Drug combinations used to treat cancer

Abstract

The present disclosure provides combination therapies for treating cancers such as leukemia, lymphoma, and triple negative breast cancer. The present invention provides a combination therapy for treating cancers using a CDK inhibitor (e.g., a CDK inhibitor represented by Formula I) or a pharmaceutically acceptable salt thereof together with a BCL-2 inhibitor or a proteasome inhibitor:.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 314,356, filed on March 28, 2016, the entire contents of which are incorporated herein by reference.

[0003] background

[0004] Numerous cancer-related therapeutic agents are under Phase I or Phase II clinical trials and evaluations at any given time; however, most of them will not advance. In fact, it is estimated that more than 90% of cancer-related therapeutic agents will fail in Phase I or Phase II clinical trial evaluations. The failure rate in Phase III trials is almost 50%, and the cost of new drug development from discovery to Phase III trials is between $800 million and $1.7 billion, and may take 8-10 years.

[0005] In addition, many patients do not even respond to the standard drugs that have been proven to be effective. Due to the reasons that are not fully understood or easily evaluated at present, each patient may not respond to standard drug therapy. In some cases, compared with the medicine used alone, the use of drug combinations may be more effective for treating cancer. These drug combinations can work synergistically to enhance the anti-cancer activity of drugs. In some cases, when combined with other drug therapies, drugs that are not particularly effective may find new and unexpected applications. SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides a method for treating blood cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a CDK inhibitor represented by Formula I or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a BCL-2 inhibitor:

[0008]

[0009] in:

[0010] R1 is optionally substituted phenyl;

[0011] R2 and R3 are each independently selected from hydroxy and OR8, wherein R8 is an optionally substituted C1-C 10 -alkyl;

[0012] R4 is optionally substituted C1-C4-alkyl; and

[0013] R9 is hydrogen or optionally substituted C1-C4-alkyl.

[0014] In certain aspects, the present invention provides a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a CDK inhibitor represented by Formula I or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a proteasome inhibitor:

[0015]

[0016] in:

[0017] R1 is optionally substituted phenyl;

[0018] R2 and R3 are each independently selected from hydroxy and OR8, wherein R8 is an optionally substituted C1-C 10 -alkyl;

[0019] R4 is optionally substituted C1-C4-alkyl; and

[0020] R9 is hydrogen or optionally substituted C1-C4-alkyl.In certain embodiments, the cancer is selected from blood cancer and triple negative breast cancer (TNBC).

[0021] In certain embodiments, the compound of Formula I is represented by Formula Ia:

[0022] , or a pharmaceutically acceptable salt thereof.

[0023] In some embodiments of the compound or salt of Formula I or Ia, R1 is optionally substituted by one or more substituents independently selected from hydroxyl, cyano, halogen, amino, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-hydroxyalkyl, C1-C4-haloalkyl and nitro. In certain embodiments, R1 is substituted by one or more substituents independently selected from halogen and C1-C4-haloalkyl. In certain embodiments, R1 is 2-chloro-4-trifluoromethylphenyl.

[0024] In certain embodiments of the compounds or salts of Formula I or Ia, R2 and R3 are each independently selected from hydroxy and OR8, wherein R8 is C1-C2 optionally substituted with one or more substituents. 10 -alkyl, the substituents being independently selected from hydroxy, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R2 and R3 are each hydroxy.

[0025] In certain embodiments of compounds or salts of Formula I or Ia, R4 is C1-C4-alkyl substituted with one or more substituents selected from hydroxyl, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R4 is C1-C4-alkyl substituted with one or more substituents selected from hydroxyl, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R4 is 2-hydroxymethyl.

[0026] In certain embodiments of compounds or salts of Formula I or Ia, R9 is C1-C4-alkyl optionally substituted with hydroxyl, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R9 is methyl. In certain embodiments, the compound of Formula I is represented by Formula Ib:

[0027] , or a pharmaceutically acceptable salt thereof.

[0028] In certain embodiments, the BCL-2 inhibitor of the method described herein is a BH3-mimetic. The BCL-2 inhibitor can specifically inhibit Bcl-2 protein. The BCL-2 inhibitor can be selected from navitoclax, venetoclax, A-1155463, A-1331852, ABT-737, obatoclax, S44563, TW-37, A-1210477, AT101, HA14-1, BAM7, sabutoclax, UMI-77, gambogic acid, maritoclax, MIM1, methylprednisolone, iMAC2, Bax inhibitor peptide V5, Bax inhibitor peptide P5, Bax channel blocker and ARRY 520 trifluoroacetate. In certain embodiments, the BCL-2 inhibitor of the method described herein is selected from navitoclax and venetoclax or a pharmaceutically acceptable salt of any one of them.

[0029] In certain embodiments, the blood cancer of the methods described herein is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic lymphoma (ALL) and chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, intravascular large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma and primary central nervous system lymphoma. The blood cancer can be diffuse large B-cell lymphoma, acute myeloid leukemia or chronic lymphocytic leukemia.

[0030] For certain methods described herein, the CDK inhibitor and the BCL-2 inhibitor may be administered concurrently. For the methods described herein, the CDK inhibitor and the BCL-2 inhibitor may be administered sequentially within about 12 hours of each other, such as within about 5 hours of each other.

[0031] For certain methods described herein, a CDK inhibitor and a BCL-2 inhibitor may be co-formulated in a pharmaceutical composition.

[0032] For certain methods described herein, the CDK inhibitor and the BCL-2 inhibitor can be administered every day, every other day, or every third day.

[0033] For certain methods described herein, the proteasome inhibitor is selected from bortezomib, marizomib, ixazomib, disulfiram, epicatechin-3-gallate, salinosporamideA, carfilzomib, ONX 0912, CEP-18770, MLN9708, epoxomicin, MG132, and a pharmaceutically acceptable salt of any one thereof. In certain embodiments, the proteasome inhibitor is selected from bortezomib, marizomib, ixazomib, and a pharmaceutically acceptable salt of any one thereof.

[0034] In certain methods described herein, a CDK inhibitor and a proteasome inhibitor are administered concurrently. The CDK inhibitor and the proteasome inhibitor may be administered sequentially within about 12 hours of each other, such as within 5 hours of each other.

[0035] In certain methods described herein, a CDK inhibitor and a proteasome inhibitor are co-formulated in a pharmaceutical composition.

[0036] In certain methods described herein, the CDK inhibitor and the BCL-2 inhibitor are administered every day, every other day, or every third day.

[0037] In certain aspects, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a CDK inhibitor represented by Formula I or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of a BCL-2 inhibitor or a proteasome inhibitor, and a pharmaceutically acceptable excipient:

[0038]

[0039] in:

[0040] R1 is optionally substituted phenyl;

[0041] R2 and R3 are each independently selected from hydroxy and OR8, wherein R8 is an optionally substituted C1-C 10 -alkyl;

[0042] R4 is optionally substituted C1-C4-alkyl; and

[0043] R9 is hydrogen or optionally substituted C1-C4-alkyl.

[0044] In certain embodiments, the compound or salt of Formula I is represented by Formula Ia: .

[0045] For the compositions described herein, for compounds or salts of Formula I or Ia, R1 can be optionally substituted with one or more substituents independently selected from hydroxyl, cyano, halogen, amino, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-hydroxyalkyl, C1-C4-haloalkyl and nitro. In certain embodiments, R1 is substituted with one or more substituents independently selected from halogen and C1-C4-haloalkyl. In certain embodiments, R1 is 2-chloro-4-trifluoromethylphenyl.

[0046] For the compositions described herein, for compounds or salts of Formula I or Ia, R2 and R3 can each be independently selected from hydroxy and OR8, wherein R8 is C1-C1 optionally substituted with one or more substituents. 10 -alkyl, the substituents being independently selected from hydroxy, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R2 and R3 are each hydroxy.

[0047] For the compositions described herein, for compounds or salts of Formula I or Ia, R4 is C1-C4-alkyl substituted with one or more substituents selected from hydroxy, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R4 is C1-C4-alkyl substituted with one or more substituents selected from hydroxy, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R4 is 2-hydroxymethyl.

[0048] For the compositions described herein, for compounds or salts of Formula I or Ia, R9 can be C1-C4-alkyl optionally substituted with hydroxy, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R9 is methyl.

[0049] For the compositions described herein, the compound of Formula I can be represented by Formula Ib: , or a pharmaceutically acceptable salt thereof.

[0050] For compositions comprising a BCL-2 inhibitor described herein, the BCL-2 inhibitor may be selected from navitoclax, venetoclax, A-1155463, A-1331852, ABT-737, obatoclax, S44563, TW-37, A-1210477, AT101, HA14-1, BAM7, sabutoclax, UMI-77, gambogic acid, maritoclax, MIM1, methylprednisolone, iMAC2, Bax inhibitor peptide V5, Bax inhibitor peptide P5, Bax channel blocker, ARRY 520 trifluoroacetate, and a pharmaceutically acceptable salt of any one thereof. The BCL-2 inhibitor may be selected from navitoclax and venetoclax or a pharmaceutically acceptable salt of any one thereof. In certain embodiments, the BCL-2 inhibitor is venetoclax.

[0051] For compositions comprising a proteasome inhibitor described herein, the proteasome inhibitor may be selected from bortezomib, marizomib, ixazomib, disulfiram, epicatechin-3-gallate, salinosporamide A, carfilzomib, ONX 0912, CEP-18770, MLN9708, epoxomicin, MG132, and a pharmaceutically acceptable salt of any one thereof. In certain embodiments, the proteasome inhibitor is selected from bortezomib, marizomib, ixazomib, and a pharmaceutically acceptable salt of any one thereof.

[0052] Incorporated by Reference

[0053] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The novel features of the present invention are particularly set forth in the appended claims. Features and advantages of the present invention will be better understood with reference to the following detailed description of exemplary embodiments in which the principles of the invention are utilized and the accompanying drawings, in which:

[0056] Figure 1 A comparison of the activities of voruciclib and flavopiradol against 38 kinases is described.

[0057] Figure 2 Single-digit nM potency of voruciclib against cyclin-dependent kinases was demonstrated.

[0058] Figures 3A-3D illustrate the synergistic effect of voruciclib in combination with venetoclax (ABT-199). Voruciclib inhibits venetoclax-induced myeloid leukemia cell differentiation protein (MLC-1) induction in NU-DHL-1 diffuse large B-cell lymphoma (DLBCL) cells. MCL-1 = red; DAPI = blue; fluorescent tracking marker (FTM) = green.

[0059] Figures 4A-4D illustrate increased apoptosis in NU-DHL-1 diffuse large B-cell lymphoma (DLBCL) cells achieved by combination treatment with voruciclib and venetoclax. Cleaved caspase-3 (CC3) = red; DAPI = blue; fluorescent tracking marker (FTM) = green.

[0060] Figures 5A-5D illustrate the synergistic effect of voruciclib in combination with navitoclax (ABT-263). Treatment of Ramos Burkitt lymphoma cell line with voruciclib and navitoclax induces apoptosis. Cleaved caspase-3 (CC3) = red; DAPI = blue; fluorescent tracking marker (FTM) = green.

[0061] Figures 6A-6E The synergistic effect of voruciclib in combination with venetoclax in five models of diffuse large B-cell lymphoma was demonstrated.

[0062] Figure 7 These results suggest that inhibition of MCL-1 by CDK9 contributes to the switch of cells into apoptosis.

[0063] Figures 8A-8D demonstrated that proteasome inhibition induces upregulation of MCL-1 in triple-negative breast cancer (TNBC). Fig. 8A Described is a list of compounds screened in the HCC1187 TNBC xenograft model. Figure 8B The mean % change in MCL-1 induction caused by different compound treatments is presented. Figure 8C-8D Depicted are CC3 staining of cells following vehicle and bortezomib treatment (shown in red).

[0064] Figures 9A-9D illustrate the synergistic effect of voruciclib and marizomib combination on NudHL1 DLBCL cells. Cleaved caspase-3 (CC3) = red; DAPI = blue; fluorescent tracking marker (FTM) = green.

[0065] Figures 10A-10D illustrate the synergistic effect of voruciclib in combination with bortezomib on NudHL1 DLBCL cells. Cleaved caspase-3 (CC3) = red; DAPI = blue; Fluorescent tracking marker (FTM) = green.

[0066] Figures 11A-11D illustrate the synergistic effect of voruciclib combined with bortezomib on triple negative breast cancer cells. Cleaved caspase-3 (CC3) = red; DAPI = blue; fluorescent tracking marker (FTM) = green.

[0067] Figures 12A-12E The synergistic effect of voruciclib and bortezomib in combination on HCC1187 triple-negative breast cancer cells was demonstrated. Figures 12A-12D Described reduced tumor volume in the HCC1187 TNBC mouse model treated with voruciclib and bortezomib. Fig.12E Western blots demonstrating reduced MCL-1 expression in HCC1187TNBC cells treated with voruciclib and bortezomib, or voruciclib and tunicamycin are depicted.

[0068] Fig.13 The effects of voruciclid and bortezomib, and the combination of voruciclib and bortezomib, on body weight in the HCC1187 TNBC mouse model were described.

[0069] Fig.14 The effects of bortezomib in combination with palbociclib, a CDK4 / 6 inhibitor, in the HCC1187 TNBC mouse model are described.

[0070] Figures 15A-15B Results showed that voruciclib reduced bortezomib-induced expression of MCL-1 and the E3 ubiquitin-protein ligase XIAP. Fig.15A A proposed model for voruciclib inhibition of CDK9 is described. Fig. 15B Western blots are presented showing that voruciclib reduces bortezomib-induced increases in the expression of MCL-1 and the E3 ubiquitin-protein ligase XIAP.

[0071] Figures 16A-16B illustrate cells resistant to bortezomib treatment.Figure 16A illustrates cells resistant to bortezomib treatment in an area where cells were otherwise depleted by bortezomib treatment. Fig. 16B It was demonstrated that cells resistant to bortezomib express GRP78, a protein expressed as part of the ER stress response.

[0072] Fig.17 Three different ER stress response pathways were described.

