TW202327595A - Combinations of azalactam compounds for the treatment of cancer - Google Patents

Abstract

This disclosure relates to a method for treating cancer by administering a compound of Formula (I) in combination with a PD-1 axis binding antagonist to a subject in need thereof.

Description

Combinations of azalactamide compounds for the treatment of cancer

The present invention relates to combination therapy suitable for the treatment of cancer. Certain embodiments relate to a combination therapy comprising an HPK1 inhibitor or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising such compound or salt, in combination with a PD-1 axis binding antagonist. The present invention also relates to related treatment methods, pharmaceutical compositions and medical uses. The methods and compositions are suitable for any indication where the therapy itself is useful for the detection, treatment and/or prevention of a disease, disorder or other condition in an individual.

Hematopoietic progenitor kinase 1 (HPK1), also known as mitogen activated protein kinase kinase kinase kinase 1 (MAP4K1), is a protein that operates through JNK and ERK signaling pathways Members of the mammalian Ste20-like serine/threonine kinase family. HPK1 is mainly expressed in hematopoietic organs and cells (such as T cells, B cells and dendritic cells), suggesting that HPK1 may be involved in the regulation of signal transduction in the hematopoietic lineage (including lymphocytes). (Shui et al., "Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses", Nature Immunology 2006, 8, 84-91).

The ability of T cells to recognize tumor-associated antigens (including neoantigens and testicular cancer antigens) is limited by HPK1 (Sawasdikosol et al., "A perspective on HPK1 as a novel immuno-oncology drug target", Elife 2020, 9; Di Bartolo et al., "A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76", J. Exp. Med. 2007, 204(3): 681-91; Shui et al. , "Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses", Nat. Immunol. 2007, 8(1):84-91). When the T-cell receptor (TCR) engages a neoantigen, the TCR-associated kinase Lck initiates a signaling cascade involving SLP76 that ultimately leads to T-cell proliferation, cytokine secretion, and/or antitumor cytotoxicity. However, Lck also phosphorylates and activates HPK1, which in turn phosphorylates SLP76 and targets SLP76 for degradation (Liou et al., "HPK1 is activated by lymphocyte antigen receptors and negatively regulates AP-1", Immunity, 2000, 12(4) : 399-408; Ling et al., "Involvement of hematopoietic progenitor kinase 1 in T cell receptor signaling", J. Biol. Chem., 2001, 276(22): 18908-14; Sauer et al., "Hematopoietic progenitor kinase 1 associates physically and functionally with the adapter proteins B cell linker protein and SLP-76 in lymphocytes”, J. Biol. Chem., 2001, 276 (48): 45207-16). HPK1-mediated loss of SLP76 blocks recognition of suboptimal stimuli.

The programmed death 1 (PD-1) receptor is expressed by activated T cells, B cells, and myeloid cells. PD-1 has two known ligands: planned death ligand 1 (PD-L1) and planned death ligand 2 (PD-L2). PD-1 is activated by PD-L1 (also known as B7-H1, B7-4, CD274, and B7-H) and PD-L2 is expressed by stromal cells, tumor cells, or both, thereby initiating T cell death and localized Immunosuppression (Dong et al., "B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion", Nat. Med. 1999; 5:1365-69; Freeman et al., "Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation", J. Exp. Med. 2000; 192:1027-34). Engagement of PD-L1 with the PD-1 receptor on T cells leads to inhibition of CD28 co-stimulatory signaling (Kamphorst et al., "Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent", Science 2017, 355 (6332):1423-7; Hui et al., “T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition”, Science 2017, 355(6332):1428-33). Conversely, inhibition of this interaction enhanced local T cell responses and mediated antitumor activity in nonclinical animal models (Iwai Y et al., "Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor Immunotherapy by PD-L1 blockade”, Proc. Natl. Acad. Sci. USA 2002; 99:12293-97).

PD-L1 is a cell surface protein and a member of the B7 family. PD-L1 is found on almost all types of lymphoid hematopoietic cells and is underexpressed by resting T cells, B cells, macrophages and dendritic cells and is further upregulated by: anti-CD40 antibodies of B cells, anti-CD40 antibodies of T cells Anti-CD3 antibody, anti-CD40 antibody of macrophages, IFNγ and granulocyte macrophage colony-stimulating factor (GM-CSF) and/or anti-CD40 antibody of dendritic cells (DC), IFNγ, IL-4, IL- 12 and GM-CSF. PD-L1 is also expressed by some non-hematopoietic cells and is overexpressed in many cancers, where its overexpression is often associated with poor prognosis (Okazaki T et al, PD-1 and PD-1 ligands: from discovery to clinical application, Intern. Immun. 2007 19(7):813) (Thompson RH et al., "Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up", Cancer Res 2006, 66(7):3381 ). Interestingly, most tumor-infiltrating T lymphocytes mainly expressed PD-1 compared with T lymphocytes in normal tissues and peripheral blood. PD-1 on tumor-reactive T cells can contribute to attenuation of the anti-tumor immune response (Ahmadzadeh et al., "Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired", Blood 2009 114 (8): 1537). This can be attributed to inappropriate utilization of PD-L1 signaling, which is mediated by PD-L1 expressing tumor cells, which interact with PD-1 expressing T cells, leading to attenuation of T cell activation and escape from immune surveillance (Sharpe et al., "The B7-CD28 superfamily", Nat. Rev. 2002) (Keir ME et al., PD-1 and its ligands in tolerance and immunity, Annu. Rev. Immunol. 2008, 26:677). Therefore, inhibition of PD-L1/PD-1 interaction promotes CD8+ T cell-mediated tumor killing.

There remains a need for new therapies for cancer using combinations of HPK1 inhibitors and PD-1 axis binding antagonists. The present invention addresses this need and others. The combination therapy of the present invention exhibits greater efficacy than treatment with the individual therapeutic agents alone.

All references, publications and patent applications disclosed herein are hereby incorporated by reference in their entirety.

The present invention relates to therapeutic methods, combinations and pharmaceutical compositions for the treatment of cancer. Combination therapies comprising a compound of the invention and other therapeutic agents are also provided. The invention also provides kits comprising one or more compositions of the invention.

In one aspect, the invention provides a method for treating cancer comprising administering to a subject in need thereof an amount of an HPK1 inhibitor in combination with an amount of a PD-1 axis binding antagonist, wherein the equal amounts are effective together Treating cancer; wherein the HPK1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: R ^1a and R ^1b are each independently (C ₁ -C ₆ ) alkyl, or R ^1a and R ^1b are formed together with the nitrogen to which they are attached via 0, 1 or 2 A 5-membered heterocycloalkyl group substituted with (C ₁ -C ₆ ) alkyl; R ^2a and R ^2b are each independently hydrogen or (C ₁ -C ₃ ) alkyl; R ^3a and R ^3b are each independently hydrogen or (C ₁ -C ₃ )alkyl; each R ⁴ is independently (C ₁ -C ₆ )alkyl substituted with 0 or 1 halogen or hydroxy; and n is 0, 1 or 2.

In some preferred embodiments of the methods as described herein, the compound is 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6 ,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methan ylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one (PF-07265028), which has the following structure: .

In some embodiments of the methods as described herein, the PD-1 axis binding antagonist comprises a PD-1 binding antagonist, a PD-L1 binding antagonist, or a PD-L2 binding antagonist.

In some embodiments, the PD-1 axis binding antagonist comprises a PD-1 binding antagonist. In some such embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In some such embodiments, the anti-PD-1 antibody is sasanlimab, nivolumab, pembrolizumab, pidilizumab, sago Cemiplimab, tislelizumab, spartalizumab, sintilimab, MEDI-0680, BGB-108, or AGEN2034 Genozumab Anti (genolimzumab), CBT-502, camrelizumab (camrelizumab), or a combination thereof. In a preferred embodiment, the anti-PD-1 antibody is saxolizumab, nivolumab or pembrolizumab. In another preferred embodiment, the anti-PD-1 antibody is saxolizumab or nivolumab.

In some embodiments of the methods as described herein, the PD-1 axis binding antagonist comprises a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.

In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a more specific embodiment, the anti-PD-L1 antibody is BMS-936559, KN035, atezolizumab, durvalumab, avelumab or a combination thereof .

In one aspect, the present invention provides a combination comprising a compound of formula (I) and a PD-1 axis binding antagonist for treating cancer in an individual in need thereof, and a pharmaceutically acceptable carrier.

In one aspect, the present invention provides a medicament comprising a compound of formula (I) for use in the treatment of cancer in combination with a PD-1 axis binding antagonist.

In one aspect, the present invention provides a use of a combination comprising a compound of formula (I) and a PD-1 axis binding antagonist for treating cancer in an individual in need thereof.

In one aspect, the present invention provides a kit comprising a compound of formula (I), a PD-1 axis binding antagonist, and a package insert, and the package insert includes instructions for using the drug to treat cancer in an individual. Such kits can be used, for example, in the methods of the invention.

The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included therein. It is to be understood that this invention is not limited to particular synthetic methods of manufacture, which can, of course, vary. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. E1. A method for treating cancer comprising administering an amount of a compound of formula (I) to an individual in need thereof: or a pharmaceutically acceptable salt thereof, wherein: R ^1a and R ^1b are each independently (C ₁ -C ₆ ) alkyl, or R ^1a and R ^1b are formed together with the nitrogen to which they are attached via 0, 1 or 2 A 5-membered heterocycloalkyl group substituted with (C ₁ -C ₆ ) alkyl; R ^2a and R ^2b are each independently hydrogen or (C ₁ -C ₄ ) alkyl; R ^3a and R ^3b are each independently hydrogen or (C ₁ -C ₄ ) alkyl; each R ⁴ is independently (C ₁ -C ₆ ) alkyl substituted by 0 or 1 halogen or hydroxyl; and n is 0, 1 or 2; and an amount of A planned death 1 protein (PD-1) axis binding antagonist, wherein the equal amounts together are effective to treat cancer. E2. The method of embodiment E1, wherein R ^1a and R ^1b are each independently methyl, ethyl or isopropyl. E3. The method of embodiment E1 or E2, wherein R ^1a and R ^1b together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl substituted by 0 or 1 substituent methyl. E4. The method of any one of embodiments E1 to E3, wherein R ^2a and R ^2b are hydrogen. E5. The method of any one of embodiments E1 to E4, wherein R ^3a and R ^3b are each independently hydrogen, methyl or ethyl. E6. The method of any one of embodiments E1 to E5, wherein R ⁴ is methyl or ethyl. E7. The method according to any one of embodiments E1 to E6, wherein n is 1. E8. The method according to any one of embodiments E1 to E7, wherein the compound of formula (I) is: 4-[1-aminopropyl]-2-{6-[5-methyl-6,7-di Hydrogen-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2 ,3-Dihydro-1H-pyrrolo[3,4-c]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro -5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2, 3-Dihydro-1H-pyrrolo[3,4-c]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro- 5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2, 3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro -5 H - pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]- 2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; or 4-[1-aminopropyl]-2-{3-[5-methyl-6,7 -Dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]- 2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; or a pharmaceutically acceptable salt thereof. E9. The method according to any one of embodiments E1 to E8, wherein the compound of formula (I) is: 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )- 5-Methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one; 4-[(1R)-1-aminoethyl Base]-2-{6-[(5S)-5-Ethyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl] Pyridin-2-yl}-6-[(2R)-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyrrolidin-1-one; 4-[(1S)-1-aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2,1-c][1, 2,4]triazol-3-yl]pyridin-2-yl}-6-[(2R)-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3 ,4-c]pyridin-1-one; 4-[(1R)-1-aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrole And[2,1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2,3-di Hydrogen-1H-pyrrolo[3,4-c]pyridin-1-one; or 4-[(1R)-1-aminopropyl]-2-{3-[(5S)-5-methyl-6 ,7-Dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino] -2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one; or a pharmaceutically acceptable salt thereof. E10. The method according to any one of embodiments E1 to E9, wherein the compound of formula (I) is: 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )- 5-Methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-1-one. E11. The method according to any one of embodiments E1 to E10, wherein the PD-1 axis binding antagonist comprises a PD-1 antagonist, a PD-L1 antagonist or a PD-L2 antagonist. E12. The method according to embodiment E11, wherein the PD-1 axis binding antagonist comprises a PD-1 antagonist. E13. The method according to embodiment E11 or E12, wherein the PD-1 binding antagonist is an anti-PD-1 antibody. E14. The method as in embodiment E13, wherein the anti-PD-1 antibody system is selected from the group consisting of: saxolizumab, nivolumab, pembrolizumab, petilizumab, cimipril Antibodies, Tislelizumab, Spartakizumab, Sintilimab, MEDI-0680, BGB-108, AGEN2034 Genozumab, CBT-502, Camrelizumab, and combinations thereof . E15. The method according to embodiment E14, wherein the anti-PD-1 antibody is saxanlimab. E16. The method according to embodiment E14, wherein the anti-PD-1 antibody is nivolumab. E17. The method according to embodiment E14, wherein the anti-PD-1 antibody is pembrolizumab. E18. The method according to embodiment E11 or E12, wherein the PD-1 axis binding antagonist comprises a PD-L1 antagonist. E19. The method according to embodiment E18, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody. E20. The method of any one of embodiments E1 to E19, wherein the individual is human. E21. The method of any one of embodiments E1 to E20, wherein the cancer is selected from the group consisting of brain cancer, head/neck cancer, prostate cancer, bladder cancer, lung cancer, breast cancer, ovarian cancer, bone cancer, colon cancer Cancer of the rectum, kidney, liver, pancreas, esophagus, stomach, gastroesophageal junction, thyroid, cervix, uterus, and kidney. E22. A combination for treating cancer in an individual in need thereof, comprising: (i) a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein: R ^1a and R ^1b are each independently (C ₁ -C ₆ ) alkyl, or R ^1a and R ^1b are formed together with the nitrogen to which they are attached via 0, 1 or 2 A 5-membered heterocycloalkyl group substituted with (C ₁ -C ₆ ) alkyl; R ^2a and R ^2b are each independently hydrogen or (C ₁ -C ₄ ) alkyl; R ^3a and R ^3b are each independently hydrogen or (C ₁ -C ₄ )alkyl; each R ⁴ is independently (C ₁ -C ₆ )alkyl substituted with 0 or 1 halogen or hydroxyl; and n is 0, 1 or 2; and (ii) PD-1 axis binding antagonists. E23. The combination according to embodiment E22, wherein the HKP1 inhibitor is 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7- Dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidine -1-yl]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-1-one, and the PD-1 axis binding antagonist comprises an anti-PD selected from the group consisting of -1 Antibodies: Sasanlizumab, Nivolumab, Pembrolizumab, Pidrolizumab, Simiprizumab, Tislelizumab, Spartakizumab, Sintilizumab Monoclonal antibody, MEDI-0680, BGB-108, or AGEN2034 genolizumab, CBT-502, camrelizumab, and combinations thereof. E24. The combination of embodiment E22 or E23, wherein the PD-1 axis binding antagonist comprises saxolizumab. E25. The combination of embodiment E22 or E23, wherein the PD-1 axis binding antagonist comprises nivolumab. E26. The combination of embodiment E22 or E23, wherein the PD-1 axis binding antagonist comprises pembrolizumab. E27. Use of the combination according to any one of embodiments E22 to E26 for the treatment of cancer in a subject in need thereof.

I. Definitions As used herein, the singular forms "a/an" and "the" include plural referents unless specified otherwise. For example, reference to "a" substituent includes one or more substituents. Where compounds, salts and the like are used in plural, this also means a single compound, salt or the like.

The disclosure described herein may suitably be practiced in the absence of any element not specifically disclosed herein. Thus, for example, herein, any of the terms "comprising", "consisting essentially of" and "consisting of" in each instance One can be replaced by either of the other two terms.

The term "about" when used to modify a parameter defined by a value means that the parameter varies by up to 10% above or below the stated value for that parameter.

The term "alkyl" refers to a saturated monovalent aliphatic hydrocarbon group, including straight and branched chain groups having the indicated number of carbon atoms. The alkyl substituents disclosed herein can be designated as having 1 to 6 carbon atoms (" _Ci - _C6 alkyl"), or 1 to 4 carbon atoms (" _C1 - _C4 alkyl"), and the like. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl , n-octyl and similar groups. Alkyl groups can be substituted or unsubstituted. In particular, unless otherwise specified, an alkyl group may be substituted with one or more (up to the total number of hydrogen atoms present on the alkyl moiety) halo group. Thus, C ₁ -C ₄ alkyl includes halogenated alkyl groups having 1 to 4 carbon atoms, and especially fluorinated alkyl groups such as trifluoromethyl or difluoroethyl (ie CF ₃ and -CH ₂ CHF ₂ ).

In other embodiments, the alkyl group is optionally substituted with one or more substituents (eg _, with 1 to 3 substituents) independently selected from the group consisting of: halo, -OH, C -C ₄ alkoxy, -CN and -NR ^x R ^y .

The term "alkoxy" refers to a monovalent -O-alkyl group wherein the alkyl portion has the indicated number of carbon atoms. Alkoxy groups typically contain 1 to 8 carbon atoms (“C ₁ -C ₈ alkoxy” or 1 to 6 carbon atoms (“C ₁ -C ₆ alkoxy”) or 1 to 4 carbon atoms (“C 1 -C 6 alkoxy”) ₁ -C ₄ alkoxy"). For example, C ₁ -C ₄ alkoxy includes methoxy, ethoxy, isopropoxy, tertiary butoxy (ie, -OCH ₃ , - OCH ₂ CH ₃ , -OCH(CH ₃ ) ₂ , -OC(CH ₃ ) ₃ ) and the like.

The term "heterocycloalkyl" refers to a non-aromatic saturated or partially unsaturated ring system containing the indicated number of ring atoms including at least one heteroatom selected from N, O and S as a ring member. 5-membered heterocycloalkyl groups may include tetrahydrofuran (tetrahydrofuranyl), tetrahydrothiophene (tetrahydrothienyl), and pyrrolidinyl (pyrrolidinyl).

As used herein, terms, including (but not limited to) "drug", "medicine", "component", "composition", "compound", "substance", "targeting agent", "targeted therapeutic agent" , "therapeutic agent" and "drug" are used interchangeably to refer to small molecule compounds of the present invention, such as HPK1 inhibitors, anti-PD-L1 antibodies, anti-PD-1 antibodies or combinations thereof.

As used herein, the terms "therapeutic antibody" and "antibody" are used interchangeably to refer to an antibody useful in the treatment of a disease or disorder. Therapeutic antibodies can have various mechanisms of action. Therapeutic antibodies bind and neutralize the normal function of the target associated with the antigen. For example, monoclonal antibodies that block the activity of proteins required for cancer cell survival cause cell death. Another therapeutic antibody can bind to and activate the normal function of the target associated with the antigen. For example, monoclonal antibodies can bind to proteins on cells and trigger apoptotic signals. Another monoclonal antibody can bind to a target antigen expressed only on diseased tissue; conjugation of a toxic payload (effective agent), such as a chemotherapeutic or radiopharmaceutical, to a monoclonal antibody can generate Drugs that can be delivered to diseased tissues to reduce damage to healthy tissues. A "biofunctional fragment" of a therapeutic antibody will exhibit at least one, if not some or all, of the biological functions attributed to an intact antibody, including at least specific binding to a target antigen.

Therapeutic antibodies can bind to any protein, including but not limited to PD-L1, PD-1. Thus, therapeutic antibodies include, but are not limited to, anti-PD-L1 antibodies, anti-PD-1 antibodies, or combinations thereof.

A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide ( ^CYTOXAN® ); alkylsulfonates such as busulfan, improsulfan ) and piposulfan; aziridines such as benzodopa, carboquone, meturdopa and uredopa; ethyleneimine and Methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine ; acetogenin (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL ^® ); beta- beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN ^® ), CPT-11 (irinotecan (irinotecan, CAMPTOSAR ^® ), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed; marine callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllotoxin acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic similar KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral α-4 integrin inhibitor; sarcodictyn; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide ), mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine (lomustine), nimustine (nimustine) and ramustine (ranimnustine); antibiotics, such as enediyne antibiotics (such as calicheamicin (calicheamicin), especially calicheamicin gamma and calicheamicin omega ( eg Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; esperamicin; And new tumor suppressor protein chromophore and related chromoprotein enediyne antibiotic chromophore)), aclacinomysins, actinomycin, authramycin, azoserine (azaserine), bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, actin Dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, N- 𠰌linyl-doxorubicin, cyano N-𠰌linyl-doxorubicin, 2-pyrrolinyl-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®) and deoxygenated cranberry), table Epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (such as mitomycin C), mycophenolate acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodomycin Rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, lerubicin (zorubicin); antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone (epothilone) and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as Fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azurine 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluorouracil Glycosides (floxuridine) and imatinib (imatinib, 2-phenylaminopyrimidine derivatives), and other c-it inhibitors; anti-adrenal agents, such as aminoglutethimide, mitotane, Trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine ); bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; Avery Elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, Such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostat Pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; ^PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; (triaziquone); 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A), bacillin A ( roridin A) and anguidine); urethan; vindesine (ELDIS1NE ^® , FILDESIN ^® ); dacarbazine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");thiotepa; Paclitaxel, such as paclitaxel (TAXOL ^® ), albumin-engineered paclitaxel nanoparticle formulation (ABRAXANE™), and doxetaxel (TAXOTERE ^® ); chlorambucil; 6-thioguanine (6-thioguanine); mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine ( ^VELBAN® ); platinum; etoposide ( etoposide) (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN ^® ); oxaliplatin; leucovovin; vinorelbine ( vinorelbine (NAVELBINE ^® ); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; Isomerase (topoisomerase) inhibitor RFS 2000; Difluoromethylornithine (difluoromethylornithine, DFMO); Retinoids, such as retinoic acid; Pharmaceutically acceptable salts and acids of any of the above or derivatives; and combinations of two or more of the above, such as CHOP (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone) and FOLFOX (oxa Abbreviation for combination regimen of ELOXATIN™ with 5-FU and leucovovin).

Other examples of chemotherapeutic agents include antihormonal agents, which are used to modulate, reduce, block or inhibit the effects of hormones that can promote cancer growth, and are usually in the form of systemic or systemic treatments. It can itself be a hormone. Examples include antiestrogens and selective estrogen receptor modulators (SERMs) including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene (EVISTA ®), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 1 1 7018, onapristone and torrex Toremifene (FARESTON®); antiprogestins; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX®); Agents used to suppress or shut down the ovaries, e.g., leutinizing hormone-releasing hormone (LHRF1) agonists such as leuprolide acetate (LUPRON® and ELIGARD®), Gosher goserelin acetate, buserelin acetate, and tripterelin; antiandrogens such as fiutamide, nilutamide, and bicalut Amines (bicalutamide); and aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, amine glutethimide, megestrol acetate (MEGASE®) , exemestane (AROMASIN®), formestanie, fadrozole, vorozole (RJVISOR®), letrozole (FEMARA®), and anatrazole anastrozole (ARIMIDEX®). Additionally, this definition of chemotherapeutic agent includes: bisphosphonates such as clodronate (eg BONEFOS ^® or OSTAC ^® ), etidronate (DIDROCAL ^® ), NE-58095 , zoledronic acid/zoledronate (ZOMETA ^® ), alendronate (FOSAMAX ^® ), pamidronate (AREDIA ^® ), alternative tiludronate (SKELID ^® ) or risedronate (ACTONEL ^® ); and troxacitabine (1,3-dioxolane nucleoside cytosine analog ); antisense oligonucleotides, especially antisense oligonucleotides that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF -R); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ^ALLOVECTIN® vaccine, ^LEUVECTIN® vaccine and ^VAXID® vaccine; topoisomerase 1 inhibitors (eg ^LURTOTECAN® ); antiestrogens such as fulvestrant group; Kit inhibitors, such as imatinib or EXEL-0862 (tyrosine kinase inhibitor); EGFR inhibitors, such as erlotinib or cetuximab; anti-VEGF inhibitors, Such as bevacizumab; arinotecan; rmRH (eg ABARELIX ^® ); lapatinib and lapatinib ditosylate (ErbB-2 and EGFR double tyrosine Kinase small molecule inhibitor, also known as GW572016); 17AAG (geldanamycin (geldanamycin) derivative, which is a heat shock protein (Hsp) 90 poison) and pharmaceutically acceptable doses of any of the above salts, acids or derivatives.

