HPK1 inhibitors and uses thereof

Abstract

The present invention provides a compound represented by structural formula (I-0) or formula (II): or a pharmaceutically acceptable salt or stereoisomer thereof, which is suitable for treating a disease (such as cancer) that can be treated by inhibiting HPK1 activity.

Description

[Technical field]

[0001] Cross-references to related applications

[0002] This application claims priority to international patent application No. PCT / CN2019 / 094634 filed on July 4, 2019. The entire contents of the above application are incorporated herein by reference.

[0003] [Prior Art]

[0004] Hematopoietic progenitor kinase 1 (HPK1), also known as Mitogen-Activated Protein Kinase Kinase Kinase Kinase 1 (MAP4K1), is a protein kinase that acts upstream of the canonical three-tiered MAPK pathway, which includes MAP3K (MAP kinase kinase kinase), which activates MAP2K (MAP kinase kinase), which in turn activates the dual Thr and Tyr MAPK family member JNK (c-Jun N-terminal kinase). HPK1 / MAP4K1, originally selected in hematopoietic progenitor cells, is mainly expressed in lymphoid organs / tissues, including bone marrow, fetal liver, lymph nodes, placenta, spleen, and thymus (Hu et al., Gene & Dev. 10(18):2251-2264, 1996; Kiefer et al., The EMBO J. 15(24):7013-7025, 1996). At the cellular level, HPK1 is expressed in all cell types in hematopoietic units, including hematopoietic progenitor cells, T cells, B cells, macrophages, dendritic cells, neutrophils, and mast cells (Hu, supra, Kiefer, supra).

[0005] HPK1 / MAP4K1 is one of the six MAP4Ks including HPK1 (MAP4K1), GCK (MAP4K2), GLK (MAP4K3), HGK / NIK (MAP4K4), KHS / GCKR (MAP4K5), and MINK (MAP4K6). In general, these MAP4Ks are members of the approximately 26 mammalian Ste20-like serine / threonine kinases that have been identified so far, which are homologs of the yeast sterile20 protein (Ste20p), a putative MAP4K that activates MAP3K in the yeast pheromone signaling pathway. These mammalian Ste20-like kinases are divided into two subgroups based on their functional domain structures: p21-activated kinases (PAKs) and germinal central kinases (GCKs). Several members of the GCK subgroup can activate the MAP3K kinase cascade, resulting in JNK activation.

[0006] MAP4Ks have a highly similar structure with an N-terminal kinase domain (KD), followed by 2-4 proline-rich motifs, and a C-terminal citron-homology domain (CNH).

[0007] The ATP-binding site of the kinase domain of HPK1 includes Lys-46. Mutation of this residue to Met (HPK1-M46) abolishes the catalytic activation of HPK1 (Hu, supra).

[0008] There are multiple conserved Ser / Thr phosphorylation sites within the kinase domain of HPK1, and a conserved Tyr phosphorylation site between its first two proline-rich motifs. HPK1 activation appears to require phosphorylation of Tyr379 (in mice, or Tyr381 in humans) by LCK / ZAP70, because in Jurkat T cells, deficiency of LCK or ZAP70 abolishes Tyr-379 phosphorylation and the kinase activity of HPK1 upon anti-CD3 stimulation (Ling et al., JBC 276(22):18908-18914, 2001; Liou et al., Immunity 12(4):399-408, 2000; Sauer et al., JBC 276(48):45207, 45216, 2001). On the other hand, Thr-355 autophosphorylation regulates the ubiquitination and degradation of HPK1. Thr-355 is a dephosphorylation site targeted to PP4; this dephosphorylation prevents CUL7 / Fbxw8-mediated ubiquitination and proteasomal degradation of activated HPK1 (Wang et al., Cancer Res. 69(3):1063-1070, 2009), and thus HPK1 is also stabilized and activated by protein phosphatase 4 (PP4) (Zhou et al., JBC 279(47):49551-49561, 2004).

[0009] The Tyr phosphorylation site is also adjacent to a caspase cleavage site (DDVD). It has been shown that full-length HPK1 can be cleaved by caspase-3 at this site in apoptotic cells, resulting in enhanced catalytic activity of the N-terminal HPK1 fragment (Chen et al., Oncogene 18:7370-7377, 1999).

[0010] Four Pro-rich motifs of HPK1 mediate the interaction between HPK1 and many SH3 domain-containing proteins (Boomer & Tan, JCB 95(1):34-44, 2005).

[0011] The CNH domain of HPK1 may be involved in HPK1-mediated lymphocyte adsorption because the citrate homology domain of another Ste20-like kinase, TNIK, binds to Rap2 and regulates the actin cytoskeleton (Taira et al., JBC 279(47):49488-49496, 2004).

[0012] MAP4K plays an important role in the immune system, especially in lymphocytes, by regulating cell signaling, immune cell activation, cell transformation, and cell migration. HPK1 knockout (KO) mice show enhanced T-cell activation, increased cytokine production, and increased antibody production after KLH immunization. HPK1 KO mice are also more susceptible to EAE induction. HPK1 KO T cells and B cells show enhanced cell activation and antigen receptor signaling. HPK1 KO dendritic cells show higher amounts of co-stimulatory molecules and pro-inflammatory cytokines (Alzabin et al., J. Immunol. 182(10):6187-6194, 2009; Shui et al., Nat. Immunol. 8(1):84-91, 2007).

[0013] Overexpression in cell lines (e.g., HEK293 and COS-1 cells, as well as hematopoietic Jurkat T cells and leukemic HL-60 cells) demonstrated that HPK1 can activate MAPK JNK (but not p38 or ERK MAP kinases) through multiple MAP3Ks (including TAK1, MEKK1, and MLK3), all of which activate MAP2K MKK4 and MKK7, which in turn activate JNK.

[0014] It is worth noting that the regulatory function of MAP4K in immune cells appears to be mediated primarily by a JNK-independent mechanism. It has been demonstrated that IKK-NF-κB activation requires HPK1 kinase activation, and it is believed that HPK1 operates by regulating CARMA1. CARMA1 is a transfer protein in the so-called CBM (CARMA1 / BCL10 / MALT1) complex that promotes IKKβ activation when stimulated by anti-CD3 in Jurkat T cells. Activated IKK cleaves IκB and releases the associated NF-κB nuclear transcription factor. Specifically, HPK1 can be induced to associate with CARMA1, directly phosphorylating CARMA1 at Ser-551, which is required for NF-κB activation (Brenner et al., PNAS USA, 196(34):14508-14513, 2009).

[0015] In T cells, upon TCR stimulation, lymphocyte protein tyrosine kinase (Lck) phosphorylates the immunoreceptor tyrosine activation motif (ITAM) on the cytosolic side of the TCR / CD3 complex. Zap-70 is subsequently recruited to the TCR / CD3 complex, where it is phosphorylated and activated. Activated ZAP-70 phosphorylates a transfer protein called SLP-76, which translocates to the plasma membrane and promotes the formation of a multi-protein signaling complex by binding to many proteins, including HPK1. These proteins collectively transmit TCR signals to different effector molecules, resulting in the activation, survival, and proliferation of T-lymphocytes.

[0016] During this process, HPK1 directly binds to the SH2 domain of SLP-76, primarily acting as a negative regulator of TCR signaling. For example, TCR signaling was enhanced in HPK1 KO naive T cells, which showed hyperproliferation and IL-2 production when TCR was ligated in vitro (Shui, supra). It is believed that HPK1 can downregulate TCR signaling by phosphorylating the SLP-76 adaptor protein at Ser-376 via a negative feedback mechanism. When Ser-376 is phosphorylated by HPK1, SLP-76 binds to 14-3-3 via the phosphorylated Ser-376 residue, resulting in ubiquitination of the Lys-30 (K30) residue of SLP-76, which then becomes a target for proteasomal degradation. HPK1 also downregulates TCR signaling at other adaptor proteins, including GADs, by similar mechanisms (eg, by phosphorylation of Thr-254 of GADs and promotion of 14-3-3 interactions).

[0017] Thus, HPK1 appears to play dual and opposing roles in JNK activation and TCR signaling. Although HPK1 has been shown to directly activate the JNK pathway via the MAP3K-MAP2K-MAPK pathway in various overexpression systems, HPK1-mediated inhibition of SLP-76 activation also results in inhibition of JNK activity in TCR signaling. This is consistent with the finding that HPK1-knocked-out naive T cells showed unaffected JNK activity (Shui, supra). Similarly, HPK1 appears to regulate IKK activation by two different and opposing mechanisms - HPK1 activates IKK by directly phosphorylating CARMA1 on the one hand, and negatively regulates IKK activation by inhibiting SLP-76 activation on the other hand. This seems to be in contrast to the dual role played by HPK1, where HPK1 is believed to promote JNK and IKK activation in the initial phase of TCR signaling, but plays an important role in hindering TCR signaling in the final phase.

[0018] HPK1 also plays a similar negative regulatory role in BCR-induced cell activation and proliferation in B cells. B cells utilize SLP-76 adaptor proteins called BLNK to transduce BCR signaling, including JNK and IKK activation. In B cells, the Tyr kinases Syk and Lyn promote Tyr phosphorylation and activation of HPK1 and result in pY379 of HPK1 mediating HPK1-BLNK binding. Negative feedback of HPK1 on BLNK occurs through Thr-152 of BLNK. pT152 is bound by 14-3-3, resulting in ubiquitination of BLNK at multiple Lys residues followed by proteasomal degradation of BLNK (thus impeding BCR signaling).

[0019] It is noteworthy that HPK1 appears to be a regulatory T cell (T reg ) is a positive regulator of the repressive function of HPK1 (Sawasdikosol et al., J Immunol. 188(supp. 1):163, 2012). + Tregs are defective in suppressing TCR-induced effector T cell proliferation, but paradoxically gain the ability to produce IL-2 upon TCR engagement (Sawasdikosol, supra). Thus, HPK1 is an important regulator of Treg function and peripheral self-tolerance.

[0020] HPK1 is also involved in PGE2-mediated CD4 + Inhibition of T cell activation (Ikegami et al., J Immunol. 166(7):4689-4696, 2001). US2007 / 0087988 shows that CD4 + When T cells are exposed to physiological concentrations of PGE2, the activity of HPK1 kinase can be increased through PGE2-induced PKA activation. The proliferation of HPK1-deficient T cells can tolerate the suppressive effects of PGE2 (US2007 / 0087988). Therefore, PGE2-mediated HPK1 activation may represent a novel regulatory pathway for regulating immune responses.