[0073] Figures 18A-18B These results suggest that voruciclib can affect the IRE1α-dependent ER stress response pathway. Fig.18A The IRE1α-dependent ER stress response pathway was elucidated. Fig.18B Western blots are presented showing that the ER stress inducer tunicamycin dramatically upregulates X-box binding protein 1 (XBP1), a pro-survival (anti-tumor cell death) protein. This effect was significantly mitigated by voruciclib. At 6 hours, only tunicamycin showed this effect, but at 24 hours, both bortezomib and tunicamycin showed this effect.

[0074] Figures 19A-19B Inhibition of bortezomib-induced XBP1 transcription by voruciclib is described. STF083010 = inhibitor of IRE1α endoribonuclease activity; Tm = tunicamycin.

[0075] Figures 20A-20D illustrate the synergistic effect of voruciclib in combination with ixazomib. Cleaved caspase 3 (CC3) = red; DAPI = blue; fluorescent tracking marker (FTM) = green.

[0076] Figures 21A-21B illustrate solid tumor sections treated with CDK inhibitors, voruciclib, palbociclib, dinaciclib, and flavopiradol in combination with ixazomib. Only voruciclib + ixazomib reproducibly resulted in significant tumor cell clearance within 24 hours.

[0077] Fig.22A -B demonstrates the synergistic effect of voruciclib and venetoclax in the SU-DHL-4 model of diffuse large B-cell lymphoma (DLBCL).

[0078] Figures 23A-23C illustrate the synergistic effect of voruciclib and venetoclax in the SU-DHL-4 model, OCI Ly10 model, and U2932 model of diffuse large B-cell lymphoma (DLBCL).

[0079] Fig.24 showed that voruciclib inhibited MCL1 expression in DLBCL xenograft tumors.

[0080] Figures 25A-25B The synergistic effect of voruciclib and venetoclax in ABC-type (RIVA) DLBCL in mice was demonstrated.

[0081] Figures 26A-26B The synergistic effects of voruciclib and venetoclax and the effects on body weight in the U2932 model of DLBCL in mice are demonstrated.

[0082] Figures 27A-27B The synergistic effect of voruciclib and venetoclax in the NUDHL1 model of DLBCL in mice was demonstrated.

[0083] Fig.28 The synergistic effect of voruciclib and venetoclax in the SUDHL4 model of GC DLBCL was demonstrated.

[0084] Fig.29 showed that voruciclib restored p53 that was abolished by venetoclax.

[0085] Figures 30A-30C show that voruciclib has single agent activity in AML cell lines.

[0086] Fig.31 showed that the combination of voruciclib and venetoclax induced synergistic cell death in AML cell lines.

[0087] Fig.32 The synergistic effect of the combination of voruciclib and venetoclax in impeding tumor growth in SKM1 AML xenografts was demonstrated.

[0088] Fig.33 These results suggest that voruciclib-induced apoptosis is associated with the inhibition of MCL. DETAILED DESCRIPTION OF THE INVENTION

[0090] The present invention provides combination therapies for treating cancer. Specifically, the present invention provides combination therapies of CDK inhibitors and other anticancer drugs for treating cancer. In one aspect, the present invention provides compositions and methods for treating cancer, which use a combination of CDK inhibitors and BCL-2 inhibitors. Such a combination will provide a synergistic effect in the treatment of cancer and especially blood cancers (e.g., leukemias and lymphomas).

[0091] In another aspect, the present invention provides compositions and methods for treating cancer using a combination of a CDK inhibitor and a proteasome inhibitor. Such a combination would provide a synergistic effect in the treatment of cancer and in particular blood cancer and triple negative breast cancer.

[0092] The general terms used above and below preferably have the following meanings in the context of the present invention, unless otherwise indicated. Thus, the definitions of the general terms used in the context of the present invention are provided below:

[0093] The singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0094] As used herein, the term "about" generally refers to an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which may depend in part on how the value is measured or determined. For example, "about" may refer to within 1 or more than 1 standard deviation. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may refer to within an order of magnitude, within 5-fold, and within 2-fold of a value.

[0095] As used herein, the term "at least one" means one or more than one. For example, the term "at least one anticancer drug" means that the combination comprises a single anticancer drug or multiple anticancer drugs.

[0096] As used herein, the term "effective amount" or "therapeutically effective amount" generally refers to an amount of a compound described herein that is sufficient to achieve the intended, predetermined, or specified application, including, but not limited to, treatment of a disease or condition. The therapeutically effective amount may vary with the application (e.g., in vitro or in vivo) or the subject being treated and the disease condition (e.g., the weight and age of the subject, the severity of the disease condition, and the mode of administration). The term may also be applied to a dose that induces a specific response in a target cell (e.g., a decrease in proliferation or downregulation of the activity of a target protein). The specific dose may vary with the following factors: the specific compound selected, the quantitative administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0097] The term "pharmaceutically acceptable" as used herein means that the carrier, diluent, excipient and / or salt must be compatible with the other ingredients of the formulation and not harmful to the recipient thereof. "Pharmaceutically acceptable" also means that the composition or dosage form is suitable for use in animals or humans within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0098] As used herein, the term "combination" or "drug combination" refers to the combined administration of anticancer drugs. The combination of the present invention includes a CDK inhibitor, e.g., a compound of Formula I, Ia or Ib, and at least one anticancer drug selected from a BCL-2 inhibitor and a proteasome inhibitor; the anticancer drugs can be administered to a subject in need thereof, e.g., concurrently or sequentially.

[0099] The term "synergy" or "synergistic effect" or "synergistic effect" as used herein generally refers to such an effect: one or more effects of the combination of the composition are greater than one or more effects of each component alone, or they can be greater than the sum of one or more effects of each component alone. The synergistic effect can be about 10%, 20%, 30%, 50%, 75%, 100%, 110%, 120%, 150%, 200%, 250%, 350% or 500% or more greater than the effect of one of the individual components on the subject or the cumulative effect of each component when administered alone. The effect can be any of the measurable effects described herein. Advantageously, when combined, such synergy between the drugs can allow the use of smaller doses of one or both drugs, may provide greater efficacy at the same dose, and may prevent or delay the accumulation of multidrug resistance. The combination index (CI) method of Chou and Talalay can be used to determine the synergistic, additive or antagonistic effects of the drugs used in combination. When the CI value is less than 1, there is a synergistic effect between the compounds used in combination; when the CI value is equal to 1, there is an additive effect between the compounds used in combination, and when the CI value exceeds 1, there is an antagonistic effect. A synergistic effect can be obtained by co-formulating the drugs of the drug combination. A synergistic effect can be obtained by administering two or more drugs as separate formulations administered simultaneously or sequentially.

[0100] Cyclin-dependent kinases (CDKs) are a family of enzymes that become active at specific stages of the cell cycle. CDKs consist of a catalytic subunit (the actual cyclin-dependent kinase or CDK) and a regulatory subunit (cyclin). There are at least 9 CDKs (CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, etc.) and at least 15 different types of cyclins (cyclin A, B1, B2, D1, D2, D3, E, H, etc.). Each step of the cell cycle is regulated by such a CDK complex: G1 / S transition (CDK2 / cyclin A, CDK4 / cyclin D1-D3, CDK6 / cyclin D3), S phase (CDK2 / cyclin A), G2 phase (CDK1 / cyclin A), G3 phase (CDK4 / cyclin D1-D3), G4 phase (CDK6 / cyclin D3). / M transition (CDK1 / cyclin B).

[0101] The term "CDK inhibitor" as used herein refers to a drug that can inhibit one or more cyclin-dependent kinases (CDKs). Abnormal expression and overexpression of these kinases are manifested in many disease conditions such as cancer. In the context of the present invention, the CDK inhibitor of the drug combination described herein can be a compound of Formula I, Ia or Ib or a pharmaceutically acceptable salt thereof. The compounds of the present invention can specifically inhibit one or more of CDK1 / cyclin B, CDK2 / cyclin E, CDK4 / cyclin D, CDK4 / cyclin D1 and CDK9 / cyclin T1. In certain embodiments, the compounds of the present invention can specifically inhibit CDK9 / cyclin T1 or CDK9.

[0102] Disclosed herein are combination therapies for treating cancer (e.g., leukemia, lymphoma, and breast cancer). The methods and compositions described herein may include a cyclin-dependent kinase (CDK) inhibitor, such as a compound of Formula I, Ia, or Ib, or a pharmaceutically acceptable salt thereof. In some cases, the combination therapy may include a CDK inhibitor in combination with a proteasome inhibitor. In other cases, the combination therapy may include a CDK inhibitor in combination with a BCL-2 inhibitor.

[0103] In certain embodiments, the CDK inhibitors of the present invention are represented by compounds disclosed in U.S. Pat. Nos. 7,271,193, 7,915,301, 8,304,449, 7,884,127, 8,563,596, the entire contents of each of which are incorporated herein by reference. In certain embodiments, the CDK inhibitors of the present invention are represented by Formula I:

[0104]

[0105] or a pharmaceutically acceptable salt thereof, wherein:

[0106] R1 is optionally substituted phenyl;

[0107] R2 and R3 are each independently selected from hydroxy and OR8, wherein R8 is an optionally substituted C1-C 10 -alkyl;

[0108] R4 is optionally substituted C1-C4-alkyl; and

[0109] R9 is hydrogen or optionally substituted C1-C4-alkyl.

[0110] In certain embodiments, the compound or salt of Formula I is represented by Formula Ia:

[0111] .

[0112] In certain embodiments of compounds or salts of Formula I or Ia, R1 is optionally substituted by one or more substituents independently selected from hydroxyl, cyano, halogen, amino, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-hydroxyalkyl, C1-C4-haloalkyl and nitro. In certain embodiments, R1 is substituted by one or more substituents independently selected from hydroxyl, cyano, halogen, C1-C4-alkyl and C1-C4-haloalkyl. In certain embodiments, R1 is substituted by one or more substituents independently selected from halogen and C1-C4-haloalkyl. In certain embodiments, R1 is 2-chloro-4-trifluoromethylphenyl.

[0113] The term "alkyl" means a straight or branched hydrocarbon chain residue consisting only of carbon and hydrogen atoms and not containing unsaturated bonds. In certain embodiments, the alkyl group comprises 1-8 carbon atoms (i.e., C1-C8 alkyl). In other embodiments, the alkyl group comprises 1-5 carbon atoms (i.e., C1-C5 alkyl). In other embodiments, the alkyl group comprises 1-4 carbon atoms (i.e., C1-C4 alkyl). In other embodiments, the alkyl group comprises 1-3 carbon atoms (i.e., C1-C3 alkyl). In other embodiments, the alkyl group comprises 1-2 carbon atoms (i.e., C1-C2 alkyl). In other embodiments, the alkyl group comprises 1 carbon atom (i.e., C1 alkyl). In other embodiments, the alkyl group comprises 5-8 carbon atoms (i.e., C5-C8 alkyl). In other embodiments, the alkyl group comprises 2-5 carbon atoms (i.e., C2-C5 alkyl). In other embodiments, the alkyl group comprises 3-5 carbon atoms (i.e., C3-C5 alkyl). In certain embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl group is connected to the rest of the molecule by a single bond. Unless otherwise specifically stated in this specification, the alkyl group is optionally substituted with one or more substituents, such as those described herein.

[0114] The term "alkoxy" denotes a residue bonded via an oxygen atom of formula -O-alkyl wherein alkyl is an alkyl chain as defined above.

[0115] The term "amino" refers to the group -NR'R", wherein R' and R" are independently selected from hydrogen; and alkyl, hydroxy, aryl, cycloalkyl, heterocycloalkyl and heteroaryl, any of which may be optionally substituted by one or more substituents such as hydroxy, cyano, halogen, amino, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-hydroxyalkyl, C1-C4-haloalkyl and nitro.

[0116] When used in conjunction with a chemical moiety such as an alkyl group, the term "C x-y " is intended to include groups containing from x to y carbons in the chain. For example, the term "C x-y "Alkyl" means a substituted or unsubstituted saturated hydrocarbon group, including straight chain alkyl and branched chain alkyl groups having from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl and the like.

[0117] The term "haloalkyl" refers to an alkyl group substituted with one or more halogen residues, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-chloromethyl-2-fluoroethyl, etc. In certain embodiments, the alkyl portion of the haloalkyl group is further optionally substituted as described herein.

[0118] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy radicals, for example, hydroxymethyl, hydroxyethyl, dihydroxymethyl, etc. In certain embodiments, the alkyl portion of the hydroxyalkyl group is further optionally substituted as described herein.

[0119] In certain embodiments of the compounds or salts of Formula I or Ia, R2 and R3 are each independently selected from hydroxy and OR8, wherein R8 is C1-C2 optionally substituted with one or more substituents. 10 In certain embodiments, R and R are hydroxyl, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R8 is selected from optionally substituted C1-C6-alkyl, such as optionally substituted C1-C4-alkyl at each occurrence. In certain embodiments, R2 and R3 are hydroxyl independently of one another.

[0120] In some embodiments of the compound or salt of Formula I or Ia, R4 is optionally substituted C1-C4-alkyl, wherein R4 is optionally substituted by one or more substituents selected from hydroxyl, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R4 is optionally substituted C1-C2-alkyl. In certain embodiments, R4 is hydroxyalkyl, for example, 2-hydroxymethyl.

[0121] In certain embodiments of compounds or salts of Formula I or Ia, R9 is a C1-C4-alkyl optionally substituted by hydroxyl, cyano, halogen, amino, =O, =S, C1-C4-alkoxy and nitro. In certain embodiments, R9 is an optionally substituted C1-C2-alkyl. In certain embodiments, R9 is methyl. In certain embodiments, R9 is hydrogen.

[0122] In certain embodiments of compounds or salts of Formula I or Ia, the compound of Formula I is a compound or pharmaceutically acceptable salt selected from the following: (+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one; (+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-chromen-4-one; and (+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.

[0123] In certain embodiments, the compound of Formula I or Ia is represented by Formula Ib:

[0124]

[0125] or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound of Formula I, Ia or Ib is in the form of an acid addition salt (such as a hydrochloride salt).