The term "immunotherapy" refers to the treatment of an individual by methods involving inducing, enhancing, suppressing or otherwise modulating an immune response.

The terms "abnormal cell growth" and "hyperproliferative disorder" are used interchangeably in this application. As used herein, unless otherwise indicated, "abnormal cell growth" refers to cell growth independent of normal regulatory mechanisms (eg, loss of contact inhibition). Abnormal cell growth can be benign (noncancerous) or malignant (cancerous).

A "disorder" is any condition that would benefit from treatment by the compounds of the invention. Such conditions include chronic and acute conditions or diseases, including those pathological conditions that predispose an individual to the conditions in question.

As used herein, the term "antibody" refers to an antibody capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Immunoglobulin molecule. As used herein, the term encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, bispecific antibodies, bispecific antibodies, bifunctional antibodies, trispecific antibodies, multispecific antibodies, bispecific heterodimeric bifunctional Antibodies, bispecific heterodimeric IgG, labeled antibodies, humanized antibodies, human antibodies, and fragments thereof (such as Fab, Fab', F(ab') ₂ , Fv), single chain (scFv) and domain antibodies (including eg shark and camel antibodies), fusion proteins comprising antibodies, any other modified configuration of immunoglobulin molecules comprising antigen recognition sites, and antibody-like binding peptide mimetics (ABiPs). Antibodies include antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and the antibody need not be of any particular class. Immunoglobulins are assigned to different classes by the antibody amino acid sequence of the constant region of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these immunoglobulins can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG- 3. IgG-4, IgA1 and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two consensus light (L) chains and two consensus heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units plus an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2 to 5 basic 4-chain units that can polymerize to combine with The J chains combine to form multivalent aggregates. In the case of IgG, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) (for each of the α and γ chains) and four CH domains (for the μ and ε isotypes ). Each L chain has a variable domain (VL) at its N-terminus followed by a constant domain at its other end. VL is aligned with VH and CL is aligned with the first constant domain (CHI) of the heavy chain. Certain amino acid residues are believed to form the interface between the light chain variable domain and the heavy chain variable domain. The VH and VL pair together form a single antigen binding site. For the structure and properties of different classes of antibodies see, eg, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Basic and Clinical Immunology, 8th Edition, 1994, p. 71 and Chapter 6. L chains from any vertebrate species can be assigned to one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the heavy-chain (CH) constant domains of an immunoglobulin, they can be assigned to different classes, or isotypes.

As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to a population of antibodies that is substantially homogeneous, that is, the amine groups of the antibody molecules that make up the population are free except for possible naturally occurring mutations that may be present in minor amounts. The acid sequence is identical. In contrast, conventional (polyclonal) antibody preparations usually include a number of different antibodies with different amino acid sequences in the variable domains, especially in their CDRs, usually specific for different epitopes. The modifier "monoclonal" indicates characteristics of an antibody, such as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used according to the present invention can be prepared by the fusionoma method first described by Kohler et al., Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 1975, 256: 495; or by Made by recombinant DNA methods (eg, US Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described in, for example, Clackson et al., Making antibody fragments using phage display libraries, Nature 1991, 352: 624-628 and Marks et al., By-passing Immunization: human antibodies from V-gene libraries displayed on phage, J. Mol. Biol . 1991, 222: 581-597. See also Presta, Selection, design, and engineering of therapeutic antibodies, J. Allergy Clin. Immunol. 2005 , 116:731.

"Chimeric antibody" refers to an antibody in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species (e.g., human) or belonging to a specific antibody class or subclass, and the The remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity That's it.

"Human antibody" refers to an antibody comprising only human immunoglobulin sequences. The human antibody may contain murine carbohydrate chains if the human antibody is produced in a mouse, in a mouse cell, or in a fusion tumor derived from a mouse cell. Similarly, a "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.

"Humanized antibody" refers to a form of antibody that contains sequences from non-human (eg, murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one, and usually two variable domains, in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FRs The regions are FR regions of human immunoglobulin sequences. A humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. When it is necessary to distinguish a humanized antibody from the parental rodent antibody, the prefix "hum", "hu" or "h" is added to the antibody clonal designation. Humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parent rodent antibody, but may include certain amino acid substitutions to improve affinity, increase stability of the humanized antibody, or for other reasons.

As used herein, the phrases "substantially reduce", "substantially different" or "substantially inhibit" mean the difference between two values (generally, one related to a molecule and the other to a reference/comparator molecule) The degree is high enough that a person skilled in the art would consider the difference between two values to be statistically significant in the context of the biological characteristic being measured by those values (eg, Kd values). The difference between the two values relative to the value of the reference/comparator molecule is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50%.

As used herein, the term "specifically binds" or "specific for" refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, in the presence of heterogeneous molecules (including biomolecules) The presence of the population determines the presence of the target. For example, an antibody that specifically binds to a target (which may be an epitope) binds this target with greater affinity, avidity, ease and/or duration than it binds to other Antibodies against the target. In one embodiment, the degree of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured by, for example, radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In certain embodiments, antibodies specifically bind to epitopes on proteins that are conserved among proteins of different species. In another example, specific binding can include, but need not necessarily be exclusive binding.

As used herein, the term "immunoadhesin" refers to an antibody-like molecule that has a combination of the binding specificity of a heterologous protein ("adhesin") and the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises the fusion of an amino acid sequence with the desired binding specificity (different (i.e., "heterologous") from the antigen recognition and binding site of the antibody) to an immunoglobulin constant domain sequence things. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site for a receptor or ligand. Immunoglobulin constant domain sequences in immunoadhesins can be obtained from any immunoglobulin, such as IgG-1, IgG-2 (including IgG2A and IgG2B), IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. Ig fusions preferably comprise replacing at least one variable region within an Ig molecule with a domain of a polypeptide or antibody as described herein. In an especially preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3 or hinge, CH1, CH2 and CH3 regions of an IgG-1 molecule. See also US Patent No. 5,428,130, issued June 27, 1995, for producing immunoglobulin fusions. Immunoadhesin combinations of Ig Fc and the ECD of cell surface receptors are sometimes referred to as soluble receptors.

"Fusion protein" and "fusion polypeptide" refer to a polypeptide having two parts covalently linked together, wherein each part is a polypeptide with different properties. A property may be a biological property, such as in vitro or in vivo activity. A property can also be a single chemical or physical property, such as binding to a target molecule, catalysis of a reaction, and the like. The two moieties can be linked directly by a single peptide bond or via a peptide linker, but be in reading frame with each other.

As described herein, a "PD-1 oligopeptide", "PD-L1 oligopeptide" or "PD-L2 oligopeptide" is a protein that binds (preferably specifically binds) to a PD-1, PD-L1 or PD-L2 negative receptor. An oligopeptide of a co-stimulatory polypeptide, comprising a receptor, ligand or signaling component, respectively. Such oligopeptides can be chemically synthesized using known oligopeptide synthesis methods or can be prepared and purified using recombinant techniques. Such oligopeptides typically have a length of at least about 5 amino acids, or at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99 or 100 amino acids in length or longer. Such oligopeptides can be identified using well known techniques. In this regard, it should be noted that techniques for screening oligopeptide libraries for oligopeptides capable of specifically binding to polypeptide targets are well known in the art (e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871 , Nos. 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 1984/0003506 and WO 1984/0003564; Geysen et al., Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid, Proc. Natl. Acad. Sci . 1984, 81:3998-4002; Geysen et al ., Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised aga inst the native protein, Proc. Natl. Acad. Sci. 1985, 82:178-182; Geysen et al., A priori delineation of a peptide which mimics a discontinuous antigenic determinant, Synthetic Peptides as Antigens , 1986, 130-149; Geysen, et al. People, Strategies for epitope analysis using peptide synthesis, J. Immunol. Meth. 1987, 102, 259-274; Schoofs et al., Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution, J. Immunol , 1988, 140 :611-616, Cwirla, SE et al., Peptides on phage: a vast library of peptides for identifying ligands., Proc. Natl. Acad. Sci. 1990, 87:6378; Lowman, HB et al., Selecting high-affinity binding proteins by monovalent phage display, Biochemistry , 1991, 30:10832; Clackson, T. et al., Making antibody fragments using phage display libraries, Nature , 1991, 352: 624; Marks, JD et al., By-passing immunization: human antib odies from V-gene libraries displayed on phage, J. Mol. Biol , 1991, 222:581; Kang, et al., Linkage of Recognition and Replication Functions by Assembling Combinatorial Antibody Fab Libraries Along Phage Surfaces, PNAS, 1991, Vol. 88, pp. 4363-4366, and Smith, GP Surface presentation of protein epitopes using bacteriophage expression systems , Curr. Opin. Biotechnol. 1991, 2:668.

An "antagonist" antibody or a "blocking" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The anti-PD-L1 antibodies of the present invention block signaling through PD-1 in order to restore T cells from a dysfunctional state to a functional response to antigenic stimulation (eg, proliferation, cytokine production, target cell killing).

An "agonist" or "activating antibody" is an antibody that enhances or initiates signaling by binding to an antigen. In some embodiments, agonist antibodies cause or activate signaling in the absence of native ligands.

The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune response to antigenic stimulation. The term includes a common component of depletion and/or anergy, where antigen recognition can occur but the subsequent immune response is ineffective in controlling infection or tumor growth.

As used herein, the term "dysfunctional" also includes difficulty or unresponsiveness to antigen recognition, in particular, the translation of antigen recognition into downstream T cell effector functions such as proliferation, cytokine production, and/or target cell kill) is weakened.

The term "anergy" refers to a state of unresponsiveness to antigenic stimulation caused by incomplete or insufficient signaling through T cell receptors (eg, elevated intracellular Ca+2 in the absence of ras activation). T cell anergy can also develop following antigen stimulation in the absence of co-stimulation, resulting in cells that become refractory to subsequent activation by antigen even in the presence of co-stimulation. The presence of interleukin-2 can often override anergy. Anergic T cells do not undergo clonal expansion and/or acquire effector functions.

The term "exhausted" refers to exhaustion of T cells in a state of T cell dysfunction due to persistent TCR signaling that occurs during various chronic infections and cancers. It is distinguished from anergy in that it is not caused by incomplete or insufficient signaling, but by persistent signaling. It is defined as adverse effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from functional effector or memory T cells. Depletion prevents optimal control of infection and tumors. Depletion can be caused by both extrinsic negative regulatory pathways, such as immunomodulatory cytokines, as well as cell intrinsic negative regulatory (co-stimulatory) pathways.

"Enhancing T cell function" means inducing, causing or stimulating T cells to have sustained or expanded biological functions, or renewing or reactivating exhausted or dysfunctional T cells. Examples of enhanced T cell function include: enhanced CD4+ or CD8+ T cell secretion of gamma interferon, enhanced proliferation, increased survival, enhanced differentiation, enhanced antigenic response (e.g. viral, pathogen or tumor clearance) (relative to such level). In some embodiments, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Ways to measure this enhancement are known to those of ordinary skill in the art.

As used herein, "metastasis" or "metastatic" means the spread of cancer from its primary site to other locations in the body. Cancer cells can break away from the primary tumor, infiltrate into lymph and blood vessels, circulate through the bloodstream, and grow in distant lesions (metastasize) among normal tissues elsewhere in the body. Metastasis can be local or distant. Depending on the tumor cells breaking away from the primary tumor, metastasis is a continuous process that travels through the bloodstream and stops at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecules within tumor cells regulate this behavior, and in distant sites, significant interactions between tumor cells and host cells also occur.

The terms "cancer", "cancerous" or "malignant" refer to or describe the physiological condition in mammals, often characterized by unregulated cell growth. The term "cancer" includes, but is not limited to, primary cancer originating in a specific part of the body, metastatic cancer that has spread from its original location to other parts of the body, recurrence of the original primary cancer after remission, and first cancer that is a new primary cancer in an individual. Two primary cancers, where the previous cancer in the medical history is different from the latter cancer type. Examples of cancers include (but are not limited to): brain cancer, head/neck cancer (including squamous cell carcinoma of the head and neck (SCCHN)), prostate cancer, bladder cancer (including urothelial carcinoma) , also known as transitional cell carcinoma (TCC)), lung cancer (including squamous cell carcinoma, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) ), breast cancer, ovarian cancer, bone cancer, colorectal cancer, kidney cancer, liver cancer (including hepatocellular carcinoma (HCC)), pancreatic cancer, esophageal cancer (including squamous cell carcinoma (SCC) ), cancer of the stomach, gastroesophageal junction, thyroid, cervix, uterus, and/or kidney. In some embodiments, the cancer is melanoma.

The term "subject" or "subject in need" refers to a human or non-human subject including veterinary subjects, such as cattle, sheep, cats, dogs, horses, primates, rabbits, and rodents (e.g., mice) in need of treatment. and rats). In one embodiment, the individual is a human and may be referred to as a patient or individual, eg, a human suffering from, at risk of, or potentially capable of developing cancer. Those skilled in the medical arts can readily identify individual patients suffering from cancer.

As used herein, the term "treat/treating" cancer means administering the combination therapy of the invention to an individual suffering from or diagnosed with cancer to achieve at least one positive therapeutic effect, such as reducing the number of cancer cells ; reduce tumor size; reduce the rate of cancer cell infiltration into peripheral tissues or reduce the rate of tumor metastasis or tumor growth; exacerbation or severity of a condition or disease, but not necessarily the complete elimination of all disease-related signs, symptoms, conditions or conditions. Within the meaning of the present invention, the term "treat/treating" also means to arrest, delay the onset (i.e. the time period preceding the clinical manifestation of the disease or the symptoms of the disease) and/or reduce the risk of the development or worsening of the symptoms of the disease .

As used herein, "in combination with," "administration in combination," or "co-administration" refers to the administration of one agent plus at least one other agent, where the administration of one agent can be administered to an individual Before, during or after at least one other agent. In some embodiments, co-administration of two or more agents may be useful for treating individuals with cancers that have primary or acquired resistance to ongoing therapy. Combination therapies provided herein may be useful for improving the efficacy and/or reducing the side effects of cancer therapy in individuals responding to such therapy.

As used herein, the terms "simultaneously", "simultaneous administration/administered simultaneously", "concurrently" or "concurrent administration" mean that the agents are administered at the same point in time in separate dosage forms or as part of the same dosage form , or immediately after each other, but the agents may be administered in any order. For example, in the latter case, two or more agents are administered at sufficiently close times that the observed results are indistinguishable from those achieved when the agents were administered at the same time point.

The agents of the invention may be administered entirely separately or in one or more separate compositions. For example, during the course of treatment, the agents may be administered separately at different times (in a chronologically staggered manner, especially in a sequence-specific manner) at intervals during which the combination therapy is effective in treating the cancer.

As used herein, the term "sequential/sequentially" or "administered sequentially/sequential administration" refers to the administration of each of the agents of the combination therapy of the invention either alone or in the form of one following the other, The agents can be administered in any order. When the therapeutic agents in combination therapy are in different dosage forms (e.g., one agent is a lozenge and the other is a sterile liquid), and/or the agents are administered according to different dosing schedules (e.g., one agent is administered daily, and Sequential administration may be especially useful when the second agent is administered less frequently, such as weekly.

As used herein, the term "single formulation" refers to a single carrier or vehicle formulated to deliver effective amounts of two therapeutic agents to a subject. A single vehicle is designed to deliver an effective amount of each agent together with a pharmaceutically acceptable carrier or excipient. In some embodiments, the vehicle is a tablet, capsule, pill or patch. In other embodiments, the vehicle is a solution or suspension.

The term "unit dose" is used herein to mean that two agents are administered together in one dosage form to the subject being treated simultaneously. In some embodiments, a unit dose is a single formulation. In certain embodiments, a unit dose includes one or more vehicles, such that each vehicle includes an effective amount of at least one pharmaceutical agent and a pharmaceutically acceptable carrier and excipient. In some embodiments, the unit dosage is one or more lozenges, capsules, pills, or patches administered to a subject at the same time.

"Oral dosage form" includes unit dosage forms ordered or intended for oral administration.

As used herein, the term "advanced" as it refers to breast cancer includes locally advanced (non-metastatic) disease as well as metastatic disease.

The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredients to be effective and that is free of other components that are unacceptably toxic to the subject to whom the formulation is administered. Such formulations are sterile. "Pharmaceutically acceptable" carriers or excipients (vehicles, additives) are those which can reasonably be administered to a subject to provide an effective dosage of the active ingredient employed.

The combinations provided herein can be formulated by a variety of methods apparent to those skilled in the art of pharmaceutical formulation. The various release profiles described above can be achieved in a number of different ways. Suitable formulations include, for example, tablets, capsules, compression-coated formulations and other formulations that are easy to administer.

"Instructions" means the instructions normally included in commercial packages of pharmaceutical products, which contain warnings regarding the indications, usage, dosage, administration, contraindications, other pharmaceutical products to be combined with the packaged product, and/or warnings concerning the use of such pharmaceutical products and other information.

"Effective dose", "effective amount", "therapeutically effective amount" of a drug, medicament, component, composition, compound, substance, targeting agent, targeted therapeutic agent, therapeutic antibody, therapeutic agent, drug or pharmaceutical composition Or a "therapeutically effective dose" is an amount that affects any one or more beneficial or desirable (including biochemical, histological and/or behavioral) symptoms of the disease, its complications and intermediate pathological phenotypes presented during disease development.

For therapeutic purposes, a therapeutically effective amount refers to the administered amount of a drug, medicament, component, composition, compound, substance, targeting agent, targeted therapeutic agent, therapeutic antibody, therapeutic agent, drug or pharmaceutical composition, This amount will alleviate to some extent one or more symptoms of the condition being treated, such as reducing one or more symptoms of the disease, improving the quality of life of the affected person, reducing the dose of other drugs needed to treat the disease, enhancing another drug The effect of treatment (such as by targeting, delaying disease progression and/or prolonging survival).

With respect to the treatment of cancer, a therapeutically effective amount refers to the amount of a drug, medicament, component, composition, compound, substance, targeting agent, targeted therapeutic agent, therapeutic antibody, therapeutic agent, drug or pharmaceutical composition, which amount is in Administration of one or more therapies is effective to achieve one or more of the following results: (1) reduction in tumor size, (2) reduction in number of cancer cells, (3) inhibition (i.e., slowing to some extent, preferably stop) cancer cell infiltration into peripheral organs, (4) inhibit (ie, slow to some extent, preferably stop) tumor metastasis, (5) inhibit (ie, slow to some extent, preferably stop ) tumor growth or tumor invasion, (6) alleviation (ie, to some extent, preferably removal) of one or more signs or symptoms associated with cancer, (7) reduction in doses of other drugs needed to treat the disease, ( 8) Enhance the effect of another drug treatment, (9) delay disease progression, (10) improve or increase disease-free, recurrence-free, deterioration-free and/or overall survival, duration or rate, (11) increase response rate, Durability of response or number of patients who respond or are in remission, (12) reduce hospitalization rate, (13) reduce length of hospital stay, (14) maintain tumor size without increase or increase less than 10%, preferably less than 5% , preferably less than 4%, preferably less than 2%; (12) increase the number of patients in remission, (15) increase the length or duration of remission, (16) reduce the recurrence rate of cancer; (15) reduce cancer Number of recurrences and (17) improvement in cancer-related symptoms and/or quality of life.

An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount is an amount sufficient to accomplish prophylactic or therapeutic treatment, either directly or indirectly. An effective amount of a drug, compound or pharmaceutical composition may or may not be combined with another drug, agent, component, composition, compound, substance, targeting agent, targeted therapeutic agent, therapeutic antibody, as understood in the clinical context. A therapeutic agent, drug or pharmaceutical composition is achieved.

An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment, either directly or indirectly.

An effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition, as understood in a clinical setting. Thus, an "effective amount" may be considered in the context of the administration of one or more therapeutic agents, and an effective amount of a single agent may be considered administered if, in combination with one or more other agents, the desired result is or has been achieved.

A therapeutic amount can also refer to a dose of a drug product that has been approved for use by a regulatory agency. As used herein, "subtherapeutic dose" refers to a dose of a drug product that is significantly lower than the approved dose.

The terms "treatment regimen", "dosing regimen" and "administration regimen" are used interchangeably to refer to the dosage and timing of administration of each therapeutic agent in a combination of the invention.

The term "ameliorating" or "amelioration" in reference to a disorder, disease or condition refers to any observable beneficial therapeutic effect. Treatment need not be absolutely beneficial to the individual. For example, alleviating means reducing or ameliorating one or more symptoms of a disease, disorder or condition as compared to not administering the therapeutic agent in a method or therapy of the invention. Amelioration also includes shortening or reducing the duration of symptoms.

The term "biosimilar" refers to a biological product that is highly similar to an FDA-approved biological product (reference product) and does not have clinically meaningful differences from the reference product in terms of pharmacokinetics, safety and efficacy.

The term "bioequivalent" refers to a biological product that is pharmaceutically equivalent and has a similar bioavailability to an FDA-approved biological product (reference product). For example, according to the FDA, the term bioequivalence is defined as "the amount of active ingredient or active fraction in a pharmaceutical equivalent or pharmaceutical substitute when administered at the same molar dose under similar conditions in an appropriately designed study." There are no significant differences in the rate and extent of becoming available at the drug site of action" (US Food and Drug Administration, "Guidance for Industry: Bioavailability and Bioequalence Studies for Orally Administered Drug Products - General Considerations", 2003, Drug Evaluation and Research center).

"Tumor" when applied to an individual diagnosed with or suspected of having cancer means a malignant or potentially malignant neoplasm or mass of tissue of any size and includes primary tumors and secondary neoplasms. Solid tumors are abnormal growths or masses of tissue that usually do not contain cysts or areas of fluid. Examples of solid tumors are sarcomas, carcinomas and lymphomas.

"Tumor burden" also known as "tumor load" refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of the tumor throughout the body (including lymph nodes and bone marrow). Tumor burden can be determined by various methods known in the art, such as using calipers, or while in the body using imaging techniques such as ultrasound, bone scans, computed tomography (CT) or magnetic resonance imaging (MRI) scanning.

