embodiments, the compounds inhibit JAK1 preferentially over JAK2 (eg, have a JAK1/JAK2 IC50 ratio of >1). In some embodiments, the compounds or salts are approximately 10-fold more selective for JAK1 over JAK2. In some embodiments, the compounds or salts are about 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold more selective for JAK1 over JAK2, as calculated by measuring IC ₅₀ at 1 mM ATP ( for example, see example A).

In some embodiments, the JAK1 and/or JAK2 inhibitor is 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile. In some embodiments, the JAK1 and/or JAK2 inhibitor is (3R)-3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile (ruxolitinib; also known as INCB018424). Ruxolitinib has an IC ₅₀ of less than 10 nM at 1 mM ATP (assay A) for JAK1 and JAK2. 3Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile and ruxolitinib can be prepared using the procedure described in US application 7,598,257 ( Example 67), filed December 12, 2006, which is incorporated herein by reference in its entirety. In some embodiments, the JAK1 and/or JAK2 inhibitor is a (3R)-3cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl salt ]propanenitrile with phosphoric acid.

In some embodiments, the JAK1 and/or JAK2 inhibitor is a compound from Table. 1 or a pharmaceutically acceptable salt thereof. Connections from the table. 1 are selective inhibitors of JAK1 (selective over JAK2, JAK3 and TYK2). The IC ₅₀ values obtained by the method according to Assay A at 1 mM ATP are shown in table. 1.

Table 1

No. Rev. Name Structure JAK1 IC ₅₀ (nM) JAK2/ JAK1 1 Example 1 ((2R,5S)-5-{2-[(lR)-l- hydroxyethyl]-1 H-imide azo[4.5 c1]thieno[3,2-b]pyridin-1yl}tetrahydro-2H-pyran-2yl)acetonitrile ^ ^OH 0 >10 2 Example 2 4-[3-(cyanomethyl)-3-(3',5'-dimethyl1H, 1'H-4,4'-bipyrazol-1 yl)azetidin-1 -yl]-2,5-difluoro-N[( 18)-2,2,2-trifluoro-1 methylethyl]benzamide ϊ ^FFF ι) ΗΝ-Ν +++ >10 3 US 2010/0298334 (Example 2) ^a 3 -[ 1 -(6-chloropyridin-2yl)pyrrolidin-3-yl]-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1-yl]propanenitrile Ν ν'^Γ^ ^{ν y Ν} II 1 7 + >10

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4 US 2010/0298334 (Example 13c) 3 -(1 - [1,3]oxazolo[5,4-b]pyridin-2 ylpyrrolidin-3-yl)-3 -[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl) - 1Hpyrazo l-1 -yl]propanenitrile IE NN VJ Λ λ ^ΙΊ Η + >10 5 US 2011/0059951 (Example 12) 4-[(4-{3-cyano-2-[4-(7Hpyrrolo [2,3-d]pyrimidin-4-yl)-1Hpyrazol-1-yl]propyl } piperazin-1yl)carbonyl] - 3-fluorobenzonitrile ⁰ /—\ y_/~ ^F Ν-Ν Νθ' ιι JL / ^Ν Η + >10 6 US 2011/0059951 (Example 13) 4-[(4-{3-cyano-2-[3-(7Hpyrrolo [2,3-d]pyrimidin-4-yl) -1Hpyrrho l-1-yl]propyl } piperazin-1yl)carbonyl] - 3-fluorobenzonitrile ζ ο '& + >10 7 US 2011/0224190 (Example 1) {1-{1-[3-Fluoro-2- (trifluoromethyl)isonicotinoyl] pipe eridin-4 -yl } -3 - [4-(7H-pyrrolo [2,3d] pyrimidin-4-yl) -1 H-pyrazol-1 yl] azetidin-3 -yl } acetonitrile 1 1 ^CF 3 Q ^F N /С-^N NN // X n^V^NHJ 7 N ^N H + >10

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8 US 2011/0224190 (Example 154) 4-{ 3 -(Cyanomethyl)-3 -[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin-1-yl} -N-[4 fluoro -2- (trifluoromethyl)phenyl]piperidine-1 carboxamide ΟγΝΗ QN X^_^N NN [1 J 7 N ^{N N} H + >10 9 US 2011/0224190 (Example 85) [3 -[4-(7H-pyrrolo[2,3 d]pyrimidin-4-yl)-1H-pyrazo l-1 yl]-1-(1-{[2(trifluoromethyl)pyrimidin-4-yl]carbonyl } piperidin-4yl)azetide in-3-yl]acetonitrile О 7=\ p ^CF = r—N NN '— // λ 03 ^N H + >10 10 US 2012/0149681 (Example 7b) [trans-1 -[4-(7H-pyrrolo[2,3d]pyrimidin-4-yl) -1 H-pyrazo l-1 - il]-3-(4-{[2- (trifluoromethyl)pyrimidin-4 yl] carbonyl } piperazin-1 yl) cyclobutyl] acetonitrile p nS o N NN ' and λ N γΆ ^N H + >10

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eleven US 2012/0149681 (Example 157) {trans-3-(4-{[4-[(3hydroxyazetide in-1 -yl) methyl]-6(trifluoromethyl)pyridin-2yl]oxy} piperidin-1 -yl) -1 - [4 -(7Hpyrrolo [ 2,3-d] pyrimidin-4 -yl) -1Hpyrazol-1yl] cyclobutyl } acetonitrile G1 ^he N- ¹ O ^N F NN PA GP ^N Y + >10 12 US 2012/0149681 (Example 161) {trans -3-(4-{ [4-{[(2S)-2- (hydroxymethyl)pyrrolidin-1 yl]methyl}-6(trifluoromethyl)pyridin-2yl] oxy } piperidin-1 -yl) -1 - [4 -(7Hpyrrolo [2,3-d]pyrimidin-4 -yl) - 1Hpyrazol-1yl] cyclobutyl } acetonitrile A i ^OH Af cP r ^N^ NN (/ X ^N it X / nn H + >10 13 US 2012/0149681 (Example 162) {trans-3 -(4-{ [4-{ [(2R)-2(hydroxymethyl)pyrrolidin-1 yl]methyl}-6(trifluoromethyl)pyridin-2yl] oxy } piperidin-1 -yl) -1 - [ 4 -(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1yl]cyclobutyl}acetonitrile n/'/ i '-ON A ^ir N ^/ ' ^F O ^IN F NN (/ A N ψλ it Λ > nn H + >10

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14 US 2012/0149682 (Example 20) ^b 4-(4- { 3 - [(d imethylamino)methyl] -5 fluorophenoxy } piperidin-1 -yl) -3 [4-(7H-pyrrolo[2,3-c1]pyrimidin-4yl) -1 H- pyrazol l-1 -yl]butanenitrile F'λ N + >10 15 US 2013/0018034 (Example 18) 5 - { 3 -(cyanomethyl)-3 -[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin-1-yl } -Nisopropylpyrazin-2-carboxamide N=—\ λ N=\ O N-N N HN—( %NH + >10 16 US 2013/0018034 (Example 28) 4-{3-(cyanomethyl)-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin-1-yl}-2,5 difluoro-M -[(18)-2,2,2-trifluoro-1methylethyl]benzamide 1- . /=4/ NN^ , ^NH O ^f ........ F 1 1) + >10 17 US 2013/0018034 (Example 34) 5 - { 3 -(cyanomethyl) -3 -[4-( 1Hpyrrolo [2,3 -b] pyrid in-4-yl)- 1Hpyrazo l-1 -yl] azetidin-1 -yl } -Nisopropylpyrazin-2-carboxamide ABOUT \ CO + >10 18 US 2013/0045963 (Example 45) {1 -(cis -4-{[6-(2-hydroxyethyl)-2(trifluoromethyl)pyrimidin-4 yl]oxy}cyclohexyl)-3 -[4-(7Hpyrrolo[2,3-d]pyrimidin-4 -yl) -1Hpyrazol-1 -yl] azetidin-3 yl}acetonitrile in + >10 19 US 2013/0045963 (Example 65) {1 -(cis-4-{ [4-[(ethylamino)methyl]-6(trifluoromethyl)pyridin-2yl]oxy}cyclohexyl)-3 -[4-(7Hpyrrolo[2,3-d]pyrimidine-4 -yl) -1Hpyrazol-1 -yl] azetidin-3 yl}acetonitrile f=\ iz X L f O TZ) + >10 20 US 2013/0045963 (Example 69) {1 -(cis-4- {[4-( 1 -hydroxy-1 methylethyl)-6(trifluoromethyl)pyridin-2yl] oxy} cyclohexyl)-3 -[4-(7Hpyrrolo[2,3-d]pyrimidine -4 -yl) -1Hpyrazol-1 -yl] azetidin-3 yl}acetonitrile N G Y* J. J » 1 Xf'N'W nJ NN O F ^F 4% + >10

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21 US 2013/0045963 (Example 95) {1-(cis-4-{[4-{[(3K)-3hydroxypyrrolidin-1-yl]methyl} 6-(trifluoromethyl)pyridin-2yl]oxy}cyclohexyl)-3 -[4-(7Hpyrrolo [2 ,3-d] pyrimidin-4 -yl) -1Hpyrazol-1 -yl] azetidin-3 yl}acetonitrile __A^on / n-Ν \—n a ^{g 47} NN IL X > ^N И + >10 22 US 2013/0045963 (Example 95) {1-(cis-4-{[4-{[(38)-3hydroxypyrrolidin-1-yl]methyl} 6-(trifluoromethyl)pyridin-2yl]oxy}cyclohexyl)-3 -[4-(7Hpyrrolo[ 2,3-d] pyrimidin-4 -yl) -1Hpyrazol-1 -yl] azetidin-3 yl}acetonitrile /-\ 'nT J νΆ ν a ^r ΟΛ J ⁴⁷ NN - (. Λ ^N ϋ 1 > ^N H + >10 23 US 2014/0005166 (Example 1) {trans-3 -(4-{ [4-( {[(18)-2-hydroxy1 -methylethyl] amino } methyl) -6(trifluoromethyl)pyridine-2- yl] oxy } piperidin-1 -yl) -1 - [4 - (7Hpyrrolo [2,3-d]pyrimidin-4 -yl) -1Hpyrazol-1yl] cyclobutyl } acetonitrile OH NH 0 ^>N H A / ¹ NN (/4 Ν tA it X > Ν N H + >10 24 US 2014/0005166 (Example 14) {trans-3 -(4-{[4-({[(2R)-2hydroxypropyl] amino } methyl) -6(trifluoromethyl)pyridin-2yl] oxy } piperidin-1 -yl) -1 - [4 -(7Hpyrrho lo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1yl]cyclobutyl}acetonitrile ^oh NH 0 ^>N H 0/ NN (/4 ^N it X > nn H + >10