[0126] The term "substituted" means such a part: it has a substituent that replaces the hydrogen on one or more carbons or heteroatoms of the structure. It should be understood that "replacement" or "replacement with..." includes implicit conditions, that is, such substitution is based on the allowed valence of the substituted atom and the substituent, and represents a stable compound that will not easily undergo transformations such as rearrangement, cyclization, elimination, etc. It is foreseen that the term "replacement" used in this article includes all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and linear, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Permissible substituents can be one or more, the same or different for appropriate organic compounds. For the purposes of the present invention, the heteroatom (such as nitrogen) can have a hydrogen substituent and / or any permissible substituent of the organic compound described herein, which satisfies the valence of the heteroatom.

[0127] Substituents may include any substituent described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, carbocyclyl, heterocyclyl, cycloalkyl, heterocycloalkyl, aromatic and heteroaromatic moieties. In certain embodiments, substituents may include any substituent described herein, for example, halogen, hydroxyl, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a 2. -R b -N(R a 2. -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a 2. -R b -OR c -C(O)N(R a 2. -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (where t is 1 or 2), -R b -S(O) t R a (where t is 1 or 2), -R b -S(O) t OR a (where t is 1 or 2) and -R b -S(O) t N(R a)2 (wherein t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl and heteroarylalkyl, any of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, hydroxy, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a 2. -R b -N(R a 2. -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a 2. -R b -OR c -C(O)N(R a 2. -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (where t is 1 or 2), -R b -S(O) t R a (where t is 1 or 2), -R b -S(O) t OR a (where t is 1 or 2) and -R b -S(O) t N(R a )2 (where t is 1 or 2); where each R a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, wherein each R aThe following groups may be optionally substituted (when valence permits): alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2. -R b -N(R a )2. -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2. -R b -OR c -C(O)N(R a )2. -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (where t is 1 or 2), -R b -S(O) t R a (where t is 1 or 2), -R b -S(O) t OR a (where t is 1 or 2) and -R b -S(O) t N(R a )2 (where t is 1 or 2); and where each R b is independently selected from a direct bond or a straight or branched alkylene, alkenylene or alkynylene chain, and each R c is a straight or branched alkylene, alkenylene or alkynylene chain.

[0128] Procedures for preparing compounds of Formula I, Ia and Ib or pharmaceutically acceptable salts thereof may be found in PCT Patent Publication No. WO2004004632 (corresponding to US Pat. No. 7,271,193) and PCT Patent Publication No. WO2007148158.

[0129] The present invention provides pharmaceutically acceptable salts of any compound described herein (e.g., a compound of Formula I, Ia, Ib), a BCL-2 inhibitor, and a proteasome inhibitor. Pharmaceutically acceptable salts include, for example, acid addition salts and base addition salts. The acid added to the compound to form an acid addition salt can be an organic acid or an inorganic acid. The base added to the compound to form a base addition salt can be an organic base or an inorganic base. In some cases, the pharmaceutically acceptable salt is a metal salt. In some cases, the pharmaceutically acceptable salt is an ammonium salt.

[0130] Acid addition salts can be produced by adding an acid to the compounds described herein. In some cases, the acid is an organic acid. In some cases, the acid is an inorganic acid. Non-limiting examples of suitable acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, phosphoric acid, nicotinic acid, isonicotinic acid, lactic acid, salicylic acid, 4-aminosalicylic acid, tartaric acid, ascorbic acid, gentisic acid, gluconic acid, glucaronic acid, saccaric acid, acid), formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, citric acid, oxalic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, glycolic acid, malic acid, cinnamic acid, mandelic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, pamoic acid, phenylacetic acid, N-cyclohexylaminosulfonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2-phosphoglyceric acid, 3-phosphoglyceric acid, glucose-6-phosphate and amino acids.

[0131] Metal salts can be produced by adding an inorganic base to the compounds of the invention. The inorganic base consists of a metal cation paired with a basic counterion (e.g., hydroxide, carbonate, bicarbonate, or phosphate). The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In certain embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

[0132] In certain embodiments, the metal salt is a lithium salt, sodium salt, potassium salt, cesium salt, cerium salt, magnesium salt, manganese salt, iron salt, calcium salt, strontium salt, cobalt salt, titanium salt, aluminum salt, copper salt, cadmium salt, or zinc salt.

[0133] Ammonium salts can be produced by adding ammonia or an organic amine to the compounds described herein. Non-limiting examples of suitable organic amines include triethylamine, diisopropylamine, ethanolamine, diethanolamine, triethanolamine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, pipyrazine, ethylenediamine, N,N'-dibenzylethylenediamine, procaine, chloroprocaine, choline, dicyclohexylamine, and N-methylglucamine.

[0134] Non-limiting examples of suitable ammonium salts include triethylamine salts, diisopropylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, morpholine salts, N-methylmorpholine salts, piperidine salts, N-methylpiperidine salts, N-ethylpiperidine salts, dibenzylamine salts, piperazine salts, pyridine salts, pyrrazole salts, pipyrrazole salts, imidazole salts, pyrazine salts, pipyrazine salts, ethylenediamine salts, N,N'-dibenzylethylenediamine salts, procaine salts, chloroprocaine salts, choline salts, dicyclohexylamine salts, and N-methylglucamine salts.

[0135] Non-limiting examples of suitable acid addition salts include hydrochloride, hydrobromide, hydroiodide, nitrate, nitrite, sulfate, sulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate, bicarbonate, nicotinate, isonicotinate, lactate, salicylate, 4-aminosalicylate, tartrate, ascorbate, gentisate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, pantothenate, acetate, propionate, butyrate, fumarate, succinate, citrate, oxalate, maleate, salts, hydroxymaleate, methylmaleate, glycolate, malate, cinnamate, mandelate, 2-phenoxybenzoate, 2-acetoxybenzoate, pamoate, phenylacetate, N-cyclohexylsulfamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, ethane-1,2-disulfonate, 4-methylbenzenesulfonate, naphthalene-2-sulfonate, naphthalene-1,5-disulfonate, 2-phosphoglycerate, 3-phosphoglycerate, glucose-6-phosphate, and amino acid salts.

[0136] The compounds described herein (e.g., compounds and salts of Formula I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors) may exist as diastereomers, enantiomers or other stereoisomeric forms in some cases. The compounds presented herein include all diastereomeric forms, enantiomeric forms and epimeric forms and their appropriate mixtures. Separation of stereoisomers can be performed by chromatography, or by forming diastereomers and separating by recrystallization or chromatography, or any combination thereof (Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981, the disclosure of which is incorporated herein by reference). Stereoisomers can also be obtained by stereoselective synthesis.

[0137] The compounds described herein (e.g., compounds and salts of Formula I, Ia, Ib, BCL-2 inhibitors, and proteasome inhibitors) include the use of amorphous forms as well as crystalline forms (also referred to as polymorphs). The compounds described herein may be in the form of pharmaceutically acceptable salts. Likewise, active metabolites of these compounds having similar activity are included within the scope of the present invention. In addition, the compounds described herein may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents (such as water, ethanol, etc.). The solvated forms of the compounds presented herein are also considered to be disclosed herein.

[0138] The compounds described herein (e.g., compounds and salts of Formula I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors) include compounds that exhibit their natural isotopic abundance, and compounds in which one or more atoms are artificially enriched with a particular isotope having the same atomic number, but whose atomic mass or mass number is different from the atomic mass or mass number that predominantly occurs in nature. All isotopic variations of the compounds of the invention, whether radioactive or not, are included within the scope of the invention. For example, hydrogen has three naturally occurring isotopes, called 1 H (hydrogen), 2 H (deuterium) and 3 H (tritium). Protium is the most abundant isotope of hydrogen in nature. Deuterium enrichment can provide certain therapeutic advantages, such as increased in vivo half-life and / or exposure, or can provide compounds that can be used to study in vivo pathways of drug elimination and metabolism. Isotopically enriched compounds can be prepared.

[0139] The compounds described herein (e.g., compounds and salts of Formula I, Ia, Ib, BCL-2 inhibitors, and proteasome inhibitors, wherein the compounds have a carbon-carbon double bond or a carbon-nitrogen double bond) can be used as appropriate. Z -or E - Form (or Cis- or Trans- In addition, some chemical entities may exist in different tautomeric forms. Unless otherwise indicated, the chemical entities described herein are also intended to include all Z -、 E - and tautomeric forms.

[0140] In some cases, the compounds described herein can be prodrugs, for example, where the carboxylic acid present in the parent compound exists as an ester. The term "prodrug" is intended to include compounds that are converted into the pharmaceutical drug of the present invention, i.e., the parent compound, under physiological conditions. A method for preparing a prodrug is to include one or more selected parts that are hydrolyzed under physiological conditions to expose the desired molecule. In certain embodiments, the prodrug is converted by an enzyme activity of a host animal, such as an enzyme activity of a specific target cell in the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention.

[0141] Prodrugs are often useful because, in some cases, they can be more easily administered than the parent drug. They can be, for example, bioavailable by oral administration, whereas the parent is not. Prodrugs can help enhance the cell permeability of the compound relative to the parent drug. For example, a prodrug can have improved cell permeability compared to the parent compound. A prodrug can also have improved solubility in a pharmaceutical formulation compared to the parent drug. In certain embodiments, the design of a prodrug can increase the lipophilicity of a pharmaceutical drug. In certain embodiments, the design of a prodrug can increase effective water solubility.

[0142] In certain embodiments, a cyclin-dependent kinase (CDK) inhibitor (e.g., a compound or salt of Formula I, Ia, or Ib) can be used in combination with an inhibitor of one or more proteins in the BCL-2 family. Inhibitors of proteins in the BCL-2 anti-apoptotic family alter at least one cell survival pathway. Apoptosis activation can occur via the following pathways: an extrinsic pathway triggered by activation of cell surface death receptors, or an intrinsic pathway triggered by developmental cues and different intracellular stresses. The intrinsic pathway (also known as the stress pathway or mitochondrial pathway) is primarily regulated by the BCL-2 family, a class of key regulators of caspase activation consisting of: anti-apoptotic (pro-survival) proteins with BH1-BH4 domains (BCL-2, i.e., BCL-2 protein members of the BCL-2 anti-apoptotic protein family), BCL-xL, BCL-w, A1, MCL-1, and BCL-B); pro-apoptotic proteins with BH1, BH2, and BH3 domains (BAX, BAK, and BOK); and pro-apoptotic BH3-only proteins (BIK, BAD, BID, BIM, BMF, HRK, NOXA, and PUMA) ( See, for example, Cory et al., Nature Reviews Cancer 2:647-56 (2002); Cory et al., Cancer Cell 8:5-6 (2005); Adams et al., Oncogene 26:1324-1337 (2007)). The anti-apoptotic protein BCL-2 blocks the activation of the pro-apoptotic multidomain proteins BAX and BAK ( See, for example, Adams et al., Oncogene 26:1324-37 (2007)).

[0143] The term "BCL-2 inhibitor" as used herein refers to a drug that can inhibit one or more of the anti-apoptotic proteins of the BCL-2 family (e.g., BCL-2, BCL-xL, and BCL-w). In certain embodiments, the BCL-2 inhibitor of the present invention selectively inhibits a protein of the BCL-2 family, for example, the BCL-2 inhibitor can selectively inhibit BCL-2 and will not inhibit BCL-xl or BCL-w.

[0144] The BCL-2 inhibitors described herein can inhibit one or more of BCL-2, BCL-xL, and BCL-w. In certain embodiments, inhibitors of proteins of the BCL-2 anti-apoptotic family inhibit BCL-2. In certain embodiments, inhibitors of proteins of the BCL-2 anti-apoptotic family inhibit BCL-2 and do not inhibit other members of the BCL-2 family, for example, do not inhibit BCL-xL or BCL-w. In certain embodiments, the BCL-2 inhibitor is a BH3-mimetic.

[0145] In certain embodiments, the BCL-2 inhibitors of the present invention inhibit BCL-xL function. In addition to the inhibition of BCL-xL, the inhibitors may also interact with BCL-2 and / or inhibit one or more functions of BCL-2, e.g., BCL-xL / BCL-2 inhibitors. In certain embodiments, the BCL-2 inhibitors of the present invention inhibit each of BCL-xL and BCL-w. In certain embodiments, the BCL-2 inhibitors of the present invention inhibit BCL-xL, BCL-2, and BCL-w.

[0146] In certain embodiments, the BCL-2 inhibitor interferes with the interaction between a BCL-2 anti-apoptotic protein family member and one or more ligands or receptors to which the BCL-2 anti-apoptotic protein family member binds in the absence of the inhibitor. In other embodiments, an inhibitor of one or more BCL-2 anti-apoptotic protein family members (wherein the inhibitor specifically inhibits at least one BCL-2 protein) binds only to one or more of BCL-xL, BCL-2, BCL-w, and does not bind to other Bcl-2 anti-apoptotic Bcl-2 family members, such as Mcl-1 and BCL2A1.

[0147] The binding affinity of a BCL-2 inhibitor to a BCL-2 family protein can be measured. As an example, the binding affinity of a BCL-xL inhibitor can be determined using a competitive fluorescence polarization assay, in which a fluorescent BAK BH3 domain peptide is incubated with a BCL-xL protein (or other BCL-2 family protein) in the presence or absence of increasing concentrations of a BCL-XL inhibitor as described previously ( See, for example, U.S. Patent Publication 20140005190; Park et al., Cancer Res . 73:5485-96(2013); Wang et al., Proc. Natl. Acad. Sci USA 97:7124-9 (2000); Zhang et al., Anal. Biochem. 307:70-5 (2002); Bruncko et al., J. Med. Chem. 50:641-62 (2007)). The percentage of inhibition can be determined by the following equation: 1-[(mP value of well - negative control) / range)] x 100%. By using the method described in Bruncko et al., J. Med. Chem . 50:641-62 (2007) (also See, Wang, FEBS Lett The inhibition constant (K) was determined by the following formula as described in . i )value: K i = [ I ] 50 / ([L] 50 / K d +[ P ]0 / K d +1).

[0148] Examples of BCL-2 inhibitors include ABT-263 (4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide or IUPAC, (R)-4-(4-((4'-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylsulfanyl)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide) ( See, for example, Park et al., 2008, J. Med. Chem. 51:6902; Tse et al., Cancer Res.,2008, 68:3421; International Patent Application Publication No. WO2009 / 155386; U.S. Patent Nos. 7390799, 7709467, 7906505, 8624027) and ABT-737 (4-[4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide, 4-[4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylsulfanyl)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]- or 4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) ( See, for example like, Oltersdorf et al., Nature , 2005, 435:677; U.S. Patent No. 7973161; U.S. Patent No. 7642260).