The term "additive" is used to mean that the result of combining two or more agents is not greater than the sum of the individual results of each agent. In one embodiment, the combination of agents described herein exhibits a synergistic effect. The term "synergy" or "synergy" is used to mean that the effect of the combination of two or more agents is greater than the sum of the individual effects of the agents. This improvement in the treated disease, condition or disorder is a "synergistic" effect. A "synergistic amount" is an amount of two or more agents combined that produces a synergistic effect as "synergistic" is defined herein. "Synergistic combination" refers to a combination of agents that produces a synergistic effect measured according to the methods described herein, in vivo or alternatively in vitro.

To determine the synergistic interaction between two or more agents, different dose ranges and/or dose ratios of the agents can be administered to the individual in need of treatment to clearly measure the optimal range and absolute effect of each agent to achieve the effect Dosage range. However, as described herein, observation of synergy in in vitro or in vivo models can predict the effect in humans and other species as well as exist in in vitro or in vivo models to measure synergy. The results of such studies can also be used to predict desired effective doses and plasma concentration ratio ranges and absolute doses and plasma concentrations in humans and other species, such as using pharmacokinetic and/or pharmacodynamic methods.

As used herein, "non-standard clinical administration regimen" refers to a regimen of administering a substance, agent, compound, or composition that differs from the amount, dose, or schedule. "Non-standard clinical dosing regimen" includes "non-standard clinical dosage" or "non-standard dosing schedule".

As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Some embodiments also pertain to pharmaceutically acceptable acid addition salts of the compounds described herein. Suitable acid addition salts are formed from acids which form non-toxic salts. Non-limiting examples of suitable acid addition salts (i.e., salts containing a pharmacologically acceptable anion) include, but are not limited to, acetate, acid citrate, adipate, aspartate, Benzoate, Benzenesulfonate, Bicarbonate/Carbonate, Bisulfate/Sulfate, Bitartrate, Borate, Camphorsulfonate, Citrate, Cyclamate, Ethylenedi Sulfonate, ethanesulfonate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, seabenzoic acid Salt (hibenzate), hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate salt, methanesulfonate, methylsulfate, naphthalene dicarboxylate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate salt, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, glucarate, stearate, succinate, tannate, tartrate, p-toluene Sulfonate, trifluoroacetate and xinofoate.

Other embodiments pertain to base addition salts of the compounds described herein. Suitable base addition salts are formed from bases which form non-toxic salts. Non-limiting examples of suitable base salts include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium , sodium, tromethamine and zinc salts.

The compounds described herein, which are basic in nature, are capable of forming a variety of salts with various inorganic and organic acids. Acids useful in the preparation of pharmaceutically acceptable acid addition salts of such basic compounds described herein are those acids which form non-toxic acid addition salts containing, for example, pharmacologically Salts of the above acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogensulfate, phosphate, acid phosphate, isonicotinate, acetate, Lactate, Salicylate, Citrate, Acid Citrate, Tartrate, Pantothenate, Bitartrate, Ascorbate, Succinate, Maleate, Gentisate, Trans Butenoate, Gluconate, Glucuronate, Glucarate, Formate, Benzoate, Glutamate, Methanesulfonate, Ethanesulfonate, Benzenesulfonic Acid salt, tosylate and pamoate [ie 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)]. Compounds described herein that include a basic moiety, such as an amine group, can form pharmaceutically acceptable salts with various amino acids other than those described above.

Chemical bases useful as reagents in the preparation of the pharmaceutically acceptable base salts having acidic properties of the compounds described herein are those bases which form non-toxic base salts with the compounds. Such non-toxic base salts include, but are not limited to, those derived from pharmacologically acceptable such cations (such as alkali metal cations such as potassium and sodium) and alkaline earth metal cations such as calcium and magnesium), ammonium or water-soluble Amine addition salts such as N-methylglucamine (meglumine) and lower alkanolammoniums, and other base salts of pharmaceutically acceptable organic amines. Half-salts of acids and bases may also be formed, such as the hemisulfate and hemicalcium salts.

For a review of suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds described herein are known to those skilled in the art.

As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is nontoxic to cells or subjects exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are usually aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin protein, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, Disaccharides and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants agents such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™.

The compounds described herein can also exist in unsolvated as well as solvated forms. Accordingly, some embodiments pertain to hydrates and solvates of the compounds described herein. As used herein, the term "solvate" describes a molecular complex comprising a compound described herein and one or more pharmaceutically acceptable solvent molecules such as water (hydrate) and ethanol.

Compounds described herein that contain one or more asymmetric carbon atoms may exist in two or more stereoisomeric forms. When the compounds described herein contain alkenyl or alkenylene groups, cis/trans (or Z/E) geometric isomers may exist. Tautomerization ("interconversion") can occur when structural isomers are interconvertible via low energy barriers. Tautomerism may be in the form of proton tautomerism in compounds described herein containing, for example, imino, keto or oxime groups, or in so-called valence tautomerism in compounds containing aromatic moieties. A single compound may exhibit more than one isomeric type.

The compounds of the embodiments described herein include all stereoisomers (e.g., cis and trans isomers) and all optical isomers (e.g., R and S enantiomers) of the compounds described herein and such Racemic, diastereomeric and other mixtures of isomers. While all stereoisomers are contemplated within the scope of our claims, those skilled in the art will recognize that certain stereoisomers may be preferred.

In some embodiments, the compounds described herein can exist in several tautomeric forms, including enol and imine forms, and keto and enamine forms, as well as geometric isomers and mixtures thereof. All such tautomeric forms are included within the scope of the present embodiments. Tautomers exist in solution as a mixture of sets of tautomers. In solid form, usually one tautomer predominates. Although one tautomer may be described, the embodiments of the invention include all tautomers of the compounds of the invention.

Included within the scope of the embodiments of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds described herein, including compounds exhibiting more than one isomeric type, and mixtures of one or more thereof. Also included are acid addition or base salts in which the counterion is photoactive, such as d-lactate or 1-lysine, or racemic, such as dl-tartrate or dl-arginine.

Cis/trans isomers can be separated by conventional techniques well known to those skilled in the art, such as chromatography and fractional crystallization.

Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors or separation of racemates (or salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). racemate) for analysis.

Alternatively, the racemate (or racemic precursor) can be combined with a suitable photoactive compound such as an alcohol, or where the compounds described herein contain an acidic or basic moiety, a base or an acid such as 1-phenylethyl amine or tartaric acid) reaction. The resulting mixture of diastereomers can be separated by chromatography and/or fractional crystallization, and one or both of the diastereomers can be converted to the corresponding pure ones by methods well known to those skilled in the art. mirror image isomers.

Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

II. HPK1 Inhibitors Embodiments of the present invention include compounds of formula I: or a pharmaceutically acceptable salt thereof, wherein: R ^1a and R ^1b are each independently (C ₁ -C ₆ ) alkyl, or R ^1a and R ^1b are formed together with the nitrogen to which they are attached via 0, 1 or 2 A 5-membered heterocycloalkyl group substituted with (C ₁ -C ₆ ) alkyl; R ^2a and R ^2b are each independently hydrogen or (C ₁ -C ₄ ) alkyl; R ^3a and R ^3b are each independently hydrogen or (C ₁ -C ₄ )alkyl; each R ⁴ is independently (C ₁ -C ₆ )alkyl substituted with 0 or 1 halogen or hydroxy; and n is 0, 1 or 2.

In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof, wherein R ^1a and R ^1b are each independently methyl, ethyl, isopropyl, and wherein R ^2a , R ^2b , R ^3a , R ^3b , R ⁴ and n are as defined in any of the embodiments described herein. In some embodiments, one of R ^1a and R ^1b is methyl. In some embodiments, one of R ^1a and R ^1b is ethyl. In some embodiments, one of R ^1a and R ^1b is isopropyl. In some embodiments, R ^1a and R ^1b are both methyl. In some embodiments, one of R ^1a and R ^1b is methyl, and the other of R ^1a and R ^1b is ethyl. In some embodiments, one of R ^1a and R ^1b is methyl, and the other of R ^1a and R ^1b is isopropyl.

In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof, wherein R ^1a and R ^1b are linked together with the nitrogen to which they are attached form 0, 1 or 2 (C ₁ -C ₆ ) alkane and wherein R ^2a , R ^2b , R ^3a , R ^3b , R ⁴ and n are as defined in any of the embodiments described herein. In some embodiments, R ^1a and R ^1b together with the nitrogen to which they are attached form a 5 membered heterocycloalkyl substituted with 0 or 1 methyl.

In certain embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, wherein R ^2a and R ^2b are both hydrogen, and wherein R ^1a , R ^1b , R ^3a , R ^3b and R ⁴ are as described herein as defined in any of the examples. In certain embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, wherein one of R ^2a and R ^2b is hydrogen and the other of R ^2a and R ^2b is methyl, and wherein R ^1a , R ^1b , R ^3a , R ^3b , R ⁴ and n are as defined in any of the embodiments described herein.

In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof, wherein R ^3a and R ^3b are each independently hydrogen, methyl or ethyl, and wherein R ^1a , R ^1b , R ^2a , R ^2b , R ⁴ and n are as defined in any of the embodiments described herein. In some embodiments, one of R ^3a and R ^3b is hydrogen. In some embodiments, one of R ^3a and R ^3b is methyl. In some embodiments, one of R ^3a and R ^3b is ethyl. In some embodiments, one of R ^3a and R ^3b is hydrogen and the other of R ^3a and R ^3b is methyl. In some embodiments, one of R ^3a and R ^3b is hydrogen and the other of R ^3a and R ^3b is ethyl. In some embodiments, both R ^3a and R ^3b are methyl.

In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof, wherein R ⁴ is methyl or ethyl, n is 1, and wherein R ^1a , R ^1b , R ^2a , R ^2b , R ^3a and R ^3b are as defined in any of the embodiments described herein. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl. In some embodiments, ^R4 is methyl substituted with one halo. In some embodiments, R ⁴ is methyl substituted with one hydroxy. In some embodiments, ^R4 is attached to the carbon adjacent to the N atom.

In certain embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof, wherein each R ⁴ is methyl or ethyl, n is 2, and wherein R ^1a , R ^1b , R ^2a , R ^2b , R ^3a and R ^3b are as defined in any of the embodiments described herein. In some embodiments, both R ⁴ are methyl.

In certain embodiments, the present invention provides compounds of formula II: or a pharmaceutically acceptable salt thereof, wherein R ^2a , R ^2b , R ^3a and R ⁴ are as defined in any of the embodiments described herein.

In some embodiments, the present invention provides a compound having absolute stereochemistry with respect to the orientation of R ² , ^{R 3a and} R ^3b when R ^3a is different from R 3b, as shown in Formula IIa: or a pharmaceutically acceptable salt thereof, wherein R ^2a , R ^2b , R ^3a and R ⁴ are as defined in any of the embodiments described herein.

In some embodiments, the present invention provides a compound of formula I, II, IIa or a pharmaceutically acceptable salt thereof, wherein R ^2a and R ^2b are both hydrogen, R ^3a is hydrogen, R ^3b is methyl or ethyl, And R ⁴ is methyl.

In some embodiments, the present invention provides an HPK1 inhibitor, wherein the HPK1 inhibitor is the compound 4-[1-aminopropyl]-2-{6-[5-methyl-6,7-dihydro-5H- Pyrrolo[2,1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2,3-di Hydrogen-1H-pyrrolo[3,4-c]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5H-pyrrole And[2,1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2,3-dihydro -1H-pyrrolo[3,4-c]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro- 5H -pyrrole And[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2,3-dihydro -1 H -pyrrolo[3,4- c ]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5 H - Pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2,3- Dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; or 4-[1-aminopropyl]-2-{3-[5-methyl-6,7-dihydro- 5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-2,3- Dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; or a pharmaceutically acceptable salt thereof.

For example, the HPK1 inhibitor of the present invention is the compound 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7-dihydro- 5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1- Base]-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; 4-[(1 R )-1-aminoethyl]-2-{6-[( 5 S )-5-Ethyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6 -[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; 4-[(1 S )-1-aminoethyl]-2-{6-[(5 S )-5-ethyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4 ]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1 H -pyrrolo[3, 4- c ]pyridin-1-one; 4-[(1 R )-1-aminoethyl]-2-{6-[(5 S )-5-ethyl-6,7-dihydro-5 H -Pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2,3 -Dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; or 4-[(1 R )-1-aminopropyl]-2-{3-[(5 S )-5 -Methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2 -yl)amino]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-1-one; or a pharmaceutically acceptable salt thereof.

Preferably, the HPK1 inhibitor of the present invention is 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl ]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-1-one; or a pharmaceutically acceptable salt thereof.

III. PD-1 Axis Binding Antagonists Embodiments of the invention include PD-1 axis binding antagonists.

As used herein, the term "PD-1 axis binding antagonist" or "PD-1 axis antagonist" refers to the inhibition of the binding of a PD-1 axis binding partner (e.g., PD-1, PD-L1, PD-L2) to it. Molecules that interact with one or more of the partners, e.g., in order to resolve or partially resolve T cell dysfunction caused by signaling on the PD-1 signaling axis - resulting in restoration, partial restoration or enhancement of T cell function (eg proliferation, cytokine production, target cell killing, survival). As used herein, a PD-1 axis binding antagonist includes one or more of (i) a PD-1 antagonist, (ii) a PD-L1 antagonist, and/or (iii) a PD-L2 antagonist.

i) PD-1 antagonists As used herein, the term "PD-1 antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the production of PD-1 by one of its binding partners (such as PD-L1, PD-L2) or Signal transduction caused by the interaction of multiple agents. In some embodiments, a PD-1 antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In a specific aspect, a PD-1 antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. In certain embodiments, the PD-1 antagonist inhibits the binding of PD-1 to PD-L1. In another embodiment, the PD-1 antagonist inhibits the binding of PD-1 to PD-L2. In another embodiment, the PD-1 antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. For example, PD-1 antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and agents that reduce, block, inhibit, eliminate, or interfere with the interaction between PD-1 and PD-L1 and / or other molecules of signal transduction caused by the interaction of PD-L2. In some embodiments, the PD-1 antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes mediated through PD-1 signaling, In order to make it difficult for dysfunctional T cells to restore normal function. In some embodiments, the PD-1 antagonist is an anti-PD-1 antibody (αPD-1).

In some embodiments, the PD-1 antagonist blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells or NKT cells) and preferably also blocks cancer cells The combination of PD-L2 expressed above and PD-1 expressed by immune cells. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279, and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H for PD-L1 and PDCD1L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. When treating a human subject with any of the treatment methods, medicaments and uses of the present invention, the PD-1 antagonist blocks the binding of human PD-L1 to human PD-1 and blocks human PD-L1 and PD-L2 Binding to human PD-1.

PD-1 antagonists suitable for any one of the treatment methods, medicines and uses of the present invention include monoclonal antibodies (mAbs) or antigen-binding fragments thereof, which specifically bind to PD-1 or PD-L1, and are more The best specific binding to human PD-1 or human PD-L1. mAbs can be human, humanized, or chimeric antibodies, and can include human constant regions. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab') ₂ , scFv, and Fv fragments.

Examples of mAbs that bind to human PD-1 and are suitable for use in the therapeutic methods, medicaments and uses of the invention are described in US Patent Nos. 7,488,802, 7,521,051, 8,008,449, 8,354,509, 8,168,757, PCT Patent Publication WO 2004/004771, WO 2004/072286, WO 2004/056875 and US Patent Publication No. 2011/0271358. Specific anti-human PD-1 mAbs suitable for use as PD-1 antagonists in the treatment methods, medicines and uses of the present invention include: saxenlimab (RN888, PF-06801591, Pfizer Inc.), nivolumab ( OPDIVO®, ONO-4538, BMS-936558, MDX1106, Bristol-Myers Squibb Company), pembrolizumab (KEYTRUDA®, MK-3475, lambrolizumab, Merck & Co., Inc .), Pidilizumab (CT-011), Cimiprizumab (LIBTAYO®, REGN2810, Regeneron Pharmaceuticals, Inc.), Tislelizumab (BGB-A317, BeiGene Ltd./Celgene Corporation ), spartacuzumab (PDR001, Novartis AG), sintilimab (IBI308, Innovent Biologics, Inc.), MEDI-0680 (AMP-514, AstraZeneca PLC), BGB-108, or AGEN2034 (Agenus Inc .), Genozumab (CBT-501, CBT Pharmaceuticals), CBT-502 (CBT Pharmaceuticals), Camrelizumab (SHR-1210, Incyte Corporation), or a combination thereof.

In certain embodiments, the PD-1 antagonist is an anti-PD-1 antibody.

Preferably, the anti-PD-1 antibody is pembrolizumab, nivolumab or saxolizumab. In some preferred embodiments, the anti-PD-1 antibody is nivolumab. In some preferred embodiments, the anti-PD-1 antibody is pembrolizumab. In some preferred embodiments, the anti-PD-1 antibody is saxolizumab. Sarsanlimab is a humanized immunoglobulin G4 (IgG4) monoclonal antibody (mAb) that binds to the PD-1 receptor. By blocking the interaction of the receptor with PD-L1 and PD-L2, the inhibition of the immune response mediated by the PD-1 pathway is released, causing an anti-tumor immune response. Clinical antitumor activity of saxanrimumab has been found against a group of anti-PD1 sensitive solid tumor types including non-small cell lung cancer and urothelial carcinoma. Sarsanlimab is described, eg, in US Patent No. US 10,155,037, which is hereby incorporated for all purposes.

Other exemplary PD-1 antagonists include those described in US Patent Application Publication 20130280265, US Patent Application Publication 20130237580, US Patent Application Publication 20130230514, US Patent Application Publication 20130109843, US Patent Application Publication 20130108651, US Patent Application Publication 20130017199, US Patent Application Publication 20120251537, US Patent Application Publication 20110271358, European Patent EP2170959B1, PCT Publication No. WO 2011/066342, PCT Publication No. WO 2015/035606, PCT Publication No. Publication No. WO 2015/085847, PCT Publication No. WO 2015/112800, PCT Publication No. WO 2015/112900, PCT Publication No. WO 2016/092419, PCT Publication No. WO 2017/017623, PCT Publication No. Publication No. WO 2017/024465, PCT Publication No. WO 2017/054646, PCT Publication No. WO 2017/071625, PCT Publication No. WO 2017/019846, PCT Publication No. WO 2017/132827, PCT Publication No. Publication No. WO 2017/214092, PCT Publication No. WO 2018/013017, PCT Publication No. WO 2018/053106, PCT Publication No. WO 2018/055503, PCT Publication No. WO 2018/053709, PCT Publication No. Publication No. WO 2018/068336 and PCT Publication No. WO 2018/072743, the entire disclosures of which are incorporated herein by reference. Other exemplary PD-1 antagonists are described in Curran et al., PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors, PNAS, 2010, 107, 4275; Topalian et al. , Safety, activity, and immune correlates of anti-PD-1 antibody in cancer, New Engl. J. Med. 2012, 366, 2443; Brahmer et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer , New Engl. J. Med. 2012, 366, 2455; Dolan et al., PD-1 pathway inhibitors: changing the landscape of cancer immunotherapy, Cancer Control 2014, 21, 3; and Sunshine et al., Pd-1/Pd- L1 Inhibitors, Curr. Opin. in Pharmacol. 2015, 23.

ii) PD-L1 antagonists The term "PD-L1 antagonist" as used herein refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the production of PD-L1 by one of its binding partners (such as PD-1, B7-1) or Signal transduction caused by the interaction of multiple agents. In some embodiments, the PD-1 axis antagonist comprises a PD-L1 antagonist. In some embodiments, a PD-L1 antagonist inhibits the binding of PD-L1 to its binding partner. In a specific aspect, a PD-L1 antagonist inhibits the binding of PD-L1 to PD-1. In another specific aspect, the PD-L1 antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In another specific aspect, the PD-L1 antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.

In some embodiments, PD-L1 antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and agents that reduce, block, inhibit, eliminate, or interfere with the binding of PD-L1 to it. Other molecules of signal transduction caused by the interaction of one or more of substances (such as PD-1 and/or B7-1). In some embodiments, the PD-L1 antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes mediated through PD-L1 signaling, In order to make it difficult for dysfunctional T cells to restore normal function. In some embodiments, the PD-L1 antagonist is an anti-PD-L1 antibody (αPD-L1). In some embodiments, the PD-L1 antibody is a biosimilar or bioequivalent thereof.

In some embodiments, the PD-1 antagonist is an anti-PD-1 antibody.

In some embodiments, anti-PD-1 antibodies suitable for use in the present invention include BMS-936559 (Bristol-Myers Squibb Company), KN035 (Jiangsu Alphamab biopharmaceuticals Co., Ltd.), atezolizumab (TECENTRIQ®, MPDL3280A, Roche Holding AG), durvalumab (IMFINZI®, AstraZeneca PLC) and/or avelumab (BAVENCIO®, Merck & Co., Inc. and Pfizer, Inc.).

Other exemplary PD-L1 antagonists include those described in US Patent Application Publication 20090055944, US Patent Application Publication 20100203056, US Patent Application Publication 20120039906, US Patent Application Publication 20130045202, US Patent Application Publication Publication 20130309250, U.S. Patent Application Publication US20130034559, U.S. Patent Application Publication US20150282460, U.S. Patent Application Publication 20160108123, PCT Publication No. WO 2011/066389, PCT Publication No. WO 2016/000619, PCT Publication No. WO 2016/094273, PCT Publication No. WO 2016/061142, PCT Publication No. WO 2016/149201, PCT Publication No. WO 2016/149350, PCT Publication No. WO 2016/179576, PCT Publication No. WO 2017/020801, PCT Publication No. WO 2017/103147, PCT Publication No. WO 2017/112741, PCT Publication No. WO 2017/205213, PCT Publication No. WO 2017/054646, PCT Publication No. WO 2017/084495, PCT Publication No. WO 2017/161976, PCT Publication No. WO 2018/005682, PCT Publication No. WO 2018/053106, PCT Publication No. WO 2018/085469, PCT Publication No. WO 2018/111890 and PCT Publication No. WO 2018/106529, the entire disclosures of which are incorporated herein by reference. Other exemplary PD-L1 binding antagonists are described in Sunshine et al., 2015.

iii) PD-L2 antagonists The term "PD-L2 antagonist" as used herein refers to a molecule that reduces, blocks, inhibits, abolishes or interferes with the interaction of one or more of PD-L2 with its binding partners, such as PD-1. Signal transduction caused by action. In some embodiments, a PD-L2 antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, a PD-L2 antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and agents that reduce, block, inhibit, eliminate, or interfere with the binding of PD-L2 to it. Other molecules of signal transduction caused by the interaction of any one or more of these substances, such as PD-1. In some embodiments, the PD-L2 antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes that are mediated through PD-L2 signaling, In order to make it difficult for dysfunctional T cells to restore normal function. In some embodiments, the PD-L2 antagonist is a PD-L2 immunoadhesin.

IV. Methods, Uses and Drugs General Methods Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (2nd edition 1982 and 1989, 3rd edition 2001) Molecular Cloning, A Laboratory Manual; Sambrook and Russell Molecular Cloning, 3rd edition, 2001; Wu, Recombinant DNA, vol. 217. Standard methods are also found in Ausbel et al., Current Protocols in Molecular Biology, Volumes 1-4, 2001, which describe the colonization and induction of DNA mutagenesis in bacterial cells (Volume 1), and in mammalian cells and yeast ( Volume 2), Glycoconjugates and Protein Expression (Volume 3), and Bioinformatics (Volume 4).