- 9 045057

25 US 2014/0005166 (Example 15) {trans -3-(4-{ [4-({[(2S)-2hydroxypropyl] amino } methyl) -6(trifluoromethyl)pyridin-2yl] oxy } piperidin-1 -yl) -1 - [4 -(7Hpyrrolo [2,3-d]pyrimidin-4-yl)-1Hpyrazol-1yl]cyclobutyl}acetonitrile ^on NH Af A // ^N NN (/Λ ^Ν ιΛ Ν Ν Η + >10 26 US 2014/0005166 (Example 20) {trans-3 -(4-{ [4-(2-hydroxyethyl)-6(trifluoromethyl)pyridine-2- yl] oxy } piperidin-1 -yl) -1 - [4 - (7Hpyrrolo [2,3-d] pyrimidin-4 -yl)- 1Hpyrazol-1yl] cyclobutyl } acetonitrile BUT Ο ^Ν F ^F ^N^ γι// NN (/ Λ OE ^N th + >10

+ means <10 nM (for assay conditions see Example A) ++ means <100 nM (for assay conditions see Example A) +++ means <300 nM (for assay conditions see Example A) aData for enantiomer 1 ^b Data for enantiomer 2

In some embodiments, the JAK1 and/or JAK2 inhibitor is {1-{1-[3-fluoro2-(trifluoromethyl)isonicotinoyl] piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d ]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin-3-yl}acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 and/or JAK2 inhibitor is a salt of {1-{1-[3fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin-3-yl}acetonitrile and adipic acid.

In some embodiments, the JAK1 and/or JAK2 inhibitor is 4-{3(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]azetidin-1-yl}-2,5-difluoro-N [(1S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 and/or JAK2 inhibitor is selected from the group consisting of (R)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-ul]-3-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl]propanenitrile, (R)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidine -3 -yl)-3 - [4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1 H-pyrazol-1 -yl] propanenitrile, (R)-4- [(4-{3 -cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-1H-pyrazol-1 -yl]propyl} piperazin-1 -yl)carbonyl]-3-fluorobenzonitrile, (R) -4-[(4-{3-cyano-2-[3-(7Hpyrrolo [2,3 -d]pyrimidin-4-yl)-1H-pyrrol-1 -yl]propyl}piperazin-1 -yl) carbonyl]-3-fluorobenzonitrile, or (R)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy} piperidin-1-yl)-3 - [4-(7H-pyrrolo [2, 3-d]pyrimidin-4yl)-1H-pyrazol-1-yl]butanenitrile, (S)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-( 7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, (S)-3-(1-[1,3] oxazolo[5,4-b]pyridin-2 -ylpyrrolidin3-yl)-3 - [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1 -yl]propanenitrile, (S)-4-[(4- {3-cyano-2-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile, ( S)-4-[(4-{ 3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl} piperazin- 1 yl)carbonyl]-3-fluorobenzonitrile, (S)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)3-[4-(7H-pyrrolo [2 ,3-d]pyrimidin-4-yl)-lH-pyrazol-1-yl]butanenitrile; and pharmaceutically acceptable

- 10 045057 salts of any of the above compounds.

In some embodiments of the connection from the table. 1 is prepared using the synthesis procedures described in US Patent Publication No. 2010/0298334, filed May 21, 2010, US Patent Publication No. 2011/0059951, filed August 31, 2010, US Patent Publication No. 2011/0224190, filed March 9 2011, US Patent Publication No. 2012/0149681, filed November 18, 2011, US Patent Publication No. 2012/0149682, filed November 18, 2011, US Patent Publication No. 2013/0018034, filed June 19, 2012, Patent US Publication No. 2013/0045963, filed August 17, 2012, and US Patent Publication No. 2014/0005166, filed May 17, 2013, each of which is incorporated herein by reference in its entirety.

In some embodiments, the JAK1 and/or JAK2 inhibitor is selected from the group consisting of compounds according to US Patent Publication No. 2010/0298334, filed May 21, 2010, US Patent Publication No. 2011/0059951, filed August 31, 2010, Patent US Patent Publication No. 2011/0224190, filed March 9, 2011, US Patent Publication No. 2012/0149681, filed November 18, 2011, US Patent Publication No. 2012/0149682, filed November 18, 2011, US Patent Publication No. 2013/ 0018034, filed June 19, 2012, US Patent Publication No. 2013/0045963, filed August 17, 2012, and US Patent Publication No. 2014/0005166, filed May 17, 2013, each of which is incorporated herein by reference in in full.

The PI3Kδ inhibitors described by the authors can be selective. By selective is meant that the compound binds to or inhibits a kinase with greater affinity or activity, respectively, than at least one other kinase. In some embodiments, the compounds we disclose are selective inhibitors of PI3Kδ (eg, upstream of PI3Ka, PI3Ke, and PI3Ky). In some embodiments, the selectivity may be at least about 2 times, 5 times, 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times greater , at least about 500 times or at least about 1000 times. Selectivity is measured by methods well known to those skilled in the art. In some embodiments, selectivity is tested at the Km ATP concentration of each enzyme. In some embodiments, the selectivity of the compounds disclosed herein may be determined by cellular assays related to the activity of a particular PI3K kinase.

In some embodiments, the PI3Kδ inhibitor is a compound shown in table. 2. Connections from table. 2 were tested in Assay B and were found to be PI3Kδ inhibitors with IC ₅₀ values presented in Table. 2.

- 11 045057

table 2

No. Rev. Name Structure PI3K5 IC ₅₀ (nM) 27 US 2011/0015212 (Example 10) 7-( 1 -(9H-purin-6-ylamino)ethyl)6-(3-fluorophenyl)-3-methyl-5Nthiazolo[3,2-a]pyrimidin-5-one /φί θ ή у* ΗΝχ,Ν. HELL N/C-NH + 28 US 2011/0015212 (Example 15) (S)-7-( 1 -(9H-purin-6 ylamino)ethyl)-6-(3-fluorophenyl)3 -methyl-5 H-thiazolo[3,2a]pyrimidin-5-one s ν^Ύ'' ΗΝ^,Ν. s N/NH + 29 US 2013/0059835 (Example 269) 4-[1-(4-amino-3-methyl-1Hpyrazolo[3,4-c1]pyrimidin-1 yl)ethyl]-6-chloro-2-{ l-[(2S)-2hydroxypropyl]azetidine-3 - yl } 3-methoxybenzonitrile β^ΟΗ N NCxIyOMe - ^N VM / \=N h ₂ n + thirty US 2013/0059835 (Example 268) 4-[1-(4-amino-3-methyl-1Hpyrazolo [3,4-c1]pyrimidin-1 yl)ethyl] -6 -chloro -2- [ 1 -(2hydroxyethyl)azetidin-3 -yl] -3 methoxybenzonitrile β^ΟΗ N NCxJ^xOMe Cl O' / \= N h ₂ n +

- 12 045057

31 US 2013/0059835 (Example 314) 5 - { 3 - [ 1 -(4-amino-3-methyl-1Hpyrazolo[3,4-d]pyrimidin-1 yl)ethyl]-6-cyano-2-is xy-5 methylphenyl} -Ν,Ν dimethylpyr idine-2-carboxamide 1 0 / 'ύ ¹⁴ νη ₂ + 32a, 32b, 32c, 32d US 2013/0059835 (Example 345-348 (four diastereomers)) Compounds 32a, 32b,32c and 32d are Examples 345, 346, 347, and 348, respectively 4-{3-[1-(4-amino-3-methyl-1Hpyrazolo[3,4-d]pyrimidin-1yl)ethyl]-5-chloro-2-ethoxy-6fluorophenyl}pyrrolidin-2-one ο 'Κνη FxJ\>DEt / \=Ν η ₂ ν 32a (++), 32b (+) 32c (+) 32d (++) 33 US 2011/0183985 (Example 17 - one enantiomer) Ν-{1-[5-χπορ-8-(3fluorophenyl)cinnolin-7yl]ethyl}-9H-purine-6-amine (I XX XX ^f Ν=\ ^hn x^W ^NH II 1 Ν^Ν + 34 US 2012/0157430 4-chloro-3'-fluoro-3-methyl-6-[1-(9Hpurin-6-ylamino)ethyl]biphenyl2-carbonitrile Ν ill κκ Τ ί Ί TT^ ^F CU ΗΝ ο ΙΨ \---- +++

+ means <50 nM ++ means 50 nM to 200 nM +++ means 50 nM to 100 nM

In some embodiments, the ΡΙ3Κδ inhibitor is selected from compounds that include:

(S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one;

(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one;

(S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one;

(R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one;

N-{(1S)-1-[5-chloro-8-(3-fluorophenyl)cinnolin-7-yl]ethyl}-9H-purin-6-amine;

and pharmaceutically acceptable salts of any of the above compounds.

In some embodiments, the PI3Kδ inhibitor is (S)-7-(1-(9H-purin-6ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin- 5-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the PI3K5 inhibitor is 4-[1-(4-amino-3-methyl-1Hpyrazolo[3,4-d]pyrimidin-1 -yl)ethyl]-6-chloro-2-{1-[(2S )-2-hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the PI3K5 inhibitor is 4-[1-(4-amino-3-methyl-1H-13 045057 pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-[1 -(2-hydroxyethyl)azetidin-3-yl]-3-methoxybenzonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the PI3Kδ inhibitor is 5-{3-[1-(4-amino-3-methyl-1Hpyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyano-2-ethoxy-5 -methylphenyl}-N,N-dimethylpyridine-2carboxamide or a pharmaceutically acceptable salt thereof.