[0149] In other embodiments, the BCL-2 inhibitor is a quinazoline sulfonamide compound ( See, for example, Sleebs et al., 2011, J. Med. Chem. 54:1914). In another embodiment, the BCL-inhibitor is a small molecule compound described in Zhou et al., J. Med. Chem., 2012, 55:4664 ( See, for example, Compound 21 (R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonylamino)phenyl)piperazin-1-yl)phenyl)-5-ethyl-1-methyl-1H-pyrrole-2-carboxylic acid) and Zhou et al., J. Med. Chem., 2012, 55:6149 ( See, for example,Compound 14 (R)-5-(4-chlorophenyl)-4-(3-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonylamino)phenyl)piperazin-1-yl)phenyl)-1-ethyl-2-methyl-1H-pyrrole-3-carboxylic acid; Compound 15 (R)-5-(4-chlorophenyl)-4-(3-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonylamino)phenyl)piperazin-1-yl)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic acid). In other embodiments, the BCL-inhibitor is a BCL-2 / BCL-xL inhibitor such as BM-1074 ( See, for example, Aguilar et al., 2013, J. Med. Chem .56:3048); BM-957 ( See, for example, Chen et al., 2012, J. Med. Chem. 55:8502); BM-1197 ( Ginseng See, for example, Bai et al. PLoS One 2014 Jun 5;9(6):e99404. Doi: 10.1371 / journal.pone.009904); U.S. Patent Application No. 2014 / 0199234; N-Acylsulfonamide Compounds ( See, for example, International Patent Application Publication No. WO 2002 / 024636, International Patent Application Publication No. WO 2005 / 049593, International Patent Application Publication No. WO2005 / 049594, U.S. Patent No. 7767684, U.S. Patent No. 7906505). In another embodiment, the BCL-2 inhibitor is a small molecule macrocyclic compound ( See, for example, International Patent Application Publication No. WO 2006 / 127364, U.S. Patent No. 7777076). In another embodiment, the BCL-2 inhibitor is an isoxazolidine compound ( See, for example like, International Patent Application Publication No. WO 2008 / 060569, U.S. Patent No. 7851637, U.S. Patent No. 7842815). In another embodiment, the BCL-2 inhibitor is S44563 ( See, for example, Loriot et al. Cell Death and Disease, 2014, 5, e1423). In one embodiment, the BCL-2 inhibitor is (R)-3-((4'-chloro-[1,1'-biphenyl]-2-yl)methyl)-N-((4-(((R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenyl)sulfonyl)-2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoline-8-carboxamide. In another embodiment, the BCL-2 inhibitor is a small molecule heterocyclic compound ( See, for example, U.S. Patent No. 9,018,381).

[0150] In some cases, a BCL-2 inhibitor is used in combination with a compound or salt of Formula I, Ia or Ib. Any BCL-2 inhibitor can be used and can exhibit a synergistic effect when used in combination with a compound or salt of Formula I, Ia or Ib. A BCL-2 family inhibitor can inhibit one or more members of the BCL-2 family, including Bcl-2, Bcl-xL, Bcl-w, BAK1, BAX, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL2L10, BCL2L13, BCL2L14, BOK, and MCL1. In certain embodiments, a compound or salt of Formula I, Ia or Ib is used in combination with any of the following: navitoclax, venetoclax, A-1155463, A-1331852, ABT-737, obatoclax, TW-37, A-1210477, AT101, HA14-1, BAM7, sabutoclax, UMI-77, gambogic acid, maritoclax, MIM1, methylprednisolone, iMAC2, Bax inhibitor peptide V5, Bax inhibitor peptide P5, Bax channel blockers, and ARRY 520 trifluoroacetate. In certain embodiments, voruciclib is used in combination with navitoclax. In certain embodiments, voruciclib is used in combination with venetoclax.

[0151] In certain embodiments, a BCL-2 inhibitor is used in combination with a CDK inhibitor of the invention (e.g., a compound of Formula I, Ia, or Ib) for the treatment of a blood cancer. In certain embodiments, the blood cancer is a leukemia, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic lymphoma (ALL), and chronic lymphocytic leukemia (CLL). In certain embodiments, the blood cancer is a non-Hodgkin lymphoma, such as a B-cell or T-cell lymphoma. B-cell lymphomas include diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, intravascular large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, and primary central nervous system lymphoma. T-cell lymphomas include precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, adult T-cell lymphoma with smoldering, chronic, acute, and lymphoma subtypes, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, nasal type, enteropathy-associated intestinal T-cell lymphoma (EATL) with subtypes I and II, and anaplastic large cell lymphoma (ALCL). The combinations of the invention, e.g., the combination of a CDK inhibitor and a BCL-2 inhibitor described herein, can be used to treat the blood cancers described herein.

[0152] As used herein, the term "treating" can include alleviating, eliminating, or ameliorating symptoms of a disease or condition, preventing additional symptoms, ameliorating or preventing the underlying cause of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving conditions caused by the disease or condition, or prophylactically and / or therapeutically halting symptoms of the disease or condition.

[0153] The present invention provides a method for preventing or reducing the recurrence of cancer in a subject in need thereof. In certain embodiments, the term "prevention" related to a disease or condition may refer to a compound or combination that reduces the occurrence of a disease or condition in a treated sample compared to an untreated control sample in a statistical sample, or delays the onset of one or more symptoms of a disease or condition or alleviates the severity of the symptom compared to an untreated control sample. The method includes administering a combination therapy described herein to treat minimal residual disease and / or as a maintenance therapy, for example, as an extension or extended therapy after cessation of another cancer treatment. For example, the combination therapy may be administered after cessation of another cancer therapy, such as chemotherapy, radiation therapy, and / or surgery.

[0154] In certain aspects, proteasome inhibitors can be combined or used in combination with CDK inhibitors of the present invention (e.g., compounds or salts of any one of Formula I, Ia or Ib). In eukaryotic cells, the ubiquitin (Ub)-proteasome pathway (UPS) comprises Ub modification and subsequent degradation of protein substrates. UPS controls the levels of many cell regulatory proteins, including transcription factors, cell cycle regulatory proteins, and factors involved in a variety of cellular processes. A common feature of the UPS pathway is that highly conserved Ub is covalently linked to a target protein by a series of enzymes (i.e., E1 Ub-activating enzymes, E2 Ub-conjugating enzymes, and E3 Ub ligases). E1 first activates Ub and transfers it to E2. From the E2 enzyme, Ub is transferred directly to the target protein or indirectly to the target protein by the E3 Ub ligase. Polyubiquitinated proteins are recognized and degraded by the 26S proteasome (a large complex with a variety of proteolytic activities).

[0155] The term "proteasome inhibitor" as used herein refers to drugs that block the action of the proteasome. Proteasome inhibition can prevent the degradation of pro-apoptotic factors (such as p53 protein), allowing the activation of programmed cell death in neoplastic cells (dependent on the inhibition of pro-apoptotic pathways).

[0156] Any proteasome inhibitor may be used and may exhibit a synergistic effect when used in combination with a CDK inhibitor (e.g., a compound or salt of Formula I, Ia or Ib). Non-limiting examples of proteasome inhibitors may include: bortezomib, marizomib, ixazomib, disulfiram, epicatechin-3-gallate, salinosporamide A, carfilzomib, ONX 0912, CEP-18770, MLN9708, epoxomicin, and MG132.

[0157] In certain embodiments, a proteasome inhibitor is used in combination with a CDK inhibitor of the invention (eg, a compound of Formula I, Ia, or Ib) for the treatment of blood cancers, such as diffuse large B-cell lymphoma or triple-negative breast cancer.

[0158] In some aspects, the combination described herein, for example, a combination of a CDK inhibitor with a BCL-2 inhibitor or a proteasome inhibitor, can be used to treat cancer. The combined therapy described herein can reduce the possibility of metastasis in a subject in need thereof. In certain embodiments, the metastasis is a solid tumor. In certain embodiments, the metastasis is a liquid tumor. Cancers that are liquid tumors can be those that occur in, for example, blood, bone marrow, and lymph nodes, and can include, for example, leukemia, myeloid leukemia, lymphocytic leukemia, lymphoma, Hodgkin's lymphoma, melanoma, and multiple myeloma. Leukemias include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and hairy cell leukemia. Cancers that are solid tumors include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer, squamous cell skin cancer, kidney cancer, head and neck cancer, laryngeal cancer, squamous cancer that forms on the moist mucosal lining of the nose, mouth, throat, bladder cancer, osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In certain embodiments, the condition treated by the methods described herein is a metastasis of a melanoma cell, a prostate cancer cell, a testicular cancer cell, a breast cancer cell, a brain cancer cell, a pancreatic cancer cell, a colon cancer cell, a thyroid cancer cell, a stomach cancer cell, a lung cancer cell, an ovarian cancer cell, a Kaposi's sarcoma cell, a skin cancer cell, a kidney cancer cell, a head or neck cancer cell, a throat cancer cell, a squamous cell carcinoma cell, a bladder cancer cell, an osteosarcoma cell, a cervical cancer cell, an endometrial cancer cell, an esophageal cancer cell, a liver cancer cell, or a kidney cancer cell.

[0159] The methods described herein can also be used to inhibit the progression of metastatic cancer tumors. Non-limiting examples of cancer include adrenocortical carcinoma, childhood adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendix cancer, basal cell carcinoma, childhood basal cell carcinoma, bladder cancer, childhood bladder cancer, bone cancer, brain tumors, childhood astrocytomas, childhood brain stem gliomas, childhood atypical teratoid / rhabdoma of the central nervous system, childhood embryonal tumors of the central nervous system, childhood central nervous system germ cell tumors, childhood craniopharyngioma brain tumors, childhood ependymoma brain tumors, breast cancer, childhood bronchial tumors, carcinoid tumors, childhood carcinoid tumors, gastrointestinal carcinoid tumors, cancer of unknown primary, childhood cancer of unknown primary, childhood heart tumors, cervical cancer, childhood cervical cancer, childhood Chordoma, chronic myeloproliferative disorder, colon cancer, colorectal cancer, childhood colorectal cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, esophageal cancer, childhood esophageal cancer, childhood esophageal esophageal blastoma, eye cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, childhood gastric cancer, gastrointestinal stromal tumor (GIST), childhood gastrointestinal stromal tumor (GIST), childhood ectocranial cell tumor, gonadal ectodermal tumor, gestational trophoblastic tumor, glioma, head and neck cancer, childhood head and neck cancer, hepatocellular carcinoma, hypopharyngeal cancer, kidney cancer, renal cell carcinoma, Wilms tumor, childhood kidney tumor, Langerhans cell histiocytosis, laryngeal cancer, childhood laryngeal cancer, leukemia, acute lymphoblastic leukemia All, acute myeloid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (cml), hairy cell leukemia, lip cancer, liver cancer (primary), childhood liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma (CNS), melanoma, childhood melanoma, intraocular melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, metastatic squamous neck cancer with unknown primary, midline tract cancer involving NUT gene, Oral cancer, childhood multiple endocrine neoplasms syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic tumor, myeloproliferative tumor, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, childhood nasopharyngeal cancer, neuroblastoma, oral cancer, childhood oral cancer, oropharyngeal cancer, ovarian cancer, childhood ovarian cancer, epithelial ovarian cancer, low-grade malignant potential ovarian cancer, pancreatic cancer, childhood pancreatic cancer, pancreatic neuroendocrine tumor (islet cell tumor), childhood papilloma, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell tumor, childhood pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvic transitional cell carcinoma, retinoblastoma, salivary gland cancer,Childhood salivary gland cancer, tumors of the Ewing sarcoma family, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, childhood skin cancer, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, childhood squamous cell carcinoma, testicular cancer, childhood testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, childhood thymoma and thymic carcinoma, thyroid cancer, childhood thyroid cancer, ureteral transitional cell carcinoma, urethral cancer, endometrial cancer, vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia.

[0160] The combined therapy described herein can be used together with other therapies (such as radiation therapy). Chemotherapy and radiotherapy treatment regimens can include a limited number of on-drug treatments followed by cycles of off-drug treatments, or be included in a limited time frame in which chemotherapy or radiotherapy is administered. The regimen can be determined by clinical trials, drug markers, and clinical staff in conjunction with the subject to be treated. The number of cycles of chemotherapy or radiotherapy or the total time length of chemotherapy or radiotherapy regimens can vary with the subject's response to cancer therapy. After the chemotherapy or radiotherapy treatment regimen has ended, pharmaceutical drugs described herein can be administered.

[0161] In some aspects, the combinations described herein can be used to treat subjects in need thereof. In some cases, the subject to be treated by the methods and compositions disclosed herein can be a human subject. The subject to be treated by the methods and compositions disclosed herein can be a non-human animal. Non-limiting examples of non-human animals can include non-human primates, livestock animals, domestic pets, and laboratory animals.

[0162] In certain embodiments, the combination therapies described herein can be administered as separate drugs, or can be combined into a single pharmaceutical composition. For example, a combination of a CDK inhibitor (e.g., a compound or salt of Formula I, Ia or Ib) and a BCL-2 inhibitor (e.g., venetoclax or navitoclax) can be formulated as two separate pharmaceutical compositions, or the two drugs can be co-formulated into a single pharmaceutical composition.

[0163] In certain embodiments, a CDK inhibitor (e.g., a compound or salt of Formula I, Ia, or Ib) is co-formulated with a BCL-2 inhibitor or a proteasome inhibitor. In some cases, a compound of Formula I, Ia, or Ib is co-formulated with any one of navitoclax, venetoclax, bortezomib, marizomib, or ixazomib, or a combination thereof.