[0021] Different from TCR and BCR, HPK1 also transduces signals downstream of TGF-R (transforming growth factor receptor) (Wang et al., JBC 272(36):22771-22775, 1997) or Gs-coupled PGE2 receptors (EP2 and EP4) (Ikegami et al., J Immunol. 166(7):4689-4696, 2001).

[0022] HPK1 negatively regulates immune cell attachment. In T cells, TCR activation also induces integrin activation, resulting in T-cell attachment and immune synapse formation. This is achieved by SLP-76 binding to the degradation-promoting adapter protein (ADAP), which is necessary for TCR-induced integrin activation (Wang et al., J. Exp. Med. 200(8):1063-1074, 2004), but its constitutively related SKAP55 protein targets the activated small GTPase Rap1 to the plasma membrane, resulting in integrin activation (Kliche et al., MCB 26(19):7130-7144, 2006). In other words, the SLP-76 / ADAP / SKAP55 ternary complex relays TCR signal transduction to the integrin family of attachment molecules, thereby promoting T-cell attachment. HPK1 not only downregulates SLP-76 (as mentioned above), but also negatively regulates this pathway by competing with ADAP for the same SH2 binding site of SLP-76, thereby hindering the activity of Rap1, a downstream effector of ADAP (Patzak et al., Eur. J. Immunol. 40(11):3220-3225, 2010).

[0023] HPK1 also negatively regulates integrin activation and cell attachment in B cells. Among them, HPK1 is associated with a SKAP55 homolog called SKAP-HOM (Konigsberger et al., PloS One 5(9).pii:e12468, 2010), which is required for B-cell attachment (Togni et al., MCB 25(18):8052-8063, 2005). It is believed that HPK1 induces negative phosphorylation sites of SKAP-HOM, thereby suppressing Rap1 activation.

[0024] However, in neutrophils, HPK1 positively regulates their attachment. Neutrophil trafficking, including slow rolling, tight binding, cell spreading, and diapedesis, is controlled by outside-in signaling of β2-integrin activation, inducing interaction between actin and HIP-55 (HPK1-interacting protein of 55 kDa). This reinforces the high-affinity conformation of β2-integrin, which facilitates neutrophil attachment (Hepper et al., J. Immunol. 188(9):4590-4601, 2012; Schymeinsky et al., Blood 114(19):4209-4220, 2009). When β2-integrin mediates attachment, HPK1 co-localizes with HIP-55 and actin at the lamellipodia of neutrophils (Jakob et al., Blood 121(20):4184-4194, 2013). CXCL1-mediated neutrophil attachment is impaired by HPK1 deficiency or HIP-55 deficiency in vitro and in vivo (Jakob, supra; Schymeinsky, supra).

[0025] Consistent with the role of HPK1 in downregulating TCR and BCR function, HPK1 negatively regulates acquired immune responses, and loss of HPK1-mediated regulation of T-cell activation and immune responses may be an important mechanism of autoimmune pathogenesis. In HPK1 KO mice, although T and B cell development appears unaffected (Shui, supra), T cells from these animals show a dramatic increase in TCR proximal signaling and downstream ERK activation when stimulated in vitro with anti-CD3, resulting in hyperproliferation of these cells (Shui, supra). T cells from immunized HPK1-deficient mice overreact when stimulated with antigen-specific stimulation, producing significantly higher amounts of inflammatory cytokines such as IL-2, IFN-γ, and IL-4. These mice also produce higher amounts of IgM and IgG isotypes, indicating enhanced B cell function in which HPK1 has been knocked out (Shui, supra).

[0026] HPK1 also negatively controls autoimmunity in mice, as HPK1 KO mice are more susceptible to induction of experimental autoimmune encephalomyelitis (EAE) (Shui, supra). HPK1 attenuation also contributes to abnormal T- and B-cell activation and autoimmunity in human patients. HPK1 is downregulated in peripheral blood mononuclear cells of patients with psoriatic arthritis or in T cells of patients with systemic lupus erythematosus (SLE).

[0027] The physiological functions of HPK1 are not limited to lymphocytes. HPK1 also negatively regulates the maturation and activation of dendritic cells (DC) through unknown mechanisms (Alzabin, such as the above-mentioned literature). In HPK1KO mice, bone marrow-derived dendritic cells (BMDC) show enhanced levels of co-stimulatory molecules CD80 / CD86 and increase the production of proinflammatory cytokines (Alzabin, such as the above-mentioned literature). As a result, the antigen presentation activity of dendritic cells in HPK1KO mice is more effective (Alzabin, such as the above-mentioned literature). More importantly, the efficacy of tumor eradication mediated by CTL response mediated by HPK1KO BMDC is higher than that of wild-type BMDC (Alzabin, such as the above-mentioned literature). In addition, HPK1 can also control anti-tumor immunity using T- and B-lymphocyte-dependent mechanisms. It has been shown that adoptive transfer of HPK1-deficient T cells is more effective than wild-type T cells in controlling tumor growth and metastasis (Alzabin et al., Cancer Immunol Immunother 59(3):419-429, 2010). Likewise, BMDCs from HPK1 knockout mice were more effective than wild-type BMDCs in providing T cell responses to eradicate Lewis lung carcinoma (Alzabin et al., J Immunol. 182(10):6187-6194, 2009).

[0028] Therefore, there is a need for HPK1 inhibitory compounds to treat diseases or disorders by modulating HPK1 activity. [Summary of the invention]

[0029] Described herein are compounds of formula (I-0), (I-1), (I-2), (I), (II), (II-1), (II-2), or (II-2'), and example compounds (collectively referred to as "compounds of the present invention") that inhibit HPK1 activity, and pharmaceutically acceptable salts thereof.

[0030] In one aspect, the present invention provides a compound represented by structural formula (I-0):

[0031]

[0032] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0033] X is CR 2 R 3 or NR 3 ;

[0034] A is CR 2 or N;

[0035] R is an 8-10 membered bicyclic nitrogen-containing heteroaryl group or an 8-10 membered bicyclic nitrogen-containing heterocyclic group, which may be substituted with a pendant oxy group as needed, wherein the nitrogen-containing heteroaryl group or nitrogen-containing heterocyclic group represented by R has 1 to 3 heteroatoms selected from: N, O, and S, and may be substituted with 1 to 4 R as needed. a Substitution, and wherein R is connected to the pyrimidine ring by a nitrogen ring atom, or R is represented by the following structural formula:

[0036]

[0037] R 1 H, deuterium, halogen, OH, CN, NH 2 、NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, phenyl, 5-6 membered heteroaryl, C 3-6 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 1 On behalf of or by R 1 The alkyl, alkenyl, alkynyl, alkoxy, phenyl, heteroaryl, cycloalkyl, or heterocyclic group in the represented group may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 alkyl,

[0038] C 1-4 Alkoxy, C 1-4 Haloalkyl, C 1-4 Haloalkoxy, and NR 11 R 12 ;or

[0039] R and R 1 Together with the carbon atoms to which they are attached they form the following representative ring:

[0040]

[0041] in The bonding system connects the pyrimidine rings;

[0042] R 2 Each of the examples is independently H, deuterium, halogen, OH, CN, NH 2 、NO 2 , C1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, P(=O)R 11 R 12 、S(=O) 2 R 11 , or S(=O) 2 NR 11 R 12 , where R 2 On behalf of or by R 2 The alkyl, alkenyl, alkynyl, or alkoxy group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 ;

[0043] R 3 H, C 1-6 Alkyl, C(O)C 1-6 Alkyl, C 3-6 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 3 On behalf of or by R 3 The alkyl, cycloalkyl or heterocyclic group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, and NR 11 R 12 ;

[0044] R 11 and R 12 Each instance of is independently H or C 1-6 Alkyl, wherein R 11 or R 12The alkyl group represented by the present invention may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6 Cycloalkyl, and 3-7 membered heterocyclic group, or

[0045] R 11 and R 12 Together with the nitrogen atom or phosphorus atom to which they are attached, they form a 3-7 membered heterocyclic group, which may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, and C 1-4 Haloalkoxy;

[0046] R a Each of the examples is independently H, deuterium, halogen, OH, CN, NH 2 、NO 2 , COOH, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)C 1-6 Alkyl, C(O)OR 11 NR 11 C(O)R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 , C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, or 5-6 membered heteroaryl group, wherein R a On behalf of or by R aThe alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, or heteroaryl groups in the represented groups may be substituted as needed by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 , and 5-6 membered heteroaryl; or

[0047] Two R's a Together with the carbon atoms (groups) to which they are attached, they form C 3-6 Cycloalkyl or 3-7 membered heterocyclic group, wherein the cycloalkyl or heterocyclic group may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, and C 1-4 Haloalkoxy;

[0048] R b Each of the examples is independently H, deuterium, halogen, OH, CN, NH 2 、NO 2 , COOH, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R12 、C(O)C 1-6 Alkyl, C(O)OR 11 NR 11 C(O)R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 , C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, or 5-6 membered heteroaryl group, wherein R b On behalf of or by R b The alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, or heteroaryl groups in the represented groups may be substituted as needed by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 , and 5-6 membered heteroaryl;

[0049] R c Each example of is independently phenyl, a 5-6 membered monocyclic heterocyclic group having 1 to 3 heteroatoms selected from N and O; a 5-6 membered monocyclic heteroaryl group having 1 to 3 heteroatoms selected from N and O; wherein R c The phenyl, heterocyclic or heteroaryl represented by phenyl may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 , and P(O)-C 1-6 alkyl;

[0050] R a ' is C 1-6 Alkyl, which may optionally be OH, CN, 5-6 membered heteroaryl, C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 replace;

[0051] m is 0, 1, 2, or 3;

[0052] n is 0, 1, 2, 3, or 4;

[0053] p is 1, 2, or 3;

[0054] q is 1, 2, or 3; and p+q≤4.