In some embodiments, the PI3K8 inhibitor is selected from compounds that include:

4-[(R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-{1-[(2S) -2hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile;

4-[1(R)-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-[1-(2-hydroxyethyl) azetidin-3-yl]-3-methoxybenzonitrile;

5-{3-[1 (R)-(4-amino-3 -methyl-1 H-pyrazolo[3,4-d]pyrimidin-1 -yl)ethyl]-6-cyano-2-ethoxy-5- methylphenyl} H,K-dimethylpyridine-2-carboxamide;

4-[(S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-{1-[(2S) -2hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile;

4-[1(S)-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-[1-(2-hydroxyethyl) azetidin-3-yl]-3-methoxybenzonitrile;

5-{3-[1(S)-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyano-2-ethoxy-5-methylphenyl }-K,K-dimethylpyridine-2-carboxamide;

and pharmaceutically acceptable salts of any of the above compounds.

In some embodiments, the PI3Kδ inhibitor is a compound according to US Patent Publication No. US 2011/0015212, filed June 28, 2010, US Patent Publication No. 2013/0059835, filed August 31, 2013, US Patent Publication No. 2011/0183985, filed 17 December 2010, or US Patent Publication No. 2012/0157430, filed December 19, 2011, each of which is incorporated herein by reference in its entirety.

In some embodiments of the connection from the table. 2 is obtained using methods according to US Patent Publication No. US 2011/0015212, filed June 28, 2010, US Patent Publication No. 2013/0059835, filed August 31, 2013, US Patent Publication No. 2011/0183985, filed December 17, 2010 ., or US Patent Publication No. 2012/0157430, filed December 19, 2011, each of which is incorporated herein by reference in its entirety.

In some embodiments, the JAK1 and/or JAK2 inhibitor is (3R)-3cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof; and (7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5Hthiazolo[3,2-a]pyrimidin-5-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 and/or JAK2 inhibitor is {1-{1-[3-fluoro-2(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1yl]azetidin-3-yl}acetonitrile or a pharmaceutically acceptable salt thereof; and the PI3Kδ inhibitor is 7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin5-one or pharmaceutical thereof acceptable salt.

In some embodiments, the JAK1 and/or JAK2 inhibitor is 4-{3(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl ]azetidin-1-yl}-2,5-difluoro-N[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof; and the PI3Kδ inhibitor is 7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2a]pyrimidin-5-one or pharmaceutical thereof acceptable salt.

In some embodiments, this application provides a method of treating diffuse large B-cell lymphoma in patients in need, which includes administering to the patient (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d ]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof; and the PI3Kδ inhibitor is (7-(1-(9H-purin-6ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one or pharmaceutical thereof acceptable salt.

In some embodiments, this application provides a method of treating diffuse large B-cell lymphoma in patients in need thereof, which comprises administering to the patient {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl } -3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile or a pharmaceutically acceptable salt thereof; and 7-(1-(9H-purin6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one or a pharmaceutically acceptable salt thereof.

In some embodiments, this application provides a method of treating diffuse large B cell lymphoma in patients in need, which includes administering to the patient 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof; and 7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one or a pharmaceutically acceptable salt thereof .

- 14 045057

The compounds described by the authors may be asymmetric (for example, having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise noted. Compounds containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials are known to those skilled in the art, for example, resolution of racemic mixtures or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, etc. may also be present in the compounds described herein, and all of these stable isomers are contemplated by the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be isolated as mixtures of isomers or in individual isomeric forms.

In some implementations, the connection has an (R)-configuration. In some implementations, the connection has a ^-configuration.

Separation of racemic mixtures of compounds is accomplished by any of many methods known to those skilled in the art. An example method involves fractional recrystallization using a chiral resolving acid, which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization processes are, for example, optically active acids such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or various optically active camphorsulfonic acids such as β -camphorsulfonic acid. Other resolving agents suitable for fractional crystallization processes include stereoisomerically pure forms of α-methylbenzylamine (e.g., S and R forms or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2diaminocyclohexane, etc. .P.

Separation of racemic mixtures can also be accomplished by elution on a column packed with an optically active resolving agent (eg dinitrobenzoylphenylglycine). The suitable composition of the elution solvent is determined by one skilled in the art.

The compounds described by the authors also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond, together with the concomitant movement of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states that have the same empirical formula and overall charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs and ring forms in which the proton can occupy two or more positions in the heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4triazole, 1H- and 2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms can remain in equilibrium or be sterically confined to one form by appropriate substitution.

The compounds described by the authors may also include all atomic isotopes occurring in the intermediate or final compounds. Isotopes are atoms that have the same atomic number but different mass numbers. For example, tritium and deuterium are isotopes of hydrogen.

The term compound as used herein includes all stereoisomers, geometric isomers, tautomers and isotopes of the structures described. Compounds identified by name or structure as one particular tautomeric form by the authors include other tautomeric forms unless otherwise noted.

All compounds and their pharmaceutically acceptable salts can exist together with other substances, such as water and solvents (eg, hydrates and solvates) or can be isolated.

In some embodiments, the compounds or salts thereof described by the authors are essentially isolated. Essentially isolated means that the compound is at least partially or substantially separated from the environment in which it was formed or discovered. The partial separation may include, for example, a composition enriched with the compounds described by the authors. The substantial separation may include compositions comprising at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or whatever at least about 99% by weight of the compounds or their salts described by the authors. Methods for isolating compounds and their salts are familiar to those skilled in the art.

The phrase pharmaceutically acceptable is used by the authors to refer to compounds, materials, compositions and/or dosage forms that are medically suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problem or complication, according to with a reasonable benefit/risk ratio.

The expressions used by the authors, ambient temperature and room temperature or rt, will be understood by those skilled in the art and generally refer to temperature, for example reaction temperature, which is approximately equal to the air temperature in the room in which the reaction is carried out.

- 15 045057 reaction, for example, at a temperature of from about 20°C to about 30°C.

The present invention also includes pharmaceutically acceptable salts of the compounds described by the authors. As used herein, pharmaceutically acceptable salts mean derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acidic or basic moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to: salts with mineral or organic acids of basic moieties such as amines; alkaline or organic salts of acid residues such as carboxylic acids; and so on. Pharmaceutically acceptable salts of the present invention include conventional non-toxic salts of the parent compound, formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from a starting compound that contains a basic or acidic moiety using conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the corresponding base or acid in water or an organic solvent or a mixture thereof; Non-aqueous media such as ether, ethyl acetate, alcohols (eg methanol, ethanol, isopropanol or butanol) or acetonitrile (ACN) are generally preferred. A list of suitable salts is contained in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

As used herein, the term subject or patient is used interchangeably and refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or primates, most preferably humans.

In some embodiments, the inhibitors are administered in a therapeutically effective amount. As used herein, the phrase therapeutically effective amount means the amount of an active compound or pharmaceutical agent that produces a biological or medical response desired by a researcher, veterinarian, physician, or clinician in a tissue, system, animal, subject, or human. In some embodiments, the dose of a compound or a pharmaceutically acceptable salt thereof that is administered to a patient or subject is from about 1 mg to about 2 g, from about 1 mg to about 1000 mg, from about 1 mg to about 500 mg, from about 1 mg to about 200 mg, from about 1 mg to about 100 mg, from about 1 mg to 50 mg, or from about 50 mg to about 500 mg.

As used herein, the term treatment means one or more of the following: (1) control of disease; for example, containing a disease, condition or disorder in a subject who is experiencing or exhibiting pathology or symptomatology of the disease, condition or disorder (ie, delaying further development of the pathology and/or symptomatology); and (2) weakening of the disease; for example, alleviating a disease, condition or disorder in a subject experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder (ie, reversing the pathology and/or symptoms) for example, reducing the severity of the disease.

Types of combination therapy

One or more additional pharmaceutical agents, such as, for example, chemotherapeutic agents, anti-inflammatory drugs, steroids, immunosuppressants, and Bcr-Abl, Flt-3, EGFR, HER2, c-MET, VEGFR, PDGFR, cKit, IGF- 1R, RAF, FAK, Akt mTOR, PIM and AKT (eg AKT1, AKT2 or AKT3) kinases, such as, for example, those described in WO 2006/056399, or other agents such as therapeutic antibodies, can be used in combinations with compounds of the present invention for the treatment of PI3K-related diseases, disorders or conditions. One or more additional pharmaceutical agents are administered to the patient simultaneously or sequentially.

Examples of antibodies for use in combination therapy include, but are not limited to: Trastuzumab (eg, anti-HER2), ranibizumab (eg, anti-VEGF-A), Bevacizumab (trade name Avastin, eg, anti-VEGF, panitumumab (eg, anti-VEGF) -EGFR), cetuximab (eg anti-EGFR), Rituxan (anti-CD20) and antibodies targeting c-MET.

One or more of the following agents may be used in combination with the compounds of the present invention and are presented as a non-limiting list: cytotoxic agent, cisplatin, doxorubicin, taxotere, taxol, etoposide, irinotean, camptostar, topotecan, paclitaxel, docetaxel, epothilones, tamoxifen, 5- fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH 66336, R115777, L778,123, BMS 214662, Iressa, Tarceva, anti-EGFR antibodies, Gleevec™, intron, ara-C, adriamycin, cytoxan, gemcitabine, uracil nitrogen-yprite derivative , chlormethine, ifosfamide , melphalan, Chlorambucil, pipobromane, triethylenemelamine, triethylenethiophosphoramine, busulfan, Carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6thioguanine, fludarabine phosphate, oxaliplatin, leucovirin, ELOKCATIN™, pentostatin, winbl astine, vincristine, vindesine, bleomycin , dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubi

- 16 045057 cin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide, 17a-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltesto sterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide , estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, goserelin, cisplatin, carboplatin, hydroxyurea, Amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, navelbene, anastrazole, letrazole, capecitabine, reloxafine, droxafine, hexamethylmelamine, Avastin, herceptin, bexxar, velcade , Zevalin, Trisenox, Xeloda, Vinorelbine, Porfimer, Erbitux, Liposomal, Thiotepa, Altretamine, Melphalan, Trastuzumab, Lerozol, Fulvestrant, Exemestane, Ifsfomide, Rituximab, C225, Campath, Clofarabine, Cladribine, Aphidicolon, Rituxan, Sunitinib, Dasatinib, Tesa citabine , Sml1, fludarabine, pentostatin, triapine, didox, trimidox, amidox, 3AP, MDL-101,731, bendamustine (Treanda), ofatumumab, or GS-1101 (also known as CAL-101).