[0164] In certain embodiments, the present invention provides a pharmaceutical composition, e.g., for oral or parenteral administration, comprising a compound or salt of Formula I, Ia or Ib. In certain aspects, the pharmaceutical composition comprises a compound or salt of Formula I, Ia or Ib in an amount of at least about 1 mg to about 1000 mg, about 100 mg to about 400 mg, about 100 mg to about 200 mg, about 200 mg to about 400 mg, or about 250 mg to about 350 mg. For example, a pharmaceutical composition of the invention may include about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg of a compound of Formula I, Ia, or Ib. For compounds described herein (e.g., compounds of Formula Ib) formulated in a pharmaceutical composition as a salt, the amount of the compound may reflect the weight of the free base, not the weight of the salt form. In certain embodiments, the pharmaceutical composition of a compound or salt of Formula I, Ia, or Ib does not include an additional anticancer agent, e.g., a BCL-2 inhibitor or a proteasome inhibitor. In certain embodiments, the pharmaceutical composition includes an additional anticancer drug, for example, a BCL-2 inhibitor or a proteasome inhibitor.

[0165] The therapeutically effective amount of a compound of the invention (e.g., a compound or salt of Formula I, Ia or Ib) can be expressed as mg compound / kg of subject body weight. In some cases, the dosage of the therapeutically effective amount can be at least about 0.1 mg / kg to about 20 mg / kg, for example, about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, or about 20 mg / kg. For compounds described herein (e.g., compounds of Formula Ib) formulated in a pharmaceutical composition in the form of a salt, the therapeutically effective amount of the compound can reflect the free base weight, rather than the weight of the salt form.

[0166] In certain embodiments, the present invention provides a pharmaceutical composition, e.g., for oral or parenteral administration, comprising a BCL-2 inhibitor, e.g., venetoclax or navitoclax. The pharmaceutical composition may comprise at least about 1 mg to about 1000 mg, about 100 mg to about 1000 mg, about 100 mg to about 800 mg, about 200 mg to about 800 mg, or about 300 mg to about 8000 mg of the BCL-2 inhibitor. For example, the pharmaceutical composition of the present invention can include about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, about 500 mg, about 520 mg, about 540 mg, about 560 mg, about 580 mg, about 600 mg, about 620 mg, about 640 mg, about 660 mg, about 680 mg, about 700 mg, about 720 mg, about 740 mg, about 760 mg, about 780 mg, about 800 mg, about 820 mg, about 840 mg, about 860 mg, about 880 mg In some embodiments, the present invention relates to an agent that is administered at least about 100 mg, about 900 mg, about 920 mg, about 940 mg, about 960 mg, about 980 mg, or about 1000 mg of a BCL-2 inhibitor, such as, for example, venetoclax or navitoclax.

[0167] In certain embodiments, the present invention provides a pharmaceutical composition, e.g., for oral or parenteral administration, comprising a proteasome inhibitor, e.g., bortezomib, marizomib, or ixazomib. The pharmaceutical composition may comprise a proteasome inhibitor in an amount of at least about 0.5 mg to about 50 mg, about 1 mg to about 30 mg, about 1 mg to about 20 mg, about 1 mg to about 10 mg, or about 1 mg to about 5 mg. For example, a pharmaceutical composition of the present invention may comprise about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg of a proteasome inhibitor, e.g., bortezomib, marizomib, or ixazomib.

[0168] In certain embodiments, a formulation of the invention comprises a compound or salt of Formula I, Ia or Ib, a BCL-2 inhibitor, or a proteasome inhibitor, wherein the compound or salt is about 70% to about 99.99%, about 80% to about 99.9%, about 85% to about 99%, about 90% to about 99%, about 95% to about 99%, about 97% to about 99%, about 98% to about 99%, about 98% to about 99.9%, about 99% to about 99.99%, about 99.5% to about 99.99%, about 99.6% to about 99.99%, about 99.8 to about 99.99%, or about 99.9% to about 99.99% free of impurities.

[0169] In certain embodiments, a pharmaceutical composition of the invention comprises an amount (such as the amounts described herein) of a compound or salt of Formula I, Ia or Ib and a BCL-2 inhibitor, for example, a pharmaceutical composition containing 100-400 mg of a compound or salt of Formula Ib and 200-800 mg of a BCL-2 inhibitor (e.g., venetoclax).

[0170] In certain embodiments, a pharmaceutical composition of the invention comprises an amount (such as the amounts described herein) of a compound or salt of Formula I, Ia or Ib and a proteasome inhibitor, for example, a pharmaceutical composition containing 100-400 mg of a compound or salt of Formula Ib and 1-10 mg of a proteasome inhibitor (e.g., ixazomib).

[0171] The pharmaceutical compositions disclosed herein may be in the form of liquid preparations, solid preparations, or combinations thereof. Non-limiting examples of preparations may include tablets, capsules, pills, gels, pastes, liquid solutions, and creams. In some cases, the therapeutic agent (e.g., a compound or salt of Formula I, Ia or Ib, a BCL-2 inhibitor, or a proteasome inhibitor) may be in a crystalline form. In a pharmaceutical composition comprising two or more therapeutic agents, each drug may be crystallized separately and then combined, or they may be crystallized together. The composition may comprise two or more therapeutic agents in one or more physical states. For example, the composition may be a tablet comprising a therapeutic agent in a solid preparation and another therapeutic agent or drug in a gel preparation. In certain embodiments, the composition is a single pharmaceutical composition comprising a compound or salt of Formula I, Ia or Ib in a first physical state and a BCL-2 family inhibitor or a proteasome inhibitor in a second physical state.

[0172] Compositions of the present invention may further comprise excipients or additives. Excipients may include any and all solvents, coating agents, chelating agents, flavoring agents, coloring agents, lubricants, disintegrants, preservatives, sweeteners, defoamers, buffers, polymers, antioxidants, adhesives, diluents and vehicles (or carriers). Typically, the excipients are compatible with therapeutic compositions of the present invention.

[0173] Liquid preparations for oral administration can be in the form of, for example, solutions, syrups or suspensions, or they can be presented as dry products for reconstitution with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional protocols with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, methylcellulose or hydrogenated edible fats), emulsifiers (for example, lecithin or gum arabic), non-aqueous vehicles (for example, almond oil, oily esters or ethanol), preservatives (for example, methylparaben or propylparaben or sorbic acid) and artificial or natural coloring agents and / or sweeteners.

[0174] The present invention further includes anhydrous compositions and dosage forms comprising active ingredients, because water can promote the degradation of certain compounds. Using anhydrous or low-water-containing ingredients and low moisture or low humidity conditions, anhydrous compositions and dosage forms of the present invention can be prepared. If it is foreseen that substantial contact with moisture and / or humidity is expected during production, packaging and / or storage, compositions and dosage forms of the present invention containing lactose can be made anhydrous. Anhydrous compositions can be prepared and stored so that its anhydrous properties are maintained. Therefore, materials known to prevent exposure to water can be used to package anhydrous compositions so that they can be included in suitable prescription drug boxes. Examples of suitable packaging include, but are not limited to, airtightly sealed foil, plastics, unit dose containers, blister packs, and strip packs.

[0175] The ingredients described herein can be combined with pharmaceutical carriers in a close mixture according to conventional pharmaceutical compounding techniques. The carrier can be in a variety of forms, depending on the form of the preparation required for administration. When preparing a composition for oral dosage forms, any commonly used pharmaceutical medium can be used as a carrier, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, colorants are used in the case of oral liquid preparations (such as suspensions, solutions and elixirs) or aerosols; or carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants can be used in the case of oral solid preparations, and in certain embodiments, lactose is not used. For example, for solid oral preparations, suitable carriers include powders, capsules and tablets. If necessary, the tablets can be coated by standard aqueous or non-aqueous techniques.

[0176] Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, etc. and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0177] Suitable binders for use in the dosage form include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, tragacanth powder, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, calcium carboxymethylcellulose, sodium carboxymethylcellulose), polyvinylpyrrolidone, methylcellulose, pregelatinized starch, hydroxypropyl methylcellulose, microcrystalline cellulose, and mixtures thereof.

[0178] Examples of suitable fillers for use in the compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextran, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof.

[0179] When aqueous suspensions and / or elixirs are desired for oral administration, the active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, with emulsifying and / or suspending agents and diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof.

[0180] In one embodiment, the composition may include a solubilizing agent to ensure good dissolution and / or dissolution of the compound of the invention and to minimize precipitation of the compound of the invention. This may be particularly important for compositions for non-oral use, e.g., compositions for injection. A solubilizing agent may also be added to increase the solubility of a hydrophilic drug and / or other components (such as a surfactant), or to maintain the composition as a stable or uniform solution or dispersion.

[0181] Pharmaceutical compositions described herein may be suitable for oral administration to a subject in need thereof. In some cases, a sustained-release formulation for oral administration may be prepared to achieve controlled release of an active agent in contact with the body fluids in the gastrointestinal tract, and to provide substantially constant and effective active agent levels in blood plasma. For this purpose, crystalline forms may be embedded in a polymer matrix and an optional suitable surfactant of a biodegradable polymer, a water-soluble polymer or a mixture thereof. In this context, embedding may refer to micronized particles being incorporated into a polymer matrix. Controlled-release formulations may also be obtained by wrapping dispersed micronized particles or emulsified droplets via known dispersion or emulsion coating techniques.

[0182] In certain embodiments, the composition can be formulated in a food composition. For example, the composition can be a beverage or other liquid, a solid food, a semi-solid food, with or without a food carrier. For example, the composition can include black tea supplemented with any composition described herein. The composition can be a dairy product supplemented with any composition described herein. In certain embodiments, the composition can be formulated in a food composition. For example, the composition can include a beverage, a solid food, a semi-solid food, or a food carrier.

[0183] In certain embodiments, the pharmaceutical preparation can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and can contain formulated drugs such as suspending agents, stabilizers and / or dispersants. Pharmaceutical preparations for parenteral administration include: for example, aqueous solutions of active compounds in water-soluble form. Suspensions of active compounds can be prepared as, for example, oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, isopropyl palmitate or medium chain triglycerides, or liposomes. In a preferred embodiment, the preparation for parenteral administration is an aqueous suspension.

[0184] The compounds described herein can be present in the composition within a concentration range, the range being defined by an upper value and a lower value selected from any of the above concentrations. For example, the compounds or salts of the present invention can be present in the formulation at a concentration of about 1 nM to about 100 mM, about 10 nM to about 10 mM, about 100 nM to about 1 mM, about 500 nM to about 1 mM, about 1 mM to about 50 mM, about 10 mM to about 40 mM, about 20 mM to about 35 mM, or about 20 mM to about 30 mM.

[0185] Methods for preparing compositions comprising compounds described herein may include formulating the compounds with one or more inert, pharmaceutically acceptable excipients. Liquid compositions include, for example, solutions in which the compounds are dissolved, emulsions containing the compounds, or solutions containing liposomes, micelles or nanoparticles containing the compounds disclosed herein. These compositions may also contain small amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers and other pharmaceutically acceptable additives.

[0186] The preparation for injection can be present in a unit dosage form, for example, in an ampoule or in a multiple dose container, and a preservative is added. The composition can be in the form of a suspension, solution or emulsion such as in an oily or aqueous vehicle, and can contain a formulated drug such as a suspending agent, a stabilizer and / or a dispersant. The composition can be present in a unit dose or multiple dose container (e.g., sealed ampoules and vials), and can be stored in powder form or under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, for example, saline or sterile pyrogen-free water, just before use. Instant injection solutions and suspensions can be prepared from the aforementioned sterile powders, granules and tablets.

[0187] Pharmaceutical preparations for parenteral administration include aqueous and nonaqueous (oily) sterile injection solutions of the active compound, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which may include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils (e.g., sesame oil) or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides) or liposomes.

[0188] The compositions described herein, e.g., compounds or salts of Formula I, Ia or Ib, or pharmaceutical compositions of BCL-2 inhibitors or proteasome inhibitors, or co-formulations of compounds of Formula I, Ia or Ib with BCL-2 inhibitors or proteasome inhibitors, can be administered once or more than once a day. The composition can be administered continuously (e.g., administered daily without interruption for the duration of the treatment regimen). In some cases, the treatment regimen can be less than 1 week, 1 week, 2 weeks, 3 weeks, 1 month, or more than 1 month. In some cases, the composition of the present invention is administered in a stage of at least 12 weeks. In other cases, the composition is administered for 1 day, at least 2 consecutive days, at least 3 consecutive days, at least 4 consecutive days, at least 5 consecutive days, at least 6 consecutive days, at least 7 consecutive days, at least 8 consecutive days, at least 9 consecutive days, at least 10 consecutive days, or at least more than 10 consecutive days. In some cases, a therapeutically effective amount may be administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, ten times a week, eleven times a week, twelve times a week, thirteen times a week, fourteen times a week, fifteen times a week, sixteen times a week, seventeen times a week, eighteen times a week, nineteen times a week, twenty times a week, twenty-five times a week, thirty times a week, thirty-five times a week, forty-six times a week, or more than forty times a week. In some cases, a therapeutically effective amount may be administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, ten times a day, or more than ten times a day. In some cases, the composition is administered at least twice a day. In other cases, the composition is administered at least every hour, at least every 2 hours, at least every 3 hours, at least every 4 hours, at least every 5 hours, at least every 6 hours, at least every 7 hours, at least every 8 hours, at least every 9 hours, at least every 10 hours, at least every 11 hours, at least every 12 hours, at least every 13 hours, at least every 14 hours, at least every 15 hours, at least every 16 hours, at least every 17 hours, at least every 18 hours, at least every 19 hours, at least every 20 hours, at least every 21 hours, at least every 22 hours, at least every 23 hours, or at least every day.

[0189] The pharmaceutical composition of the present invention can be used acutely or chronically. The pharmaceutical composition of the present invention can be used as a single treatment or as a course of treatment. The treatment can be applied once a day, twice a day, three times a day, in the morning, in the evening, before going to bed, or continuously in a day. The treatment can be applied every day, every other day, every 3 days, twice a week, once a week, every other week, every month, every 6 weeks, every other month, every 3 months, every 6 months, every year, every other year, every 5 years, or as needed.

[0190] In certain embodiments, the dosage of the drug being administered can be temporarily reduced or temporarily stopped at a certain length of time. In certain embodiments, the patient will have a drug holiday, wherein the patient does not receive the drug or receives the drug of the amount reduced for a period of time. The drug holiday can be, for example, 2 days to 1 year, including, as an example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days or more than 28 days. The drug holiday can be about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 months. The dose reduction during the drug holiday can be, for example, 10%-100% of the original administered dose, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 100%. For other examples, the dose reduction can be between 10% and 100%, between 20% and 80%, between 30% and 70%, between 50% and 90%, between 80% and 100%, or between 90% and 100%.