[0055] In another aspect, the present invention provides a compound represented by structural formula (II):

[0056]

[0057] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0058] A 1 CR' or N;

[0059] X is –P(=O)R 3’ R 4’ ;

[0060] R' is H, deuterium, halogen, CN, NO 2 , C 1-6Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, wherein the alkyl, alkenyl, alkynyl, or alkoxy represented by R' or in the group represented by R' may be substituted with one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0061] R 1’ Each of the examples is independently H, deuterium, halogen, OH, CN, NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, C 3-7 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 1’ On behalf of or by R 1’ The alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, or heterocyclic group in the represented group may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0062] R 2’ Each of the examples is independently H, deuterium, halogen, OH, CN, NO 2 , C 1-6 Alkyl, C2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, phenyl, 5-6 membered heteroaryl, C 3-7 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 2’ On behalf of or by R 2’ The alkyl, alkenyl, alkynyl, alkoxy, phenyl, heteroaryl, cycloalkyl, or heterocyclic group in the represented group may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0063] Each R 3’ and R 4’ H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, wherein R 3’ or R 4’ The alkyl, alkenyl, or alkynyl represented by alkyl may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0064] R 11’ and R 12’ Each instance of is independently H or C 1-6 Alkyl, wherein R 11’ or R 12’ The alkyl group represented by the present invention may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6Cycloalkyl, and 3-7 membered heterocyclic group, or

[0065] R 11’ and R 12’ Together with the nitrogen atom to which they are attached, they form a 3-7 membered heterocyclic group, which may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, or C 1-4 Haloalkoxy;

[0066] m' is 0, 1, or 2; and

[0067] n' is 0, 1, or 2.

[0068] Provided herein is a pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0069] Also provided is a combination comprising a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, and one or more therapeutically active adjunct agents.

[0070] The present invention further provides a method for inhibiting HPK1 activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof.

[0071] The present invention further provides a method for treating a subject suffering from a disease or condition described herein (e.g., cancer, such as breast cancer, colorectal cancer, lung cancer, ovarian cancer, and pancreatic cancer), which comprises administering to the subject a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof.

[0072] Certain embodiments disclose compounds of the present invention or pharmaceutically acceptable salts thereof for use as medicines, such as medicines as HPK1 inhibitors.

[0073] The present invention also provides a use of a compound of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same in any of the methods of the present invention described above. In one embodiment, a compound of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same is provided for use in any of the methods of the present invention described above. In another embodiment, a use of a compound of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same is provided for use in the manufacture of a medicine for use in any of the methods of the present invention described above.

[0074] [Implementation Method]

[0075] 1. Overview

[0076] The invention described herein provides a HPK1 / MAP4K1 inhibitor, a pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof, and a method of using the same to modulate (e.g., inhibit) HPK1 / MAP4K1 activity, the method comprising administering the HPK1 / MAP4K1 inhibitor compound of the invention, or a pharmaceutically acceptable salt thereof, to a patient / subject in need thereof. In certain embodiments, the compounds of the invention or their pharmaceutically acceptable salts are suitable for medical administration to enhance, stimulate and / or increase immunity in the treatment of cancer.

[0077] For example, a method for treating a disease or condition associated with inhibiting HPK1 interaction comprises administering to a patient in need thereof a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt thereof. The compounds of the present invention can be used alone, in combination with other agents or therapies, or as an adjuvant or pre-adjuvant for treating a disease or condition, including cancer.

[0078] 2. Definition

[0079] The terms “a”, “an”, “the” and similar terms used in the present invention (especially in the claims) include both the singular and the plural, unless otherwise specified or contradicted by the context.

[0080] As used herein, "halo" means halogen and includes chloro, fluoro, bromo, and iodo.

[0081] The term "alkyl" used alone or as part of a larger moiety (such as "alkoxy" or "haloalkyl" and the like) refers to a saturated aliphatic straight-chain or branched monovalent hydrocarbon radical. Unless otherwise specified, an alkyl group typically has 1-4 or 1-6 carbon atoms, i.e., (C 1 -C 4 ) alkyl or (C 1 -C 6 ) alkyl. In this case, "(C 1 -C 4 ")alkyl" means a group having 1 to 4 carbon atoms in a straight chain or branched arrangement. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and the like.

[0082] The term "C 1-6 "Alkylene" refers to a divalent, fully saturated, branched or straight-chain monovalent hydrocarbon group having 1 to 6 carbon atoms. 1-4 Alkylene, C 1-3 Alkylene" and "C 1-2 C 1-6Representative examples of alkylene include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, isobutylene, t-butylene, n-pentylene, isopentylene, neopentylene, and n-hexylene.

[0083] The term "C optionally substituted with hydroxyl groups" 1 -C 6 "Alkyl" means a C group as defined above which may be substituted by one or more hydroxy groups. 1 -C 6 Examples include, but are not limited to, hydroxymethyl, hydroxyethyl, 1,2-dihydroxyethyl, 2,3-dihydroxy-propyl, and the like.

[0084] The term “C 1-6 "Alkylamino" refers to a group of the formula -N(R a )-R a Part of the body, each R a C is defined as above 1-6 Alkyl, which may be the same or different, is similar to the term "mono C 1-6 "Alkylamino" refers to a group such as -N(H)-R a Part of the body, where R a C is defined as above 1-6 alkyl.

[0085] The term "alkenyl" means a branched or straight-chain monovalent hydrocarbon radical containing at least one double bond. Alkenyl groups may be monounsaturated or polyunsaturated and may be in the E or Z configuration. Unless otherwise specified, alkenyl groups typically have 2 to 6 carbon atoms, i.e., (C 2 -C 6 ) alkenyl. For example, "(C 2 -C 6 The term "alkenyl" refers to a group having 2 to 6 carbon atoms in a straight chain or branched arrangement.

[0086] The term "alkynyl" refers to a branched or straight-chain monovalent hydrocarbon radical containing at least one triple bond. Unless otherwise specified, an alkynyl group generally has 2 to 6 carbon atoms, i.e., (C 2 -C 6 )alkynyl. For example, "(C 2 -C 6 The term "alkynyl" refers to a group having 2 to 6 carbon atoms in a straight chain or branched arrangement.

[0087] The term "alkoxy" refers to an alkyl group attached through an oxygen linking atom, represented by -O-alkyl. For example, "C 1 -C 6 "Alkoxy" refers to -OC 1 -C 6Alkyl, wherein alkyl is as defined above, and "(C 1 -C 4 ") Alkoxy" includes methoxy, ethoxy, propoxy, and butoxy, etc. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy-, and the like. Typically, the alkoxy has about 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 2 carbon atoms.

[0088] The terms "haloalkyl" and "haloalkoxy" refer to an alkyl or alkoxy group which may be substituted with one or more halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, and the like.

[0089] Therefore, the term "C optionally substituted by halogen" 1-6 "Alkyl" refers to a C group as defined above which may be substituted by one or more halogens. 1 -C 6 Examples include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, and 1-bromomethyl-2-bromoethyl.

[0090] The term "cycloalkyl" as used herein includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 14 carbon atoms and containing the specified number of rings and carbon atoms (e.g., C 3 -C 14 Single ring, C 4 -C 14 Double ring, C 5 -C 14 Tricyclic, or C 6 -C 14 In some embodiments, "cycloalkyl" is a monocyclic cycloalkyl. Examples of monocyclic cycloalkyl include cyclopentyl (C 5 ), cyclohexyl (C 6 ), cyclopropyl (C 3 ), cyclobutyl (C 4 ), cycloheptyl (C 7 ) and cyclooctyl (C 8 In some embodiments, "cycloalkyl" is a bicyclic cycloalkyl. Examples of bicyclic cycloalkyl include bicyclo[1.1.0]butane (C 4 ), bicyclo[1.1.1]pentane (C 5 ), spiro[2.2]pentane (C 5 ), bicyclo[2.1.0]pentane (C 5 ), bicyclo[2.1.1]hexane (C 6), bicyclo[3.3.3]undecane (C 11 ), decahydronaphthalene (C 10 ), bicyclo[4.3.2]undecane (C 11 ), spiro[5.5]undecane (C 11 ) and bicyclo[4.3.3]dodecane (C 12 In some embodiments, the "cycloalkyl" is a tricyclic cycloalkyl. Examples of tricyclic cycloalkyl include adamantane (C 12 ). Unless otherwise specified, a "cycloalkyl" group has 3 to 6 carbon atoms and is a monocyclic ring.

[0091] The term "aryl", used alone or as part of a larger moiety such as "aralkyl", "aralkyloxy", or "aryloxyalkyl", refers to a carbocyclic aromatic ring. The term "aryl" may be used interchangeably with the terms "aryl ring", "carbocyclic aromatic ring", "aryl group", and "carbocyclic aromatic group". Typically, aryl is a monocyclic, bicyclic, or tricyclic aromatic group having 6-20 carbon atoms, typically 6-14 ring carbon atoms. In addition, the term "aryl" as used herein refers to an aromatic substituent which may be a single aromatic ring or multiple aromatic rings fused together. Examples include phenyl, naphthyl, anthracenyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, and the like.

[0092] "Substituted aryl" is substituted on any one or more substitutable ring atoms which are ring carbon atoms bonded to hydrogen. Substituted aryl is usually substituted with 1-5 (e.g., one or two or three) substituents independently selected from the group consisting of hydroxyl, thiol, cyano, nitro, C 1 -C 4 Alkyl, C 1 -C 4 Alkenyl, C 1 -C 4 Alkynyl, C 1 -C 4 Alkoxy, C 1 -C 4 Sulfanyl, C 1 -C 4 Alkenyloxy, C 1 -C 4 Alkynyloxy, halogen, C 1 -C 4 Alkylcarbonyl, carboxyl, C 1 -C 4 Alkoxycarbonyl, amine, C 1 -C 4 Alkylamino, di-C 1 -C 4 Alkylamine, C 1 -C4 Alkylaminocarbonyl, di-C 1 -C 4 Alkylaminocarbonyl, C 1 -C 4 Alkylcarbonylamino, C 1 -C 4 Alkylcarbonyl, C 1 -C 4 Alkylamino, sulfonyl, sulfonamide, alkylsulfonamide, and C 1 -C 4 Alkylaminosulfonyl, wherein each of the above hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl, alkoxy residues) may be further substituted at each occurrence by one or more residues independently selected from the following: halogen, hydroxyl or C 1 -C 4 Alkoxy.