Examples of chemotherapeutic agents include proteasome inhibitors (eg, bortezomib), thalidomide, Revlimid, and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, etc.

Examples of steroids include corticosteroids such as dexamethasone or prednisone.

Examples of Bcr-Abl inhibitors include the compounds and their pharmaceutically acceptable salts of the genera and species disclosed in US Pat. No. 5,521,184, WO 04/005281, and US Application No. 60/578491.

Examples of suitable Flt-3 inhibitors include the compounds and their pharmaceutically acceptable salts disclosed in WO 03/037347, WO 03/099771 and WO 04G6120.

Examples of suitable RAF inhibitors include the compounds and their pharmaceutically acceptable salts disclosed in WO 00/09495 and WO 05/028444.

Examples of suitable FAK inhibitors include the compounds and their pharmaceutically acceptable salts disclosed in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595 and WO 01/014402.

Examples of suitable mTOR inhibitors include the compounds and their pharmaceutically acceptable salts disclosed in WO 2011/025889.

In some embodiments, the compounds of the invention may be used in combination with one or more other kinase inhibitors, including imatinib, particularly for the treatment of patients resistant to imatinib or other kinase inhibitors.

In some embodiments, the compounds of the invention may be used in combination with chemotherapeutic agents for the treatment of cancer, such as multiple myeloma, and may improve the response to treatment compared to the response to the chemotherapeutic agent alone, without increasing its toxic effects. Examples of additional pharmaceutical agents used to treat multiple myeloma include, but are not limited to, melphalan, melphalan plus prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Other additional agents used to treat multiple myeloma include Bcr-Abl, Flt-3, RAF, and FAK kinase inhibitors. the desired results of combining the PI3K inhibitor of the present invention with an additional agent are additive or synergistic effects. In addition, resistance of multiple myeloma cells to agents such as dexamethasone may be reversible after treatment with a PI3K inhibitor according to the present invention. The agents may be combined with the present compound in a single or continuous dosage form, or the agents may be administered simultaneously or sequentially as separate dosage forms.

In some embodiments, a corticosteroid, such as dexamethasone, is administered to a patient in combination with compounds of the invention, wherein the dexamethasone is administered intermittently as opposed to continuously administered.

In some other embodiments, combinations of compounds of the invention with other therapeutic agents are administered to a patient before, during, and/or after a bone marrow transplant or stem cell transplant.

Pharmaceutical compositions and dosage forms.

When used as pharmaceuticals, the compounds described by the authors can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known to those skilled in the art of pharmaceuticals and can be administered in a variety of ways depending on whether local or systemic treatment is required and the site being treated. Administration may be local (including intradermal, epidermal, ocular and mucosal, including intranasal, vaginal and rectal), pulmonary (eg, by inhalation or insufflation of powders or aerosols, including administration by an aerosol apparatus; intrathecal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; or intracranial, for example, intrathecal or intraventricular administration. Parenteral administration may be provided in the form of a single bolus dose or, for example, as a continuous perfusion. Pharmaceutical compositions and topical formulations

- 17 045057 administration may be provided in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, aerosols, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like may be necessary or desirable. This invention also covers pharmaceutical compositions containing as an active ingredient the compound described by the authors or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable carriers (excipients). In some embodiments, the composition is suitable for topical administration. In the preparation of compositions according to the invention, the active ingredient is typically mixed with the excipient, diluted with the excipient, or included in such a carrier, for example, in the form of a capsule, sachet, paper or other container. If the excipient serves as a diluent, it may be a solid, semi-solid or liquid material that serves as a base, carrier or medium for the active ingredient. Thus, the compositions may be provided in the form of tablets, pills, powders, troches, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in solid form or in a liquid carrier), ointments containing, for example, up to 10 % by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

When preparing the composition, the active compound may be milled to ensure proper particle size before combining with other ingredients. If the active compound is substantially insoluble, it may be ground to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is adjusted by grinding to ensure substantially uniform distribution in the composition, for example, about 40 mesh.

The compounds described by us are crushed using known grinding procedures, such as wet grinding, to provide a particle size suitable for tablet forming and other types of compositions. Finely divided (nanoparticle) compositions of the compounds described by the authors are prepared using processes known to those skilled in the art. For example, see International Application No. WO 2002/000196.

Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The compositions may additionally contain: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; and flavors. The compositions of the invention may be formulated to provide rapid, sustained or delayed release of the active ingredient following administration to a patient using procedures known to those skilled in the art.

The compositions may be formulated in unit dosage form, each containing from about 5 to about 1000 mg (1 g), more typically from about 100 to about 500 mg, of active ingredient. The term unit dosage form refers to physical discrete units suitable as unit dosages for humans and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in combination with a suitable pharmaceutical excipient.

In some embodiments, the compositions of the invention contain from about 5 to about 50 mg of active ingredient. One skilled in the art will appreciate that compositions can be realized containing from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 35, about 35 to about 40, about 40 to about 45, or about 45 to about 50 mg of active ingredient.

In some embodiments, the compositions of the invention contain from about 50 to about 500 mg of active ingredient. One skilled in the art will appreciate that compositions can be realized containing from about 50 to about 100, from about 100 to about 150, from about 150 to about 200, from about 200 to about 250, from about 250 to about 300, from about 350 to about 400 or about 450 to about 500 mg of active ingredient.

In some embodiments, the compositions of the invention contain from about 500 to about 1000 mg of active ingredient. One skilled in the art will appreciate that compositions may be realized containing from about 500 to about 550, from about 550 to about 600, from about 600 to about 650, from about 650 to about 700, from about 700 to about 750, from about 750 to about 800, about 800 to about 850, about 850 to about 900, about 900 to about 950, or about 950 to about 1000 mg of active ingredient.

Similar doses of the compounds described by the authors can be used according to the methods of application of the invention.

The active compound can be effective over a wide range of dosages and is typically administered in a pharmaceutically effective amount. It should be understood, however, that the actual amount of compound administered will generally be determined by the physician depending on the relevant circumstances, including the condition being treated, the route of administration chosen, the particular compound administered, the age, weight and response of the particular patient, the severity of the patient's symptoms, and etc.

For preformed solid compositions, such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a solid composition candidate containing a homogeneous mixture of the compound of the present invention. In the case of homogeneous composition candidates, the active ingredient is typically dispersed uniformly throughout the composition so that the composition can be readily divided into equally effective unit dosage forms such as tablets, pills and capsules. This solid composition candidate is then divided into unit dose forms of the type described above, which contain, for example, from about 0.1 to about 1000 mg of the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form capable of sustained release. For example, a tablet or pill may include an inner and an outer dose component, the latter being provided in the form of a shell over the former. The two components may be separated by an enteric layer, which prevents disintegration in the stomach and allows the internal component to pass intact into the duodenum or delay its release. Various materials are used for such enteric layers or coatings, for example, materials including many polymer acids and mixtures of polymer acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Liquid forms in which the compounds and compositions of the present invention may be formulated for oral or injection administration include aqueous solutions, flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil , coconut oil or peanut butter, as well as elixirs and similar pharmaceutical bases.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures and powders thereof. Liquid or solid compositions may contain appropriate pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. The compositions are nebulized by using inert gases. Nebulized solutions may be inhaled directly from the nebulization device, or the nebulization device may be attached to a face mask, tent, or intermittent positive pressure breathing apparatus. The solution, suspension or powder compositions can be administered orally or nasally from devices appropriately delivering the composition.

Compositions for topical use may contain one or more conventional carriers. In some embodiments, ointments may contain water and one or more hydrophobic carriers selected, for example, from liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petrolatum, and the like. Cream carrier compositions may be provided in an aqueous base in combination with glycerin and one or more other components, for example, glycerol monostearate, PEG-glycerol monostearate and cetyl stearyl alcohol. Gels can be formulated using isopropyl alcohol and water, preferably in combination with other components such as, for example, glycerin, hydroxyethylcellulose, etc. In some embodiments, the topical compositions contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5 weight percent of the disclosed compound. . Compositions for topical use may be suitably packaged in tubes, for example, 100 g, which are optionally accompanied by instructions for treatment of the selected indication, for example, psoriasis or other skin indication.

The amount of compound or composition to be administered to a patient may vary depending on the substance administered, the purpose of administration, e.g., prophylaxis or therapy, the condition of the patient, the route of administration, and the like. when used therapeutically, the compositions are administered to a patient already suffering from a disease in an amount sufficient to treat or at least partially control the symptoms of the disease and its complications. Effective doses depend on the disease state being treated and are at the discretion of the clinician depending on factors such as the severity of the disease, age, weight and general condition of the patient, etc.

The compositions administered to the patient may be provided in the form of pharmaceutical compositions described by the authors. These compositions may be sterilized by conventional sterilization methods or may be subjected to sterile filtration. Aqueous solutions may be packaged for use as is or lyophilized, the lyophilized composition being combined with a sterile aqueous vehicle prior to administration. The pH level of the compound compositions is generally from 3 to 11, more preferably from 5 to 9, and most preferably from 7 to 8. It should be understood that the use of some of the above excipients, carriers or stabilizers results in leads to the formation of pharmaceutical salts.

The therapeutic dosage of a compound of the present invention may vary, for example, depending on the specific application for which the treatment is intended, the route of administration of the compound, the medical condition of the patient, and the opinion of the prescriber. The proportion or concentration of the compound described by the authors in the pharmaceutical composition may vary depending on many factors, including dose, chemical characteristics (eg, hydrophobicity) and route of administration. For example, the compounds described by us may be provided in an aqueous physiological buffer solution containing from about 0.1 to about 10% (w/v) of the compound for parenteral administration. Some typical dosage ranges are from about 1 mcg/kg to about 1 g/kg body weight per day. In some embodiments, the dosage range is from about 0.01 mg/kg to about 100 mg/kg body weight per day. The dosage generally depends on such variables as the type and degree of progression of the disease or disorder, the general health of the individual patient, the relative biological effectiveness of the selected compound, the formulation of the excipient, and the route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro tests or animal models.

The compositions of the invention may also include one or more additional pharmaceutical agents, such as a chemotherapeutic agent, a steroid, an anti-inflammatory compound or an immunosuppressant, examples of which are listed by the authors.

Sets.