Methods for protein purification are described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization (Coligan et al., Current Protocols in Protein Science, Vol. 1, 2000, John Wiley and Sons, Inc., New York). Describe chemical analysis, chemical modification, post-translational modification, fusion protein production, and protein glycosylation (eg Coligan et al., Current Protocols in Protein Science, Vol. 2, 2000; Ausubel et al., Current Protocols in Molecular Biology, Vol. 3 , 2001, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. Products for Life Science Research, 2001, pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, pp. 384-391). The production, purification and fragmentation of polyclonal and monoclonal antibodies are described (Coligan et al., Current Protocols in Immunology, Vol. 1, 2001; Harlow and Lane, Using Antibodies, 1999). Standard techniques for characterizing ligand/receptor interactions are available (eg Coligan et al., Current Protocols in Immunology, Vol. 4, 2001).

Monoclonal, polyclonal and humanized antibodies can be produced (eg Sheperd and Dean (eds) Monoclonal Antibodies, 2000; Kontermann and Dubel (eds) Antibody Engineering, 2001; Harlow and Lane, Antibodies A Laboratory Manual, 1988, pp. 139-243 Page; Carpenter et al., Non-Fc receptor-binding humanized anti-CD3 antibodies induce apoptosis of activated human T cells, J. Immunol. 2000, 165:6205; He et al., Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E-and P-selectin, J. Immunol. 1998, 160:1029; Tang et al., Use of a peptide mimotope to guide the humanization of MRK-16, an anti-P-glycoprotein monoclonal antibody , J. Biol. Chem. 1999, 274:27371-27378; Baca et al., Antibody humanization using monovalent phage display, J. Biol. Chem. 1997, 272:10678-10684; Chothia et al., Conformations of immunoglobulin hypervariable regions, Nature 1989, 342:877- 883; Foote and Winter Antibody framework residues affecting the conformation of the hypervariable loops, J. Mol. Biol. 1992, 224:487-499; US Patent No. 6,329,511).

An alternative approach to humanization is to use human antibody libraries displayed on phage or in transgenic mice (Vaughan et al., Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library, Nature Biotechnol . 1996, 14:309-314; Barbas , Synthetic human antibodies, Nature Medicine 1995, 1:837-839; Mendez et al., Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, N Nature Genetics 1997, 15: 146-156; Hoogenboom and Chames, Natural and designer binding sites made by phage display technology, Immunol. Today 2000, 21:371-377; Barbas et al., Phage Display: A Laboratory Manual, 2001; Kay et al., Phage Display of Peptides and Proteins: A Laboratory Manual, 1996; de Bruin et al., Selection of high-affinity phage antibodies from phage display libraries, Nature Biotechnol. 1999, 17:397-399).

Purification of antigen is not required for antibody production. Animals can be immunized with cells carrying the antigen of interest. Splenocytes can then be isolated from the immunized animal and fused with a myeloma cell line to generate a fusionoma (e.g. Meyaard, L. et al ., LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes, Immunity 1997 , 7:283-290; Wright et al., Inhibition of chicken adipocyte differentiation by in vitro exposure to monoclonal antibodies against embryonic chicken adipocyte plasma membranes, Immunity 2000, 13:233-242; Preston et al., The leukoc yte/neuron cell surface antigen OX2 binds to a ligand on macrophages), Eur. J. Immunol. 1997, 27:1911-1918, Kaithamana et al., Induction of experimental autoimmune Graves' disease in BALB/c mice, J. Immunol . 1999, 163:5157- 5164).

Antibodies can be conjugated to, for example, small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are suitable for therapeutic, diagnostic, kit or other purposes and include those conjugated to, for example, dyes, radioisotopes, enzymes or metals such as colloidal gold (e.g. Le Doussal et al., Enhanced in vivo targeting of an asymmetric bivalent hapten to double-antigen-positive mouse B cells with monoclonal antibody conjugate cocktails, J. Immunol . 1991, 146:169-175; Gibellini et al., Extracellular HIV-1 Tat protein induces the rapid Ser133 phosphorylation and activation of CR EB transcription factor in both Jurkat lymphoblastoid T cells and primary… , J. Immunol. 1998160:3891-3898; Hsing and Bishop, Requirement for nuclear factor -κ B activation by a distinct subset of CD40-mediated effector functions in B lymphocytes, J. Immunol. 1999, 162:2804-2811; Everts et al., Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate, J. Immunol. 2002, 168:883-889).

Methods are available for flow cytometry techniques, including fluorescence activated cell sorting (FACS) (eg Owens et al., Flow Cytometry Principles for Clinical Laboratory Practice, 1994; Givan Flow Cytometry, 2nd Edition; 2001; Shapiro, Practical Flow Cytometry, 2003). Fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and probes, polypeptides and antibodies) are available, which are used, for example, as diagnostic reagents (Molecular Probes, Catalog, 2003; Sigma-Aldrich, Catalog, 2003.

Standard histological methods for describing the immune system (eg Muller-Harmelink (ed.), Human Thymus: Histopathology and Pathology, 1986; Hiatt et al., Color Atlas of Histology, 2000; Louis et al., Basic Histology: Text and Atlas, 2002.

Packaged software and databases (e.g. GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, MD) for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available; GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne et al., A comparison of signal sequence prediction methods using a test set of signal peptides, Bioinformatics 2000, 16: 741-742; Menne, KML et al., A Comparison of Signal Sequence Prediction Methods USING A Test of Signal Peptides, BioInFormatics 2000, 16, 741-742; Gnal-sequence information and geenomic analysis comput. Methods Programs Biomed. 2002, 68:177-181; von Heijne, Patterns of amino acids near signal‐sequence cleavage sites, Eur. J. Biochem . 1983, 133:17-21; von Heijne, A new method for predicting signal sequence cleavage avage sites, Nucleic Acids Res . 1986, 14:4683-4690) .

Methods of Treatment and Uses The present invention provides methods of treatment and uses comprising administering to a subject a combination as described herein, optionally further in combination with other therapeutic or palliative agents.

In one aspect, the invention provides a method of treating cancer in an individual comprising administering to the individual a combination therapy of the invention. In one aspect, the invention provides a method for treating cancer comprising administering to a subject in need thereof an amount of an HPK1 inhibitor and an amount of a PD-1 axis binding antagonist, wherein the equal amounts are effective together Treating cancer, wherein the HPK1 inhibitor is the compound of formula I, II or IIa disclosed herein or a pharmaceutically acceptable salt thereof. In some such embodiments, the individual is human.

In some embodiments, the method involves using an HPK1 inhibitor described herein in combination with an anti-PD-L1 antibody.

In some embodiments, the method involves 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7-dihydro-5 H - Pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]- Use of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof in combination with an anti-PD-1 antibody selected from the group consisting of saxolizumab , Nivolumab, Pembrolizumab, Pidilizumab, Simiprizumab, Tislelizumab, Spartakizumab, Sintilimab, MEDI-0680, BGB -108, or AGEN2034 genolizumab, CBT-502, camrelizumab, or a combination thereof.

Preferably, the method involves 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7-dihydro-5 H -pyrrolo [2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-2, Use of 3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof in combination with saxanlimab.

Preferably, the method involves 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7-dihydro-5 H -pyrrolo [2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-2, Use of 3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof in combination with nivolumab.

Preferably, the method involves 4-[(1R)-1-aminopropyl]-2-{6-[(5S)-5-methyl-6,7-dihydro-5H-pyrrolo[2, 1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2R)-2-methylpyrrolidin-1-yl]-2,3-dihydro - Use of 1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof in combination with pembrolizumab.

In some embodiments, treatment results in a sustained response in the subject after cessation of treatment. The methods of the invention are useful in the treatment of conditions requiring enhanced immunogenicity, such as increasing tumor immunogenicity to treat cancer. Thus, many cancers can be treated, or their progression can be delayed.

In some embodiments, the individual has a cancer that is resistant (proven resistant) to one or more PD-1 axis binding antagonists. In some embodiments, resistance to a PD-1 axis binding antagonist comprises recurrence of cancer or refractory cancer. Recurrence can refer to recurrence of cancer at the original site or at a new site after treatment. In some embodiments, resistance to the PD-1 axis binding antagonist comprises cancer progression during treatment with the PD-1 axis binding antagonist. In some embodiments, resistance to a PD-1 axis binding antagonist comprises a cancer that is unresponsive to treatment. Cancer may be resistant when treatment begins or it may become resistant during treatment. In some embodiments, the cancer is early or advanced.

In certain embodiments, the individual has received one, two, three, four, five or more prior cancer therapies. In other embodiments, the individual is treatment naïve. In some embodiments, the individual progresses on other cancer treatments. In certain embodiments, the prior cancer therapy comprises immunotherapy. In other embodiments, the prior cancer therapy comprises chemotherapy. In some embodiments, the tumor has recurred. In some embodiments, the tumor is advanced. In some embodiments, the tumor is metastatic. In other embodiments, the tumor is not metastatic.

In some embodiments, the individual has received prior therapy to treat the tumor and the tumor is relapsed or refractory. In some embodiments, the individual has received prior immuno-oncology therapy to treat the tumor and the tumor is relapsed or refractory. In some embodiments, the individual has received more than one prior therapy to treat the tumor and the individual is relapsed or refractory.

In some embodiments of the methods as described herein, the cancer is a solid tumor.

In another embodiment, the present invention relates to a method for treating cancer, wherein the cancer is selected from the group consisting of brain cancer, head/neck cancer (including squamous cell carcinoma of the head and neck ( SCCHN)), prostate cancer, bladder cancer (including urothelial carcinoma, also known as transitional cell carcinoma (TCC)), lung cancer (including squamous cell carcinoma, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)), breast cancer , ovarian cancer, bone cancer, colorectal cancer, kidney cancer, liver cancer (including hepatocellular carcinoma (HCC)), pancreatic cancer, esophageal cancer (including squamous cell carcinoma (SCC)), gastric cancer, gastroesophageal junction cancer, Cancer of the thyroid, cervix, uterus, and/or kidney. In another embodiment, the present invention relates to a method for treating cancer, wherein the cancer is selected from the group consisting of squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), urothelial carcinoma and gastric cancer . In some embodiments, the invention relates to a method of treating cancer, wherein the cancer is melanoma.

In another embodiment, the invention provides a method for treating melanoma or breast cancer in a subject comprising administering to the subject 4-[(1 R )-1-aminopropyl]-2-{6-[( 5 S )-5-Methyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6 -[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or its pharmaceutically acceptable Salt in combination with saxolizumab, nivolumab, or pembrolizumab.

In another embodiment, the invention provides a method for treating melanoma or breast cancer in a subject comprising administering to the subject 4-[(1 R )-1-aminopropyl]-2-{6-[( 5 S )-5-Methyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6 -[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or its pharmaceutically acceptable Salt in combination with saxolizumab or nivolumab.

In one embodiment, the invention provides a method for treating melanoma in a subject comprising administering to the subject 4-[( 1R )-1-aminopropyl]-2-{6-[( 5S )-5-methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[ (2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or its pharmaceutically acceptable salt and Combinations of saxolizumab.

In another embodiment, the invention provides a method for treating melanoma or breast cancer in a subject comprising administering to the subject 4-[(1 R )-1-aminopropyl]-2-{6-[( 5 S )-5-Methyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6 -[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4- c ]pyrrolidin-1-one or its pharmaceutically acceptable A combination of salt and nivolumab.

In another embodiment, the invention provides a method for treating melanoma or breast cancer in a subject comprising administering to the subject 4-[(1 R )-1-aminopropyl]-2-{6-[( 5 S )-5-Methyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6 -[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4- c ]pyrrolidin-1-one or its pharmaceutically acceptable The combination of salt and pembrolizumab.

In some embodiments, the method may further comprise other therapies. Other therapies may be radiation therapy, surgery (such as lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, Phototherapy or a combination of the foregoing. Other therapies can be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the other therapy is the administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, the additional therapy is the administration of side effect limiting agents (eg, agents intended to reduce the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the other therapy is radiation therapy. In some embodiments, the other therapy is surgery. In some embodiments, the other therapy is a combination of radiation therapy and surgery. In some embodiments, the other therapy is immunotherapy.

In some embodiments, the methods of the invention further comprise administering chemotherapy, radiation therapy, immunotherapy, or phototherapy, or any combination thereof, to the individual.

In some embodiments of the methods, uses, compositions and kits described above and herein, the therapy further comprises administering a chemotherapeutic agent for treating or delaying the progression of cancer in the individual. In some embodiments, the individual has been treated with a chemotherapeutic agent prior to treatment with the HPK1 inhibitor in combination with the PD-1 axis binding antagonist. In some embodiments, an individual treated with a combination of an HPK1 inhibitor, a PD-1 axis binding antagonist is refractory to treatment with a chemotherapeutic agent. Some embodiments of the methods, uses, compositions and kits described throughout the application further comprise administering a chemotherapeutic agent for treating or delaying progression of cancer.

In some embodiments, combination therapies of the invention comprise administering an HPK1 inhibitor in combination with a PD-1 axis binding antagonist. In the methods provided herein, each of the HPK1 inhibitor and the PD-1 axis binding antagonist can be administered in any suitable manner known in the art. In one embodiment, the HPK1 inhibitor and the PD-1 axis binding antagonist are administered simultaneously or sequentially in either order.

In some embodiments of the foregoing, the PD-1 axis binding antagonist is: a PD-1 antagonist; a PD-L1 antagonist; or a PD-1 antagonist and a PD-L1 antagonist.

In some embodiments of the foregoing, a. the PD-1 binding antagonist and the PD-L1 binding antagonist are present in the same composition.

In one aspect, the invention provides a synergistic combination. In some such embodiments, the invention provides a synergistic combination comprising: (i) an HPK1 inhibitor of formula I, II or IIa, or a pharmaceutically acceptable salt thereof; and (ii) a PD described herein -1 axis binding antagonist for use in the treatment of cancer in a subject, wherein component (i) and component (ii) are synergistic. In some embodiments, the PD-1 axis binding antagonist is a PD-1 antagonist. In some embodiments, the PD-1 axis binding antagonist is a PD-L1 antagonist.

In some embodiments, the present invention provides a synergistic combination comprising: (i) an HPK1 inhibitor of formula I, II or IIa, or a pharmaceutically acceptable salt thereof; and (ii) saxanlimab; In the treatment of cancer in a subject, wherein component (i) and component (ii) are synergistic.

In a specific embodiment, the present invention provides a combination comprising: (i) 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl- 6,7-Dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2- Methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof; and (ii) anti-PD- 1 An antibody; for use in treating cancer in a subject.

In a specific embodiment, the present invention provides a combination comprising: a. (i) 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl Base-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )- 2-Methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof; and (ii) Sal Senlimumab; it is used in the treatment of cancer in a subject.

In another embodiment, the present invention provides a combination comprising: a. (i) 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methan Base-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )- 2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof; and (ii) nitric acid Wolumab; it is used to treat cancer in a subject.

In another specific embodiment, the present invention provides a combination comprising: a. (i) 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5- Methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R ) -2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof; and (ii) Pembrolizumab; it is used to treat cancer in a subject.

In a specific embodiment of each of the foregoing, the invention provides a combination, wherein the PD-1 antagonist is an anti-PD-1 antibody; and/or the PD-L1 antagonist is an anti-PD-L1 antibody.

In some embodiments of the methods, uses, compositions and kits described herein, the cancer is resistant to a therapeutic agent or class of treatment, including a standard of care agent or class. In some embodiments of the methods, uses, compositions and kits described herein, the cancer is characterized by innate or acquired resistance to a therapeutic agent or class of treatment, including standard of care agents or classes.

In some embodiments of the methods, uses, compositions and kits described herein, the cancer is refractory, that is, the cancer is completely unresponsive to treatment with a therapeutic agent or class of treatment (including agents or classes of standard of care) or Responds initially, but begins to grow again within a very short period of time.

In some embodiments, the methods, uses and combinations described herein are associated with therapy, adjuvant therapy, first-line therapy, second-line therapy, or third-line or later-line therapy. In some embodiments, the methods, uses and combinations described herein are related to therapy. In some embodiments, the methods, uses and combinations described herein relate to adjuvant therapy. In some embodiments, the methods, uses and combinations described herein relate to first-line therapy. In some embodiments, the methods, uses and combinations described herein relate to second line therapy. In some embodiments, the methods, uses and combinations described herein relate to third or later line therapy.

V. Dosage Forms and Therapies Administration of the compounds of the invention may be effected by any method capable of delivering the compound to the site of action. Such methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical and rectal administration.

Those skilled in the art will consider various factors including, but not limited to, the degree of disease progression, age, weight, general health, sex, diet, the compound administered, the time and route of administration, and the cancer requiring treatment. nature and exacerbations, and other drugs taken by the individual), it will be possible to determine the appropriate amount, dose or dosage form of each compound used in the combination of the invention to administer to the patient.

In some embodiments, methods of administration of the agents and combinations herein may include oral, intravenous, intramuscular, subcutaneous, topical, transdermal, intraperitoneal, by implantation, by inhalation, intrathecal, intracerebroventricular or intranasal administration.

Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus injection can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are determined by and directly dependent on: (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the individual therapeutic implications inherent in the compounding techniques of such active compounds. Sensitivity Limitations.

Accordingly, those skilled in the art will appreciate that, based on the disclosure provided herein, dosages and dosing regimens may be adjusted according to methods well known in the therapeutic art. That is, the maximum tolerable dose can be readily determined, and an effective amount to provide a detectable therapeutic benefit to the patient can also be determined, as can the temporal need to administer each agent to provide a detectable therapeutic benefit to the patient. Thus, although certain dosages and administration regimens are illustrated herein, these examples in no way limit the dosages and administration regimens that can be provided to patients in practicing the invention.

For combination therapy as described herein, the agents can be administered in their approved dosages. Treatment was continued as long as clinical benefit was observed or until unacceptable toxicity or disease progression occurred. Nonetheless, in certain embodiments, the combination therapies of the present invention may advantageously utilize lower doses of the therapeutic agents administered, thereby avoiding possible toxicity or complications associated with various monotherapies. For example, the dose of the agent administered is significantly lower than the approved dose, eg, a subtherapeutic dose of an HPK1 inhibitor is administered in combination with a subtherapeutic dose of a PD-1 axis binding antagonist. Those skilled in the art will appreciate that when the agents of the invention are used as part of a combination therapy, lower doses of the agents may be required than when the agents alone are administered to an individual, and that a synergistic therapeutic effect can be achieved through the use of the combination therapy. It also allows the use of lower doses of the agent to achieve the desired therapeutic effect.

In one embodiment, the dosage may be lower and may also be administered less frequently, thereby reducing the incidence or severity of side effects. This meets the needs and requirements of the individual to be treated.

In one embodiment, the present invention provides a pharmaceutical composition comprising amounts which, in combination, are therapeutically effective in the treatment of cancer. In this pharmaceutical composition, two or more compounds may be administered together, one after the other or separately in one combined unit dosage form or in two separate unit dosage forms.

The unit dosage form can also be a fixed combination. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated and may comprise single or multiple doses. In addition, it should be understood that for any particular individual, the particular dosage regimen should be adjusted over time according to the needs of the individual and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are exemplary only are not intended to limit the scope or practice of the compositions claimed. For example, dosages may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, such as toxic effects, and/or experimental values. Accordingly, the invention encompasses intra-patient dose escalation as determined by one skilled in the art. Determining appropriate administered doses and regimens of chemotherapeutic agents is well known in the relevant art and encompassed by the teachings disclosed herein, as understood by those skilled in the art.

The amount of an agent of the invention administered will depend on the individual being treated, the severity of the disorder or condition, the rate of administration, the formulation of the compound, and the judgment of the prescribing physician.

Effective amounts of HPK1 inhibitors and PD-1 axis binding antagonists can be administered to prevent or treat diseases. The appropriate dose of HPK1 inhibitors and PD-1 axis binding antagonists can be determined based on the following: the type of disease to be treated, HPK1 inhibitors, PD-1 axis binding antagonists, the severity and course of the disease, the individual's clinical symptoms, The individual's clinical history and response to treatment and the judgment of the attending physician. In some embodiments, the combination therapy of an HPK1 inhibitor and a PD-1 axis binding antagonist (such as an anti-PD-1 antibody or an anti-PD-L1 antibody) has a synergistic effect, thereby reducing the HPK1 inhibitor, PD- Effective doses of 1 axis binding antagonists (relative to the effective doses of each of the HPK1 inhibitor, PD1 axis binding antagonists as a single agent).

In some embodiments, the individual has an advanced or metastatic solid tumor that has progressed following systemic anticancer therapy comprising at least one checkpoint inhibitor (eg, PD-1, PD-L1, or CTLA-4). In some embodiments, the individual has an advanced or metastatic solid tumor that is resistant to one or more checkpoint inhibitors (eg, PD-1, PD-L1, or CTLA-4). In some embodiments, the individual has an advanced or metastatic solid tumor that has progressed following systemic anticancer therapy comprising at least one checkpoint inhibitor (e.g., PD-1, PD-L1, or CTLA-4), and resistance to checkpoint inhibitors. In some such embodiments, the advanced or metastatic tumor status has been histologically or cytologically confirmed.

In some embodiments of the foregoing, the solid tumor is selected from the group consisting of gastric/gastroesophageal junction (GEJ) cancer, head and neck squamous cell carcinoma (HNSCC), and urothelial carcinoma. In some embodiments of the foregoing, the solid tumor is selected from the group consisting of non-small cell lung cancer and other solid tumors. In some embodiments, the solid tumor is an advanced solid tumor. In some other embodiments, the solid tumor is a metastatic solid tumor.

In some embodiments, the PD1 axis binding antagonist is saxenlimab and may be administered for about 14 days (±2 days) or about 21 days (±2 days) or about 30 days (±2 days) throughout the course of treatment Administered subcutaneously in doses of about 1, 2, 3, 4, 5, 6, 7 or 8 mg/kg at time intervals. In some embodiments, saxenlimab is administered in a uniform dose of about 80, 150, 160, 200, 240, 250, 300, 320, 350, or 400 mg.

VI. Kits In some embodiments, the present invention provides a kit comprising: (i) a pharmaceutical composition comprising an HPK1 inhibitor described herein and a pharmaceutically acceptable carrier; (ii) comprising A pharmaceutical composition comprising the described PD-1 axis binding antagonist and a pharmaceutically acceptable carrier.

In some embodiments, the kit further comprises a package insert comprising treating or delaying progression or enhancement of cancer in a subject using an HPK1 inhibitor in combination with a PD-1 axis binding antagonist (eg, an anti-PD-1 or anti-PD-L1 antibody) Description of immune function in individuals with cancer. In another embodiment, any one of the HPK1 inhibitors and PD-1 axis binding antagonists described herein may be included in the kit. For example, in some embodiments, the HPK1 inhibitor is a compound of formula I, II or IIa, or a pharmaceutically acceptable salt thereof. In some of these embodiments, the HPK1 inhibitor is 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7-dihydro- 5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1- Base]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one or a pharmaceutically acceptable salt thereof. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of saxolizumab, nivolumab, pembrolizumab, pintilizumab, simiprizumab, tislelizumab, Badazizumab, sintilizumab, MEDI-0680, BGB-108, or AGEN2034 Genozumab, CBT-502, camrelizumab, or combinations thereof. In one embodiment, the PD-1 binding antagonist is saxenlimab. In one embodiment, the PD-1 binding antagonist is nivolumab.