[0191] Once improvement of the patient's condition has occurred, a maintenance dose may be administered if necessary. Subsequently, as symptoms change, the dose or frequency of administration, or both, may be reduced to a level at which the improved disease, disorder or condition is maintained.

[0192] Additional methods of administering the formulations described herein include, for example, limited to delivery via enteral routes (including oral, gastric or duodenal feeding tubes, rectal suppositories, rectal enemas), parenteral routes, injections, infusions, intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, intracameral, epidural, subcutaneous, inhalation, transdermal, transmucosal, sublingual, buccal, topical, intradermal, dermal, enema drops, intranasal, and vaginal administration. The compounds described herein may be administered topically to the area in need of treatment, for example, by local infusion in surgery, topical application such as creams or ointments, injections, catheters, or implants. The administration may also be by direct injection at the site of the diseased tissue or organ.

[0193] The length of the phase of administration and / or the amount of administration can be determined by a physician or any other type of clinician. The physician or clinician can observe the response of the subject to the administered composition and adjust the dosage based on the subject's performance. For example, for a subject who exhibits a reduced effect in energy regulation, the dosage can be increased to achieve the desired result.

[0194] In certain embodiments, the combination therapies described herein can be administered together at the same time in the same route, or administered separately. In certain embodiments, the components of the composition can be administered using the same or different routes of administration.

[0195] In certain embodiments, the present invention also provides methods of making the compositions described herein.In certain embodiments, the preparation of the compositions described herein comprises mixing or combining two or more components.

[0196] In certain embodiments, the composition can be combined or mixed with a pharmaceutically active agent or therapeutic agent, a carrier and / or an excipient. Examples of such components are described herein. The combined composition can be formulated into unit doses as tablets, capsules, gel capsules, slow-release tablets, etc.

[0197] In certain embodiments, the compositions are prepared so as to obtain a solid composition containing a substantially homogeneous mixture of one or more components, so that the one or more components are evenly distributed throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules.

[0198] The unit dose can be packaged in a container to be transferred to the user. The unit dose can be packaged in a test tube, cylinder, box, bottle, bag, tray, roll, bottle, syringe or can.

[0199] Another aspect of the invention provides achieving a desired effect in one or more subjects after the specified time period of the combination composition described herein is applied. For example, after applying the composition to the subject for 1, 2, 3, 4, 6, 8, 10, 12, 24 or 52 weeks, the beneficial effects of the composition described herein can be observed.

[0200] In certain embodiments, the combination therapy described herein can be administered by a combination therapy regimen. A combination therapy regimen may include a treatment regimen in which administration of a compound described herein or a pharmaceutically acceptable salt thereof is initiated before, during, or after treatment with a second drug described herein and continued until any time during treatment with the second drug or after treatment with the second drug is terminated. The present invention also includes treatments in which a compound described herein or a pharmaceutically acceptable salt thereof and a second drug used in combination are administered simultaneously or at different times and / or at decreasing or increasing intervals during a treatment phase. Combination therapy also includes periodic treatments that are started and stopped at different times to assist in the clinical management of the patient.

[0201] In certain embodiments, the combination therapy can provide a therapeutic advantage in view of the differential toxicity associated with the two treatment modalities. For example, treatment with a CDK inhibitor (such as those described herein) can result in specific toxicity not seen with the anticancer drug (e.g., a BCL-2 inhibitor or a proteasome inhibitor), and vice versa. In this way, the differential toxicity can allow each treatment to be administered at a dose where the toxicity does not occur or is minimized, so that the combination therapy provides a therapeutic dose together while avoiding the toxicity of each component in the combined drug. In addition, when the therapeutic effect achieved as a result of the combination therapy is a synergistic effect, the dose of each drug can be even further reduced, thereby reducing the relevant toxicity to an even greater extent.

[0202] The compounds described herein, or pharmaceutically acceptable salts thereof, and combination therapy may be administered before, during, or after the onset of a disease or condition, and the timing of administering a composition containing the compound may vary. The compounds described herein may be used as prophylactics and may be administered continuously to subjects with a tendency to develop a condition or disease in order to prevent the occurrence of a disease or condition. The compounds described herein and compositions thereof may be administered to a subject as soon as possible during or after the onset of symptoms. The compounds described herein may be administered as soon as feasible after the onset of a disease or condition is detected or suspected, and for a period of time necessary for the treatment of the disease. Example

[0203] The following examples are given for the purpose of illustrating different embodiments of the present invention and are not intended to limit the present invention in any way. The examples of the present invention, together with the methods described herein, currently represent preferred embodiments, which are exemplary and are not intended to be used as limitations on the scope of the present invention. Those skilled in the art will make changes therein and other uses, which are included in the spirit of the present invention as defined by the scope of the claims.

[0204] For all in vitro drug action mechanism studies, the following materials and methods were used.

[0205] The attached or suspended cell lines shown in Table 1 were treated in 6-well dishes or T10 flasks, respectively, and seeded in a total volume of 3-10 ml at a density of 1 million / ml before treatment. All drugs used for in vitro cell treatment were formulated in water / DMSO to achieve the desired concentration (as described in the individual figure labels / legends), wherein the DMSO concentration did not exceed 0.01%. In the table, the drug source is indicated under 'CIVO microinjection study'. Cells were harvested at experimental time points: 2, 6 or 24 hours after treatment as shown in each figure, cells were first washed twice in ice-cold PBS in 15 ml falcon tubes, centrifuged at 1000 rpm for 5 minutes, and then various cell pellets were lysed as described in the following section. Note: NUDHL1Dox 1000 nM and Toledo Dox 1000 nM cell lines with drug resistance were generated in Presage by culturing the parental line in a medium with a stepwise increase in doxorubicin concentrations in several passages. When resistance to 1 uM Dox was achieved, these cells were used to generate xenografts.

[0206]

[0207] For all Western blot analysis studies, the following materials and methods were used.

[0208] Protein lysate is prepared as follows: ice-cold 1X RIPA buffer (from 10X, Millipore) containing 1X-3X Halt protease and phosphatase inhibitor cocktail (100X, Pierce) is added to cell pellet, then pulse sound treatment (~ 3-5 seconds) 3 times. Lysate is clarified by centrifugation at 14K rpm for 15-20 minutes, and supernatant (lysate) is collected for further analysis. In order to prepare lysate from the xenograft tumor tissue of cryopreservation, tissue is first homogenized using a portable tissue homogenizer (ColePalmer) in the above-mentioned lysis buffer, then pulse sound treatment and centrifugation as described above. Carry out the above steps under ice-cold conditions. Use the appropriate blank control containing 1x RIPA, use Bradford to measure (Biorad) at 1:10 or higher dilution quantitative protein samples. 20-25 micrograms of protein from each sample were subjected to SDS-PAGE using BOLT 4-12% Bis-Tris gels (Thermofisher) and transferred to 0.45 or 0.2 micron pore size membranes (based on protein molecular size) and nitrocellulose (NC) or pvdf (Thermofisher) using the manufacturer's protocol, blocked in 5% milk at room temperature for 1 hour, probed with the primary antibody (Table 2) at 4°C overnight, and probed with the corresponding HRP-conjugated secondary antibody (Jackson Immunoresearch, 1:10,000-1:20,000 dilution) at room temperature for 1 hour. After each antibody incubation, 3 PBS / 0.01% Tween-20 washes (5-10 minutes each) were performed. ECL chemiluminescent substrate (Pierce or GE) and autoradiographic film (Thermofisher) were used for detection of protein signals.

[0209]

[0210] For all in vivo The following materials and methods were used for the study.

[0211] All work in mice was approved by IACUC Board of Presage Biosciences, Seattle, WA. All related operations were performed under anesthesia, and all efforts were made to minimize pain and suffering. The mice used for this study were not sick or dead before the experimental endpoint, and all mice received medication as described below, experienced routine health monitoring, and were humanely euthanized at the end of the experiment. Subcutaneous flank xenografts were produced using the following cell lines / mouse strains in the following table (Table 3). Using 1 ml syringe (BD 309659) and 27G needle (BD305109), 200ul cell suspension was injected into the right flank with a 1:1 ratio with matrigel (Corning).

[0212]

[0213] For all CIVO microinjection studies, the following materials and methods were used.

[0214] When the tumor volume reaches approximately 1000 mm 3 Mice were recruited into the CIVO drug study at 1:10 p.m. Microinjection studies were performed using the CIVO device as described previously (Klinghoffer et al., Science Translational Medicine 2015). The device was constructed with 6 injection needle sets (for 6 mm injection length) and a total volume delivery of 3 μl. A fluorescent tracking marker (FTM) was added to each drug reservoir in the vehicle for delivery along with each drug or drug combination. All microdoses were equivalent to or lower than the doses allowed under the FDA's guidance for exploratory IND (investigational new drug) studies and depended on the solubility of the drug in the vehicle. The total amount of drug injected is listed in Table 4 below.

[0215]

[0216] For all systemic drug efficacy studies, the following materials and methods were used.

[0217] When the tumor reaches 150-200 mm 3 Mice were recruited for the study when the mean tumor volume was . V = length x width x height, all three dimensions were measured using digital calipers along with body weight. Animals were removed from the study if any of the three measured tumor dimensions exceeded 2 cm, volume exceeded 2500 mm 3 , ulcer formation or body weight loss greater than 20%.

[0218] Tumor growth inhibition % (TGI) was defined as:

[0219]

[0220] The results are the mean values ​​of the tumors in each group. Wilcoxon Rank Sum or Mann-Whitney test was used as a statistical test to determine the differences between treatment groups.

[0221] The drug dosage, administration route, schedule and formulation regimen are detailed below. For oral tube feeding an Instech, Ref: FTP-20-38 feeding tube was used and for intravenous (iv) a BD insulin syringe was used.

[0222] DLBCL Research

[0223] ● RIVA: voruciclib 200 mpk (oral) 6 days on / 1 day off and ABT 199 (oral) 1 mpk 2 times / week for 4 weeks

[0224] ● SUDHL4: voruciclib 200 mpk (oral) 6 days on / 1 day off and ABT 199 (oral) 25 mpk 6 days on / 1 day off for 4 weeks

[0225] ● U2932: voruciclib 200 mpk (oral) 6 days on / 1 day off and ABT 199 (oral) 10 mpk 2 times / week for 4 weeks.

[0226] AML Research

[0227] ● SKM1: voruciclib 200 mpk (oral) 6 days on / 1 day off + ABT 199 (oral) 10 mpk 6 days on / 1 day off for 3 weeks.

[0228] TNBC Research

[0229] ● HCC1187: voruciclib 200 mpk (oral) 5 days on / 2 days off + Ixazomib / MLN2238 (oral) 3 mpk on days 3, 6, 10, 13 for 2 weeks

[0230] ● HCC1187: Palbociclib 120 mpk (oral) once a day for 14 days + bortezomib 0.42 mpk (intravenous) on days 1, 4, 8, and 11 for 2 weeks

[0231] ● HCC1187: voruciclib 200 mpk (oral) + bortezomib 0.42 mpk (intravenous) on days 1, 4, 8, and 11 for 2 weeks.

[0232] Example 1. Voruciclib is a potent inhibitor of cyclin-dependent kinase-9 (CDK9)

[0233] Voruciclib is considered to be a potent CDK4 / 6 inhibitor. The activity of voruciclib against 38 different kinases was tested in comparison with flavopiradol, a known CDK9 inhibitor. Figure 1 The structures of voruciclib and flavopiridol are depicted, along with a comparison of the inhibitory properties of voruciclib and flavopiridol. Figure 1 It was confirmed that, similar to flavopiradol, voruciclib is a potent inhibitor of CDK9 (K i <10 nM). However, voruciclib has a higher specificity for CDK than flavopiradol. Figure 1 It was shown that flavopiradol is a potent inhibitor of the serine / threonine protein kinases MAK and ICK, while voruciclib did not show potent inhibition of MAK or ICK. MAK and ICK are associated with intestinal epithelial cells and may contribute to the severe diarrhea associated with flavopiradol treatment. On the other hand, voruciclib may show fewer gastrointestinal side effects. DiscoveRx and Thermofisher screening: Percent inhibition of kinase activity at 10 uM and 50 nM for 468 kinase assays of the DiscoveRxScanMax kinase panel and 414 kinase assays of the ThermoFisherSelectScreen kinase panel. Assays were pre-weighed into silanized amber screw-cap vials at Presage and shipped to DiscoveRx and Reaction Biology. For ThermoFisher, the compounds were weighed at ambient conditions and dissolved in 10 mM DMSO prior to shipping. Upon receipt, the vials were stored at -20°C until use. The powder was dissolved in DMSO at 10 mM.

[0234] Figure 2Voruciclib showed single-digit nM potency against CDK 1, 4, 6, and 9, with the strongest inhibition of CDK9. Reaction Biology Profile: The purpose of the study was to determine the rank order of sensitivity of 48 kinases to voruciclib hydrochloride. The experimental equipment was Reaction Biology Corp. Kinase activity was measured using a filter binding assay with radioactive γ-33P-ATP as a phosphate donor. The ATP concentration approximated the Km of the various kinases. For each kinase, IC50 values ​​were calculated from a 10-point concentration curve of the test article and converted to Ki values. The 48 kinases studied here had been identified as the most promising target candidates in previous screening experiments.

[0235] Example 2. Combination of voruciclib and Bcl-2 family inhibitor venetoclax (ABT-199) shows synergistic Same effect

[0236] NU-DHL-1 diffuse large B-cell lymphoma (DLBCL) cells were treated with vehicle, voruciclib alone, venetoclax (ABT-199) alone, or a combination of voruciclib and venetoclax. Figures 3A-3D show NU-DHL-1 cells stained for the anti-apoptotic protein, induced myeloid leukemia cell differentiation protein Mcl-1 (MCL-1) (shown in red). Figure 3A shows cells treated with vehicle only. In Figure 3B, voruciclib alone reduced the expression of MCL-1, as shown by the increase in the darker area outline in outline 301. Venetoclax alone increased the expression of MCL-1, as shown in the light area within outline 302 of Figure 3C. The combination of voruciclib and venetoclax resulted in a significant decrease in the expression of MCL-1, shown in Figure 3C. Figure 3D , shown in the dark area of ​​the outline 303.