The present invention also includes pharmaceutical kits used, for example, in the treatment or prevention of PI3K-related diseases or disorders, such as cancer, and comprising one or more receptacles containing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound. Such kits may also optionally include one or more conventional pharmaceutical kit components, such as, for example, containers containing one or more pharmaceutically acceptable carriers, additional containers, and the like, which may be apparent to those skilled in the art. The kit may also include instructions, in the form of inserts or labels, indicating the quantities of components to be administered, recommendations for administration, and/or recommendations for mixing components.

Examples

Example 1. ((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro2H -pyran-2-yl)acetonitrile.

Step 1. tert-butyl (4S)-2,2-dimethyl-4-vinyl-1,3-oxazolidine-3-carboxylate.

To a suspension of methyltriphenylphosphonium bromide (5.63 g, 15.8 mmol) in tetrahydrofuran (140 ml) was added 2.5 M n-butyllithium in hexane (7.35 ml, 18.4 mmol). The deep red solution was stirred at 0°C for 1 hour. Then a solution of tert-butyl (4R)-4-formyl-2,2dimethyl-1,3-oxazolidine-3-carboxylate (from Aldrich, 3.01 g, 13.1 mmol) in tetrahydrofuran (7.3 ml). The red solution was heated to room temperature and stirred for 12 hours. A mixture of hexane isomers was added to the reaction mixture in a 4:1 (vol/volume) ratio. The suspension was filtered through celite and the filtrate was concentrated. The resulting residue was purified by flash chromatography (eluting with 10% ethyl acetate/hexane) to give the title compound as a colorless oil (1.92 g, 64%).

Step 2. tert-Butyl [(1S)-1-(hydroxymethyl)prop-2-en-1-yl]carbamate.

To a solution of tert-butyl (4S)-2,2-dimethyl-4-vinyl-1,3-oxazolidine-3-carboxylate (1.90 g, 8.36 mmol) in methanol (83 ml) was added p-toluenesulfonic acid monohydrate acid (0.80 g, 4.2 mmol) at 0°C. The mixture was slowly warmed to room temperature over the course of 24 hours. The reaction mixture was diluted with saturated NaHCO ₃ solution, concentrated and then diluted with ethyl acetate. The organic layer was washed with saturated NaHCO ₃ (2x) and brine, dried over Na ₂ SO ₄ , filtered and concentrated to give the desired product as a colorless oil (1.187 g, 76%). 1H NMR (400 MHz, CDC13) δ 5.81 (1H, m), 5.25 (2H, m), 4.90 (1H, m), 4.25 (1H, w s), 3.67 ( 2H, m), 1.45 (9H, s) ppm.

Step 3. tert-Butyl [(1S)-1-({[1-(hydroxymethyl)prop-2-en-1-yl]oxy}methyl)prop-2-en-1-yl]carbamate.

The flask was charged with tert-butyl [(1S)-1-(hydroxymethyl)prop-2-en-1-yl]carbamate (0.401 g, 2.14 mmol), tris(dibenzylideneacetone) dipalladium (0) (59 mg, 0.064 mmol), N,N'-(1S,2S)-cyclohexane-1,2-diylbis[2(diphenylphosphino)-1-naphthalimide] (150 mg, 0.19 mmol) and 4-dimethylaminopyridine (78 mg, 0. 64 mmol). The reaction mixture was purged with N2 three times and then methylene chloride (21.3 mL) and 1.0 M triethylborane in THF (130 μL, 0.13 mmol) were added sequentially. After stirring for 10 minutes, 2vinyloxirane (0.150 g, 2.14 mmol) was added and the resulting mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and saturated NaHCO ₃ solution. Organic layer

- 20 045057 was separated and dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by flash chromatography (eluting with 0-50% ethyl acetate/hexane) to give the desired product (0.271 g, 49%). 1H NMR (300 MHz, CDC13) δ 5.85 (1H, m), 5.67 (1H, m), 5.84~5.17 (4H, m), 4.83 (1H, m), 4 .30 (1H, w s), 3.83 (1H, m), 3.69 (1H, dd, J = 4.5 and 6.9 Hz), 3.54 (2H, m), 3.36 (1H, dd,

J=4.5 and 6.9 Hz), 1.45 (9H, s) ppm.

Step 4. 2-({(2S)-2-[(tert-Butoxycarbonyl)amino]but-3-en-1-yl}oxy)but-3-en-1-yl acetate.

To a mixture of tert-butyl [(1S)-1-({[1-(hydroxymethyl)prop-2-en-1-yl]oxy}methyl)prop-2-en-1yl]carbamate (268 mg, 1.04 mmol) in methylene chloride (10 ml), triethylamine (435 μl, 3.12 mmol) was added. The mixture was cooled to 0 °C and acetyl chloride (150 μL, 2.1 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours and then quenched with water. The organic layer was concentrated and the resulting residue was purified on silica gel (eluting with 20% ethyl acetate/hexane) to give the desired product (0.26 g, 85%). LCMS calculation for C ₁₀ H ₁₈ NO ₃ (M-100+H)+: m/z = 200.1; Detected: 200.1.

Step 5. {(5S)-5-[(tert-Butoxycarbonyl)amino]-5,6-dihydro-2H-pyran-2-yl}methyl acetate.

Benzylidene(dichloro)(1,3dimesitylimidazolidin-2-id-2-yl)(tricyclohexylphosphoranyl)ruthenium (38 mg, 0.044 mmol) was added to a 500 mL two-neck round bottom flask. After purging with nitrogen three times, dichloromethane (anhydrous, 8 ml) was added followed by 2-({(2S)-2-[(tert-butoxycarbonyl)amino]but-3-en-1-yl}oxy)but-3-ene -1-yl acetate (265 mg, 0.885 mmol). The reaction mixture was stirred at room temperature for 15 hours. The mixture was concentrated in vacuo. The residue was purified by flash chromatography (eluting with hexane to 25% EtOAc in hexane) to give the desired product as a brown oil (0.205 g, 85%). LCMS calculation for C ₉ H ₁₄ NO ₅ (M+H-Bu+H)+: m/z = 216.1; Detected: 216.1. ^1H NMR (300 MHz, CDCI3) δ 5.94 (0.17H, m), 5.84 (0.83H, m), 5.69 (1H, m), 4.89 (0.13H, m ), 4.70 (0.83Н, m), 4.25 (1Н, m), 4.05 (4Н, m), 3.56 (0.13Н, m), 3.38 (0.87Н, m), 2.04 (2.49H, s), 2.03 (0.5H, m), 1.38 (9H, s) ppm. (the product was a ~5:1 mixture of trans and cis isomers).

Step 6. [(5S)-5-Amino-5,6-dihydro-2H-pyran-2-yl]methyl acetate.

To a solution of {(5S)-5-[(tert-butoxycarbonyl)amino]-5,6-dihydro-2H-pyran-2-yl}methyl acetate (205 mg, 0.756 mmol) in methylene chloride (5.2 ml) was added 4.0 M hydrogen chloride in dioxane (1.5 ml, 6.0 mmol). The reaction solution was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure to obtain the desired product as a white solid. LCMS calculation for C ₈ H ₁₄ NO ₃ (M+H) ⁺ : m/z =172.1; Detected: 172.1.

Step 7. {(5S)-5-[(6-Humpomueno[3,2-b]pyridin-7-yl)amino]-5,6-dihydro-2H-pyran-2-yl} methyl acetate.

A mixture of 7-chloro-6-nitrothieno[3,2-b]pyridine (156 mg, 0.727 mmol), [(5S)-5-amino-5,6-dihydro-2Hpyran-2-yl]methyl acetate (129 mg, 0.754 mmol) and N,N-diisopropylethylamine (0.26 ml, 1.5 mmol) in isopropyl alcohol (1.7 ml) were treated at 90°C for 2 hours. The reaction mixture was concentrated and purified by flash chromatography to obtain desired product (0.21 g, 83%). LCMS calculation for C ₁₅ H ₁₆ N ₃ O ₅ S (M+ H) ⁺ : m/z = 350.1; Detected: 350.0.

Step 8. {(5S)-5-[(6-Aminothieno[3,2-b]pyridin-7-yl)amino]tetrahydro-2H-pyran-2-yl}methyl acetate.

Mixture of {(5S)-5-[(6-nitrothieno[3,2-b]pyridin-7-yl)amino]-5,6-dihydro-2H-pyran-2-yl}methyl acetate (210 mg, 0.600 mmol ) and 10% palladium on carbon (0.21 g) in methanol (4.0 ml) were subjected to balloon pressure with H2 at room temperature for 2 hours. The mixture was filtered and the filtrate was concentrated and purified by flash chromatography (eluting 15% methanol in dichloromethane) to obtain the desired product (145 mg, 75%). LCMS calculation for C ₁₅ H ₂₀ N ₃ O ₃ S (M+ H) ⁺ : m/z = 322.1; Detected: 322.0.

Step 9. (1R)-1-{1-[(3S)-6-(Hydroxymethyl)tetrahydro-2H-pyran-3-yl]-1H-imidazo[4,5-d]mueno[3,2b]pyridine -2-yl}ethanol.

A mixture of (2R)-2-hydroxypropanamide (131 mg, 1.47 mmol) and triethyloxonium tetrafluoroborate (263 mg, 1.38 mmol) in THF (2 ml) was stirred at room temperature for 2 hours. The solvent was removed and the residue was dissolved in ethanol. (0.85 ml) and added to a suspension of {(5S)-5-[(6-aminothieno[3,2b]pyridin-7-yl)amino]tetrahydro-2H-pyran-2-yl}methyl α acetate (145 mg, 0.451 mmol) in ethanol (3.1 ml). The mixture was stirred at 80°C for 1 hour. The reaction mixture was cooled to room temperature and diluted with water (1.0 ml). Lithium hydroxide (32.4 mg, 1.35 mmol) was added and the mixture was stirred for 2 hours. The reaction mixture was diluted with methanol and purified by preparative LCMS (XBridge C18 column, eluting with an acetonitrile/water gradient containing 0.1% ammonium hydroxide at flow rate 60 ml/min) to obtain the desired product as a white solid (95 mg, 63%). LCMS calculation for C ₁₆ H ₂₀ N ₃ O ₃ S (M+ H) ⁺ : m/z = 334.1; Detected: 334.0.