[0093] The term "heterocyclyl" or "heterocyclic group" means a monocyclic non-aromatic (including partially saturated) ring preferably having 3 to 10 members and preferably containing 1-4 ring heteroatoms, or a polycyclic ring preferably having 7 to 20 members and preferably 1 to 4 ring heteroatoms, wherein the polycyclic ring has one or more monocyclic non-aromatic heterocyclic rings fused to one or more aromatic or heteroaromatic rings. The heterocyclic group typically has 3 to 7, 3 to 24, 4 to 16, 5 to 10, or 5 or 6 ring atoms; of which 1 to 4, especially 1 or 2, ring atoms may be heteroatoms (so that the remaining ring atoms are carbon) as required. Each heteroatom is independently selected from: nitrogen, quaternary nitrogen, nitrogen oxide (e.g., NO); oxygen; and sulfur, including sulfoxides and sulfones. The heterocyclic group may be attached to a heteroatom or a carbon atom. Examples of heterocycles include tetrahydrofuran (THF), dihydrofuran, 1,4-dioxane, morpholine, 1,4-dithiolane, piperazine, piperidine, 1,3-dioxolane, imidazoline, imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1,3-dioxane, 1,3-dithiolane, oxathiolane, thiomorpholine, and the like.

[0094] The heterocyclic group may include fused or bridged rings, and spiro rings. In one embodiment, the heterocyclic group is a bicyclic ring having a monocyclic non-aromatic heterocyclic ring fused to a phenyl group. Examples of polycyclic heterocyclic groups include tetrahydroisoquinolinyl (e.g., 1,2,3,4-tetrahydroisoquinolin-7-yl, 2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl, 1,2,3,4-tetrahydroisoquinolin-6-yl, and 2-methyl-1,2,3,4-tetrahydroisoquinolin-6-yl), isoindolyl (e.g., 2-ethylisoindolyl-5-yl, 2-methylisoindolyl-5-yl), indolyl, tetrahydrobenz[f]oxazepanyl (e.g., 2,3,4,5-tetrahydrobenz[f][1,4]oxazepan-7-yl).

[0095] The term "heterocycle", "heterocyclic group", or "heterocyclic ring system" refers to a ring which may be substituted as required, whether saturated or partially saturated. The substituted heterocyclic group may be a heterocyclic group which is independently substituted by 1 to 4, such as one, two, three, or four substituents.

[0096] In some embodiments, the heterocyclyl group is a 3-14 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("3-14 membered heterocyclyl").

[0097] The terms "heteroaryl", "heteroaromatic", "heteroaryl ring", "heteroaryl group", "heteroaromatic ring", and "heteroaromatic group", used alone or as part of a larger moiety such as "heteroaralkyl" or "heteroarylalkoxy", refer to aromatic ring groups having 5 to 14 ring atoms selected from carbon and at least one (usually 1 to 4, more usually 1 or 2) heteroatom (e.g., oxygen, nitrogen, or sulfur). "Heteroaryl" includes monocyclic rings and polycyclic (e.g., bi- or tricyclic) rings in which a monocyclic heteroaromatic ring is fused to one or more other carbocyclic aromatic or heteroaromatic rings. Thus, "5-14 membered heteroaryl" includes monocyclic, bicyclic, or tricyclic ring systems.

[0098] Examples of monocyclic 5-6 membered heteroaryl groups include furanyl (e.g., 2-furanyl, 3-furanyl), imidazolyl (e.g., N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isocyanate, Azolyl (e.g., 3-iso Azolyl, 4-iso Azolyl, 5-iso Azolyl), Oxazolyl (e.g., 2- Oxazolyl, 5- Oxazolyl), Azolyl (e.g., 2- Azolyl, 4- Azolyl, 5- 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, pyridyl (e.g., 2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), pyridazinyl (e.g., 3-pyridazinyl), thiazolyl (e.g., 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), triazolyl (e.g., 2-triazolyl, 5-triazolyl), tetrazolyl (e.g., tetrazolyl), thienyl (e.g., 2-thienyl, 3-thienyl), pyrimidinyl, pyridinyl, and pyridazinyl.

[0099] Typically, the heteroaryl group is a 5-10 membered ring system (e.g., a 5-6 membered monocyclic ring or an 8-10 membered bicyclic ring) or a 5-6 membered ring system. Typical heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5- Azolyl, 3-, 4-, or 5-iso oxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridinyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl.

[0100] Examples of polycyclic aromatic heteroaryl groups include carbazolyl, benzoimidazolyl, benzothiophenyl, benzofuranyl, indolyl, quinolyl, benzotriazolyl, benzothiazolyl, benzo oxazolyl, benzoimidazole, isoquinolinyl, indolyl, isoindolyl, acridinyl, or benzoisoquinolinyl Azolyl.

[0101] Thus, the term "heteroaryl" also refers to a heteroaromatic ring fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, wherein the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include 1-, 2-, 3-, 5-, 6-, 7-, or 8-indolizinyl, 1-, 3-, 4-, 5-, 6-, or 7-isoindolyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-indazolyl, 2-, 4-, 5-, 6-, 7-, or 8-purinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, or 9-quinolizinyl, 2-, 3-, 4-, 5 ... 1-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 1-, 4-, 5-, 6-, 7-, or 8-phthalazinyl, 2-, 3-, 4-, 5-, or 6-naphthyridinyl, 2-, 3-, 5-, 6-, 7-, or 8-quinazolinyl, 3-, 4-, 5-, 6-, 7-, or 8-cinnolinyl, 2-, 4-, 6-, or 7-pteridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-4aH carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-carbazolyl, 1-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-carbolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-phenazone, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-acridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-acridinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenanthroline, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenothiazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenanthroline, oxazinyl, 2-, 3-, 4-, 5-, 6-, or 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-phenylisoquinolyl, 2-, 3-, 4-, or thiophene[2,3-b]furanyl, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or 11-7H-pyrazinyl[2,3-c]carbazolyl, 2-, 3-, 5-, 6-, or 7-2H-furanyl[3,2-b]-pyranyl, 2-, 3-, 4-, 5-, 7-, or 8-5H-pyridinyl[2,3-d]-o- Oxazine, 1-, 3-, or 5-1H-pyrazol[4,3-d]- oxazolyl, 2-, 4-, or 5-4H-imidazo[4,5-d]-thiazolyl, 3-, 5-, or 8-pyrazino[2,3-d]pyridazinyl, 2-, 3-, 5-, or 6-imidazo[2,1-b]-thiazolyl, 1-, 3-, 6-, 7-, 8-, or 9-furano[3,4-c]cinnolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 8-, 9-, 10-, or 11-4H-pyridino[2,3-c]carbazolyl, 2-, 3-, 8-, or 7-imidazo[1,2-b][1,2,4]triazinyl, 7-phenyl[b]thienyl, 2-, 4-, 5-, 6-, or 7-phenyl oxazolyl, 2-, 4-, 5-, 6-, or 7-benzoimidazolyl, 2-, 4-, 5-, 6-, or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-benzooxepanyl, 2-, 4-, 5-, 6-, 7-, or 8-benzoxanthyl, oxazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or 11-1H-pyrrolo[1,2-b][2]phenylazepanyl.

[0102] Typical fused heteroaryl groups include, but are not limited to, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-phenyl[b]thienyl, 2-, 4-, 5-, 6-, or 7-phenyl[b]thienyl, oxazolyl, 2-, 4-, 5-, 6-, or 7-benzoimidazolyl, and 2-, 4-, 5-, 6-, or 7-benzothiazolyl.

[0103] As used herein, the term pyridine or pyridyl optionally substituted with hydroxy (e.g., 2-pyridyl, 3-pyridyl or 4-pyridyl) refers to hydroxy-pyridine or hydroxy-pyridyl, respectively, and may include tautomerism thereof, such as pyridone or pyridone, respectively.

[0104] As used herein, the term pyridine or pyridyl, which may be optionally substituted with a pendant oxy group (e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), refers to pyridone or pyridone, respectively, and may include tautomerism thereof, such as hydroxy-pyridine or hydroxy-pyridyl, respectively, provided that such tautomerism is available. Pyridine or pyridyl, which may be optionally substituted with a pendant oxy group, may further be referred to as pyridine-N-oxide or pyridyl-N-oxide, respectively.

[0105] "Substituted heteroaryl" is substituted at any one or more substitutable ring atoms which are ring carbon or ring nitrogen atoms bonded to hydrogen.

[0106] The term "bridged bicyclic" refers to a ring system comprising two rings which share at least three adjacent ring atoms.

[0107] As used herein, many moieties (e.g., alkyl, alkylene, cycloalkyl, aryl, heteroaryl, or heterocyclyl) are referred to as "substituted" or "optionally substituted." When one of these terms is used to modify a moiety, it refers to any portion of the moiety that is known to those skilled in the art to be substituted, including one or more substituents. Where more than one substituent is present, each substituent may be independently selected. Such substitution patterns are known in the art and / or taught by the present invention. The substituents selected as desired may be any substituents suitable for attachment to the moiety.

[0108] If suitable substituents are not explicitly indicated, examples of substituents include, but are not limited to: 1 -C 5 ) alkyl, (C 1 -C 5 ) hydroxyalkyl, (C 1 -C 5 ) haloalkyl, (C 1 -C 5 ) alkoxy, (C 1 -C 5 )Haloalkoxy, halogen, hydroxyl, cyano, amine, -CN, -NO 2 、-OR c1 、-NR a1 R b1 、-S(O) i R a1 、-NR a1 S(O) i R b1 、-S(O) i NR a1 R b1 、-C(=O)OR a1 、-OC(=O)OR a1 、-C(=S)OR a1 、-O(C=S)R a1 、-C(=O)NR a1 R b1 、-NR a1 C(=O)R b1 、-C(=S)NR a1 R b1 、-C(=O)R a1 、-C(=S)R a1 NR a1 C(=S)R b1 、-O(C=O)NR a1 Rb1 、-NR a1 (C=S)OR b1 ,

[0109] -O(C=S)NR a1 R b1 、-NR a1 (C=O)NR a1 R b1 、-NR a1 (C=S)NR a1 R b1 , phenyl, or 5-6 membered heteroaryl. a1 And each R b1 are independently selected from -H and (C 1 -C 5 ) alkyl, which may be optionally substituted with a hydroxyl group or (C 1 -C 3 ) alkoxy substituted; R c1 =–H, (C 1 -C 5 ) haloalkyl or (C 1 -C 5 ) alkyl, wherein (C 1 -C 5 ) alkyl may be optionally substituted with a hydroxyl group or (C 1 -C 3 ) alkoxy substituted.