In some embodiments, the HPK1 inhibitor and the PD-1 axis binding antagonist are stored in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloys (such as stainless steel or hastelloy). In some embodiments, the container contains the formulation, and a label on or associated with the container may indicate directions for use. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the kit further includes one or more additional agents (eg, chemotherapeutics and antineoplastic agents). Suitable containers for one or more medicaments include, for example, bottles, vials, bags, and syringes.

This description is sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES The invention will be better understood with reference to the following examples. These examples are intended to represent specific embodiments of the invention and are not intended to limit the scope of the invention.

Preparation of Synthetic Intermediates For the intermediates and Examples prepared herein, where the stereochemistry is known, the stereochemistry is as drawn and the name designates the specific stereochemistry, eg ( R ) or ( S ). The stereochemistry as specified herein is known as compounds are synthesized from known, chiral starting materials or racemic mixtures, and the stereochemistry of certain examples or intermediates is confirmed using X-ray crystallography.

Intermediate 2 : ( 5S )-3-(6-bromopyridin-2-yl)-5-methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2 ,4] Triazole Step 1: Tertiary butyl {( 2S )-5-[2-(6-bromopyridine-2-carbonyl)hydrazino]-5-oxopentan-2-yl}carbamate ( 2a )

A solution of ( 4S )-4-[(tert-butoxycarbonyl)amino]valeric acid (600 mg, 2.76 mmol) in THF (13.8 mL, 0.2 M) was cooled to 0 °C. To the solution was added propylphosphonic anhydride solution (50% solution in EtOAc, 3.62 mL, 6.08 mmol) at 0 °C, then the bath was removed and the reaction mixture was stirred at room temperature for 30 min. Then, N , N -diisopropylethylamine (2.89 mL, 16.6 mmol) and 6-bromopicolinylhydrazine (656 mg, 3.04 mmol) were added and the reaction mixture was stirred at room temperature for 22 hours. LCMS analysis showed consumption of starting material. The reaction was quenched with water (15 mL) and transferred to a separatory funnel with EtOAc (20 mL). The layers were separated, and the organic phase was washed successively with 20% citric acid (20 mL), saturated NaHCO ₃ solution (20 mL) and brine (20 mL). The organic extract was then dried over MgSO ₄ , filtered and concentrated to dryness to afford the title compound ( 2a ) (1.07 g, 93% yield) as an off-white solid which was used without further purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.76 (br. s, 1H), 9.36 (br. s, 1H), 8.15 (dd, J = 0.9, 7.5 Hz, 1H), 7.77 - 7.71 (m, 1H ), 7.69 - 7.64 (m, 1H), 4.45 (br. d, J = 1.0 Hz, 1H), 3.91 (br. s, 1H), 2.47 - 2.35 (m, 2H), 2.00 - 1.89 (m, 1H ), 1.75 - 1.66 (m, 1H), 1.48 (s, 9H), 1.22 (d, J = 6.6 Hz, 3H). LCMS m/z (APCI), 315.0 (M+H-Boc) ⁺ for (C ₁₁ H ₁₅ BrN ₄ O ₂ ).

Step 2: {(2 S )-4-[5-(6-bromopyridin-2-yl)-1,3,4-oxadiazol-2-yl]butan-2-yl}carbamic acid tertiary Butyl ester ( 2b )

To {(2 S )-5-[2-(6-bromopyridine-2-carbonyl)hydrazino]-5-pentoxypent-2-yl}carbamate ( 2a ) (290 mg , 0.698 mmol) in DCM (2.8 mL, 0.25 M) was added triethylamine (0.292 mL, 2.09 mmol) and p-toluenesulfonyl chloride (160 mg, 0.838 mmol). The reaction was stirred at room temperature for 16 hours. LCMS analysis showed consumption of starting material. Ethylenediamine (0.047 mL, 0.698 mmol) was added to remove excess p-toluenesulfonyl chloride; during the addition, a precipitate formed immediately. After stirring at room temperature for 30 minutes, the reaction was washed with 20% citric acid (5 ml) and the layers were separated. The aqueous layer was extracted with DCM (5 mL), then the combined organic layers were washed with brine (10 mL), dried over MgSO ₄ , filtered and concentrated to dryness to give the title compound ( 2b ) (274 mg) as a pale yellow solid. , 98% yield), which was used without further purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.21 (dd, J = 0.9, 7.6 Hz, 1H), 7.76 - 7.69 (m, 1H), 7.68 - 7.65 (m, 1H), 4.39 (br.s, 1H ), 3.85 (br. s, 1H), 3.08 - 3.01 (m, 2H), 2.17 - 2.03 (m, 1H), 2.03 - 1.90 (m, 1H), 1.44 (s, 9H), 1.22 (d, J = 6.6 Hz, 3H). LCMS m/z (APCI), 297.0 (M+H-Boc) ⁺ for (C ₁₁ H ₁₃ BrN ₄ O).

Step 3: Intermediate 2 was loaded into a microwave vial with {(2 S )-4-[5-(6-bromopyridin-2-yl)-1,3,4-oxadiazol-2-yl]butan- Tert-butyl 2-yl}carbamate ( 2b ) (150 mg, 0.378 mmol) and trifluoroethanol (1.89 mL, 0.2 M) were sealed and then heated to 180° C. in a microwave for 30 minutes. LCMS analysis showed consumption of starting material. The reaction mixture was concentrated and the residue was purified by flash chromatography ( _Si02 , 100% heptane to 1:10 MeOH/EtOAc) to afford Intermediate 2 (74.3 mg, 71% yield) as a brown gummy solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.38 (d, J = 7.7 Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 5.22 - 5.08 (m, 1H), 3.21 - 3.16 (m, 1H), 3.16 - 3.02 (m, 2H), 2.55 - 2.44 (m, 1H), 1.60 (d, J = 6.6 Hz, 3H). LCMS m/z (APCI), 279.1 (M+H) ⁺ for (C ₁₁ H ₁₁ BrN ₄ ). By SFC (10-60% methanol (0.5% NH ₃ ) in carbon dioxide (400-450 bar), gradient time = 2 minutes, flow rate = 4 mL/min, Chiralpack IC-U 50mm*3mm*1.6µm tube column) determined as a single enantiomer. The stereochemistry of Intermediate 2 was assigned based on the use of ( 4S )-4-[(tertiary butoxycarbonyl)amino]pentanoic acid in Step 1.

Intermediate 3 : ( 5S )-3-(6-bromopyridin-2-yl)-5-ethyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2 ,4] Triazole Step 1: Tertiary butyl {( 2S )-1-[methoxy(methyl)amino]-1-oxobutan-2-yl}carbamate ( 3a )

A solution of ( 2S )-2-[(tertiary butoxycarbonyl)amino]butanoic acid (2.0 g, 9.84 mmol) in THF (49.2 mL, 0.2 M) was cooled to 0 °C. A solution of propylphosphonic anhydride (50% solution in EtOAc, 12.9 mL, 21.6 mmol) was added to the solution at 0 °C, then the bath was removed, and the reaction mixture was warmed to room temperature and stirred for 30 min. Then, N , N -diisopropylethylamine (10.3 mL, 59.0 mmol) and methoxy(methyl)amine hydrochloride (1.06 g, 10.8 mmol) were added and the reaction mixture was stirred at room temperature for 18 hours. LCMS analysis showed consumption of starting material. The reaction was quenched with water (40 mL) and transferred to a separatory funnel with EtOAc (40 mL). The layers were separated, and the organic phase was washed successively with 20% citric acid (40 mL), saturated NaHCO ₃ solution (40 mL) and brine (40 mL). The organic extract was then dried over MgSO ₄ , filtered and concentrated to dryness to give the title compound ( 3a ) (1.29 g, 53% yield) as a yellow oil which was used without further purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.28 - 5.12 (m, 1H), 4.74 - 4.52 (m, 1H), 3.79 (s, 3H), 3.23 (s, 3H), 1.85 - 1.73 (m, 1H ), 1.64 - 1.54 (m, 1H), 1.46 (s, 9H), 0.96 (t, J = 7.5 Hz, 3H). LCMS m/z (APCI), 247.1 (M+H) ⁺ for (C ₁₁ H ₂₂ N ₂ O ₄ ).

Step 2: Tertiary butyl ( S )-(1-oxobutan-2-yl)carbamate ( 3b )

Tertiary butyl {( 2S )-1-[methoxy(methyl)amino]-1-oxobutan-2-yl}carbamate ( 3a ) (1.29 g, 5.25 mmol) The solution in THF (26.2 mL, 0.2 M) was cooled to 0 °C. Lithium aluminum hydride (2.3 M solution in 2-MeTHF, 2.51 mL, 5.77 mmol) was added dropwise to the solution at 0 °C. The reaction mixture was stirred at 0°C for 40 minutes. LCMS analysis showed consumption of starting material. The reaction was quenched with EtOAc (10 mL) and 1 M HCl (10 mL) and transferred to a separatory funnel. The layers were separated and the aqueous phase was extracted with EtOAc (3 x 10 mL). The combined organic extracts were then dried over MgSO ₄ , filtered, and concentrated to dryness. The residue was purified by flash chromatography ( _Si02 , 100% heptane to 100% EtOAc) to afford the title compound ( 3b ) (0.575 g, 59% yield) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.60 (s, 1H), 5.09 (br. s, 1H), 4.22 (br. s, 1H), 2.04 - 1.90 (m, 1H), 1.73 - 1.64 (m , 2H), 1.48 (s, 9H), 0.99 (t, J = 7.5 Hz, 3H).

Step 3: ( 2E , 4S )-4-[(tertiary butoxycarbonyl)amino]hex-2-enoic acid ethyl ester ( 3c )

To a solution of tert-butyl ( S )-(1-oxobutan-2-yl)carbamate ( 3b ) (575 mg, 3.07 mmol) in DCM (6.14 mL, 0.5 M) was added ( Ethoxycarbonylmethylene)triphenylphosphine (1.60 g, 4.61 mmol). The reaction mixture was stirred at room temperature for 17 hours. LCMS analysis showed consumption of starting material. The reaction was concentrated to dryness, then isopropanol (8.5 mL, 0.36 M) and zinc chloride (1.26 g, 9.21 mmol) were added to precipitate triphenylphosphine oxide. After stirring at room temperature for 30 minutes, the reaction mixture was filtered and concentrated to dryness. The residue was purified by flash chromatography (SiO ₂ , 100% heptane to 100% EtOAc) to afford the title compound ( 3c ) as a colorless oil (653 g, 83% yield, E/Z isomer ratio > 20:1). ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.86 (dd, J = 5.4, 15.6 Hz, 1H), 5.94 (dd, J = 1.7, 15.7 Hz, 1H), 4.50 (br. s, 1H), 4.31 - 4.16 (m, 3H), 1.65 - 1.63 (m, 1H), 1.60 - 1.51 (m, 1H), 1.47 (s, 9H), 1.32 - 1.30 (m, 3H), 0.99 - 0.95 (m, 3H). LCMS m/z (APCI), 158.2 (M+H-Boc) ⁺ for (C ₈ H ₁₅ NO ₂ ).

Step 4: Ethyl ( 4S )-4-[(tertiary butoxycarbonyl)amino]hexanoate ( 3d )

To ( 2E , 4S )-4-[(tertiary butoxycarbonyl)amino]hex-2-enoic acid ethyl ester ( 3c ) (653 mg, 2.54 mmol) in methanol (12.7 mL, 0.2 M) To the solution in was added palladium on carbon (10% w/w, 270 mg, 0.254 mmol). The reaction vial was evacuated and refilled with _H2 under dynamic vacuum for 10 sec. The reaction mixture was subsequently stirred at room temperature _under 1 atm H for 18 h. The mixture was stirred at room temperature for 17 hours. LCMS analysis showed consumption of starting material. The reaction was filtered through ^Celite® and concentrated to dryness. The residue was purified by flash chromatography ( _Si02 , 100% heptane to 100% EtOAc) to afford the title compound ( 3d ) (589 g, 90% yield) as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.37 - 4.23 (m, 1H), 4.20 - 4.11 (m, 2H), 3.61 - 3.46 (m, 1H), 2.42 - 2.33 (m, 2H), 1.93 - 1.82 (m, 1H), 1.71 - 1.62 (m, 1H), 1.57 - 1.51 (m, 1H), 1.46 (s, 9H), 1.44 - 1.38 (m, 1H), 1.30 - 1.26 (m, 3H), 0.94 (t, J = 7.4 Hz, 3H).

Step 5: ( 4S )-4-[(tertiary butoxycarbonyl)amino]hexanoic acid ( 3e )

To ( 4S )-4-[(tertiary butoxycarbonyl)amino]hexanoic acid ethyl ester ( 3d ) (589 mg, 2.27 mmol) in THF (11.4 mL, 0.2 M) and MeOH (5.7 mL, 0.4 To the solution in M) was added a solution of LiOH (544 mg, 22.7 mmol) in water (2.84 mL, 0.8 M). The reaction mixture was stirred at room temperature for 24 hours. The reaction was concentrated until pH 5 was reached before adding water and 1 M HCl. The aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine, dried over MgSO ₄ , filtered and concentrated to dryness to give the title compound ( 3e ) (260 mg, 50% yield) as a light yellow oil which was used without further purification.

Step 6: Tertiary butyl {( 3S )-6-[2-(6-bromopyridine-2-carbonyl)hydrazino]-6-oxohexan-3-yl}carbamate ( 3f )

A solution of ( 4S )-4-[(tertiary-butoxycarbonyl)amino]hexanoic acid ( 3e ) (260 mg, 1.12 mmol) in THF (5.6 mL, 0.2 M) was cooled to 0 °C. To the solution was added propylphosphonic anhydride solution (50% solution in EtOAc, 1.47 mL, 2.47 mmol) at 0 °C, then the bath was removed and the reaction mixture was stirred at room temperature for 30 min. Then, N , N -diisopropylethylamine (1.17 mL, 6.74 mmol) and 6-bromopicolinylhydrazine (267 mg, 1.24 mmol) were added and the reaction mixture was stirred at room temperature for 18 hours. The reaction was quenched with water (15 mL) and transferred to a separatory funnel with EtOAc (20 mL). The layers were separated, and the organic phase was washed successively with 20% citric acid (20 mL), saturated NaHCO ₃ solution (20 mL) and brine (20 mL). The organic extracts were then dried over MgSO ₄ , filtered, and concentrated to dryness. The residue was purified by flash chromatography ( _Si02 , 100% heptane to 100% EtOAc) to afford the title compound ( 3f ) (241 g, 50% yield) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.76 (br. s, 1H), 9.29 (br. s, 1H), 8.15 (d, J = 6.8 Hz, 1H), 7.76 - 7.71 (m, 1H), 7.68 - 7.64 (m, 1H), 4.45 - 4.31 (m, 1H), 3.69 (br. s, 1H), 2.45 - 2.37 (m, 2H), 2.04 - 1.94 (m, 1H), 1.69 - 1.62 (m , 1H), 1.49 (s, 9H), 1.45 (br. d, J = 7.3 Hz, 2H), 0.99 (t, J = 7.5 Hz, 3H). LCMS m/z (APCI), 329.0 (M+H-Boc) ⁺ for (C ₁₂ H ₁₇ BrN ₄ O ₂ ).

Step 7: {(3 S )-1-[5-(6-Bromopyridin-2-yl)-1,3,4-oxadiazol-2-yl]pentan-3-yl}carbamic acid tris Grade butyl ester ( 3g )

To {(3 S )-6-[2-(6-bromopyridine-2-carbonyl)hydrazino]-6-oxohexan-3-yl}carbamic acid tertiary butyl ester ( 3f ) (241 mg, 0.561 mmol) in DCM (2.2 mL, 0.25 M) was added triethylamine (0.235 mL, 1.68 mmol) and p-toluenesulfonyl chloride (128 mg, 0.673 mmol). The reaction was stirred at room temperature for 16 hours. LCMS analysis showed consumption of starting material. Ethylenediamine (0.038 mL, 0.561 mmol) was added to remove excess p-toluenesulfonyl chloride; during the addition, a precipitate formed immediately. After stirring at room temperature for 30 minutes, the reaction was washed with 20% citric acid (5 mL) and the layers were separated. The aqueous layer was extracted with DCM (5 mL), then the combined organic layers were washed with brine (10 mL), dried over MgSO ₄ , filtered and concentrated to dryness. The residue was purified by flash chromatography ( _Si02 , 100% heptane to 100% EtOAc) to afford the title compound ( 3 g ) as a white solid (178 g, 77% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.22 (dd, J = 0.7, 7.6 Hz, 1H), 7.77 - 7.72 (m, 1H), 7.68 - 7.65 (m, 1H), 4.35 (br. d, J = 8.2 Hz, 1H), 3.65 (br. s, 1H), 3.07 (dt, J = 6.1, 10.0 Hz, 2H), 2.21 - 2.11 (m, 1H), 1.93 - 1.83 (m, 1H), 1.65 - 1.61 (m, 1H), 1.53 - 1.48 (m, 1H), 1.46 (s, 9H), 0.98 (t, J = 7.4 Hz, 3H). LCMS m/z (APCI), 311.0 (M+H-Boc) ⁺ for (C ₁₂ H ₁₅ BrN ₄ O).

Step 8 : Charge {(3 S )-1-[5-(6-bromopyridin-2-yl)-1,3,4-oxadiazol-2-yl]pentane- Tert-butyl 3-yl}carbamate ( 3 g ) (178 mg, 0.431 mmol) and trifluoroethanol (2.16 mL, 0.2 M) were sealed and then heated to 180 °C in a microwave for 60 min. LCMS analysis showed consumption of starting material. The reaction mixture was concentrated and the residue was purified by flash chromatography ( _Si02 , 100% heptane to 1:10 MeOH/EtOAc) to afford Intermediate 3 (116 mg, 91% yield) as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.26 (dd, J = 0.7, 7.7 Hz, 1H), 7.67 (t, J = 7.9 Hz, 1H), 7.49 (dd, J = 0.7, 7.8 Hz, 1H) , 4.90 - 4.83 (m, 1H), 3.07 - 2.89 (m, 3H), 2.58 - 2.49 (m, 1H), 2.04 - 1.97 (m, 1H), 1.83 - 1.70 (m, 1H), 0.96 (t, J = 7.5 Hz, 3H). LCMS m/z (APCI), 293.0 (M+H) ⁺ for (C ₁₂ H ₁₃ BrN ₄ ). By SFC (10-60% methanol (0.5% NH ₃ ) in carbon dioxide (400-450 bar), gradient time = 2 minutes, flow rate = 4 mL/min, Kromasil (R,R) Whelk-O 50mm* 3mm*1.8µm column) was determined to be 97.4% ee. The stereochemistry was assigned based on the use of ( 2S )-2-[(tertiary butoxycarbonyl)amino]butanoic acid in the first step.

Intermediate 4 : ( S , S )-2-methyl-N-[(1 R )-1-{6-[(2 R )-2-methylpyrrolidin-1-yl]-1-oxo Base-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-4-yl}ethyl]propane-2-sulfinamide Step 1: {2-Chloro-6-[( 2R )-2-methylpyrrolidin-1-yl]pyridin-4-yl}(piperidin-1-yl)methanone ( 4a )

(2,6-dichloropyridin-4-yl)(piperidin-1-yl)methanone (600 mg, 2.32 mmol) and (2 R )-2-methylpyrrolidine (591 mg, 6.95 mmol) in A solution in DMF (1.5 mL) was stirred at 100 °C for 16 hours. LCMS analysis showed consumption of starting material. The reaction was cooled to room temperature, H ₂ O (40 mL) was added, and the reaction was extracted with DCM (3×40 mL). The combined org. layers were dried over _Na2SO4 , filtered _and concentrated. The residue was purified by flash chromatography (24 g SiO ₂ , 0-20% EtOAc/heptane) to afford the title compound ( 4a ) (664 mg, 93% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.44 (d, J = 1.0 Hz, 1H), 6.21 (d, J = 1.0 Hz, 1H), 4.12 (q, J = 7.1 Hz, 1H), 3.72 - 3.62 (m, 2H), 3.54 (ddd, J = 10.5, 7.6, 2.9 Hz, 1H), 3.40 - 3.28 (m, 2H), 2.10 - 2.04 (m, 2H), 1.75 - 1.62 (m, 4H), 1.26 (t, J = 7.2 Hz, 1H), 1.21 (d, J = 6.3 Hz, 2H); m/z (APCI+), 308.2 (M+H) ⁺ for (C ₁₆ H ₂₂ ClN ₃ O).

Step 2: 2-Chloro-6-[( 2R )-2-methylpyrrolidin-1-yl]-4-(piperidine-1-carbonyl)pyridine-3-carbaldehyde ( 4b )

To a solution of DMF (473 mg, 6.47 mmol) in DCM (3.0 mL) was added _POCl3 (992 mg, 6.47 mmol). The mixture was stirred for 10 minutes, and then {2-chloro-6-[( 2R )-2-methylpyrrolidin-1-yl]pyridin-4-yl}(piperidin-1-yl)methanone ( 4a ) (664 mg, 2.16 mmol) in DCM (3.0 mL). The mixture was stirred at reflux for 15 hours. LCMS analysis showed consumption of starting material. The reaction was concentrated to dryness and poured slowly into saturated NaHCO ₃ solution (30 mL). The mixture was extracted with DCM (3 x 30 mL). The combined organics were dried _{over Na2SO4} , filtered and concentrated. The residue was purified by flash chromatography (24 g SiO ₂ , 0-40% EtOAc/heptane) to afford the title compound ( 4b ) (568 mg, 78% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.07 (s, 1H), 5.99 (s, 1H), 4.13 - 4.65 (m, 1H), 3.68 - 3.83 (m, 1H), 3.55 - 3.68 (m, 2H) ), 3.35 - 3.55 (m, 1H), 2.98 - 3.20 (m, 2H), 1.88 - 2.17 (m, 3H), 1.71 - 1.83 (m, 2H), 1.55 - 1.67 (m, 3H), 1.46 - 1.55 (m, 1H), 1.31 - 1.42 (m, 1H), 1.17 - 1.26 (m, 3H); m/z (APCI+), 336.1 (M+H) ⁺ for (C ₁₇ H ₂₂ ClN ₃ O ₂ ).

Step 3: N -[( E )-{2-chloro-6-[( 2R )-2-methylpyrrolidin-1-yl]-4-(piperidin-1-carbonyl)pyridin-3-yl }methylene]-2-methylpropane-2-sulfinamide ( 4c )

2-Chloro-6-[( 2R )-2-methylpyrrolidin-1-yl]-4-(piperidine-1-carbonyl)pyridine-3-carbaldehyde ( 4b ) (432 mg, 1.29 mmol), A mixture of ( R )-(+)-2-methyl-2-propanesulfinamide (187 mg, 1.54 mmol) and Ti(OEt) ₄ (880 mg, 3.86 mmol) in THF (10.0 mL) was Stir at 45°C for 16 hours. LCMS analysis showed about 25% starting material remaining. Additional portions of ( R )-(+)-2-methyl-2-propanesulfinamide (62.4 mg, 0.515 mmol) and Ti(OEt) ₄ (293 mg, 1.29 mmol) were added and the mixture was heated at 50 °C Stirring was continued for 16 hours. LCMS analysis showed consumption of starting material. The reaction was cooled to room temperature. The mixture was diluted with DCM (50 mL) and washed with saturated NaHCO ₃ solution (35 mL) and brine (35 mL). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to give the title compound ( 4c ) (495 mg, 88% yield) as a white gum which was used without further purification. m/z (APCI+), 440.2 (M+H) ⁺ for (C ₂₁ H ₃₁ ClN ₄ O ₂ S).