Step 10. ((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1yl}tetrahydro-2H -pyran-2-yl)methyl 4-methylbenzenesulfonate and ((2S,5S)-5-{2-[(1R)-1-hydroxyethyl]-1Nimidazo[4,5-d]thieno[3,2-b] pyridin-1-yl}tetrahydro-2H-pyran-2-yl)methyl 4-methylbenzenesulfonate.

To a solution of (1R)-1-{1-[(3 S)-6-(hydroxymethyl)tetrahydro-2H-pyran-3 -yl]-1 H-imidazo[4,5-d]thieno[3,2b] pyridin-2-yl}ethanol (100 mg, 0.300 mmol) (previous step) in methylene chloride (3.4 ml) and pyridium

- 21 045057 p-toluenesulfonyl chloride (57.2 mg, 0.300 mmol) and 4dimethylaminopyridine (1.8 mg, 0.015 mmol) were added (0.146 ml, 1.80 mmol) at 0°C. The reaction mixture was allowed to warm to room temperature overnight. The reaction mixture was concentrated, diluted with methanol and purified by preparative LCMS (XBridge C18 column, eluting with acetonitrile/water gradient containing 0.1% ammonium hydroxide at a flow rate of 60 ml/min) to obtain two peaks. With analytical HPLC (Waters SunFire C18, 2.1 x 50 mm, 5 μM; flow rate 3 ml/min; injection volume 2 μl; with a gradient from 2 to 80% B in 3 minutes (A = water with 0.025% TFA, B = acetonitrile)): first peak (45.3 mg, 31%) retention time 1.81 min, LCMS calculation for C ₂₃ H ₂₆ N ₃ O ₅ S ₂ (M+H)+: m/z = 488, 1; Found: 488.1. Second peak (8.5 mg, 5.8%) retention time 1.88 min, LCMS calculation for C ₂₃ H ₂₆ N ₃ O ₅ S ₂ (M+H) ⁺ : m/z = 488.1; Found: 488.1.

Step 11. ((2R,5S)-5-{2-[(1R)-1-Hydroksiethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1yl}tetrahydro-2H -pyran-2-yl)acetonitrile.

Mixture of ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2Hpyran -2-yl)methyl 4-methylbenzenesulfonate (from the 1st peak of the previous step, 27 mg, 0.055 mmol) and sodium cyanide (4.5 mg, 0.092 mmol) in dimethyl sulfoxide (0.4 ml) were stirred at 50 ° C in After cooling, the mixture was diluted with methanol and purified by preparative LCMS (XBridge C18 column, eluting with acetonitrile/water gradient containing 0.1% ammonium hydroxide at a flow rate of 30 ml/min) to obtain the desired product (14.5 mg, 76% ). LCMS calculation for C17H19N4O2S (M+ H) ⁺ : m/z = 343.1; Detected: 343.0. ^1H NMR (DMSOL, 500 MHz) δ 9.51 (1H, s), 8.45 (1H, d, J=5.5 Hz), 7.97 (1H, d, J=5„5 Hz) , 5.31 (1H, m), 5.20 (1H, m), 4.31 (1H, m), 4.23 (1H, m), 4.02 (1H, m), 2.96 ( 1H, dd, J=17.0 and 4.5 Hz), 2.85 (1H, dd, J=17.0 and 4.5 Hz), 2.66 (1H, m), 2.26 (1H , m), 2.09 (1H, m), 1.73 (1H, m), 1.69 (3H, dD = 6.5 Hz) ppm.

Example 1a. Hydrate ((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1yl}tetrahydro-2H-pyran -2-yl)acetonitrile.

((2R,5 S)-5-{2-[( 1R)-1-G hydroxyethyl]-1 H-imidazo[4,5-0]thieno[3,2-b]pyridin-1-yl} tetrahydro -2Hpyran-2-yl)acetonitrile (52 mg, 0.15 mmol) according to Example 25 was crystallized from a mixture of acetonitrile (8 ml) and water (4 ml). The resulting and collected prismatic crystals were suitable for X-ray crystal structure analysis.

Crystal data shows: ~0.520 x 0.180 x 0.100 mm, orthorhombic, P212121, a = 6.962(3)A, b = 11.531(4)A, c = 20.799(7)A, Vol = 1669.6(10)A ³ , Z = 4, T = -100°C, molecular weight according to the compound formula = 359.42, density = 1.430 g/cm ³ , μ(Mo) = 0.22 mm ^-1 .

Data acquisition was carried out on a Bruker SMART APEX-II CCD system, MoKalpha radiation, standard focusing tube, anode power = 50 kV x 42 mA, crystal-to-plate distance = 5.0 cm, 512 x 512 pixels/frame, beam center = (256.13, 253.14), total frames = 1151, oscillation/frame = 0.50°, exposure/frame =10.1 sec/frame, SAINT integration, hkl min./max. = ( -9, 9, -15, 15, -27, 27), shelx data entry = 17025, unique data = 3975, 2-theta range = 3.92 to 55.72°, complete to 2-theta 55.72 = 99.80%, R(int-xl) = 0.0681, SADABS correction applied.

The structure was solved using XS(Shelxtl), refined using the shelxtl software package, least squares refinement in the full matrix approximation using F ² , dispersion coefficients from Int. Tab. Vol C Tables 4.2.6.8 and 6.1.1.4, number of data = 3975, number of restrictions = 0, number of parameters = 235, data/parameter ratio = 16.91, goodness-of-fit criterion for F ² = 1.04, R-indices [I >4sigma(I)] R1 = 0.0505, wR2 = 0.1242, R-indices (all data) R1 = 0.0769, wR2 = 0.1401, peak and trough max, differences = 0.724 and -0.277 e/ A ³ , refined Flack parameter = -0.12(13), all hydrogen atoms CH were refined using the constraint model. The OH hydrogen atoms were detected by difference imaging and fully refined.

The results showed that the asymmetric unit contains one molecule and one water, as shown by thermal ellipsoids constructed at a 50% probability level. The stereochemistry at each of the three stereocenters (as indicated by the name and structure of the above compound) was confirmed. The Flack parameter, refined to 0.28(24), indicated the correct enantiomeric setting.

Example 2 4-[3-(Cyanomethyl)-3-(3',5'-dimethyl-1H,1Ή-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro -N[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

- 22 045057

Step 1. 2,4,5-Trifluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

To a solution of 2,4,5-trifluorobenzoic acid (5.00 g, 28.4 mmol) in acetonitrile (50 ml) was added N,N-dimethylformamide (40 μl), followed by the addition of oxalyl chloride (3.60 ml, 42.6 mmol). After 90 min, volatiles were removed under reduced pressure. The residue was evaporated with acetonitrile (50 ml). The residue was then dissolved in methylene chloride (50 ml). This solution was added dropwise to a cooled (ice bath) mixture of (2S)-1,1,1-trifluoropronan-2-amine hydrochloride (5.52 g, 36.9 mmol) (from Synquest, 98% ee) in toluene (100 ml) and 0.5 M aqueous sodium hydroxide solution (142 ml, 71.0 mmol). After addition, the ice bath was removed and the reaction mixture was allowed to warm to room temperature. The reaction mixture was stirred for 24 hours. The organic layer was separated. The aqueous layer was extracted with methylene chloride (50 ml). The combined organic layers were washed with 20% brine (75 ml) and water (2 x 75 ml), dried over MgSO ₄ , filtered and concentrated under reduced pressure to obtain the desired product (6.49 g, 84%), which was used directly for next stage without further purification. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 9.01 (d, J=7.6 Hz, 1H), 7.92 - 7.50 (m, 2H), 4.76 (m, 1H) , 1.31 (d, J=7.0 Hz, 3H) ppm. LCMS calculation for C ₁₀ H ₈ F ₆ NO (M+1)+: m/z = 272.0; Detected: 272.0.

Step 2. 2,5-Difluoro-4-(3-hydroxyazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

A mixture of 2,4,5-trifluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (6.39 g, 23.6 mmol), azetidin-3ol hydrochloride (3.19 g, 28. 3 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (8.81 ml, 58.9 mmol) in acetonitrile (25 ml) were stirred at 80°C for 2 hours.

The reaction mixture was diluted with EtOAc (75 ml) and washed with 1N HCl (50 ml), 1N NaHCO ₃ (60 ml), 20% saline (50 ml) and water (75 ml). The aqueous layers were extracted with EtOAc (100 ml). The organic layers were combined, dried over MgSO ₄ , filtered and concentrated under reduced pressure to yield the desired product (7.59 g, 91.8%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.38 (dd, J=8.9, 1.9 Hz, 1H), 7.27 (dd, J=12.8, 6.5 Hz, 1H), 6.38 (dd, J=12.3, 7.5 Hz, 1H), 5.71 (d, J=6.4 Hz, 1H), 4.74 (dp, J=15.3 , 7.6 Hz, 1H), 4.62 - 4.46 (m, 1H), 4.30 - 4.15 (m, 2H), 3.71 (m, 2H), 1.29 (d, J=7.1 Hz, 3H) ppm. LCMS calculation for C ₁₃ H ₁₄ F ₅ N ₂ O ₂ (M+1) ⁺ : m/z = 325.1; Detected: 325.1.

Step 3. 2,5-Difluoro-4-(3-oxoazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

To a solution of 2,5-difluoro-4-(3-hydroxyazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (7.57 g, 23.3 mmol ) in methylene chloride (93 ml) added iodobenzene diacetate (9.40 g, 29.2 mmol) and the free radical 2,2,6,6-tetramethyl-1-piperidinyloxy (1.82 g, 11.7 mmol) (TEMPO) at room temperature. The reaction mixture was stirred at room temperature for 24 hours. The mixture was diluted with EtOAc (100 ml), washed with 0.5N NaHCO ₃ (2x80 ml), 20% saline (100 ml) and water (100 ml). The aqueous layers were extracted with ethyl acetate (75 ml). The organic extracts were combined, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography eluting with 0% to 5% ethyl acetate in methylene chloride to give the crude product, which was recrystallized from MTBE (50 ml) and heptane (100 ml) to give the desired product (5.44 g, 72 %) as a colorless solid. 1H NMR (300 MHz, DMSO-d ₆ ) δ 8.52 (d, J = 8.0 Hz, 1H), 7.36 (dd, J = 12.5, 6.5 Hz, 1H), 6, 63 (dd, J=12.1, 7.6 Hz, 1H), 4.90 (d, J=2.1 Hz, 4H), 4.86 -4.68 (m, 1H), 1.31 (d, J=7.1 Hz, 3H) ppm LCMS calculation for C ₁₃ H ₁₂ F ₅ N ₂ O ₂ (M+1) ⁺ : m/z = 323.1; Detected: 323.0.