[0110] The compounds described herein may be in various tautomeric forms. The term "tautomer" or "tautomerism" refers to two or more interconvertible compounds / substituents resulting from at least one formal shift of a hydrogen atom and at least one valence change (e.g., from a single bond to a double bond, from a triple bond to a single bond, or vice versa). Examples of tautomerization include ketone to enol, amide to imide, lactam to lactimide, enamine to imine, and enamine to (different enamine) tautomerization. The present teachings include compounds in tautomeric forms, including forms where the structure is not shown. All isomeric forms of such compounds are included. If the tautomer of a compound is aromatic, then the compound is aromatic.

[0111] The compounds of any of the above formulae may be present in one or more isomers (e.g., optical, geometric, or tautomeric forms). The compounds of any of the above formulae may also be isotopically labeled. Such variations are implicit in the compounds of any of the above defined formulae because they refer to their structural features and are therefore within the scope of the present invention.

[0112] Compounds of any of the above formulae containing one or more asymmetric carbon atoms may exist as two or more stereoisomers. If a compound of any of the above formulae contains an alkene or alkenylene group, geometric cis / trans (or Z / E) isomers may occur. Tautomerism ("tautomerization") may occur if structural isomers are interconvertible across a low energy barrier. This may occur in the form of proton tautomerization in compounds of any of the above formulae containing, for example, an imino, keto, or oxime group, or in the form of so-called valence tautomerization in compounds containing aromatic moieties. Thus, a single compound may exist in more than one isomerization.

[0113] Compounds with one or more chiral centers may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their arrangement in space. Stereoisomers include all diastereoisomers, enantiomers, and diastereomeric forms, as well as racemates and mixtures thereof. The term "geometric isomer" refers to compounds having at least one double bond, wherein the double bond may be in the cis (also known as syn or entgegen (E)) or trans (also known as anti or zusammen (Z)) form, and mixtures thereof. When a compound is disclosed by name or formula without indicating stereochemistry, it is understood that the name or formula encompasses one or more possible stereoisomers, or geometric isomers, or a mixture of stereoisomers or geometric isomers.

[0114] When a geometric isomer is indicated by name or formula, it is understood that the isomer indicated by the name or formula is present in a greater degree than another isomer, that is, the geometric purity of the geometric isomer indicated by the name or formula is greater than 50%, such as at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight. The geometric purity is determined by dividing the weight of the geometric isomer indicated by the name or formula in the mixture by the total weight of all geometric isomers in the mixture.

[0115] A racemic mixture means 50% one enantiomer and 50% the corresponding enantiomer. When a compound having one chiral center is represented by a name or formula without indicating the stereochemistry of the chiral center, it is understood that the name or formula encompasses both possible enantiomeric forms of the compound (e.g., two pure enantiomers, an enriched enantiomer, or a racemic form). When a compound having two chiral centers is represented by a name or formula without indicating the stereochemistry of the chiral center, it is understood that the name or formula encompasses all possible diastereomeric forms of the compound (e.g., pure diastereomers, enriched diastereomers, and equimolar mixtures of one or more diastereomers (e.g., racemic mixtures)).

[0116] Enantiomeric and diastereomeric mixtures can be resolved into their enantiomeric or stereoisomeric components by conventional methods, such as palm-phase gas chromatography, palm-phase high performance liquid chromatography, crystallization of the compound to form a chiral salt complex, or crystallization of the compound in a chiral solvent. Enantiomers and diastereoisomers can also be prepared from pure diastereomeric or enantiomerically pure intermediates, reagents, and catalysts using conventional asymmetric synthesis methods.

[0117] When a compound is represented by a name or formula that indicates a single enantiomer, the optical purity of that compound is at least 60%, 70%, 80%, 90%, 99% or 99.9% (also referred to as "enantiomerically pure") unless otherwise indicated. Optical purity is determined by dividing the weight of the enantiomer indicated by the name or formula in the mixture by the total weight of the two enantiomers in the mixture.

[0118] When the stereochemistry of a disclosed compound is indicated by name or structural formula and the name or the structural formula shown encompasses more than one stereoisomer (e.g., as a pair of diastereomers), it is understood that it includes any one of the stereoisomers or any mixture of the included stereoisomers. It is also understood that the stereoisomeric purity of the stereoisomer indicated by the name or structural formula is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight. In this case, the stereoisomeric purity is determined by dividing the total weight of the mixture of stereoisomers encompassed by the name or structural formula by the total weight of the mixture of all stereoisomers.

[0119] Pharmaceutically acceptable salts of the compounds of any of the formulae described above may also contain optically active counterions (eg, d-lactate or l-lysine) or be racemic (eg, dl-tartrate or dl-arginine).

[0120] Cis / trans isomers can be separated by techniques known to those skilled in the art, for example, chromatography and fractional crystallization.

[0121] Techniques commonly used to prepare / isolate individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). Alternatively, the racemate (or racemic precursor) may be reacted with a suitable optically active compound (e.g., an alcohol), or, if the compound of any of the above formulae contains an acidic or basic moiety, with a base or acid (e.g., 1-phenylethylamine or tartaric acid). The resulting mixture of diastereoisomers may be separated by chromatography and / or fractional crystallization, and one or both diastereoisomers may be converted into the corresponding pure enantiomers by methods known to those skilled in the art. The chiral compound of any of the formulae described above (and its chiral precursor) can be subjected to chromatography (usually HPLC) on an asymmetric resin using a mobile phase consisting of a hydrocarbon (usually heptane or hexane) containing 0 to 50% by volume of isopropanol (usually 2% to 20%) and 0 to 5% by volume of an alkylamine (usually 0.1% diethylamine) to obtain an enriched enantiomer. The eluate is concentrated to obtain an enriched mixture. Chiral chromatography can be performed using subcritical and supercritical fluids. Chiral chromatography suitable for use in some embodiments of the present invention is known in the art (see, for example, Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography with Packed Columns), pp. 223-249 and references cited therein). The column is available from Chiral Technologies, Inc, West Chester, Pa., USA. A subsidiary of Chemical Industries, Ltd., Tokyo, Japan.

[0122] It must be emphasized that where the compounds of any of the formulae described above have been drawn in a single tautomeric form, all possible tautomeric forms are included within the scope of the present invention.

[0123] The present invention also includes all pharmaceutically acceptable isotopically labeled compounds of any of the above formulae wherein one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature.

[0124] Examples of isotopes suitable for incorporation into the compounds of the present invention include isotopes of hydrogen (e.g. 2 H and 3 H), carbon isotopes (such as 11C. 13 C and 14 C), isotopes of chlorine (such as 36 Cl), fluorine isotopes (such as 18 F), isotopes of iodine (such as 123 I and 125 I), nitrogen isotopes (such as 13 N and 15 N), oxygen isotopes (such as 15 O. 17 O and 18 O), phosphorus isotopes (such as 32 P), and sulfur isotopes (such as 35 S).

[0125] Certain isotopically labeled compounds of any of the above formulae, for example, those into which a radioactive isotope has been incorporated, are useful in drug and / or substrate tissue distribution studies. 3 H, and carbon-14, i.e. 14 C, is particularly suitable for this purpose because it is easy to incorporate and easy to detect.

[0126] Using heavier isotopes such as deuterium, 2 H, may provide some medical benefit due to greater metabolic stability, for example, prolonged in vivo half-life or reduced dosage requirements.

[0127] Instead, use positron-emitting isotopes such as 11 C. 18 F. 15 O and 13 N substitution can be used to detect substrate receptor occupancy in positron emission tomography (PET) studies.

[0128] Isotope-labeled compounds of any of the above formulae can generally be prepared by conventional techniques known to those skilled in the art or by processes similar to those described in the accompanying examples and preparations, using an appropriately labeled isotope reagent in place of the unlabeled reagent previously used.

[0129] Pharmaceutically acceptable solvates according to the present invention include those wherein the solvent of crystallization may be isotopically substituted, for example, D 2 O、d 6 -acetone, d 6 -DMSO.

[0130] It is understood that when a compound is represented by a structural formula or indicated by a chemical name herein, all other tautomeric forms that may exist for the compound are encompassed by the structural formula.

[0131] Any chemical formula shown herein is also intended to represent unlabeled and labeled isotopic forms of the compound, such as 2H. In addition, substitution with deuterium (i.e., 2H or D) may provide certain medical benefits due to higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements or improved medical indexes, and it is understood that deuterium is a substituent of the compound of formula (I-0), (I-1) or (I-2). The concentration of these heavier isotopes, specifically deuterium, can be defined by the isotopic enrichment factor. As used herein, the term "isotopic enrichment factor" means the ratio between the abundance of the isotope and the natural abundance of the specified isotope. When the substituents on the compounds of the invention are deuterium, the isotopic enrichment factors of such compounds for each designated deuterium atom are at least 3500 (52.5% deuterium incorporated at each designated deuterium atom), at least 4000 (60% deuterium incorporated at each designated deuterium atom), at least 4500 (67.5% deuterium incorporated at each designated deuterium atom), at least 5000 (75% deuterium incorporated at each designated deuterium atom), at least 5 ... (82.5% deuterium is included at each designated deuterium atom), at least 6000 (90% deuterium is included at each designated deuterium atom), at least 6333.3 (95% deuterium is included at each designated deuterium atom), at least 6466.7 (97% deuterium is included at each designated deuterium atom), at least 6600 (99% deuterium is included at each designated deuterium atom), or at least 6633.3 (99.5% deuterium is included at each designated deuterium atom).

[0132] The compounds of the invention may be used therapeutically in free form or, where appropriate, in the form of a pharmaceutically acceptable salt.

[0133] The term "salt" or "salts" as used herein refers to acid addition salts or base addition salts of the compounds of the present invention. "Salts" specifically include "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of the present invention, which are generally not biologically or otherwise undesirable. In many cases, the compounds of the present invention can form acid and / or base salts by utilizing the presence of amine and / or carboxyl groups or similar groups.

[0134] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorophylline, citrate, edisylate, fumarate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, stearate, succinate, subsalicylate, tartrate, toluenesulfonate and trifluoroacetate.

[0135] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like.

[0136] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid and the like. Pharmaceutically acceptable base addition salts can be formed from inorganic bases and organic bases. Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from Groups I to XII of the periodic table. In some specific examples, salts can be derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper. In some specific examples, suitable salts include salts such as ammonium, potassium, sodium, calcium, and magnesium.