Step 4: 4-Chloro-6-[( 2R )-2-methylpyrrolidin-1-yl]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridine-1- Ketones ( 4d )

N -[( E )-{2-chloro-6-[(2 R )-2-methylpyrrolidin-1-yl]-4-(piperidin-1-carbonyl)pyridin-3-yl}ylidene A solution of methyl]-2-methylpropane-2-sulfinamide ( 4c ) (495 mg, 1.13 mmol) in THF (15.0 mL) was cooled to 0 °C, and then LiBH ₄ solution (2.0 M in in THF, 620 mL, 1.24 mmol). The mixture was stirred at 0 °C for 2 h and then NaOMe solution (25% in MeOH, 2.5 mL, 10.1 mmol) was added. The reaction was allowed to warm to room temperature and then stirred for 16 hours. The reaction was diluted with DCM (60 mL) and washed with saturated aqueous NH ₄ Cl (60 mL) and brine (60 mL). _The organic layer was dried over _Na2SO4 , filtered and concentrated. The residue was purified by flash chromatography ( _Si02 , 50-100% EtOAc/heptane) to afford the title compound ( 4d ) (199 mg, 70% yield) as a colorless foam. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.68 (s, 1H), 6.45 (s, 1H), 4.35 (s, 2H), 4.21 - 4.14 (m, 1H), 3.58 (ddd, J = 10.5, 7.6 , 2.8 Hz, 1H), 3.39 (q, J = 8.9 Hz, 1H), 2.13 - 1.97 (m, 2H), 1.75 (dt, J = 5.2, 2.6 Hz, 1H), 1.23 (d, J = 6.3 Hz , 3H). Assuming a hydrogen atom is masked by the water peak; m/z (C ₁₂ H ₁₄ ClN ₃ O) of (APCI+), 252.3 (M+H) ⁺ .

Step 5: 6-[( 2R )-2-Methylpyrrolidin-1-yl]-1-oxo-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridine- 4-carbonitrile ( 4e )

4-Chloro-6-[( 2R )-2-methylpyrrolidin-1-yl]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridine-1 - A mixture of ketone ( 4d ) (438 mg, 1.74 mmol), Zn(CN) ₂ (306 mg, 2.6 mmol), DMF (15 mL) and Pd(PPh ₃ ) ₄ (100 mg, 0.09 mmol) was heated to 140 °C for 30 minutes. The reaction was diluted with DCM and filtered. The filter cake was washed with DCM. The filtrate was concentrated in vacuo and the crude compound was purified by flash chromatography ( _Si02 , 0 to 100% 1:1 EtOAc:DCM/heptane) to afford the title compound ( 4e ) (375 mg, 89% yield) as a yellow solid ). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.00 - 6.94 (m, 1H), 6.46 - 6.32 (m, 1H), 4.55 (s, 2H), 4.29 - 4.19 (m, 1H), 3.61 (ddd, J = 2.6, 7.6, 10.3 Hz, 1H), 3.46 - 3.34 (m, 1H), 2.22 - 2.02 (m, 3H), 1.86 - 1.72 (m, 1H), 1.25 (d, J = 6.4 Hz, 3H); m/z (APCI+), 243.1 (M+H) ⁺ for (C ₁₃ H ₁₄ N ₄ O).

Step 6: 4-Acetyl-6-[( 2R )-2-methylpyrrolidin-1-yl]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridine- 1-keto ( 4f )

6-[(2R)-2-Methylpyrrolidin-1-yl]-1-oxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine in an ice/water bath - To a solution of 4-carbonitrile ( 4e ) (355 mg, 1.47 mmol) in THF (15 mL) was added MeMgBr (4.88 mL, 14.7 mmol, 3.0 M in THF). The resulting mixture was stirred at this temperature for 10 minutes, then allowed to warm to room temperature and allowed to stir for 2 hours. The mixture was quenched with 2 N HCl (6 mL) at 0 °C and stirred at room temperature for 15 min. The mixture was neutralized with saturated NaHCO ₃ solution and extracted with DCM (3×40 mL). The combined org. layers were dried over _Na2SO4 , filtered _and concentrated. The crude compound was purified by flash chromatography ( _Si02 , 20-50% EtOAc/heptane) to afford the title compound ( 4f ) (190 mg, 50% yield) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) 6.98 (s, 1H), 6.59 (br. s, 1H), 4.70 (s, 2H), 4.28 (br. t, J = 5.7 Hz, 1H), 3.64 (ddd , J = 2.6, 7.5, 10.1 Hz, 1H), 3.48 - 3.38 (m, 1H), 2.70 (s, 3H), 2.19 - 2.10 (m, 2H), 2.08 - 2.01 (m, 1H), 1.79 (td , J = 2.6, 5.0 Hz, 1H), 1.30 (d, J = 6.2 Hz, 3H); m/z (APCI+) of (C ₁₄ H ₁₇ N ₃ O ₂ ), 260.2 (M+H) ⁺ .

Step 7: Intermediate 4 Add 4-acetyl-6-[( 2R )-2-methylpyrrolidin-1-yl]-2,3-dihydro- 1H -pyrrolo to a 40 mL vial [3,4- c ]pyridin-1-one ( 4f ) (200 mg, 0.77 mmol), ( S )-tertiary butylsulfinamide (187 mg, 1.54 mmol), THF (1.54 mL) and Ti (OEt) ₄ (1.54 mmol, 0.323 mL). The vial was capped and heated to 80°C for 48 hours. The reaction was then cooled to -78 °C (dry ice/acetone bath) and tris-dibutyllithium borohydride (1.54 mmol, 1.54 mL, 1.0 M in THF) was added dropwise. The resulting mixture was stirred at -78°C for 3 hours. The mixture was allowed to warm to room temperature and quenched dropwise with sat. NH ₄ Cl (2 mL), followed by addition of DCM (20 mL) and brine (20 mL). The mixture was filtered through ^Celite® and washed with DCM (40 mL). The organic layer was collected, and the aqueous layer was extracted with DCM (20 mL). The combined org. layers were dried over _Na2SO4 , filtered _and concentrated. The crude product was purified by flash chromatography ( _Si02 , solvent 0-10% MeOH in EtOAc) to afford intermediate 4 (127 mg, 45% yield) as a yellow foam. m/z (APCI+), 365.3 (M+H) ⁺ for (C ₁₈ H ₂₈ N ₄ O ₂ S). Stereochemistry was assigned based on the use of ( 2R )-2-methylpyrrolidine in step 1 and ( S )-tert-butylsulfinamide in step 7.

Intermediate 5 : ( S , R )-2-methyl- N -[(1 R )-1-{6-[(2 R )-2-methylpyrrolidin-1-yl]-1-oxo yl-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl}propyl]propane-2-sulfinamide Step 1: 6-[( 2R )-2-Methylpyrrolidin-1-yl]-4-propionyl-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridine- 1-keto ( 5a )

6-[(2 R )-2-methylpyrrolidin-1-yl]-1-oxo-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridine-4- A solution of forminonitrile ( 4e ) (808 mg, 3.33 mmol) in THF (8.0 mL) was cooled to 0 °C and then washed with ethylmagnesium bromide solution (3.0 M in _Et2O , 11.1 mL, 33.3 mmol) deal with. The mixture was stirred at room temperature for 1 hour. LCMS analysis showed consumption of starting material. The reaction was cooled to 0 °C and quenched by addition of 2 N HCl. The mixture was stirred at room temperature for 10 min, neutralized by addition of saturated NaHCO ₃ solution, and extracted with DCM (2×30 mL). The combined org. layers were dried over _Na2SO4 , filtered _and concentrated. The residue was purified by flash chromatography (24 g _Si02 , 0-100% EtOAc/heptane) to afford the title compound ( 5a ) (646 mg, 71% yield) as a yellow oil. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.01 (s, 1H), 6.41 (br. s, 1H), 4.73 (s, 2H), 4.39 - 4.22 (m, 1H), 3.66 (ddd, J = 2.6 , 7.4, 10.1 Hz, 1H), 3.52 - 3.38 (m, 1H), 3.30 - 3.14 (m, 2H), 2.26 - 2.10 (m, 2H), 2.06 - 2.01 (m, 1H), 1.81 (td, J = 2.5, 5.1 Hz, 1H), 1.31 (d, J = 6.2 Hz, 3H), 1.24 (t, J = 7.3 Hz, 3H); m/z of (C ₁₅ H ₁₉ N ₃ O ₂ ) (APCI+) , 274.2 (M+H) ⁺ .

Step 2: ( S , S )-2-methyl- N -[( 1E )-1-{6-[( 2R )-2-methylpyrrolidin-1-yl]-1-oxo -2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl}propylene]propane-2-sulfinamide ( 5b )

To 6-[(2 R )-2-methylpyrrolidin-1-yl]-4-propionyl-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridine-1- To a solution of ketone ( 5a ) (640 mg, 2.34 mmol) in THF (2.0 mL) was added ( S )-(−)-2-methyll-2-propanesulfinamide (568 mg, 4.68 mmol) and Ti(OEt) ₄ (2.14 g, 9.37 mmol). The mixture was stirred at 90°C for 23 hours. LCMS analysis showed consumption of starting material. The mixture was cooled to room temperature and then brine (40 mL) and DCM (30 mL) were added. The mixture was stirred for 10 minutes and then filtered through ^Celite® . Separate the layers. The aqueous layer was extracted with DCM (30 mL). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated to give the title compound ( 5b ) (779 mg, 88% yield) as a white foam, which was used directly in the next step. m/z (APCI+), 377.2 (M+H) ⁺ for (C ₁₉ H ₂₈ N ₄ O ₂ S).

Step 3: Intermediate 5 converts ( S , S )-2-methyl- N -[( 1E )-1-{6-[( 2R )-2-methylpyrrolidin-1-yl]-1 -Oxy-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl}propylene]propane-2-sulfinamide ( 5b ) (654 mg, 1.40 mmol) in THF (12.0 mL) was cooled to -78 °C and then treated dropwise with lithium tris-dibutylborohydride solution (1.0 M in THF, 2.5 mL, 2.5 mmol). The mixture was stirred at -78°C for 1.5 hours. Additional lithium tris-secondary butylborohydride (1.0 M, 0.417 mL, 0.417 mmol) was added and the mixture was stirred at -78 °C for an additional 1.5 h. The mixture was quenched with MeOH, diluted with brine (50 mL), and extracted with DCM (2×50 mL). The combined org. layers were dried over _Na2SO4 , filtered _and concentrated. The residue was purified by flash chromatography (12 g _Si02 , 0-10% MeOH/DCM) to afford intermediate 5 (300 mg, 57% yield) as a yellow gum. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.66 (s, 1H), 6.48 (s, 1H), 5.14 (d, J = 6.6 Hz, 1H), 4.35 (s, 2H), 4.27 - 4.14 ( m, 2H), 3.59 - 3.44 (m, 1H), 3.29 - 3.18 (m, 1H), 2.14 - 2.01 (m, 2H), 1.98 - 1.81 (m, 3H), 1.73 - 1.60 (m, 1H), 1.20 (d, J = 6.2 Hz, 3H), 1.07 (s, 9H), 0.86 - 0.73 (m, 3H); m/z (APCI+) of (C ₁₉ H ₃₀ N ₄ O ₂ S), 379.2 (M +H) ⁺ .

Stereochemistry based on the use of ( 2R )-2-methylpyrrolidine in step 1 and ( S )-(−)-2-methyl-2-propanesulfinamide in step 2 of the synthesis of intermediate 4 to specify.

Intermediate 10 : ( S , S )-2-methyl-N-[( 1R )-1-{6-[methyl(propan-2-yl)amino]-1-oxo-2, 3-Dihydro-1 H -pyrrolo[3,4- c ]pyridin-4-yl}propyl]propane-2-sulfinamide Step 1: 6-[Methyl(propan-2-yl)amino]-1-oxo-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridine-4-carbonitrile

4-Chloro-6-[methyl(propan-2-yl)amino]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-1-one ( 9d ) (706 mg, 2.95 mmol), Zn(CN) ₂ (519 mg, 4.42 mmol), DMF (10 mL) and Pd(PPh ₃ ) ₄ (170 mg, 0.147 mmol) was heated to 140°C for 30 min . The reaction was diluted with DCM and filtered. The filter cake was washed with DCM. The filtrate was concentrated in vacuo to afford the title compound ( 10a ) (544 mg, 80% yield) as a yellow solid, which was used without further purification. ¹ H NMR (400 MHz, DMSO- d ₆ ) 9.01 (s, 1H), 7.13 (s, 1H), 4.80 (td, J = 6.7, 13.4 Hz, 1H), 4.47 (s, 2H), 2.89 (s , 3H), 1.15 (d, J = 6.7 Hz, 6H); m/z (APCI+), 231.2 (M+H) ⁺ for (C ₁₂ H ₁₄ N ₄ O).

Step 2: 6-[Methyl(propan-2-yl)amino]-4-propionyl-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-1-one ( 10b )

6-[methyl(propane-2-yl)amino]-1-oxo-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridine-4-carbonitrile ( 10a ) (511 mg, 2.22 mmol) in THF (20.0 mL) was cooled to 0 °C and then treated with ethylmagnesium bromide solution (3.0 M in _Et2O , 7.40 mL, 22.2 mmol). The mixture was stirred at room temperature for 1 hour. LCMS analysis showed consumption of starting material. The reaction was cooled to 0 °C and quenched by addition of saturated _NH4Cl (60 mL), then extracted with DCM (2 x 80 mL). The combined org. layers were dried over _Na2SO4 , filtered _and concentrated. The residue was purified by flash chromatography (24 g _Si02 , 0 to 100% EtOAc/heptane) to afford the title compound ( 10b ) (125 mg, 22% yield) as a yellow foam. m/z (APCI+), 262.2 (M+H) ⁺ for (C ₁₄ H ₁₉ N ₃ O ₂ ).

Step 3: Intermediate 10 to 6-[methyl(propan-2-yl)amino]-4-propionyl-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridine- To a solution of 1-ketone ( 10b ) (124 mg, 0.475 mmol) in THF (6.0 mL) was added ( S )-(−)-2-methyl-2-propanesulfinamide (92 mg, 0.759 mmol ) and Ti(OEt) ₄ (433 mg, 1.90 mmol). The mixture was stirred at reflux for 42 hours. LCMS analysis showed consumption of starting material. The mixture was cooled to -78 °C and then treated dropwise with a solution of lithium tris-dibutylborohydride (1.0 M in THF, 1.9 mL, 1.90 mmol). The mixture was stirred at -78°C for 4 hours. The mixture was quenched with MeOH, diluted with brine (30 mL), and extracted with DCM (20 x 2 mL). The combined org. layers were dried over _Na2SO4 , filtered _and concentrated. The residue was purified by flash chromatography (12 g _Si02 , 0-10% MeOH/DCM) to afford intermediate 10 (72 mg, 41% yield) as a yellow solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.67 (s, 1H), 6.64 (s, 1H), 5.25 (d, J = 6.7 Hz, 1H), 4.94 (quin, J = 6.7 Hz, 1H) , 4.34 (s, 2H), 4.21 (q, J = 6.7 Hz, 1H), 2.84 (s, 3H), 1.98 - 1.80 (m, 2H), 1.13 (dd, J = 3.6, 6.7 Hz, 6H), 1.06 (s, 9H), 0.83 (t, J = 7.3 Hz, 3H). m/z (APCI+), 367.2 (M+H) ⁺ for (C ₁₈ H ₃₀ N ₄ O ₂ S). The stereochemistry was assigned based on the use of ( S )-(−)-2-methyl-2-propanesulfinamide in step 2.

Intermediate 13 : ( S , S )-2-methyl- N -[( 1R )-1-{6-[methyl(propan-2-yl)amino]-1-oxo-2, 3-Dihydro-1 H -pyrrolo[3,4- c ]pyridin-4-yl}ethyl]propane-2-sulfinamide Step 1: 4-Acetyl-6-(isopropyl(methyl)amino)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one

Methylmagnesium bromide (9.11 g, 76.4 mmol, 25.9 mL, 3.0 M) was added to 6-(isopropyl(methyl)amino)-1-oxo-2,3-di Hydrogen- 1H -pyrrolo[3,4- c ]pyridine-4-carbonitrile ( intermediate 10a ) (1.76 g, 7.64 mmol) in THF (70 mL, c=0.11 M) in solution. The mixture was stirred at this temperature for 10 minutes and then warmed to room temperature and stirred for 3 hours. The mixture was then quenched dropwise with 2 M HCl (7 mL) at room temperature and stirred for 30 min. A saturated solution of NaHCO ₃ (70 mL) was added to neutralize the solution and extracted with DCM (3×100 mL). The combined organic layers were dried _{over Na2SO4} and concentrated in vacuo. The resulting residue was purified by flash chromatography (40 g _Si02 , 10-40% EtOAc/heptane) to afford the title compound ( 13a ) (553 mg, 29% yield) as a yellow solid. ¹ H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H) 7.06 (s, 1H) 4.88 (br. d, J = 6.72 Hz, 1H) 4.49 (s, 2H) 2.94 (s, 3H) 2.61 (s, 3H) 1.19 (d, J = 6.60 Hz, 6H); m/z (APCI+), 248.2 (M+H) ⁺ for (C ₁₃ H ₁₇ N ₃ O ₂ ).

Step 2: Intermediate 13 4-Acetyl-6-[methyl(propan-2-yl)amino]-2,3-dihydro-1 H -pyrrolo[3,4-c]pyridine-1 -A mixture of ketone ( 13a ) (8.10 g, 32.75 mmol), ( S )-tertiary butylsulfinamide (7.94 g, 65.5 mmol) in Ti(OEt) ₄ (29.9 g, 131 mmol) in THF (100 mL) at 90 °C under _N2 for 72 h, and the reaction was monitored by LCMS. The reaction was then cooled to 0°C and lithium tris-dibutylborohydride (1 M in THF, 131 mmol, 131 mL) was added dropwise at 0°C. The mixture was stirred at 0°C for 3 hours. The mixture was quenched with saturated _NH4Cl (200 mL) and brine (200 mL) at 0 to 5 °C. The suspension was filtered through a pad of ^Celite® , and the filter cake was washed with DCM (500 mL). The filtrate layers were separated and the aqueous layer was extracted with DCM (2 x 100 mL). The combined organic layers were washed with brine (250 mL), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by flash chromatography ( _Si02 , 0-10% MeOH/DCM) to afford intermediate 13 (10 g, 87%) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.83 (br. s, 1H), 6.75 (s, 1H), 4.89 - 4.76 (m, 1H), 4.48 - 4.39 (m, 1H), 4.37 - 4.26 (m , 3H), 2.83 (s, 3H), 1.55 (d, J = 6.6 Hz, 3H), 1.19 - 1.07 (m, 15H). m/z (APCI+), 353.2 (M+H) ⁺ for (C ₁₇ H ₂₈ N ₄ O ₂ S). Stereochemistry is assigned based on the use of ( S )-tert-butylsulfinamide in Step 1.

Intermediate 14 : ( S , R )-2-methyl- N -[( 1S )-1-{6-[( 2R )-2-methylpyrrolidin-1-yl]-1-oxo Base-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-4-yl}ethyl]propane-2-sulfinamide

4-Acetyl-6-[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyrrolidin-1-one A mixture of ( 4f ) (4000 mg, 15.43 mmol), ( R )-2-methylpropane-2-sulfinamide (2240 mg, 18.5 mmol) in Ti(OEt) ₄ (20 mL) in N ₂ The mixture was stirred at 100°C for 20 hours. LCMS analysis showed consumption of starting material. The yellow solution was cooled to 0 °C and diluted with THF (50 mL). Lithium tris-secondary butylborohydride (38.6 mmol, 38.6 mL, 1M in THF) was added dropwise at 0 °C. The mixture was then stirred at 10°C for 20 hours. LCMS showed starting material remaining. Additional lithium tris-secondary butylborohydride (38.6 mmol, 38.6 mL, 1M in THF) was added dropwise at 0°C and the mixture was stirred at 10°C for another 2 hours. TLC analysis showed consumption of starting material. The mixture was quenched with saturated aqueous _NH4Cl (200 mL) at 0 to 5 °C. The suspension was filtered through a pad of ^Celite® , and the filter cake was washed with EtOAc (2 x 50 mL). The filtrate layers were separated and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine (30 mL), dried over Na ₂ SO ₄ , filtered and concentrated to give a residue. The residue was first purified by flash chromatography (SiO ₂ , EtOAc/MeOH=10:1) and then by preparative HPLC (ACSSH-CA; method column: YMC Triart C18 250*50 mm*7 μm; water ( 0.05 v/v% ammonium hydroxide)-ACN to give intermediate 14 (2.5 g, 44%) as an off-white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.70 (s, 1H), 6.47 (br s, 1H), 4.57 - 4.34 (m, 4H), 4.20 (br. t, J = 5.9 Hz, 1H), 3.64 - 3.53 (m, 1H), 3.47 - 3.35 (m, 1H), 2.20 - 1.99 (m, 3H), 1.77 (br. dd, J = 2.5, 4.7 Hz, 1H), 1.64 (d, J = 6.5 Hz, 3H), 1.28 (d, J = 6.2 Hz, 3H), 1.22 (s, 9H). m/z (ESI) for (C ₁₈ H ₂₈ N ₄ O ₂ S), 365.1 (M+H) ⁺ . Stereochemistry was assigned based on the use of ( R )-tertiary butylsulfinamide.

Example 1 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro- 1 H -pyrrolo[3,4- c ]pyridin-1-one Step 1: ( S , S )-2-methyl-N-[(1R)-1-(2-{6-[( 5S )-5-methyl-6,7-dihydro- 5H- Pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]- 1-oxo-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-4-yl)propyl]propane-2-sulfinamide

( S , S )-2-methyl- N -[(1 R )-1-{6-[(2 R )-2-methylpyrrolidin-1-yl]-1-oxo-2, 3-Dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl}propyl]propane-2-sulfinamide ( Intermediate 5 ) (63.0 mg, 0.17 mmol), (5 S )-3-(6-bromopyridin-2-yl)-5-methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazole ( Intermediate 2) (147 mg, 0.528 mmol), K ₃ PO ₄ (336 mg, 1.59 mmol), Pd ₂ (dba) ₃ (30.4 mg, 0.0528 mmol) and 4,5-bisdiphenylphosphine-9, A mixture of 9-dimethylxanthene (XantPhos) (61.1 mg, 0.106 mmol) in 1,4-dioxane (4.5 mL, c=0.1 M) in a 40 mL vial (capped) at 100°C (heat block temperature) for 18 hours. The mixture was filtered through ^Celite® , washing with DCM. The filtrate was concentrated in vacuo and the crude compound was purified by flash chromatography ( _Si02 , 0 to 10% MeOH/DCM) to afford the title compound (194 mg, 64% yield) as a pale yellow foam. m/z (APCI+) for, 577.3 (M+H) ⁺ for (C ₂₉ H ₄₀ N ₈ O ₂ S).