Step 4. 4-[3-(Cyanomethylene)azetudin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

Diethylcyanomethylphosphonate (1.95 mL, 11.8 mmol) was added dropwise to a cooled (ice bath) solution of 1.0 M potassium tert-butoxide in THF (11.8 mL, 11.8 mmol), which was diluted with tetrahydrofuran (12 ml). The bath was removed and the reaction mixture was warmed to room temperature and stirred for 90 minutes. The reaction solution was cooled again using an ice bath. The previously prepared solution was then added over 12 min to a cooled (ice bath) solution of 2,5difluoro-4-(3-oxoazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1- methylethyl]benzamide (4.00 g, 12.4 mmol) in tetrahydrofuran (50 ml). The reaction mixture was stirred for 30 minutes. The ice bath was removed and the reaction mixture was stirred at room temperature overnight, then quenched by adding 20% saline (75 ml) and ethyl acetate (75 ml). The organic layer was separated. The aqueous layer was extracted with ethyl acetate (50 ml). The combined organic layers were dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column with ethyl acetate in a mixture of hexane isomers (0% to 30%) to give the desired product (2.6 g). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.59 - 8.37 (m, 1H), 7.33 (dd, J=12.5, 6.4 Hz, 1H), 6.59 (dd , J = 12.0, 7.4 Hz, 1H), 5.88 (m, 1H), 4.94 - 4.75 (m, 4H), 4.76 (m, 1H), 1.31 ( d,

- 23 045057

J=7.1 Hz, 3H) ppm, LCMS calculation for C ₁₅ H ₁₃ F ₅ N ₃ O (M+1)+: m/z = 346.1; Found: 346.1.

Step 5. 4-{3-(Cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1yl]azetidin-1 -yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

Mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.00 g, 5.15 mmol), 4-[3(cyanomethylene)azetidine -1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (1.78 g, 5.15 mmol) and 1,8-diazabicyclo[ 5.4.0]undec-7-ene (0.31 ml, 2.1 mmol) in acetonitrile (20.2 ml) was heated at 50°C for 24 hours. After cooling, the solvent was removed under reduced pressure. The residue was used in the next step without further purification. LCMS calculation for C ₂₄ H ₂₈ BF ₅ N ₅ O ₃ (M+1) ⁺ : m/z = 540.2; Detected: 540.1.

Step 6. 4-[3-(Cyanomethyl)-3-(3', 5'-dimethyl-1H, 1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5difluoro -N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

Mixture of 4-{3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1yl]azetidin-1-yl }-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (329 mg, 0.610 mmol), 4bromo-3,5-dimethyl-1H-pyrazole (206 mg , 1.18 mmol), tetrakis(triphenylphosphine)palladium(0) (110 mg, 0.098 mmol) and sodium carbonate (320 mg, 3.0 mmol) in 1,4-dioxane (10 ml)/water (5 ml) purged with nitrogen and stirred at 110°C for 1 hour. The reaction mixture was diluted with EtOAc, washed with water and brine, and concentrated. The residue was purified first with silica gel (eluting with 0-100% EtOAc/hexane mixture followed by 10% methanol/dichloromethane) and then by preparative LCMS (XBridge C18 column, eluting with a gradient of acetonitrile/water containing 0.1% ammonium hydroxide at flow rate 60 ml/min) to obtain the desired product (30 mg, 9.7%). 1H NMR (500 MHz, DMSO-d6) δ 12.17 (1H, s), 8.45 (1H, d, J=8.0 Hz), 8.10 (1H, s), 7.70 (1H , s), 7.34 (1H, m), 6.61 (1H, s), 4.77 (1H, m), 4.62 (2H, d, J=9.0 Hz), 4.39 (1H, d, J=9.0 Hz), 3.64 (2H, s), 2.22 (6H, s), 1.31 (6H, d, J=7.0 Hz) ppm. , LCMS calculation for C ₂₃ H ₂₃ F ₅ N ₇ O (M+H)+: m/z = 508.2; Detected: 508.0.

Example A. In vitro JAK kinase assay.

These compounds were tested for inhibitory activity of JAK entities according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94 104. Catalytic domains of human JAK1 (aa 837-1142), JAK2 (a .a. 828-1132) and JAK3 (a.a. 781-1124) were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2, or JAK3 was analyzed by measuring the phosphorylation of the biotinylated peptide. Phosphorylated peptide was detected using homogeneous time-resolved fluorescence (HTRF). IC ₅₀ values of compounds were measured for each kinase in 40 μl reaction mixtures that contained enzyme, ATP and 500 nM peptide in 50 mM Tris buffer (pH 7.8) with 100 mM NaCl, 5 mM DTT and 0.1 mg/ml (0.01%) BSA. For IC ₅₀ 1 mM measurements, the ATP concentration in the reaction mixtures was 1 mM. Reactions were carried out at room temperature for 1 hour and then terminated with 20 μl of 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, MA). Binding to the europium-labeled antibody was carried out for 40 minutes and the HTRF signal was measured on a PHERAstar plate reader (BMG, Cary, NC). Data for JAK1 and/or JAK2 inhibitors were obtained by testing the compound by assay according to Example A at 1 mM ATP.

Example B: PI3Kδ Proximity Scintillation Assay.

Materials.

[y- ³³ P]ATP (10 mCi/ml) was purchased from Perkin-Elmer (Waltham, MA). Lipid kinase substrate, D-myo-phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho-linked (PIP2), CAS 204858-53-7, purchased from Echelon Biosciences (Salt Lake City, UT). PI3Kδ (p110δ/p85a) was purchased from Millipore (Bedford, MA). ATP, MgCl2, DTT, EDTA, MOPS, and CHAPS were purchased from Sigma-Aldrich (St. Louis, MO). Wheat germ agglutinin (WGA), YSi SPA scintillation beads were purchased from GE Healthcare Life Sciences (Piscataway, NJ).

The kinase reaction was performed in a white polystyrene 384-well plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first serially diluted in DMSO and added to the wells of the plate before adding other reaction components. The final concentration of DMSO in the assay was 0.5%. PI3K assays were performed at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl ₂ , 5 mM DTT, and CHAPS 0.03%. Reactions were started by adding ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi[y- ^33P ]ATP, 4 nM PI3Kδ. The reaction mixtures were incubated for 210 min and the reaction was terminated by adding 40 μl of SPA beads suspended in quenching buffer: 150 mM potassium phosphate, pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After sealing, the plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, and the radioactivity of the product was determined by scintillation counting on a Topcount (Perkin-Elmer). The determination of IC ₅₀ was carried out by adjusting the percentage control activity curve by the logarithm of the inhibitor concentration using the Graph-24 045057 program

Pad Prism 3.0. Data for ΡΙ3Κδ inhibitors were obtained by testing compounds by assay according to Example B.

Example C. Pfeiffer model for lymphoma.

Ways.

Female SCID mice (5 to 8 weeks old, Charles River Laboratories, Wilmington, MA) were inoculated with 1x107 tumor cells (Pfeiffer, ATCC #CRL-2632, Manassas, VA) and Matrigel (BD Biosciences #354234) in 0.2 ml sterile saline solution. Inoculation was carried out subcutaneously in the side. Tumor tissue fragments (about 3 mmx3 mm) were collected 3-6 weeks after inoculation of cultured cells and implanted subcutaneously instead of cell inoculation. Tissue fragments were implanted as solid tumor fragments using blunt forceps. Treatment of tumor-bearing mice began 15–25 days after tumor inoculation, depending on tumor size. Animals were sorted to obtain approximately equal mean tumor volumes in each group. The minimum mean tumor volume in all groups was 150 mm3 on the first day of treatment, and the groups consisted of 7 animals. The experimental therapeutic agent of Example 347 was administered orally (PO) to mice. Treatment frequency was 2 times daily for at least 14 days to ensure effectiveness. The size of subcutaneous tumors was measured 2 to 3 times per week using a digital caliper. Tumor volume was calculated by measuring the tumor in 2 dimensions and using the equation: volume = [length x (width2)]/2; where the larger number meant length and the smaller number meant width. If multiple tumors formed, the final volume was the sum of the individual tumors according to the same equation: for example, for 2 tumors, volume = {[L1x(W1)2]/2}+{[L2x(W2)2]/2}. The effect on tumor growth was expressed as percentage tumor growth inhibition (%TGI). The percentage of TGI was calculated using the equation: (1-(Tx-volume/control, volume))*100, where control volume meant the volume of the base-treated or untreated tumor on that day, and Tx-volume meant the volume of the tumor in any experimental group on that day. same day. Statistical differences between experimental and control groups were determined using ANOVA: one-way test.

Results.

Compound 32c (see Table 2 above) was evaluated as a single agent in the Pfeiffer xenograft model of human diffuse large B-cell lymphoma, NHL subtype. Pfeiffer cancer cells were sensitive to the antiproliferative effect of Example 347 in vitro. Thus, a tumor model was established by subcutaneously inoculating tumor cells into immunocompromised SCID mice, and the tumor-bearing mice received oral doses of 0.3, 1, 3, or 10 mg/kg of vehicle or compound 32c twice daily. within 14 days. Treatment with compound 32c inhibited tumor growth by 22%, 24%, 36% and 58% (percent tumor growth inhibition) with increasing dose.

Example D: Western blot analysis.

The following materials and methods were used in the Western blot analysis presented below. Cells (5 million) were lysed in a volume of 300 μl of lysis buffer. Soluble fractions were collected by centrifugation. 25 μl of cell lysate was loaded onto Tris-glycine polyacrylamidamide gels and subjected to electrophoresis. Proteins were transferred to a nitrocellulose membrane and probed with antibodies from Cell Signaling Technology for the following proteins: phospho-Stat3 Y705, phospho-Akt S473, pim1, pim2, pim3, c-myc, phospho-p7086K phosho-S6, phospho-Bad S112, and actin.

Example E: IL6 and IL10 Levels in Cell Lines.