[0137] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, bile salts, diethanolamine, diethylamine, lysine, methylglucamine, piperazine, and trishydroxymethylmethylamine.

[0138] The pharmaceutically acceptable salts of the present invention can be synthesized from basic or acidic moieties using conventional chemical methods. Generally, the preparation of these salts can be achieved by reacting the free acid form of these compounds with a stoichiometric amount of an appropriate base (such as a hydroxide, carbonate, bicarbonate or the like of Na, Ca, Mg, or K), or by reacting the free base form of these compounds with a stoichiometric amount of an appropriate acid. These reactions are usually carried out in water or in an organic solvent, or in a mixture of the two. Generally, if feasible, it is desirable to use a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. For a list of other suitable salts, see, for example, the 20th edition of "Remington's Pharmaceutical Sciences", Mack Publishing Company, Easton, PA, (1985); and Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use", (Wiley-VCH, Weinheim, Germany, 2002).

[0139] The terms "composition" and "formulation" are used interchangeably.

[0140] A "subject" is a mammal, preferably a human, but may also be an animal in need of veterinary treatment, such as pet animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like), and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

[0141] As used herein, an individual is "in need of" a treatment if such individual would benefit biologically, medically, or in quality of life from such treatment.

[0142] The terms "administering", "dosing" or "administration" refer to providing a compound of the invention or a composition thereof into or onto a subject. Such methods include, but are not limited to, intraarticularly (into a joint), intravenously, intramuscularly, intratumorally, intradermally, intraperitoneally, subcutaneously, orally, topically, intrathecally, inhaled, transdermally, rectly, and the like. Administration techniques that can be used with the formulations and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition; Pergamon; and Remington, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pennsylvania.

[0143] As used herein, the terms "inhibit," "inhibitory," or "inhibition" refer to the reduction or suppression of a specified symptom, condition, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0144] The terms "treat", "treat" and "treatment" refer to reversing, slowing down, or inhibiting the progression of a disease described herein. In some embodiments, treatment (i.e., medical treatment) may be administered after one or more signs or symptoms of a disease have developed or been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment (i.e., preventive treatment) may be administered before the onset of symptoms in a susceptible individual (e.g., based on a history of symptoms and / or based on the pathogens to which the patient has been exposed). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent relapse.

[0145] The terms "disease," "disease," and "disorder" are used interchangeably.

[0146] Generally, the effective amount of the compounds taught herein varies depending on various factors, such as the specified drug or compound, pharmaceutical formulation, route of administration, disease or disorder pattern, characteristics of the individual or host to be treated, and similar factors, but is routinely determined by those skilled in the art. The effective amount of the compounds taught herein is easily determined by those skilled in the art using routine methods known in the art.

[0147] The term "effective amount" means an amount that, when administered to a subject, can result in a beneficial or desired outcome, including clinical outcomes, such as inhibition, suppression or reduction of symptoms of the disorder in the treated subject compared to a control group. For example, an effective amount can be provided in a unit dosage form (e.g., 1 mg to about 50 g per day, e.g., 1 mg to about 5 g per day).

[0148] The term "therapeutically effective amount" of a compound of the present invention refers to an amount of a compound of the present invention that can induce a biological or medical response in a subject, such as reducing or inhibiting enzyme or protein activity, or alleviating symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease, etc. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present invention that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent and / or alleviate (i) a condition or disorder or disease mediated by HPK1, or (ii) associated with HPK1 activity, or (iii) characterized by HPK1 activity (normal or abnormal); or (2) reduce or inhibit HPK1 activity; or (3) reduce or inhibit HPK1 expression; or (4) modify the protein level of HPK1. In another non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present invention that, when administered to a cell, or tissue, or non-cellular biological material, or medium, is effective to at least partially reduce or inhibit HPK1 activity; or partially or completely reduce or inhibit HPK1 expression.

[0149] All methods described herein can be performed in any suitable order, unless otherwise indicated or otherwise clearly stated herein. The use of any examples and other examples provided herein, or exemplary statements (e.g., "such as") are only intended to better illustrate the present invention, and unless otherwise claimed, are not intended to limit the scope of the present invention.

[0150] The general chemical terms used in the above chemical formulas have their general definitions.

[0151] As used herein, “h” or “hr” means hour, “min” means minute, “MCL” means mantle cell lymphoma, “AML” means acute myeloid leukemia, “CML” means chronic myeloid leukemia, “Boc” means N-tert-butyloxycarbonyl, “EA” means ethyl acetate, “DCM” means dichloromethane, “DMSO” means dimethyl sulfoxide, “DMA” means dimethylacetamide, “THF” means tetrahydrofuran, “MtBE” means methyl tert-butyl ether, “TEA” means triethylamine, “FBS” means fetal bovine serum, “PBS” means phosphate-buffered saline, “BSA” means bovine serum albumin, “RT” means room temperature, “mpk” means milligrams per kilogram, and “po” means oral administration. “qd” means once a day, “HPLC” means high performance liquid chromatography, “q2d” means a single dose administered every 2 days, “q2dx10” means a single dose administered every 2 days multiplied by 10, “VSMC” means vascular smooth muscle cells, and “XRD” means X-ray diffraction.

[0152] The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes and the like and combinations thereof, which are known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329). Unless any commonly used carrier is incompatible with the active ingredient, it can be used in a medical or pharmaceutical composition.

[0153] In addition, the compounds of the present invention, including their salts, may also be prepared in the form of hydrates or include other solvents used for crystallization. The compounds of the present invention may be solvates themselves or may be designed to form solvates with pharmaceutically acceptable solvents (including water); therefore, the present invention is intended to include both solvate and non-solvate forms. The term "solvate" refers to a complex formed by the compounds of the present invention (including their pharmaceutically acceptable salts) and one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical art and are known to be harmless to the recipient, for example, water, ethanol and the like. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0154] The compounds of the present invention, including their salts, hydrates and solvates, may themselves be polymorphs or may be designed to form polymorphs. In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated for specific routes of administration, such as oral administration, parenteral administration, and rectal administration, etc. In addition, the pharmaceutical composition of the present invention can be prepared in solid form (including, but not limited to capsules, tablets, pills, granules, powders, or suppositories), or in liquid form (including, but not limited to solutions, suspensions or emulsions). The pharmaceutical composition can be subjected to commonly used pharmaceutical operations, such as sterilization and / or may contain commonly used inert diluents, lubricants, or buffers, and adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers, etc.

[0155] Typically, the pharmaceutical composition is a tablet or a capsule containing the active ingredient, together with a) a diluent, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycerol; b) a lubricant, for example, silica, talc, stearic acid, its magnesium or calcium salt, and / or polyethylene glycol; and c) a binder for tablets, for example, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if necessary, d) a disintegrant, for example, starch, agar, alginic acid or its sodium salt, or an effervescent mixture; and / or e) an absorbent, a colorant, a flavoring agent and a sweetener. The tablet can be film-coated or enteric-coated according to known methods in the art.

[0156] Compositions suitable for oral administration include an effective amount of a compound of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral administration are prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and these compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives to produce pharmaceutically aesthetic and palatable preparations. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating agents and disintegrating agents such as corn starch, or alginic acid; binders such as starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or may be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained long-term effect. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used. Oral formulations may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0157] Some injection compositions are aqueous isotonic solutions or suspensions, and suppositories are preferably made of fat emulsions or suspensions. Such compositions may be sterilized and / or contain adjuvants, such as preservatives, tranquilizers, wetting agents, or emulsifiers, pro-solubility agents, salts for regulating osmotic pressure, and / or buffers. In addition, they may also contain other substances with medical value. Such compositions are prepared according to general mixing, granulation, or coating methods, respectively, and contain about 0.1-75% or about 1-50% of active ingredients. Compositions suitable for transdermal administration include an effective amount of the compound of the present invention and a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmaceutically acceptable solvents to facilitate passage through the host skin. For example, a transdermal device is in the form of a bandage comprising a backing member, a reservoir containing the compound and, optionally, a carrier, optionally a rate-controlling barrier to provide for long-term delivery of the compound to the skin of the host at a controlled and predetermined rate, and means for securing the device to the skin.

[0158] Compositions suitable for topical (e.g., administration to the skin and eyes) administration include aqueous solutions, suspensions, ointments, creams, gels, or spray formulations, e.g., delivered by aerosol, or the like. Such topical surface delivery systems are particularly suitable for skin administration, e.g., for the treatment of skin cancer, e.g., for preventive use in sunscreens, lotions, sprays, and the like. They are therefore particularly suitable for topical, including cosmetic formulations known in the art. These may contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0159] As used herein, topical administration also relates to inhalation or intranasal administration, which are suitably delivered as dry powders (alone, as a mixture (e.g., a dry blend with lactose, or with, for example, phospholipids to form mixed component particles) from a dry powder inhaler, or as an aerosol spray from a pressurized container, pump, spray bottle, nebulizer or aerosol, with or without a suitable propellant.

[0160] The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients, since water may promote the degradation of certain compounds.

[0161] The anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low moisture content ingredients under low moisture and low humidity conditions. Anhydrous pharmaceutical compositions can be prepared and stored so that their anhydrous nature is maintained. Therefore, the anhydrous compositions are packaged using materials known to prevent exposure to moisture so that they can be contained in a suitable formulation kit. Examples of suitable packaging include, but are not limited to, airtight foil packages, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0162] The present invention further provides a pharmaceutical composition and dosage form, which comprises one or more agents that can reduce the degradation rate of the compounds of the present invention as active ingredients. Such agents are referred to herein as "stabilizers" and include (but are not limited to) antioxidants such as ascorbic acid, pH buffers, or salt buffers, etc.