Step 2: Example 1 A solution of 4 N HCl in 1,4-dioxane (1.01 mmol, 0.252 mL, 4 M) was added to ( S , S )-2-methyl-N-[(1R)- 1-(2-{6-[( 5S )-5-methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazole-3- Base]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-1-oxo-2,3-dihydro-1 H -pyrrolo[3, 4- c ]pyridin-4-yl)propyl]propane-2-sulfinamide (194 mg, 0.336 mmol) in suspension in MeOH (12 mL). The resulting mixture was stirred at room temperature for 2 hours. Volatiles were removed under reduced pressure. The crude product was purified by chiral SFC (Phenomenex Lux cellulose-1 4.6×100 mm 3 µm column, 30% MeOH + 10 mM NH ₃ in CO ₂ at 120 bar, 4 mL/min) to give Example 1 (146 mg, 92% yield, >99% de) as a yellow solid. ¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.48 (d, J = 8.3 Hz, 1H), 8.01 (t, J = 8.0 Hz, 1H), 7.91 (d, J = 7.7 Hz, 1H), 6.67 (s, 1H), 5.22 - 5.03 (m, 3H), 4.33 - 4.21 (m, 2H), 3.60 - 3.50 (m, 1H), 3.41 - 3.32 (m, 1H), 3.06 - 2.98 (m, 1H) , 2.97 - 2.89 (m, 1H), 2.88 - 2.76 (m, 1H), 2.37 - 2.27 (m, 1H), 2.09 - 1.86 (m, 5H), 1.69 - 1.62 (m, 1H), 1.44 (d, J = 6.4 Hz, 3H), 1.15 (d, J = 6.2 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H); m/z (APCI+) for (C ₂₆ H ₃₂ N ₈ O), 473.2 (M+H) ⁺ ; [α]D ₂₂ = +37.0° (c=0.1 M, MeOH) .

Example 2 4-[(1 R )-1-aminoethyl]-2-{6-[(5 S )-5-ethyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro- 1 H -pyrrolo[3,4- c ]pyridin-1-one Step 1: ( S , S )-N-[(1 R )-1-(2-{6-[(5 S )-5-ethyl-6,7-dihydro- 5H -pyrrolo[2 ,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-1-oxo Base-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl)ethyl]-2-methylpropane-2-sulfinamide

Towards ( S , S )-2-methyl-N-[(1 R )-1-{6- _[ (2 R )-2-methylpyrrolidin-1-yl]-1-side under N 2 Oxy-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl}ethyl]propane-2-sulfinamide ( Intermediate 4) (50 mg, 0.14 mmol ), (5 S )-3-(6-bromopyridin-2-yl)-5-ethyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4 ] To a mixture of triazole ( intermediate 3) (42.2 mg, 0.144 mmol) and K ₃ PO ₄ (87.4 mg, 0.412 mmol) in 1,4-dioxane (3 mL) was added Pd ₂ (dba) ₃ (12.6 mg, 0.014 mmol) and 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (15.9 mg, 0.027 mmol). After the addition, the mixture was bubbled through _N2 for 2 min. The resulting mixture was sealed and stirred at 85°C for 18 hours. The reaction mixture was concentrated in vacuo, and the residue was purified by flash chromatography ( _Si02 , 10% EtOAc/MeOH) to afford the title compound (60 mg, 76% yield) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.70 (d, J = 8.2 Hz, 1H), 8.18 (d, J = 7.4 Hz, 1H), 7.93 (t, J = 8.0 Hz, 1H), 6.76 (s , 1H), 5.11 - 5.02 (m, 2H), 4.99 - 4.93 (m, 1H), 4.61 - 4.49 (m, 2H), 4.34 - 4.27 (m, 1H), 3.67 - 3.57 (m, 1H), 3.46 - 3.35 (m, 1H), 3.11 - 3.01 (m, 3H), 2.65 (td, J = 3.7, 7.9 Hz, 1H), 2.19 - 2.08 (m, 3H), 2.07 (s, 1H), 1.86 - 1.79 (m, 1H), 1.79 (br. d, J = 2.3 Hz, 1H), 1.69 (d, J = 6.4 Hz, 3H), 1.31 - 1.28 (m, 3H), 1.22 (s, 9H), 1.02 ( t, J = 7.5 Hz, 3H); m/z (ESI+) of (C ₃₀ H ₄₀ N ₈ O ₂ S), 577.5 (M+H) ⁺ ; [α] D ₂₂ = +85.3° (c=0.1 M, MeOH).

Step 2: Example 2 at 0 ° C to ( S , S )-N-[(1 R )-1-(2-{6-[(5 S )-5-ethyl-6,7-dihydro- 5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1- Base]-1-oxo-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl)ethyl]-2-methylpropane-2-sulfinamide (60 mg, 0.10 mmol) in EtOAc (5 mL) was added dropwise with 4 M HCl in EtOAc (3 mL). The mixture was stirred at 20°C for 1 hour. The reaction mixture was concentrated in vacuo. EtOAc (10 mL) and water (10 mL) were added to the residue. The aqueous layer was basified with saturated NaHCO ₃ and extracted with DCM (10 mL×3). The combined organic layers were washed with brine (20 mL), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was lyophilized for 16 hours to afford Example 2 (47 mg, 95%) as a pale yellow solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.59 (d, J = 8.3 Hz, 1H), 8.09 - 8.02 (m, 1H), 8.02 - 7.97 (m, 1H), 6.58 (s, 1H), 5.33 (d, J = 17.1 Hz, 1H), 5.10 (d, J = 17.1 Hz, 1H), 4.99 (br. t, J = 6.1 Hz, 1H), 4.29 - 4.19 (m, 1H), 4.12 (q , J = 6.9 Hz, 1H), 3.55 (br. t, J = 8.0 Hz, 1H), 3.04 - 2.85 (m, 3H), 2.58 - 2.55 (m, 1H), 2.11 - 1.96 (m, 4H), 1.79 - 1.66 (m, 2H), 1.36 (d, J = 6.5 Hz, 3H), 1.24 - 1.20 (m, 3H), 0.93 (t, J = 7.4 Hz, 3H); (C ₂₆ H ₃₂ N ₈ O ), m/z (ESI+), 473.4 (M+H) ⁺ .

Example 3 4-[( 1S )-1-aminoethyl]-2-{6-[( 5S )-5-ethyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-2,3-dihydro- 1 H -pyrrolo[3,4- c ]pyridin-1-one Step 1: ( S , R )-N-[(1 S )-1-(2-{6-[(5 S )-5-ethyl-6,7-dihydro- 5H -pyrrolo[2 ,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1-yl]-1-oxo Base-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl)ethyl]-2-methylpropane-2-sulfinamide

Towards ( S , R )-2-methyl-N-[(1 S )-1-{6-[( ₂ R )-2-methylpyrrolidin-1-yl]-1-side under N 2 Oxy-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl}ethyl]propane-2-sulfinamide ( intermediate 14 ) (2000 mg, 5.487 mmol ), (5 S )-3-(6-bromopyridin-2-yl)-5-ethyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4 ] To a suspension of triazole ( intermediate 3 ) (1610 mg, 5.49 mmol) and K ₃ PO ₄ (3490 mg, 16.5 mmol) in 1,4-dioxane (30 mL) was added Pd ₂ (dba) ₃ (502 mg, 0.549 mmol) and 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (635 mg, 1.10 mmol). After the addition, the mixture was bubbled with argon for 2 minutes. The resulting mixture was sealed and stirred at 85°C for 18 hours. TLC analysis showed consumption of starting material. The mixture was diluted with _H2O (100 mL) with stirring and the resulting suspension was filtered. The aqueous filtrate was separated. The solid was washed with DCM (100 mL). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to give a yellow residue (3 g), which was purified by flash chromatography (SiO ₂ , 0 to 10% MeOH/EtOAc) to give a yellow solid. The solid was dissolved in DCM (50 mL) and silica-SH (4 g) was added. The yellow mixture was refluxed for 20 minutes. The mixture was filtered, and the filter cake was washed with DCM/MeOH (10:1). The filtrate was concentrated. Treatment with silica-SH was repeated three more times to afford the title compound (2.3 g, 72.7%) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.71 (dd, J = 0.7, 8.4 Hz, 1H), 8.18 (dd, J = 0.7, 7.6 Hz, 1H), 7.97 - 7.87 (m, 1H), 6.76 ( s, 1H), 5.19 - 5.11 (m, 1H), 5.03 - 4.94 (m, 2H), 4.65 - 4.57 (m, 1H), 4.57 - 4.51 (m, 1H), 4.24 (br t, J = 6.1 Hz , 1H), 3.66 - 3.56 (m, 1H), 3.48 - 3.39 (m, 1H), 3.13 - 2.98 (m, 3H), 2.71 - 2.59 (m, 1H), 2.21 - 2.03 (m, 4H), 1.87 - 1.75 (m, 2H), 1.68 (d, J = 6.5 Hz, 4H), 1.30 (d, J = 6.2 Hz, 3H), 1.24 (s, 9H), 1.02 (t, J = 7.5 Hz, 3H) . m/z (ESI), 577.5 (M+H) ⁺ for (C ₃₀ H ₄₀ N ₈ O ₂ S).

Step 2: Example 3 at 0 ° C to ( S , R )-N-[(1 S )-1-(2-{6-[(5 S )-5-ethyl-6,7-dihydro- 5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1- Base]-1-oxo-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl)ethyl]-2-methylpropane-2-sulfinamide (2300 mg, 3.988 mmol) in EtOAc (10 mL) was added 4 M HCl in EtOAc (20 mL). After the addition, the mixture was stirred at 15°C for 1 hour. The reaction was monitored by LCMS. The resulting yellow suspension was concentrated to give a residue which was dissolved in H ₂ O (30 mL) and extracted with EtOAc (25 mL). The aqueous layer was basified to about pH 8 with saturated NaHCO ₃ solution, and extracted with DCM (3×35 mL). The combined organic layers were washed with brine (20 mL), dried over Na ₂ SO ₄ , filtered and concentrated to give a solid, to which DCM (50 mL) was added. The solution was filtered four times through a glass microfibre filter GF/F (approximately 0.7 µm). The filtrate was concentrated to obtain a yellow solid, which was analyzed by preparative HPLC (column: Agela DuraShell C18 150*40mm*5 μm, water (0.05 v/v% ammonium hydroxide)-CAN, gradient time 12 minutes, flow rate 25 mL/min ) to purify. This provided Example 3 (1.5 g, 79.6%) as a yellow solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.55 (d, J = 8.0 Hz, 1H), 8.08 - 7.90 (m, 2H), 6.51 (s, 1H), 5.26 (d, J = 16.8 Hz, 1H), 5.08 - 4.92 (m, 2H), 4.23 (br. t, J = 5.8 Hz, 1H), 4.09 (q, J = 6.5 Hz, 1H), 3.53 (br. t, J = 7.5 Hz, 1H ), 3.30 - 3.23 (m, 1H), 3.07 - 2.83 (m, 3H), 2.62 - 2.53 (m, 1H), 2.16 - 1.85 (m, 6H), 1.77 - 1.64 (m, 2H), 1.33 (d , J = 6.5 Hz, 3H), 1.21 (d, J = 6.0 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H). m/z (ESI), 473.4 (M+H) ⁺ for (C ₂₆ H ₃₂ N ₈ O).

Example 4 4-[(1 R )-1-aminoethyl]-2-{6-[(5 S )-5-ethyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro- 1H -pyrrole And[3,4- c ]pyridin-1-one Step 1: ( S , S )-N-[(1 R )-1-(2-{6-[(5 S )-5-ethyl-6,7-dihydro- 5H -pyrrolo[2 ,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-1-oxo-2, 3-Dihydro-1 H -pyrrolo[3,4- c ]pyridin-4-yl)ethyl]-2-methylpropane-2-sulfinamide

( S , S )-2-methyl-N-[( 1R )-1-{6-[methyl(propan-2- _yl )amino]-1-oxo-2 ,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl}ethyl]propane-2-sulfinamide ( intermediate 13 ) (627 mg, 1.78 mmol), (5 S )-3-(6-bromopyridin-2-yl)-5-ethyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazole ( Intermediate 3 ) (521 mg, 1.78 mmol), K ₃ PO ₄ (1.13 mg, 5.34 mmol), Pd ₂ (dba) ₃ (102 mg, 0.178 mmol) and 4,5-bisdiphenylphosphine-9, A solution of 9-dimethylxanthene (206 mg, 0.356 mmol) in 1,4-dioxane (17.8 mL, c=0.1 M) was heated in a 100 mL flask with a condenser at 100 °C for 18 Hour. The mixture was cooled to room temperature, filtered and washed with DCM (20 mL). The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (24 g, _Si02 , 0 to 10% MeOH/DCM) to afford the title compound (808 mg, 80%) as a light yellow solid. m/z (APCI+), 565.3 (M+H) ⁺ for (C ₂₉ H ₄₀ N ₈ O ₂ S).

Step 2: Example 4 A 4 N solution of HCl in 1,4-dioxane (1.07 mL, 4.29 mmol) was added to ( S , S )-N-[( 1R )-1-(2-{6 -[(5 S )-5-Ethyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl }-6-[methyl(propan-2-yl)amino]-1-oxo-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-4-yl)ethyl Di]-2-methylpropane-2-sulfinamide (808 mg, 1.43 mmol) in MeOH (14.3 mL, c=0.1 M). The mixture was stirred at room temperature for 2 hours. Volatile materials were removed under reduced pressure. The crude product was purified by chiral SFC (Phenomenex Lux cellulose-1 21×250 mm column, 30% MeOH + 10 mM NH ₃ in CO ₂ , maintained at 120 bar, 100 mL/min) to give Example 4 (345 mg, 52% yield, >99% de, >99% pure) as a yellow solid. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.56 (dd, J = 1.0, 8.1 Hz, 1H), 8.07 - 8.01 (m, 1H), 8.00 - 7.93 (m, 1H), 6.78 (s, 1H ), 5.34 - 5.28 (m, 1H), 5.17 - 5.10 (m, 1H), 5.02 - 4.95 (m, 1H), 4.89 (td, J = 6.7, 13.4 Hz, 1H), 4.18 (q, J = 6.7 Hz, 1H), 3.01 - 2.94 (m, 2H), 2.93 (s, 3H), 2.62 - 2.54 (m, 2H), 2.09 - 1.97 (m, 1H), 1.84 - 1.72 (m, 1H), 1.41 ( m/z of (C ₂₅ H ₃₂ N ₈ O) ( APCI +) , 461.3 (M+H) ⁺ . [α]D ₂₂ = +120.5° (c=0.1 M, MeOH) .

Example 5 4-[(1 R )-1-aminopropyl]-2-{3-[(5 S )-5-methyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro- 1H -pyrrolo[3 ,4- c ]pyridin-1-one Step 1: ( S,S )-2-Methyl-N-[( 1R )-1-(2-{3-[( 5S )-5-methyl-6,7-dihydro- 5H -Pyrrolo[2,1- c ][1,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-1-side oxy- 2,3-Dihydro-1 H -pyrrolo[3,4- c ]pyridin-4-yl)propyl]propane-2-sulfinamide

( S , S )-2-methyl- N- (1-{6-[methyl(propan-2-yl)amino]-1-oxo-2,3-dihydro-1 H - Pyrrolo[3,4- c ]pyridin-4-yl}propyl]propane-2-sulfinamide ( Intermediate 10 ) (63 mg, 0.17 mmol), ( 5S )-3-(3-bromo Phenyl)-5-methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazole ( Intermediate 2 ) (48 mg, 0.172 mmol), K ₃ PO ₄ (109 mg, 0.516 mmol), Pd ₂ (dba) ₃ (9.88 mg, 0.0172 mmol) and 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (19.9 mg, 0.0344 mmol) in 1,4-dioxane (4.5 mL, c=0.1 M) was heated in a 40 mL vial (capped) at 100 °C for 18 h. The volatiles were removed under reduced pressure. The residue was borrowed from Purification by flash chromatography (12 g, SiO ₂ , 0-10% MeOH/DCM) afforded the title compound (74.0 mg, 76%) as a pale yellow foam. (C ₂₉ H ₄₀ N ₈ O ₂ S) m/z (APCI+), 565.3 (M+H) ⁺ .

Step 2: Example 5 A 4 N solution of HCl in 1,4-dioxane (0.164 mL, 0.655 mmol) was added to ( S,S )-2-methyl-N-[( 1R )-1- (2-{3-[(5 S )-5-methyl-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl] Phenyl}-6-[methyl(propan-2-yl)amino]-1-oxo-2,3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-4-yl )Propyl]propane-2-sulfinamide (74 mg, 0.13 mmol) in MeOH (5.0 mL, c=0.026 M). The mixture was stirred at room temperature for 2 hours. Volatiles were removed under reduced pressure. The crude product was filtered by chiral SFC (Phenomenex Lux cellulose-1 4.6 x 100 mm 3 µm column with 5-60% MeOH + 10 mM NH ₃ in CO ₂ over 3.0 min at 120 bar, 4 mL /min) to afford Example 5 (11.6 mg, 19% yield, >99% de, >95% pure) as a pale yellow solid. ¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.54 (d, J = 8.3 Hz, 1H), 8.35 (br. s, 2H), 8.07 (t, J = 8.0 Hz, 1H), 8.00 - 7.90 ( m, 1H), 6.91 (d, J = 0.9 Hz, 1H), 5.27 - 5.07 (m, 3H), 5.01 (br. s, 1H), 4.37 (br. s, 1H), 3.14 - 2.97 (m, 2H), 2.94 (d, J = 1.5 Hz, 3H), 2.92 - 2.85 (m, 1H), 2.42 - 2.35 (m, 1H), 2.06 - 1.91 (m, 2H), 1.50 (d, J = 6.1 Hz , 3H), 1.19 (d, J = 6.6 Hz, 3H), 1.15 (d, J = 6.6 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H); (C ₂₅ H ₃₂ N ₈ O) m/z (APCI+), 461.3 (M+H) ⁺ . [α]D ₂₂ = +75.9° (c=0.2 M, MeOH) .

Example 6 In Vitro Growth Inhibition Study of the Combination of the HPK1 Inhibitor PF - 07265028 and the Anti -PD1 Antibody Sasanlimab and evaluating the potential of PF-07265028 as a cancer treatment option in the presence or absence of the anti-PD1 antibody (saxolizumab) of the present invention.

Materials and Methods Cell Culture : To generate tumor-reactive T cells, CD4 or CD8 T cells were isolated from PBMC using the EasySep Human T Cell Isolation Kit (obtained from StemCell Technologies) protocol and incubated with mitomycin C-treated A375 Cells (treated with 10 μg/mL Mitomycin C for 2 hours; Mitomycin C was obtained from StemCell Technologies) were cultured in X-VIVO™ 15 medium (obtained from Lonza Walkersville, Inc.). After 10 minutes, 20 ng/mL recombinant human IL-2 (IS) and 10 ng/mL recombinant human IL-7 (obtained from Miltenyi Biotec) were added to expand the activated T cells, and the expansion continued in the G-Rex culture vessel. Add 21 days. The resulting cells were cryopreserved in CryoStor CS10 (obtained from StemCell).

A375 human melanoma cells were passaged in Gibco's Modified Eagle Medium (DMEM) containing 10% FBS and 1% penicillin/streptomycin. Prior to co-culture analysis, A375 cell lines were transduced with Incucyte NucLight green lentivirus (nucGFP) (from Sartorius) and selected by 1 μg/mL puromycin (from Sigma).

Tumor Killing Assay : A375 cells expressing nucGFP were seeded in ImageLock 96-well culture plates at 5,000 to 10,000 cells per well in 100 μL of XVIVO™ 15 (assay medium) with 10% human serum AB. Cells were incubated for 24 hours at 37°C and 5% CO ₂ in a humidified incubator. The next day, frozen CD4 and CD8 cells were thawed in assay medium at 37°C and allowed to settle for 2 hours. At the same time, the medium of the pre-inoculated A375 cells was replaced with 50 μL of PBS containing 3× phospholipid binding protein V red staining reagent and 3 mM CaCl ₂ . PF-07265028 at a concentration of approximately 8 to 300 nM in 50 μL assay medium and/or 2 μg/mL saxenlimab was added to each well, and T cells present in 50 μL assay medium were also added. Equal amounts of CD4 and CD8 T cells were mixed and then added to each well. Wells contained 2, 4, 5, 10 or 20 T cells per tumor cell, depending on the ratio specified. Plates were then transferred to an Incucyte S3 imager and monitored for 4 days.

Data Analysis : The Incucyte S3 imager monitored A375 proliferation via nuclear GFP and apoptosis via phospholipase V red stain and reported the average number of green and red targets per field.

Calculate the A375 clearance percentage using the following formula:

Calculate the percent apoptosis of A375 cells using the following formula:

Results : Tumor reactive T cells against the A375 cell line were generated and then co-cultured with A375 tumor cells expressing nucGFP in the presence of an apoptotic marker (phospholipid binding protein V red, obtained from Sartorius). Expansion and loss of A375 cells were monitored over a four day period by imaging the frequency of GFP positive cells. A375-nucGFP cells were counted by examining the number of green nuclei by fluorescence microscopy. Cells were treated with 111 nM PF-07265028 alone, 2 μg/mL saxanlimab alone, or a combination of these agents. Controls were A375 cells grown in the presence of no T cells or mock-treated T cells (in DMSO). The effect of PF-07265028 alone and in combination with saxolizumab on the growth of A375 tumor cells is shown in FIG. 1 . Inoculate according to the ratio of six T cells for each tumor cell. Each value is the mean of six replicate experiments, where error bars represent SEM.

The calculation of % clearance of A375 is described in Methods and reports the reduction of A375-nucGFP cells at 96 hours. The increase in PF-07265028 alone or in combination with 2 μg/mL saxolizumab was assessed. Figure 2 shows the dose response of A375 tumor cell growth in T cell co-cultures to PF-07265028 alone and in combination with saxenlimab. Control groups show growth of A375 cells in the presence of DMSO treatment (mock) or DMSO with saxenlimab. Each value is the mean of six replicate experiments, where error bars represent SEM.

To assess the potential benefit of the combination of PF-07265028 and saxenlimab, co-cultures of T cells and tumor cells were evaluated in the presence of saxenlimab and increasing concentrations of PF-07265028. Tumor-reactive T cells were co-cultured with A375-nucGFP cells in the presence of an apoptotic marker (phospholipase V red) as described above. Figure 3 shows the effect of PF-07265028 alone and in combination with saxolizumab on the growth of A375 apoptotic tumor cells in T cell co-cultures. In co-cultures of T cells and tumor cells, PF-07265028 increased the number of apoptotic tumor cells. Apoptotic A375-nucGFP tumor cells were imaged by phospholipase V red stain, and % A375 apoptotic cells after 96 hours of co-culture were calculated as described in Methods. The increase in PF-07265028 alone or in combination with 2 μg/mL saxolizumab was assessed. Control groups show growth of A375 cells in the presence of DMSO treatment (mock) or DMSO with saxenlimab. Each value is the mean of six replicate experiments, with error bars depicting SEM.