High levels of IL6 and IL10 were observed in various DLBCL cell lines (Fig. 1A). IL6 and IL10 also demonstrated activation of JAK/STAT signaling, with IL10 being a stronger activator of JAK/STAT signaling compared with IL6 across the panel of DLBCL cell lines ( Fig. 1B ). High levels of IL6 and IL10 are present in the serum of patients with DLBCL and correlate with shorter event-free survival and higher International Prognostic Index scores.

Example F: IL10 confers Pfeiffer cells resistance to PI3Kδ inhibition, which can be reversed by blocking JAK1/2 or JAK1.

Cell proliferation assay.

Diffuse large B-cell lymphoma cells were seeded at 2000 cells/well in 96-well culture plates in the absence or presence of 10 ng/ml IL10. Compounds were added to these cells after dilution first in DMSO and then in culture medium (4x concentration). Cells were cultured in an incubator for 3 days with 5% CO ₂ . Cell proliferation was determined using the Cell Titer-Glow assay (Promega, Madison, WI). Cell proliferation assays were performed first on Pfeiffer cells (germinal center B-cell (GCB)-diffuse large B-cell lymphoma (GCB-DLBCL)) and HBL-1 cells (activated B-cell-like (ABC) diffuse large B-cell lymphoma (ABC) -DLBCL)).

Fig. 2A shows the % inhibition in the cell proliferation assay on Pfeiffer cells as a function of the concentration of compound 28 (ΡΙ3Κδ inhibitor) with base (DMSO), DMSO+IL10 and DMSO+IL10+ruxolitinib (JAK1/JAK2 inhibitor). Fig. 2B shows the % inhibition in the Pfeiffer cell proliferation assay as a function of the concentration of compound 28 (PI3Kδ inhibitor) with backbone (DMSO), DMSO+IL10, and DMSO+IL10+compound 7 (selective JAK1 inhibitor). The results indicate that IL10 confers resistance to PI3Kδ inhibition in Pfeiffer cells, but this resistance can be reversed by blocking JAK1 and/or JAK2 signaling. Thus, there is a synergistic effect on Pfeiffer cell proliferation when a PI3Kδ inhibitor and a JAK1 and JAK2 inhibitor are used in combination. Synergism was also observed without IL10. Induction of apoptosis was also observed with this combination.

Similar results were observed in HBL-1 cells with ruxolitinib. Accordingly, FIG. 3 shows the % inhibition in the cell proliferation assay in HBL-1 cells as a function of the concentration of compound 28 (PI3Kδ inhibitor) with backbone (DMSO), DMSO+IL10 and DMSO+IL10+ruxolitinib (JAK1/JAK2 inhibitor).

Example G: IL10-induced Pim2 expression is blocked by a JAK1/2 inhibitor.

Pfeiffer cells were treated for 24 hours with vehicle (DMSO), ruxolitinib (18424), compound 28, or compound 28 and ruxolitinib (18424) with or without IL10, and then subjected to Western blotting to test for the following proteins: phospho-Stat3 Y705, phospho-Akt S473, Pim2, c-Myc, phospho-p7086K, phospho-S6, phospho-Bad S112 and actin. Fig. 4 shows that IL10-induced Pim2 expression is blocked by ruxolitinib (a JAK1/JAK2 inhibitor). IL6 and IL10 promote cell survival through Pim2 expression, which is dependent on JAK1 activity. Fig. 4 also shows a synergistic decrease in c-Myc and P-S6 in the presence of combination treatment with compound 28 and ruxolitinib. The decrease in c-Myc protein may be responsible for the synergistic effect for the combination treatment.

Example H: IL10-induced Pim2 expression is blocked by a selective JAK1 inhibitor.

Pfeiffer cells were treated for 24 hours with vehicle (DMSO), compound 7, compound 28, or compound 28 and compound 7 with or without IL10 and then Western blotted to test for the following proteins: phospho-Stat3 Y705, phospho-Akt S473 , Pim2, c-Myc, phospho-p7086K, phospho-S6, phospho-Bad S112 and actin. Fig. 5 shows that IL10-induced Pim2 expression is blocked by a selective JAK1 inhibitor (compound 7).

Example I: Enhancing the Efficacy of a PI3Kδ Inhibitor with a Selective JAK1 Inhibitor

To test the effects of IL-10 on cell growth and sensitivity to BCR pathway inhibition, a Pfeiffer cell line was used as a DLBCL model system. Pfeiffer cells belong to the germinal center B cell (GCB) subtype of DLBCL, have demonstrated PI3Kδ expression, are sensitive to PI3Kδ inhibition, and activate the JAK/STAT pathway in response to multiple cytokines, as shown above. Pfeiffer cells were treated for 3 days with various concentrations of Compound 28 in the presence or absence of IL-10 and 1 μM Compound 16 and cell growth was measured using an ATP readout (see table below). As shown in FIG. 6, the presence of IL-10 changed the activity of compound 28 by ~10 times (IC ₅₀ =0.67 μM, - IL-10; IC ₅₀ =6.36 μM, + IL-10). Addition of the JAK1 inhibitor, Compound 16, reversed this effect and thus the combination was ~50-fold more potent. In this system, only the JAK1 inhibitor had no effect (IC ₅₀ > 1 μM). Moreover, as shown in FIG. 7 (showing Annexin-V staining of Pfeiffer cells treated for 3 days in 10% FBS + IL10; compound 16 tested at 1 μM), inhibition of PI3Kδ together with the JAK1 signal resulted in increased apoptosis, whereas neither agent alone did not have a significant impact.____________________

10% FBS+IL10 Connection 28 Connection 16 Connection 28+Connection 16 1C ₅ 0 (µM) 6.36 >1 0.035

Example J. Effects of combined JAK1 and PI3Kδ treatment on STAT3 phosphorylation and pAKT inhibition.

To assess the effect on downstream signaling pathways, Pfeiffer cells were treated with compound 28 ± compound 16 for 4 hours and then stimulated with IL-10 for 15 minutes. Extracts were analyzed by Western blotting for pAKT and pSTAT3. As shown in FIG. 8, the AKT pathway was constitutively activated in Pfeiffer cells. Blocking the PI3Kδ signal resulted in complete inhibition of pAKT, whereas treatment with a JAK1 inhibitor had no effect. At the same time, compound 16 led to inhibition of STAT3 phosphorylation, while the PI3K8 inhibitor did not have such an effect. A combination of two compounds was required to block both paths.

All patents, patent publications and journal articles mentioned above are incorporated herein by reference in their entirety.

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Claims (15)

CLAIM 1. A method of treating myelofibrosis in a patient in need thereof, comprising administering to said patient: (a) a JAK1/2 inhibitor, which is [3-[4-(7H-pyrrolo[2,3-d]pyrimidin4-yl)- 1H-pyrazol-1-yl]-3-cyclopentylpropanenitrile or a pharmaceutically acceptable salt thereof; and (b) a PI3Kδ inhibitor, which is 4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4d]pyrimidin-1-yl)ethyl]-5-chloro-2- ethoxy-6-fluorophenyl}pyrrolidin-2-one or a pharmaceutically acceptable salt thereof. 2. The method according to claim 1, wherein the JAK1/2 inhibitor is a pharmaceutically acceptable salt of [3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1 -yl] -3-cyclopentylpropanenitrile. 3. The method according to claim 1, wherein the JAK1/2 inhibitor is a salt of [3-[4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-3-cyclopentylpropanenitrile and phosphoric acid. 4. The method according to claim 1, characterized in that said PI3Kδ inhibitor is selected from: (S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one; (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one; (S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one; and (R)-4-(3-((R)-1 -(4-amino-3-methyl-1 H-pyrazolo[3,4-d]pyrimidin-1 -yl)ethyl)-5-chloro- 2-ethoxy-6fluorophenyl)pyrrolidin-2-one; or a pharmaceutically acceptable salt of any of the above compounds. 5. Method according to claim 1, characterized in that the PI3Kδ inhibitor is (S)-4-(3-((S)-1-(4amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine -1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2one or a pharmaceutically acceptable salt thereof. 6. Method according to claim 1, characterized in that the PI3Kδ inhibitor is (R)-4-(3-((S)-1-(4amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine -1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one or a pharmaceutically acceptable salt thereof. 7. Method according to claim 1, characterized in that the PI3Kδ inhibitor is (S)-4-(3-((R)-1-(4amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine -1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one or a pharmaceutically acceptable salt thereof. 8. The method according to claim 1, characterized in that said PI3Kδ inhibitor is (R)-4-(3-((R)1-(4-amino-3-methyl-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2one or a pharmaceutically acceptable salt thereof. 9. The method of claim 1, wherein said JAK1/2 inhibitor and said PI3Kδ inhibitor are administered simultaneously. 10. The method of claim 1, wherein said JAK1/2 inhibitor and said PI3Kδ inhibitor are administered sequentially. 11. A method of treating myelofibrosis in a patient in need thereof, comprising administering to said patient: (a) a JAK1/2 inhibitor, which is (3R)-3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-d] pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof and (b) a PI3Kδ inhibitor which is selected from: (S)-4-(3-((S)-1-(4 -amino-3-methyl-1H-pyrazolo[3,4d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one; (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one; (S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2- ethoxy-6fluorophenyl)pyrrolidin-2-one; and (R)-4-(3-((R)-1 -(4-amino-3-methyl-1 H-pyrazolo[3,4-d]pyrimidin-1 -yl)ethyl)-5-chloro- 2-ethoxy-6fluorophenyl)pyrrolidin-2-one; or a pharmaceutically acceptable salt of any of the above compounds. 12. The method according to claim 11, characterized in that the PI3Kδ inhibitor is (S)-4-(3-((S)-1-(4amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine -1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2one or a pharmaceutically acceptable salt thereof. 13. The method according to claim 11, characterized in that the PI3Kδ inhibitor is (R)-4-(3-((S)-1-(4αmino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine -1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one or a pharmaceutically acceptable salt thereof. 14. The method according to claim 11, characterized in that the PI3Kδ inhibitor is (S)-4-(3-((R)-1-(4αmino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine -1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one or a pharmaceutically acceptable salt thereof. 15. The method according to claim 11, characterized in that the PI3Kδ inhibitor is (R)-4-(3-((R)-1-(4amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidine -1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one -