[0163] 3. Compounds

[0164] Specific examples of compounds having the general structural formula of formula (I-0) or (II) are disclosed herein:

[0165]

[0166] The first embodiment of the present invention provides a compound represented by formula (I-0) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0167] X is CR 2 R 3 or NR 3 ;

[0168] A is CR 2 or N;

[0169] R is an 8-10 membered bicyclic nitrogen-containing heteroaryl group or an 8-10 membered bicyclic nitrogen-containing heterocyclic group, which may be substituted with a pendant oxy group as needed, wherein the nitrogen-containing heteroaryl group or nitrogen-containing heterocyclic group represented by R has 1 to 3 heteroatoms selected from: N, O, and S, and may be substituted with 1 to 4 R as needed. a Substitution, and wherein R is connected to the pyrimidine ring by a nitrogen ring atom or R is represented by the following structural formula:

[0170]

[0171] R 1 H, deuterium, halogen, OH, CN, NH 2 、NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, phenyl, 5-6 membered heteroaryl, C 3-6 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 1 On behalf of or by R 1 The alkyl, alkenyl, alkynyl, alkoxy, phenyl, heteroaryl, cycloalkyl, or heterocyclic group in the represented group may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Haloalkyl, C 1-4 Haloalkoxy, and NR 11 R 12 ;or

[0172] R and R 1 Together with the carbon atoms to which they are attached they form the following representative ring:

[0173]

[0174] in The bonding system connects the pyrimidine rings;

[0175] R 2 Each of the examples is independently H, deuterium, halogen, OH, CN, NH2 、NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, P(=O)R 11 R 12 、S(=O) 2 R 11 , or S(=O) 2 NR 11 R 12 , where R 2 On behalf of or by R 2 The alkyl, alkenyl, alkynyl, or alkoxy group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 ;

[0176] R 3 H, C 1-6 Alkyl, C(O)C 1-6 Alkyl, C 3-6 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 3 On behalf of or by R 3 The alkyl, cycloalkyl or heterocyclic group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, and NR 11 R 12 ;

[0177] R 11 and R 12 Each instance of is independently H or C 1-6 Alkyl, wherein R 11 or R 12The alkyl group represented by the present invention may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6 Cycloalkyl, and 3-7 membered heterocyclic group,

[0178] R 11 and R 12 Together with the nitrogen atom or phosphorus atom to which they are attached, they form a 3-7 membered heterocyclic group, which may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, and C 1-4 Haloalkoxy;

[0179] R a Each of the examples is independently H, deuterium, halogen, OH, CN, NH 2 、NO 2 , COOH, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)C 1-6 Alkyl, C(O)OR 11 NR 11 C(O)R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 , C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, or 5-6 membered heteroaryl group, wherein R a On behalf of or by R aThe alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, or heteroaryl groups in the represented groups may be substituted as needed by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 , and 5-6 membered heteroaryl; or

[0180] Two R's a Together with the carbon atoms (groups) to which they are attached, they form C 3-6 Cycloalkyl or 3-7 membered heterocyclic group, wherein the cycloalkyl or heterocyclic group may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, and C 1-4 Haloalkoxy;

[0181] R b Each of the examples is independently H, deuterium, halogen, OH, CN, NH 2 、NO 2 , COOH, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R12 、C(O)C 1-6 Alkyl, C(O)OR 11 NR 11 C(O)R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 , C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, or 5-6 membered heteroaryl group, wherein R b On behalf of or by R b The alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, or heteroaryl groups in the represented groups may be substituted as needed by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 , and 5-6 membered heteroaryl;

[0182] R c Each example of is independently phenyl, a 5-6 membered monocyclic heterocyclic group having 1 to 3 heteroatoms selected from N and O; a 5-6 membered monocyclic heteroaryl group having 1 to 3 heteroatoms selected from N and O; wherein R c The phenyl, heterocyclic or heteroaryl represented by phenyl may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 , and P(O)-C 1-6 alkyl;

[0183] R a ' is C 1-6 Alkyl, which may optionally be OH, CN, 5-6 membered heteroaryl, C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 replace;

[0184] m is 0, 1, 2, or 3;

[0185] n is 0, 1, 2, 3, or 4;

[0186] p is 1, 2, or 3;

[0187] q is 1, 2, or 3; and p+q≤4.

[0188] In the second embodiment of the present invention, the compound is represented by structural formula (I-1):

[0189]

[0190] or a pharmaceutically acceptable salt or stereoisomer thereof, and the remaining variables are as defined in the first embodiment.

[0191] In the third embodiment of the present invention, the compound is represented by structural formula (I-2):

[0192]

[0193] The fourth specific example of the present invention provides a compound of formula (I-0), (I-1), or (I-2), or

[0194] Its pharmaceutically acceptable salt or stereoisomer, wherein R is

[0195]

[0196] in The bond is to the pyrimidine ring, and the remaining variables are as defined in the first, second and / or third embodiment.

[0197] In a fifth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R a Each of the examples is independently H, halogen, OH, CN, C 1-4 Alkyl, C 1-4 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)C 1-4 Alkyl, C(O)OR 11 NR 11 C(O)C 1-4 Alkyl, NR 11 S(=O) 2 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 、P(=O)R 11 R 12 , C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, or 5-6 membered heteroaryl group, wherein R a On behalf of or by R a The alkyl, alkoxy, cycloalkyl, heterocyclic or heteroaryl groups in the represented groups may be substituted as needed by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 , and 5-6 membered heteroaryl, and the remaining variables are as defined in the first, second, third and / or fourth embodiments.

[0198] In a sixth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R 1 H, halogen, CN, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, wherein R 1 On behalf of or by R 1 The alkyl, alkenyl, or alkoxy group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Haloalkyl, C 1-4 Haloalkoxy, and NR 11 R 12 , and the remaining variables are defined as in the first, second, third, fourth and / or fifth specific examples.

[0199] In the seventh embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein each R 2 Each independently represents H, halogen, OH, CN, NH 2 、NO 2 , C 1-4 Alkyl, C 1-4 Alkoxy, NR11 R 12 、C(O)NR 11 R 12 、C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, NR 11 C(O)C 1-4 Alkyl, P(=O)R 11 R 12 、S(=O) 2 R 11 , or S(=O) 2 NR 11 R 12 , where R 2 On behalf of or by R 2 The alkyl or alkoxy group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 , and the remaining variables are defined as in the first, second, third, fourth, fifth and / or sixth specific examples.

[0200] In an eighth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R 3 H, C 1-6 Alkyl, or C(O)C 1-6 Alkyl, wherein R 3 On behalf of or by R 3 The alkyl group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 , and the remaining variables are defined as in the first, second, third, fourth, fifth, sixth, and / or seventh specific examples.

[0201] In the ninth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein each R a are independently H, halogen, OH, CN, C 1-4 Alkyl, C 3-6 Cycloalkyl, C(O)NR11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 、N(R 11 )S(=O) 2 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 、P(O)R 11 R 12 , or a 5-6 membered heteroaryl group, wherein R a On behalf of or by R a The alkyl or cycloalkyl group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)NR 11 OR 12 、C(O)NR 11 S(=O) 2 R 12 、C(O)OR 11 NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 , and 5-6 membered heteroaryl; each example R 11 and R 12 Each is independently H or C 1-6 Alkyl, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, and / or eighth embodiment.

[0202] In the tenth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R 1H, halogen, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 1-4 Alkoxy, or C 1-4 Haloalkoxy, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, and / or ninth embodiment.

[0203] In the eleventh embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R is in The bond is to the pyrimidine ring, and n is 0 to 4, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth embodiment.

[0204] In the twelfth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R is in The bond is to the pyrimidine ring, and n is 0 to 2, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth embodiment.

[0205] In the thirteenth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R is in The bond is to the pyrimidine ring, and n is 0 to 2, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth embodiment.

[0206] In a fourteenth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein

[0207] R a Each of the examples is independently H, halogen, C 1-4 Alkyl, -(CHR aa ) k OH, -(CHR aa ) k CN, -(CHR aa ) k C(O)OR 11 、-(CHR aa )k C(O)NR 11 R 12 、-(CHR aa ) k C(O)NR 11 OR 12 、-(CHR aa ) k S(=O) 2 R 11 、-(CHR aa ) k C(O)NR 11 S(=O) 2 R 12 、-(CHR aa ) k S(=O) 2 NR 11 R 12 、-(CHR aa ) k NR 11 S(=O) 2 R 12 、-(CHR aa ) k -5-6 membered heteroaryl, or -(CHR aa ) k P(=O)R 11 R 12 ;

[0208] R aa are independently H or optionally halogen-substituted C 1-3 alkyl;

[0209] R 11 and R 12 Each is independently H or C 1-4 Alkyl; and

[0210] k is 0 or 1, and the remaining variables are defined as in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and / or thirteenth embodiments.

[0211] In a fifteenth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R 1 H, F, Cl, CN, or CF 3 , and the remaining variables are defined as in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and / or fourteenth specific examples.

[0212] In the sixteenth embodiment, there is provided a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R is in The bond is to the pyrimidine ring, and n is 0 to 4, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, and / or fifteenth embodiment.

[0213] In the seventeenth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein

[0214] R is in The bond system connects the pyrimidine ring, R a1 H, C 1-4 Alkyl, or C 1-4 Hydroxyalkyl;

[0215] R a1 'are independently -(CHR aa ) k OH, -(CHR aa ) k CN, -(CHR aa ) k C(O)OR 11 、-(CHR aa ) k S(=O) 2 R 11 、-(CHR aa ) k C(O)NR 11 R 12 、-(CHR aa ) k C(O)NR 11 OR 12 、-(CHR aa ) k C(O)NR 11 S(=O) 2 R 12 、-(CHR aa ) k S(=O) 2 NR 11 R 12 、-(CHR aa ) k NR 11 S(=O) 2 R 12 、-(CHRaa ) k -5-6 membered heteroaryl, or -(CHR aa ) k P(=O)R 11 R 12 ;

[0216] R aa are independently H or optionally halogen-substituted C 1-3 alkyl;

[0217] R a are independently H, F, or Cl;

[0218] R 11 and R 12 Each is independently H or C 1-4 alkyl;

[0219] k is 0 or 1, and the remaining variables are defined as in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and / or tenth embodiments.

[0220] In the eighteenth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein

[0221] R a1 H, CH 3 , or CH 2 OH;

[0222] R a1 'are independently -(CHR aa ) k OH, -(CHR aa ) k CN, -(CHR aa ) k C(O)OR 11 、-(CHR aa ) k S(=O) 2 R 11 、-(CHR aa ) k C(O)NR 11 R 12 、-(CHR aa ) k C(O)NR 11 OR 12 、-(CHR aa ) k C(O)NR 11 S(=O) 2 R 12、-(CHR aa ) k S(=O) 2 NR 11 R 12 、-(CHR aa ) k NR 11 S(=O) 2 R 12 、-(CHR aa ) k -tetrazolyl, or -(CHR aa ) k P(=O)R 11 R 12 ;

[0223] R aa H, CH 3 , or CF 3 ;

[0224] R 11 and R 12 Each is independently H or C 1-2 Alkyl; and

[0225] k is 0 or 1, and the remaining variables are defined as in the seventeenth specific example.