In co-cultures of T cells and tumor cells, PF-07265028 increased the amount of IFNγ produced by T cells. After 96 hours of co-cultivation, supernatants were collected and analyzed by MSD quantitative immunoassay. The increase in PF-07265028 alone or in combination with 2 μg/mL saxolizumab was assessed. Figure 4 shows the effect of PF-07265028 alone and in combination with saxenlimab on IFNγ production in T cells co-cultured with tumor cells. Control groups show growth of A375 cells in the presence of DMSO treatment (mock) or DMSO with saxenlimab. Each value is the mean of six replicate experiments, where error bars represent SEM.

Control of tumor cell growth by T cells and PF - 07265028 It was observed that addition of 300 nM PF-07265028 resulted in 33.9% A375 clearance after 96 hours, whereas mock-treated co-cultures only cleared 9.4% A375 ( Table 1 , Fig . 1 ). Clearance of A375 cells was due in part to increased apoptosis of A375 cells as measured by phospholipid binding protein V red stain. While mock-treated T cells produced 22.2% A375 apoptosis after 96 hours, addition of 300 nM PF-07265028 produced 31.9% ( Table 1 ). Consistent with increased T cell activity, an increase in IFNγ was observed from 8,165 pg/mL in the presence of mock-treated T cells to 15,651 pg/mL after 300 nM PF-07265028 ( Table 1 ). Antitumor dose responses were observed after incubation with PF-07265028 , the values of which are listed in Table 1 and plotted in Figures 2 , 3 and 4 . Table 1 : Enhanced control of tumor cells by T cells in the presence of PF-07265028 at 96 hours Concentration of PF-07265028 (nM) A375 Clearance % SEM A375 cell apoptosis % SEM IFNγ (pg/mL) SEM 0 9.4 2.4 22.2 1.0 8165 488 8 13.6 2.8 24.1 0.6 8640 410 12 15.0 1.9 24.0 1.0 8208 346 25 14.3 5.0 24.4 2.4 8567 357 50 17.9 2.2 23.8 0.8 9656 295 83 28.6 3.3 29.2 1.7 10856 502 111 23.4 2.7 26.7 1.6 11924 517 300 33.9 4.9 31.9 3.2 15651 272 The values listed in the above table are plotted in Figure 2 , Figure 3 and Figure 4 and represent the mean and SEM of six replicate experiments. A375 % clearance and % apoptosis were based on live cell imaging of A375 tumor cells and calculated as described in Methods. IFNγ was measured by MSD immunoassay.

Effects of PF - 07265028 and saxenlimab in controlling tumor cell growth. Sassenlimab alone increased the clearance of A375 cells from 9.4% to 29.5% at 96 hours ( Table 2 , Figure 1 ). Addition of 300 nM PF-07265028 further increased A375 clearance to 52.6%. The % apoptosis of A375 cells increased from 30.2% to 45.6% for saxenlimab alone and the combination of saxenlimab and 300 nM PF-07265028 ( Table 2 , Figure 3 ). Consistent with this, IFNγ increased from 14,428 to 26,082 pg/mL ( Table 2 , Figure 4 ). PF-07265028 dose responses are listed in Table 1 and graphically presented in Figures 2 , 3 and 4 , and demonstrate that there is a combination benefit from 8 to 300 nM PF-07265028. Table 2 : Combination benefit of PF-07265028 and saxolizumab at 96 hours Concentration of PF-07265028 (nM) A375 Clearance % SEM A375 cell apoptosis % SEM IFNγ (pg/mL) SEM 0 29.5 3.6 30.2 1.4 14428 695 8 37.5 2.8 33.7 2.5 17447 835 12 32.7 1.7 30.6 0.9 14648 422 25 34.0 3.0 32.0 1.9 15572 940 50 38.3 3.0 34.7 2.6 18543 566 83 42.7 1.3 36.9 1.3 19231 291 111 53.1 5.4 47.5 5.3 23621 914 300 52.6 3.2 45.6 2.9 26082 693 The values listed in the table above are plotted in Figures 2 and 3 and represent the mean and SEM of six replicate experiments. Add saxolizumab at approximately 2 μg/mL. A375 % clearance and % apoptosis were based on live cell imaging of A375 tumor cells and calculated as described in Methods. IFNγ was measured by MSD immunoassay.

Example 7 In Vitro Growth Inhibition Study of Combination of HPK1 Inhibitor PF - 07265028 and Anti -PD1 Antibody Nivolumab Potential as a cancer treatment option does not exist.

This example was performed according to the same protocol as described in Example 6, except that nivolumab 10 μg/mL was substituted for saxolizumab 2 μg/mL. Flow cytometric analysis techniques were performed as described below.

Materials and Methods Flow Cytometry : For cytometry, T cells were washed from co-cultures at 60 hours and blotted with antibodies clones SK7 (BD 564001), SK3 (BD 612887), RPA-T8 according to the manufacturer's protocol. (Biolegend 301028), FN50 (Biolegend 310932), B56 (BD 561284) and B27 (BD 554702) stain for CD3, CD4, CD8, CD69, Ki67 and IFNγ. Viable cells were identified by Live/Dead Near-IR stain (ThermoFischer L10119). Stained cells were imaged on a Cytek Aurora CS, and data were analyzed on FlowJo (version 10).

Results : Figure 5 shows the effect of PF-07265028 alone and in combination with nivolumab on the growth of A375 tumor cells. Figure 6 shows the dose response of PF-07265028 alone and in combination with nivolumab for A375 tumor cell depletion in T cell co-cultures. Figure 7 shows the effect of PF-07265028 alone and in combination with nivolumab on T cell proliferation as measured by Ki67. Figure 8 shows the effect of PF-07265028 alone and in combination with nivolumab on IFNγ production per activated T cell. PF-07265028 increased the proliferation of T cells co-cultured with tumor cells, while nivolumab increased the amount of IFNγ produced by each T cell. When PF-07265028 was combined with nivolumab, there was a synergistic increase in T cell proliferation and IFNγ production, resulting in greater control of tumor growth.

Example 8 Combination of HPK1 Inhibitor PF-07265028 and Anti -PD1 Antibody Nivolumab Produces IFNγ in T Cells Co-cultured with Breast Cancer Cells in Test Tube The purpose of this example is to evaluate the effect of HPK1 inhibitor PF-07265028 on tumor reactive T cells Effects on the ability to produce IFNγ in response to co-culture with MDA-MB-231 breast cancer tumor cells, another oncology indication, and assessing the potential of PF-07265028 as a cancer treatment option in the presence or absence of anti-PD1 antibodies.

Materials and Methods Cell Culture : To generate tumor-reactive T cells, PBMCs (from two independent human donors, referred to as "Individual 1" and "Individual 1" and "Individual 1" were obtained using the EasySep Human T Cell Isolation Kit (obtained from StemCell Technologies) protocol. 2') CD4 or CD8 T cells were isolated and treated with mitomycin-treated MDA-MB-231 cells (10 μg/mL Mitomycin C for 2 hours; Mitomycin C was obtained from StemCell Technologies ) were mixed in X-VIVO™ 15 medium (obtained from Lonza Walkersville, Inc.) with 10% human serum AB (obtained from Fisher Scientific). After 10 minutes, 20 ng/mL recombinant human IL-2 (IS) and 10 ng/mL recombinant human IL-7 (obtained from Miltenyi Biotec) were added to expand the activated T cells, and the expansion continued in the G-Rex culture vessel. Add 21 days. The resulting cells were cryopreserved in a solution of 90% fetal bovine serum (FBS, obtained from Fisher Scientific) and 10% DMSO.

MDA-MB-231 human breast cancer cells were subcultured in Gibco Roswell Park Memorial Institute 1640 (RPMI) medium containing 10% FBS and 1% penicillin/streptomycin. Prior to co-culture analysis, the MDA-MB-231 cell line was transduced with Incucyte NucLight green lentivirus (nucGFP) (from Sartorius) and selected by 1 μg/mL puromycin (from Sigma).

Tumor co-culture analysis : MDA-MB-231 cells expressing nucGFP were seeded in ImageLock 96-well plates (from Sartorius) at 5,000 to 10,000 cells per well in 100 μL RPMI (assay medium) with 10% FBS . Cells were incubated for 18 hours at 37°C and 5% CO ₂ in a humidified incubator. The next day, frozen CD4 and CD8 cells were thawed in assay medium at 37°C and allowed to settle for 2 hours. At the same time, the culture medium of the pre-inoculated A375 cells was replaced with 50 μL assay medium containing 3× phospholipid-binding protein V red staining reagent and 3 mM CaCl ₂ . 50 μL of PF-07265028 at a concentration of 333 nM (3×final) and/or nivolumab (purchased from Selleck Chemicals) at a concentration of 30 μg/mL (3×final) in assay medium was added to each well, and also Add 50 µL of T cells in assay medium. Human T cells from Individual 1 and Individual 2 were analyzed independently. For individual 1, equal amounts of CD4 and CD8 T cells were mixed and then added to each well. For individual 2, only CD8 T cells were added to each well. Cells contained a total of 10 T cells per tumor cell with an E:T ratio of 10:1. The plates were then transferred to a 37°C and 5% _CO2 Incucyte S3 incubator and monitored for 4 days.

Data analysis : After 96 hours of co-culture, 50 μL of cell culture supernatant was collected from each well, transferred to individual 96-well plates, and stored overnight at -20°C. The next day, supernatant samples were thawed at room temperature and analyzed by MSD Quantitative Immunoassay (Human IFNγ Kit, Meso Scale Diagnostics) according to the manufacturer's protocol. The concentration of IFNγ in the supernatant (in pg/mL) was quantitatively determined using a calibrator of specified concentration provided by the manufacturer to establish a standard curve.

Results : To assess the potential benefit of combining PF-07265028 with an anti-PD1 antibody, co-cultures of T cells and tumor cells were evaluated in the presence of nivolumab and PF-07265028. Tumor-reactive T cells against the MDA-MB-231 cell line from two human donors (Individual 1 and Individual 2) were generated and subsequently co-existed with MDA-MB-231 tumor cells as described above in Materials and Methods nourish. Cells were treated with 111 nM PF-07265028 alone, 10 μg/mL nivolumab alone, or a combination of these agents. Control groups were MDA-MB-231 cells cultured without T cells or with mock-treated T cells (in DMSO). After 96 hours of co-cultivation, supernatants were collected and analyzed by MSD quantitative immunoassay as described above in Materials and Methods. The effect of PF-07265028 alone and in combination with the anti-PD1 antibody nivolumab on the amount of IFNγ produced by Subject 1 T cells co-cultured with MDA-MB-231 tumor cells is shown in FIG. 9 . The control group shows the amount of IFNγ produced by Subject 1 T cells co-cultured with MDA-MB-231 tumor cells in the presence of DMSO treatment (mock) or DMSO with nivolumab. Inoculate at the rate of ten T cells for each tumor cell. Bar graphs depict the mean of triplicate experiments, with error bars representing SEM and individual data points plotted.

The effect of PF-07265028 alone and in combination with the anti-PD1 antibody nivolumab on the amount of IFNγ produced by Individual 2 T cells co-cultured with MDA-MB-231 tumor cells is shown in FIG. 10 . The control group shows the amount of IFNγ produced by Individual 2 T cells co-cultured with MDA-MB-231 tumor cells in the presence of DMSO treatment (mock) or DMSO with nivolumab. Inoculate at the rate of ten T cells for each tumor cell. Bar graphs depict the mean of triplicate experiments, with error bars representing SEM and individual data points plotted.

Combination benefit of PF - 07265028 and anti -PD1 antibody nivolumab in enhancing IFNγ production by T cells co-cultured with breast cancer cells Addition of 111 nM PF-07265028 resulted in increased IFNγ production, consistent with increased T cell activity. Addition of 111 nM PF-07265028 to Individual 1 T cells co-cultured with MDA-MB-231 tumor cells increased IFNγ production from 9,114 pg/mL to 17,473 pg/mL in mock-treated cells ( Table 3 , Fig . 9 ). In contrast, adding only 10 μg/mL of the anti-PD1 antibody nivolumab to individual 1 T cells co-cultured with MDA-MB-231 tumor cells resulted in IFNγ production of 9,114 pg/mL from mock-treated cells. mL increased to 16,012 pg/mL ( Table 3 , Figure 9 ). Addition of 111 nM PF-07265028 in combination with 10 μg/mL nivolumab further increased IFNγ production to 29,795 pg/mL ( Table 3 , Figure 9 ). Addition of 111 nM PF-07265028 to Individual 2 T cells co-cultured with MDA-MB-231 tumor cells increased IFNγ production from 231 pg/mL to 816 pg/mL in mock-treated cells ( Table 3 , Fig. 10 ). In contrast, adding only 10 μg/mL of the anti-PD1 antibody nivolumab to individual 2 T cells co-cultured with MDA-MB-231 tumor cells resulted in IFNγ production from 231 pg/mL of mock-treated cells. mL increased to 398 pg/mL ( Table 3 , Figure 10 ). Addition of 111 nM PF-07265028 in combination with 10 μg/mL nivolumab further increased IFNγ production to 1,106 pg/mL ( Table 3 , Figure 10 ). All quantitative results from two human donors are listed in Table 3 and shown graphically in Figures 9 and 10 , and demonstrate that PF-07265028 and the anti-PD1 antibody nivolumab enhance T cell production co-cultured with breast cancer tumor cells The IFNγ aspect has combinatorial benefits. Table 3 : Combination benefit of PF-07265028 and anti-PD1 at 96 hours T cell donor PF-07265028 Concentration (nM) Nivolumab (concentration μg/mL) IFNγ (pg/mL) SEM Individual 1 0 0 9,114 879 Individual 1 111 0 17,473 380 Individual 1 0 10 16,012 435 Individual 1 111 10 29,795 1,543 Individual 2 0 0 231 10.0 Individual 2 111 0 816 64.5 Individual 2 0 10 398 13.0 Individual 2 111 10 1,106 14.3 The values listed in the above table are plotted in Figures 9 and 10 and represent the mean and SEM of three replicate experiments. IFNγ was measured by MSD immunoassay as described in Methods.

All references cited herein, including patent applications and patent publications cited in this specification, are incorporated herein by reference in their entirety. Although the foregoing disclosure has been described in considerable detail by way of illustration and example, it will be apparent to those of ordinary skill in the art from the teachings of the present invention that, without departing from the spirit or scope of the appended claims, subject to certain changes and modifications.

The foregoing description and examples detail certain specific embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may be presented in text, the invention may be practiced in many ways and that the invention should be construed in accordance with the appended claims and any equivalents thereof.

Various embodiments of the invention will be described below with reference to the figures in which: Figure 1 shows the effect of PF-07265028 alone and in combination with the anti-PD1 antibody saxanlimab on the growth of A375 tumor cells according to certain embodiments of the invention . Figure 2 shows the dose response of A375 tumor cell growth in T cell co-cultures to PF-07265028 alone and in combination with the anti-PDl antibody saxolizumab, according to certain embodiments of the invention. Figure 3 shows the effect of PF-07265028 alone and in combination with the anti-PDl antibody saxenlimab on the growth of A375 apoptotic tumor cells in T cell co-cultures, according to certain embodiments of the present invention. Figure 4 shows the effect of PF-07265028 alone and in combination with the anti-PD1 antibody saxenlimab on IFNγ production in T cell co-cultures with tumor cells, according to certain embodiments of the present invention. Figure 5 shows the effect of PF-07265028 alone and in combination with the anti-PD1 antibody nivolumab on the growth of A375 tumor cells, according to certain embodiments of the present invention. Figure 6 shows the dose response of A375 tumor cell growth in T cell co-cultures to PF-07265028 alone and in combination with nivolumab, according to certain embodiments of the invention. 7 shows the effect of PF-07265028 alone and in combination with the anti-PD1 antibody nivolumab on the percentage of Ki67 - positive CD8 T cells in A375 tumor cell co-cultures, according to certain embodiments of the present invention. Figure 8 shows the effect of PF-07265028 alone and in combination with the anti-PD1 antibody nivolumab on IFNγ production per activated T cell in A375 tumor cell co-cultures, according to certain embodiments of the present invention. Figure 9 shows the effect of PF-07265028 alone and in combination with anti-PD1 antibody nivolumab on IFNγ produced by human donor "Individual 1" T cells co-cultured with MDA-MB-231 tumor cells according to certain embodiments of the present invention The role of quantity. Figure 10 shows the effect of PF-07265028 alone and in combination with the anti-PD1 antibody nivolumab on IFNγ produced by human donor "Individual 2" T cells co-cultured with MDA-MB-231 tumor cells according to certain embodiments of the present invention The role of quantity.

Claims (27)

A method for treating cancer comprising administering an amount of a compound of formula (I) to an individual in need thereof: or a pharmaceutically acceptable salt thereof, wherein: R ^1a and R ^1b are each independently (C ₁ -C ₆ ) alkyl, or R ^1a and R ^1b are formed together with the nitrogen to which they are attached via 0, 1 or 2 A 5-membered heterocycloalkyl group substituted with (C ₁ -C ₆ ) alkyl; R ^2a and R ^2b are each independently hydrogen or (C ₁ -C ₄ ) alkyl; R ^3a and R ^3b are each independently hydrogen or (C ₁ -C ₄ ) alkyl; each R ⁴ is independently (C ₁ -C ₆ ) alkyl substituted by 0 or 1 halogen or hydroxyl; and n is 0, 1 or 2; and an amount of A combination of planned death 1 protein (PD-1) axis binding antagonists, wherein the equal amounts together are effective to treat cancer. The method of claim 1, wherein R ^1a and R ^1b are each independently methyl, ethyl or isopropyl. The method according to claim 1 or 2, wherein R ^1a and R ^1b together with the nitrogen to which they are attached form a 5-membered heterocycloalkyl group substituted with 0 or 1 methyl substituent. The method according to any one of claims 1 to 3, wherein R ^2a and R ^2b are hydrogen. The method according to any one of claims 1 to 4, wherein R ^3a and R ^3b are each independently hydrogen, methyl or ethyl. The method according to any one of claims 1 to 5, wherein R ⁴ is methyl or ethyl. The method according to any one of claims 1 to 6, wherein n is 1. The method according to any one of claims 1 to 7, wherein the compound of formula (I) is: 4-[1-aminopropyl]-2-{6-[5-methyl-6,7-dihydro- 5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2,3 -Dihydro-1H-pyrrolo[3,4-c]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5H -Pyrrolo[2,1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2,3- Dihydro-1H-pyrrolo[3,4-c]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[2-methylpyrrolidin-1-yl]-2,3- Dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; 4-[1-aminoethyl]-2-{6-[5-ethyl-6,7-dihydro-5 H - pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2, 3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; or 4-[1-aminopropyl]-2-{3-[5-methyl-6,7-di Hydrogen-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-2, 3-dihydro-1 H -pyrrolo[3,4- c ]pyridin-1-one; or a pharmaceutically acceptable salt thereof. The method according to any one of claims 1 to 8, wherein the compound of formula (I) is: 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5- Methyl-6,7-dihydro- 5H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R ) -2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one; 4-[(1R)-1-aminoethyl] -2-{6-[(5S)-5-Ethyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl]pyridine- 2-base}-6-[(2R)-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4-c]pyrrolidin-1-one; 4- [(1S)-1-aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2, 4] Triazol-3-yl] pyridin-2-yl}-6-[(2R)-2-methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4 -c] pyridin-1-one; 4-[(1R)-1-aminoethyl]-2-{6-[(5S)-5-ethyl-6,7-dihydro-5H-pyrrolo[ 2,1-c][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[methyl(propan-2-yl)amino]-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridin-1-one; or 4-[(1R)-1-aminopropyl]-2-{3-[(5S)-5-methyl-6,7 -Dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl]phenyl}-6-[methyl(propan-2-yl)amino]-2 , 3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-one; or a pharmaceutically acceptable salt thereof. The method according to any one of claims 1 to 9, wherein the compound of formula (I) is 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl Base-6,7-dihydro-5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )- 2-Methylpyrrolidin-1-yl]-2,3-dihydro-1H-pyrrolo[3,4- c ]pyridin-1-one. The method according to any one of claims 1 to 10, wherein the PD-1 axis binding antagonist comprises a PD-1 antagonist, a PD-L1 antagonist or a PD-L2 antagonist. The method according to claim 11, wherein the PD-1 axis binding antagonist comprises a PD-1 antagonist. The method according to claim 11 or 12, wherein the PD-1 binding antagonist is an anti-PD-1 antibody. The method of claim 13, wherein the anti-PD-1 antibody system is selected from the group consisting of: sasanlimab, nivolumab, pembrolizumab, and pidelitizumab pidilizumab, cemiplimab, tislelizumab, spartalizumab, sintilimab, MEDI-0680, BGB -108, AGEN2034 genolimzumab, CBT-502, camrelizumab, and combinations thereof. The method according to claim 14, wherein the anti-PD-1 antibody is saxenlimab. The method according to claim 14, wherein the anti-PD-1 antibody is nivolumab. The method according to claim 14, wherein the anti-PD-1 antibody is pembrolizumab. The method according to claim 11 or 12, wherein the PD-1 axis binding antagonist comprises a PD-L1 antagonist. The method according to claim 18, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody. The method according to any one of claims 1 to 19, wherein the individual is human. The method according to any one of claims 1 to 20, wherein the cancer is selected from the group consisting of brain cancer, head/neck cancer, prostate cancer, bladder cancer, lung cancer, breast cancer, ovarian cancer, bone cancer, colorectal cancer , kidney cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, gastroesophageal junction cancer, thyroid cancer, cervical cancer, uterine cancer and kidney cancer. A combination for treating cancer in an individual in need thereof, comprising: (i) a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein: R ^1a and R ^1b are each independently (C ₁ -C ₆ ) alkyl, or R ^1a and R ^1b are formed together with the nitrogen to which they are attached via 0, 1 or 2 A 5-membered heterocycloalkyl group substituted with (C ₁ -C ₆ ) alkyl; R ^2a and R ^2b are each independently hydrogen or (C ₁ -C ₄ ) alkyl; R ^3a and R ^3b are each independently hydrogen or (C ₁ -C ₄ )alkyl; each R ⁴ is independently (C ₁ -C ₆ )alkyl substituted with 0 or 1 halogen or hydroxyl; and n is 0, 1 or 2; and (ii) PD-1 axis binding antagonists. The combination of claim 22, wherein the HKP1 inhibitor is 4-[(1 R )-1-aminopropyl]-2-{6-[(5 S )-5-methyl-6,7-dihydro -5 H -pyrrolo[2,1- c ][1,2,4]triazol-3-yl]pyridin-2-yl}-6-[(2 R )-2-methylpyrrolidin-1 -yl]-2,3-dihydro- 1H -pyrrolo[3,4- c ]pyridin-1-one, and the PD-1 axis binding antagonist comprises an anti-PD-1 antibody, the anti-PD-1 The antibody is selected from the group consisting of: saxolizumab, nivolumab, pembrolizumab, piderolizumab, simiprizumab, tislelizumab, spartakizumab , Sintilimab, MEDI-0680, BGB-108, or AGEN2034 Genozumab, CBT-502, Camrelizumab, and combinations thereof. The combination of claim 22 or 23, wherein the PD-1 axis binding antagonist comprises saxanlimab. The combination of claim 22 or 23, wherein the PD-1 axis binding antagonist comprises nivolumab. The combination of claim 22 or 23, wherein the PD-1 axis binding antagonist comprises pembrolizumab. 1. Use of the combination according to any one of claims 22 to 26 for the treatment of cancer in an individual in need thereof.