[0237] Figures 4A-4D demonstrate the association between MCL-1 inhibition and synergistic induction of apoptosis. NU-DHL-1 diffuse large B-cell lymphoma (DLBCL) cells were treated with vehicle, voruciclib alone, venetoclax (ABT-199) alone, or a combination of voruciclib and venetoclax. Cells were stained for cleaved caspase-3 (CC3) (shown in red), a marker of apoptosis. Figure 4A shows cells treated with vehicle alone. Voruciclib alone, shown in Figure 4B, showed slightly increased expression of CC3, as shown in the light-colored area within outline 401. Similarly, venetoclax alone, shown in Figure 4C, showed slightly increased expression of CC3, as shown in the light-colored area within outline 402. In Figure 4D The combination of voruciclib and venetoclax in 404 demonstrated synergistically increased expression of CC3, as shown in the large light-colored area within outline 403, suggesting increased apoptosis induction in the treated cells.

[0238] Figures 6A-6E The synergistic effect of voruciclib and venetoclax on apoptosis was demonstrated to be reproducible in multiple DLBCL models. Briefly, RIVA, Toledo, Toledo / Dox1000, NUDHL, and NUDHL / Dox1000 cells were treated with voruciclib alone, venetoclax alone, or a combination of voruciclib and venetoclax, respectively, as described in Fig. 6A , Figure 6B , Figure 6C , Fig.6D and Fig. 6E Voruciclib alone and venetoclax alone had a mild effect on CC3 expression, however, the combination of voruciclib and venetoclax demonstrated synergistic induction of CC3 expression across all five DLBCL models.

[0239] Figure 7 A proposed model for this synergistic effect is depicted. By inhibiting MCL-1 through CDK9, voruciclib can switch cells to apoptosis when combined with Bcl-2 inhibition.

[0240] Example 3. Combination of voruciclib and Bcl-2 family inhibitor navitoclax exhibits synergistic effect

[0241] Ramos Burkitt lymphoma cells were treated with vehicle, voruciclib alone, navitoclax (ABT-263) alone, or a combination of voruciclib and navitoclax. Figures 5A-5D show Ramos Burkitt lymphoma cells stained for cleaved caspase-3 (CC3) (shown in red), a marker of apoptosis. Figure 5A shows cells treated with vehicle alone. Voruciclib alone, as shown in Figure 5B, showed a slight increase in the expression of CC3, as shown in the light-colored area of ​​outline 501. Similarly, venetoclax alone, as shown in Figure 5C, showed a slight increase in the expression of CC3, as shown in the light-colored area of ​​outline 502. The combination of voruciclib and navitoclax showed a significant increase in the expression of CC3, as shown in Figure 5A. Figure 5D The large amount of light-colored areas within the outline 503 are shown, suggesting increased apoptosis induction in the treated cells.

[0242] Example 4. Combination of voruciclib and proteasome inhibitors exhibits synergistic effects

[0243] Perform drug screens to test multiple drugs simultaneously. Fig. 8A Shown is a list of different compounds injected into the HCC1187 triple negative breast cancer (TNBC) xenograft model. Figure 8B Mean % change in MCL-1 induction in xenografts is shown. The proteasome inhibitor bortezomib showed an increase in MCL-1 expression. Figure 8C Xenografts treated with vehicle were confirmed. Fig.8D Xenografts treated with bortezomib and stained for MCL-1 expression (shown in red) are shown. Bortezomib significantly increased MCL-1 expression in these cells, as shown by the increase in the light-colored area within the outline 801.

[0244] Example 5. Combination of voruciclib and proteasome inhibitor marizomib exhibits synergistic effect

[0245] NudHL1 DLBCL xenografts were treated with vehicle, voruciclib alone, marizomib alone, or voruciclib and marizomib in combination for 6 hours. Figures 9A-9D depict cells treated as above and stained for cleaved caspase-3 (CC3) (shown in red) as a marker of apoptosis. Figure 9A shows cells treated with vehicle alone. As shown in Figure 9B, cells treated with voruciclib alone had little to no increase in CC3 expression, as evidenced by the small amount of light-colored areas within outline 901. Similarly, as shown in Figure 9C, cells treated with marizomib alone had little to no increase in CC3 expression, as evidenced by the small amount of light-colored areas within outline 902. Cells treated with voruciclib and marizomib in combination showed a significant increase in CC3 expression, as shown in Figure 9A. Fig.9D This is evidenced by the large amount of light-colored areas within the outline 903, suggesting a synergistic effect on the induction of apoptosis in these cells.

[0246] Example 6. Combination of voruciclib and proteasome inhibitor bortezomib exhibits synergistic effect

[0247] Briefly, NudHL1 DLBCL xenografts were treated with vehicle, voruciclib alone, bortezomib alone, or voruciclib and bortezomib in combination for 6 hours. Figures 10A-10D depict cells treated as above and stained for cleaved caspase-3 (CC3) (shown in red) as a marker of apoptosis.

[0248] Figure 10A shows cells treated with vehicle only. As shown in Figure 10B, cells treated with voruciclib alone had little to no increase in CC3 expression. Similarly, as shown in Figure 10C, cells treated with bortezomib alone had little to no increase in CC3 expression, as evidenced by the small amount of light-colored area within outline 1001. Cells treated with the combination of voruciclib and bortezomib showed a significant increase in CC3 expression, as shown in Figure 10C. Fig. 10D This is evidenced by the large amount of light-colored areas within the outline 1002, suggesting a synergistic effect on the induction of apoptosis in these cells.

[0249] Figure 11 depicts a similar experiment performed on a HCC1187 triple negative breast cancer (TNBC) xenograft model. In short, cells were treated with vehicle, voruciclib alone, bortezomib alone, or a combination of voruciclib and bortezomib. Cells were then stained for CC3 expression (shown in red) as a marker of apoptosis. Figure 11A shows cells treated with vehicle alone. As shown in Figure 11B, cells treated with voruciclib alone had less to no increase in CC3 expression. Similarly, as shown in Figure 11C, cells treated with bortezomib alone had less to no increase in CC3 expression. Cells treated with a combination of voruciclib and bortezomib showed a significant increase in CC3 expression, as shown in Figure 11A. Fig.11D This is evidenced by the large amount of light-colored areas within the outline 1101, suggesting a synergistic effect on the induction of apoptosis in these cells.

[0250] HCC1187 triple negative breast cancer (TNBC) xenograft mouse model treated with vehicle, voruciclib (200 mg / kg), bortezomib (0.42 mg / kg), or a combination of voruciclib (200 mg / kg) and bortezomib (0.42 mg / kg). Tumor volume in each mouse was measured over time and the results are shown in Figures 12A-12D Mice treated with a combination of voruciclib and bortezomib ( Fig.12D ) demonstrated a significant decrease in normalized tumor volume over time compared to mice treated with voruciclib alone ( FIG. 12B ) or bortezomib alone ( FIG. 12C ). Fig.12E Western blots of MLC-1 protein expression are shown. Cells treated with voruciclib had reduced MCL-1 protein expression compared to untreated samples, whereas cells treated with bortezomib had increased MCL-1 protein expression compared to untreated samples. Furthermore, voruciclib reduced the increase in MCL-1 expression induced by bortezomib.

[0251] Fig.13 It was confirmed that treatment with voruciclib alone, bortezomib alone, or a combination of voruciclib and bortezomib did not affect the body weight of mice. This data may suggest that the combination therapy of voruciclib and bortezomib may exhibit less to no toxicity.

[0252] Furthermore, voruciclib abolished bortezomib-induced upregulation of the prosurvival protein MCL-1 and the E3 ubiquitin-protein ligase XIAP. Fig.15A A proposed model for voruciclib inhibition of CDK9 is depicted. Fig. 15B Shown are MCL-1 and XIAP protein levels in cells treated with voruciclib alone, bortezomib alone, or voruciclib and bortezomib in combination. Western blot demonstrated that voruciclib reduced the upregulation of MCL-1 and XIAP proteins induced by bortezomib in these cells.

[0253] Example 7. Palbociclib (a CDK 4 / 6 inhibitor) does not show synergistic effect in combination with bortezomib answer

[0254] The HCC1187 triple negative breast cancer (TNBC) xenograft mouse model was treated with vehicle, palbociclib (120 mg / kg), bortezomib (0.42 mg / kg), or a combination of palbociclib (120 mg / kg) and bortezomib (0.42 mg / kg). Tumor volume in each mouse was measured over time and the results are shown in Fig.14 Unlike voruciclib, palbociclib combined with bortezomib did not show a synergistic effect on tumor volume. Palbociclib is a specific CDK4 / 6 inhibitor, while voruciclib targets CDK9. Thus, this difference may be CDK9-specific.

[0255] Example 8. Possible role of the endoplasmic reticulum (ER) stress response pathway in the synergistic effect of voruciclib and bortezomib combination Should play a role

[0256] Figure 16A shows a tissue section treated with bortezomib. Bortezomib induces apoptosis in certain cells, while some cells appear to be resistant to bortezomib treatment. The cells were stained for the ER chaperone protein GRP178 / BiP. Fig. 16B It was demonstrated that cells resistant to bortezomib-induced apoptosis express GRP178 / BiP.

[0257] Next, HCC1187 triple-negative breast cancer cells were treated with voruciclib alone, bortezomib alone, tunicamycin (an ER stress inducer) alone, or in combination. Tunicamycin alone strongly induced X-box binding protein 1 (XBP1), an ER stress protein of the IRE1α branch of the ER stress pathway, at 6 hours. In addition, tunicamycin and bortezomib strongly induced XBP1 expression at 24 hours ( Fig.18B ). The combination of voruciclib with bortezomib or tunicamycin reduced XBP1 upregulation. These results suggest that voruciclib may inhibit the IRE1α branch of the ER stress pathway ( Fig.18B voruciclib also reduced bortezomib-induced transcriptional induction of XBP1 ( Figures 19A-19B ). HCC1187 cells were treated with voruciclib (1.5uM), bortezomib (10nM), STF083010 (IRE1a endoribonuclease activity inhibitor) (Millipore) (60uM) or tunicamycin (Sigma) (100nM) for 4 hours. mRNA was harvested using Qiagen RNAeasy Kit and quantified using Nanodrop spectrophotometer. cDNA was generated using 1 ug mRNA from each condition using High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems) and manufacturer's protocol. PCR assays were performed using 100ng cDNA to amplify unspliced ​​or spliced ​​forms of XBP1 (Thermofisher PCR Supermix) - 94℃ for 2 minutes, (94℃ for 15 seconds, 55℃ for 30 seconds, 72℃ for 1 min) x 30 cycles. 72℃ for 7 minutes, hold at 4℃. Spliced ​​and unspliced ​​variants of XBP1 were detected.

[0258] Example 9. Combination of voruciclib and proteasome inhibitor ixazomib exhibits synergistic effect

[0259] Figure 20A-20D depicts a breast cancer (TNBC) xenograft model of HCC1187 triple negatives. In short, cells were treated with vehicle, voruciclib alone, ixazomib alone, or voruciclib and ixazomib combined. Cells were then stained for CC3 expression (shown in red) as a marker of apoptosis.

[0260] Figure 20A shows cells treated with vehicle only. As shown in Figure 20B, cells treated with voruciclib alone had little to no increase in CC3 expression, as evidenced by the small amount of light-colored area within outline 2001. Similarly, as shown in Figure 20C, cells treated with ixazomib alone had little to no increase in CC3 expression, as evidenced by the small amount of light-colored area within outline 2002. Cells treated with the combination of voruciclib and ixazomib showed a significant increase in CC3 expression, as evidenced by the small amount of light-colored area within outline 2003. Fig.20D This is evidenced by the large amount of light-colored areas within the outline 2003, suggesting a synergistic effect on the induction of apoptosis in these cells.

[0261] Various CDK inhibitors were tested in combination with ixazomib, as shown in Figures 21A and 21B. Only voruciclib in combination with ixazomib exhibited a synergistic effect on apoptosis of tumor cells, as demonstrated by the light-colored areas within outlines 2101 and 2102. Vehicle alone, ixazomib alone, palbociclib and ixazomib, dinaciclib and ixazomib, and flavopiradol and ixazomib did not exhibit a synergistic effect on apoptosis of tumor cells as the combination of voruciclib and ixazomib did.

[0262] Example 10. Combination of voruciclib and venetoclax exhibits synergistic effect

[0263] Fig.22A -B illustrates the synergistic effect from voruciclib and venetoclax in the SU-DHL-4 model of DLBCL. Cleaved PARP (cPARP) can be used as a biomarker of apoptosis. To evaluate the combined efficacy of voruciclib and venetoclax together, low doses of each (150 nM of voruciclib and 20 nM of venetoclax) were examined as single agents or as a combination in the SUDHL4 model of DLBCL. Cells were exposed to the drugs for 24 h, then lysed and assayed for PARP, MCL-1, C-MYC, and β-actin (as Fig.22A protein loading control) and BCL2 (as Fig. 22B The cleavage form of the other protein (loading control) was checked by western blotting with an antibody specific for the cleavage form. Fig.22A showed that 1.5 uM of voruciclib inhibited MCL1 in a DLBCL model.

[0264] Fig. 22BA 0.15 uM concentration of voruciclib was used, which is 1 / 10 of the concentration that has been shown to be effective in inducing apoptosis as a single agent. This level of voruciclib maintains a certain efficacy in inhibiting MCL-1 expression (compare Fig. 22B 1 and 2), while exposure of cells to venetoclax induced MCL-1 expression (compare Fig. 22B Low vorucicilib exposure also inhibited venetoclax-induced MCL-1 (compare Fig. 22B Lanes 3 and 4).

[0265] Figure 23A-C illustrates the synergistic effect of voruciclib and venetoclax in the SU-DHL-4 model, OCI Ly10 model, and U2932 model of DLBCL. Cleaved PARP (cPARP) can be used as a biomarker for apoptosis. To evaluate the combined efficacy of voruciclib and venetoclax, low doses of each were examined as a single agent or as a combination in three models of DLBCL. Cells were exposed to the drugs for 24 h, then lysed and examined by Western blot using antibodies specific to the cleaved forms of PARP and β-actin (as a protein loading control).

[0266] Figure 23A shows that the combination of 0.15 uM voruciclib and 20 nM venetoclax exhibits synergistic effects to induce apoptosis in the SUDHL4 model of DLBCL, where neither compound as a single agent induces apoptosis. Figure 23B shows that the combination of 5 uM voruciclib and 50 nM venetoclax exhibits synergistic effects to induce apoptosis in the OCI Ly10 model of DLBCL, where neither compound as a single agent significantly induces apoptosis. Fig.23C showed that the combination of 5 uM voruciclib and 50 nM venetoclax exhibited synergistic effects to induce apoptosis in the U2932 model of DLBCL, with the combination of the two compounds being significantly greater than the simple addition of each compound as a single agent.

[0267] Example 11. Voruciclib inhibits MCL1 expression in DLBCL xenograft tumors in mice

[0268] Fig.24It is shown that voruciclib inhibits the expression of MCL1 in DLBCL xenograft tumors in mice. Mice carrying DLBCL xenograft tumors (OCILY10) were given voruciclib or vehicle control by oral gavage every day for 5 days, and the tumors were harvested 4 h after the final dose for analysis of MCL-1 expression. The tumor samples were cut in half, one half was dissolved in lysis buffer for western blot analysis, and the other half was fixed in formalin and prepared for immunohistochemistry. Western blots of tumor lysates of 3 individual mice in each treatment group were performed with antibodies specific to MCL-1 or beta actin (protein loading control). The figure shows that voruciclib at 100 mpk inhibits the expression of MCL1 in DLBCL xenograft tumors as a single drug.

[0269] Example 12. Voruciclib and venetoclax inhibit the growth of DLBCL xenograft tumors in mice

[0270] Fig.25A -B demonstrates that the combination of voruciclib and venetoclax impedes the growth of DLBCL xenograft tumors. Immunodeficient NOD SCID mice were implanted with the RIVA (activated B-cell, or ABC) model of DLBCL. Xenograft tumors were allowed to grow in the NOD SCID hosts until they reached 200 mm 3 , at which time xenografted animals were randomized to 4 study groups: 1. Vehicle control; 2. 200 mpk of voruciclib; 3. 1 mpk of venetoclax; and 4. a combination of 200 mpk of voruciclib and 1 mpk of venetoclax. Voruciclib was administered daily by oral gavage 6 days per week with a 1-day drug rest between cycles. Venetoclax was administered by oral gavage on days 1 and 4 of each weekly cycle. Drug effects were assessed by twice-weekly tumor volume measurements by a technician who was blinded to the treatment of each subject in the study. N=5-6 animals / treatment group.

[0271] Fig.25A The mean tumor volume (mm) for each of the 4 study groups is shown. 3 ): Vehicle, single-agent voruciclib, single-agent venetoclax, and a combination of voruciclib and venetoclax. Single-agent treatment saw reductions in tumor volume over the longer study day phase. Combination treatment saw much higher reductions in tumor volume size, reaching a mean tumor volume reduction of more than 2-fold before week 3 of the study.

[0272] Fig.25BDepicted are tumor images of animals from each of the four study groups: vehicle, single agent voruciclib, single agent venetoclax, and a combination of voruciclib and venetoclax. Vehicle tumor size is the largest, as indicated by outline 2501. Tumors with single agent voruciclib are smaller, as indicated by outline 2502. Tumors with single agent venetoclax are also smaller than vehicle, as indicated by outline 2503. Combination therapy tumors are represented by outline 2504, which are significantly smaller than tumors with either vehicle or single agent therapy, suggesting a synergistic effect.

[0273] Fig.26A -B demonstrates that the combination of voruciclib and venetoclax impedes the growth of DLBCL xenograft tumors. Immunodeficient NOD SCID mice were implanted with the U2932 model of DLBCL. Xenograft tumors were allowed to grow in the NOD SCID hosts until they reached 200 mm 3 , at which time xenografted animals were randomized to 4 study groups: 1. Vehicle control; 2. 200 mpk of voruciclib; 3. 10 mpk of venetoclax; and 4. a combination of 200 mpk of voruciclib and 10 mpk of venetoclax. Voruciclib was administered daily by oral gavage 6 days per week with a 1-day drug rest between cycles. Venetoclax was administered by oral gavage on days 1 and 4 of each weekly cycle. Drug effects were assessed by tumor volume measurements twice weekly by a technician who was blinded to the treatment of each subject in the study. N=5-6 animals / treatment group.

[0274] Fig.26A The mean tumor volume (mm) for each of the 4 study groups is shown. 3 ). Single-drug treatment saw a reduction in tumor volume over the longer study day period. Combination treatment saw little tumor growth, with mean tumor volume remaining consistent over the 29-day study period.

[0275] Fig.26B The mean body weight in grams (g) for the subjects in each of the four study groups is shown. All four study groups exhibited stable and consistent body weights, a general indication of health and safety.

[0276] Fig.27A-B demonstrates that the combination of voruciclib and venetoclax impedes the growth of DLBCL xenograft tumors. Immunodeficient NOD SCID mice were implanted with the NUDHL1 model of DLBCL. Xenograft tumors were allowed to grow in the NOD SCID hosts until they reached 200 mm 3 , at which time xenografted animals were randomized to 4 study groups: 1. Vehicle control; 2. 200 mpk of voruciclib; 3. 50 mpk of venetoclax; and 4. a combination of 200 mpk of voruciclib and 50 mpk of venetoclax. Voruciclib was administered daily by oral gavage 6 days per week, with a 1-day drug withdrawal between cycles. Venetoclax was administered by oral gavage on days 1 and 4 of each weekly cycle. Drug effects were assessed by tumor volume measurements twice weekly by a technician who was blinded to the treatment of each subject in the study. N=5-6 animals / treatment group.

[0277] Fig.27A Normalized tumor volume (mm) for each of the 4 study groups is shown. 3 ). Single drug treatment saw reductions in tumor volume over the longer study day period. Combination treatment saw much higher reductions in tumor volume size, reaching a mean tumor volume reduction of more than 2-fold before week 3 of the study.

[0278] Fig.27B Depicted are tumor images of animals from each of the four study groups: vehicle, single agent voruciclib, single agent venetoclax, and the combination of voruciclib and venetoclax. Vehicle tumor size is the largest, as shown by outline 2701. Tumors with single agent voruciclib are smaller, as shown by outline 2702. Tumors with single agent venetoclax are also smaller than vehicle, as shown by outline 2703. Combination therapy tumors are represented by outline 2704, which are significantly smaller than tumors with either vehicle or single agent therapy, suggesting a synergistic effect.

[0279] Fig.28 The combination of voruciclib and venetoclax was demonstrated to impede the growth of DLBCL xenograft tumors. Immunodeficient NOD SCID mice were implanted with the SUDHL4 model of DLBCL. Xenograft tumors were allowed to grow in the NOD SCID hosts until they reached 200 mm 3, at which time xenografted animals were randomized to 4 study groups: 1. Vehicle control; 2. 200 mpk of voruciclib; 3. 25 mpk of venetoclax; and 4. A combination of 200 mpk of voruciclib and 25 mpk of venetoclax. Voruciclib was administered daily by oral gavage 6 days per week with a 1-day break between cycles. Venetoclax was administered daily by oral gavage 6 days per week with a 1-day break between cycles. Drug effects were assessed by tumor volume measurements twice weekly by a technician who was blinded to the treatment of each subject in the study. N=5-6 animals / treatment group. Fig.28 Normalized tumor volume (mm) for each of the 4 study groups is shown. 3 ). Single drug treatment saw an increase in tumor volume over the study days. Combination treatment saw less tumor growth than single drug treatment and vehicle treatment.

[0280] Fig.29 Each lane represents a single SUDHL4 xenograft. Western blots together show that voruciclib restores p53 that was abolished by venetoclax. Tumors were harvested 4 hours after treatment on day 26. Each tumor was cut into 2 halves. One half was used for FFPE-based IHC analysis and the other half for Western blot analysis.

[0281] Example 13. Voruciclib inhibits MCL1 expression in AML cell lines

[0282] Figures 30A-30C show that voruciclib has single agent activity in AML cell lines. AML cell lines (SKM-1, MV-4-11, and OCI-AML-3) were left untreated or exposed to clinically achievable levels of voruciclib (1.5 and 5.0 micromolar) for 2, 6, or 24 hours. At given time points, cells were lysed and analyzed by Western blot with antibodies that specifically recognize phosphorylated RNA pol II (pSer2), MCL-1, cleaved PARP, and β-actin. In all three AML cell lines (SKM-1, MV-4-11, and OCI-AML-3), voruciclib inhibited MCL-1 and induced apoptosis, as shown within outlines 3001, 3003, and 3005. Cleaved PARP (cPARP) can be used as a biomarker for apoptosis. In all three AML cell lines (SKM-1, MV-4-11, and OCI-AML-3), voruciclib induced apoptosis as shown within outlines 3002, 3004, and 3006.

[0283] Example 14. Combination of voruciclib and venetoclax induces cell death in AML cell lines

[0284] Fig.31 The combination of voruciclib and venetoclax was shown to induce synergistic cell death in AML cell lines. AML cell lines (SKM-1, MV-4-11, and OCI-AML-3) were left untreated or exposed to voruciclib (1.5 micromolar), venetoclax (100 nM), or a combination of the two drugs at these sub-effective concentrations for 24 h at levels previously shown to be below those required to induce apoptosis with a single drug. At given time points, cells were lysed and analyzed by Western blot with antibodies that specifically recognize phosphorylated RNA pol II (pSer2), MCL-1, cleaved PARP, and β-actin. As shown within contour lines 3101 and 3102, the combination of voruciclib and venetoclax induced apoptosis (PARP) when either drug alone did not induce apoptosis. It was observed that venetoclax induced upregulation of MCL-1 in all three cell lines. Voruciclib inhibited resistance mechanisms driven by MCL-1 in all three cell lines.

[0285] Example 15. Voruciclib and venetoclax combination impedes tumor growth in AML xenografts

[0286] Fig.32 The combination of voruciclib and venetoclax was shown to impede tumor growth in SKM1 AML xenografts. Venetoclax monotherapy had normalized tumor volumes similar to vehicle. Voruciclib monotherapy demonstrated slightly lower tumor volumes, while the combination of voruciclib and venetoclax greatly impeded tumor growth, resulting in tumor volumes that were approximately half of those of vehicle by day 22 of the study.

[0287] Example 16. Voruciclib as a single agent induces apoptosis in a DLBCL model

[0288] Fig.33 It was shown that voruciclib-induced apoptosis is associated with the inhibition of MCL-1. Three DLBCL models were used: SUDHL4, RIvA, and U2932. Cleaved PARP (cPARP) can be used as a biomarker for apoptosis. Western blot showed that apoptosis increased with increasing concentrations of voruciclib. Similarly, voruciclib inhibited MCL-1 and induced apoptosis with increasing concentrations of voruciclib.

[0289] Although preferred embodiments of the present invention have been shown and described herein, it will be appreciated by those skilled in the art that such embodiments are provided only as examples. Those skilled in the art will now make numerous variations, changes and substitutions without departing from the present invention. It should be understood that the various alternatives of the embodiments of the invention described herein may be used to practice the present invention. The following claims are intended to define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A therapeutically effective amount of a CDK inhibitor represented by Formula Ib: Or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of venetoclax or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a blood cancer selected from acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL).

2. The use according to claim 1, wherein the blood cancer is acute myeloid leukemia (AML).

3. The use according to claim 1, wherein the blood cancer is diffuse large B-cell lymphoma (DLBCL).

4. The use according to claim 1, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are concurrently administered to a subject in need thereof.

5. The use according to claim 1, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are administered sequentially to a subject in need thereof within about 12 hours of each other.

6. The use according to claim 5, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are administered sequentially to a subject in need thereof within about 5 hours of each other.

7. The use according to claim 1, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are co-formulated in a pharmaceutical composition.

8. The use according to claim 1, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are administered to a subject in need thereof every day, every other day, or every three days.

9. A therapeutically effective amount of a CDK inhibitor represented by Formula Ib: or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a proteasome inhibitor in the preparation of a medicament for treating cancer, wherein: The proteasome inhibitor is bortezomib or a pharmaceutically acceptable salt thereof and the cancer is diffuse large B-cell lymphoma (DLBCL) or triple negative breast cancer (TNBC), or The proteasome inhibitor is marizomib or a pharmaceutically acceptable salt thereof and the cancer is diffuse large B-cell lymphoma (DLBCL), or The proteasome inhibitor is ixazomib or a pharmaceutically acceptable salt thereof and the cancer is triple negative breast cancer (TNBC).

10. The use according to claim 9, wherein the proteasome inhibitor is bortezomib or a pharmaceutically acceptable salt thereof and the cancer is diffuse large B-cell lymphoma (DLBCL).

11. The use according to claim 9, wherein the proteasome inhibitor is bortezomib or a pharmaceutically acceptable salt thereof and the cancer is triple negative breast cancer (TNBC).

12. The use according to claim 9, wherein the proteasome inhibitor is marizomib or a pharmaceutically acceptable salt thereof and the cancer is diffuse large B-cell lymphoma (DLBCL).

13. The use according to claim 9, wherein the proteasome inhibitor is ixazomib or a pharmaceutically acceptable salt thereof and the cancer is triple negative breast cancer (TNBC).

14. The use according to claim 9, wherein the CDK inhibitor and the proteasome inhibitor are administered concurrently.

15. The use of claim 9, wherein the CDK inhibitor and the proteasome inhibitor are administered sequentially to a subject in need thereof within about 12 hours of each other.

16. The use according to claim 15, wherein the CDK inhibitor and the proteasome inhibitor are administered sequentially to a subject in need thereof within about 5 hours of each other.

17. The use according to claim 9, wherein the CDK inhibitor and the proteasome inhibitor are co-formulated in a pharmaceutical composition.

18. The use according to claim 9, wherein the CDK inhibitor and the proteasome inhibitor are administered to a subject in need thereof every day, every other day, or every three days.

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