[0226] In the nineteenth embodiment of the present invention, there is provided a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each R a are independently H or F, and the remaining variables are as defined in the eighteenth embodiment.

[0227] In the twentieth embodiment of the present invention, there is provided a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 is Cl, and the remaining variables are defined as in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, and / or nineteenth specific examples.

[0228] In the twenty-first embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R and R 1 Together with the carbon atoms to which they are attached they form the following representative ring:

[0229]

[0230] in The bond is to the pyrimidine ring, and the remaining variables are as defined in the first, second, and / or third embodiment.

[0231] In the twenty-second embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein

[0232] R b Each of the examples is independently H, halogen, OH, CN, NH 2 , COOH, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Hydroxyalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Hydroxyalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, C(O)NR 11 OR 12 、S(=O) 2 R 11 、S(=O) 2 NR 11 R 12 NR 11 (S=O) 2 R 12 、C(O)NR 11 S(=O) 2 R 12 、P(=O)R 11 R 12 , 5-6 membered heteroaryl, or NR 11 C(O)C 1-4 Alkyl; and

[0233] R c Each of the examples is phenyl or pyridyl, each of which may be substituted with one or two substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 , and P(=O)-C 1-6 Alkyl, and the remaining variables are as defined in the first, second, third, and / or twenty-first embodiment.

[0234] In the twenty-third embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R and R 1 Together with the carbon atoms to which they are attached they form the following representative ring:

[0235] in The bond is to the pyrimidine ring, and the remaining variables are as defined in the first, second, third, twenty-first and / or twenty-second embodiment.

[0236] In the twenty-fourth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein

[0237] R b Each of the examples is independently H, halogen, CN, COOH, C 1-2 Alkyl, or C 1-2 Haloalkyl;

[0238] R c Each of the examples is phenyl or pyridyl, each of which may be substituted with one or two substituents independently selected from the group consisting of the following: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、C(O)NR 11 R 12 , and P(=O)-C 1-6 Alkyl, and

[0239] R 11 and R 12 Each instance of is independently H or C 1-6 Alkyl, and the remaining variables are as defined in the first, second, third, twenty-first, twenty-second, and / or twenty-third embodiment.

[0240] In the twenty-fifth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein

[0241] R b Each instance of is independently H, CN, or COOH; and

[0242] R c Each example is phenyl or pyridyl, each of which may be substituted with one or two substituents independently selected from the group consisting of the following: 1-4Alkyl, C(O)N(CH 3 ) 2 , and P(=O)(CH 3 ) 2 , and the remaining variables are defined as in the first, second, third, twenty-first, twenty-second, twenty-third, and / or twenty-fourth specific examples.

[0243] In the twenty-sixth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein m is 0 or 1, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, and / or twenty-fifth embodiment.

[0244] In the twenty-seventh embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein for and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, and / or twenty-sixth specific examples.

[0245] In the twenty-eighth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R 2 Each example is H, halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy; and R 3 H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, or COCH 2 NR 11 R 12 , and the remaining variables are defined as in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, and / or twenty-seventh specific examples.

[0246] In the twenty-ninth embodiment, a compound of formula (I-0), (I-1), or (I-2), or a pharmaceutically acceptable salt or stereoisomer thereof is provided, wherein R 2 Each example is H, F, Cl, or OCH 3 ; and R 3 H or C 1-4 Alkyl, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, and / or twenty-eighth embodiment.

[0247] In the 30th embodiment of the present invention, there is provided a compound represented by formula (II), or a pharmaceutically acceptable salt or stereoisomer thereof,

[0248] in

[0249] A 1 CR' or N;

[0250] X is –P(=O)R 3’ R 4’ ;

[0251] R' is H, deuterium, halogen, CN, NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, wherein the alkyl, alkenyl, alkynyl, or alkoxy represented by R' or in the group represented by R' may be substituted with one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0252] R 1’ Each of the examples is independently H, deuterium, halogen, OH, CN, NO 2 , C1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, C 3-7 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 1’ On behalf of or by R 1’ The alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, or heterocyclic group in the represented group may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0253] R 2’ Each of the examples is independently H, deuterium, halogen, OH, CN, NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, phenyl, 5-6 membered heteroaryl, C 3-7 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 2’ On behalf of or by R 2’ The alkyl, alkenyl, alkynyl, alkoxy, phenyl, heteroaryl, cycloalkyl, or heterocyclic group in the represented group may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0254] Each R 3’ and R 4’ H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, wherein R 3’ or R 4’ The alkyl, alkenyl, or alkynyl represented by alkyl may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0255] R 11’ and R 12’ Each instance of is independently H or C 1-6 Alkyl, wherein R 11’ or R 12’ The alkyl group represented by the present invention may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6 Cycloalkyl, and 3-7 membered heterocyclic group, or

[0256] R 11’ and R 12’ Together with the nitrogen atom to which they are attached, they form a 3-7 membered heterocyclic group, which may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, or C 1-4 Haloalkoxy;

[0257] m' is 0, 1, or 2; and

[0258] n' is 0, 1, or 2.

[0259] In the thirty-first specific example of the present invention, the compound of formula (II) is represented by structural formula (II-1):

[0260]

[0261] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R' is H, halogen, NR 11’ R 12’ , or C1-6 Alkyl, which may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ , and the remaining variables are defined as in the 30th specific example.

[0262] In the thirty-second embodiment, the compound of formula (II) or (II-1), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3’ and R 4’ Each is independently H or C 1-6 Alkyl, which may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ , and the remaining variables are defined as in the 30th and / or 31st specific examples.

[0263] In the thirty-third embodiment, the compound of formula (II) or (II-1), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0264] R 1’ Each of the examples is independently H, halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 1’ The alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclic group represented by alkyl may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0265] R 2’ Each of the examples is independently H, halogen, OH, NO 2 , C 1-6 Alkyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 alkyl, 5-6 membered heteroaryl, or 3-7 membered heterocyclic group, wherein R 2’ On behalf of or by R 2’ The alkyl, alkoxy, heteroaryl, or heterocyclic group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ , and the remaining variables are defined as in the 30th, 31st, and / or 32nd specific examples.

[0266] In the thirty-fourth specific example, the compound of formula (II) or (II-1) is represented by the structural formula (II-2) or (II-2'):

[0267]

[0268] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R' is H, halogen, or NH 2 , and the remaining variables are as defined in the 30th, 31st, 32nd, and / or 33rd specific examples.

[0269] In the thirty-fifth embodiment, the compound of formula (II), (II-1), (II-2) or (II-2'), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2’ Each of the examples is independently H, halogen, OH, C 1-4 Alkyl (which may be optionally substituted with halogen or OH or NR 11’ R 12’ Replace), C 1-4 Alkoxy, C(O)OC 1-6 Alkyl, C(O)NR 11’ R 12’ ,NR 11’ R 12’ ,NR 11’ C(O)C 1-6 alkyl, 5-6 membered heterocyclic group, or 5-6 membered heteroaryl; and each example R 1’ Each is independently H or C 1-4 Alkyl, and the remaining variables are as defined in the 30th, 31st, 32nd, 33rd, and / or 34th embodiment.

[0270] In the thirty-sixth embodiment, the compound of formula (II), (II-1), (II-2) or (II-2'), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3’ and R 4’ Each is independent of C 1-6 Alkyl; and each R 2’ are independently H, halogen, OH, C 1-4 Alkyl (which may be optionally substituted with halogen or OH or NR 11’ R 12’ Replace), C 1-4 Alkoxy, C(O)OC 1-4 Alkyl, C(O)NR 11’ R 12’ NR 11’ R 12’ 、NHC(O)C 1-6 Alkyl, pyrrolidinyl, pyrrolidin-2-one, Azoles, where R 11’ and R 12’ Each instance of is independently H or C 1-4 Alkyl, and the remaining variables are as defined in the 30th, 31st, 32nd, 33rd, 34th, and / or 35th embodiment.

[0271] In the thirty-seventh embodiment, the compound of formula (II), (II-1), (II-2) or (II-2'), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein X is -P(O)(CH 3 ) 2 and R 2’ Each of the examples is independently H, F, Cl, OH, CH 3 NH 2 , or NHCOCH 3 , and the remaining variables are as defined in the thirtieth, thirty-first, thirty-second, thirty-third, thirty-fourth, thirty-fifth, and / or thirty-sixth specific examples.

[0272] 1. A compound represented by structural formula (I):

[0273]

[0274] or a pharmaceutically acceptable salt thereof, wherein

[0275] A is CR 2 or N;

[0276] R is an 8-10 membered bicyclic nitrogen-containing heteroaryl group or an 8-10 membered bicyclic nitrogen-containing heterocyclic group, which may be substituted with a pendant oxy group as needed, wherein the nitrogen-containing heteroaryl group or nitrogen-containing heterocyclic group represented by R has 1 to 3 heteroatoms selected from: N, O, and S, and may be substituted with 1 to 4 R as needed.a replace;

[0277] R 1 H, deuterium, halogen, OH, CN, NH 2 、NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, phenyl, 5-6 membered heteroaryl, C 3-6 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 1 On behalf of or by R 1 The alkyl, alkenyl, alkynyl, alkoxy, phenyl, heteroaryl, cycloalkyl, or heterocyclic group in the represented group may be substituted with one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 ;or

[0278] R and R 1 Together with the carbon atoms to which they are attached they form the following representative ring: in The bonding system connects the pyrimidine rings;

[0279] R 2 H, deuterium, halogen, OH, CN, NH 2 、NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, -P(=O)R 11 R 12,-S(=O) 2 R 11 , or -S(=O) 2 NR 11 R 12 , where R 2 On behalf of or by R 2 The alkyl, alkenyl, alkynyl, or alkoxy group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 ;

[0280] R 3 H, C 1-6 Alkyl, C(O)C 1-6 Alkyl, C 3-6 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 3 On behalf of or by R 3 The alkyl, cycloalkyl, or heterocyclic group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6 Cycloalkyl, 3-7 membered heterocyclic group, and NR 11 R 12 ;

[0281] R 11 and R 12 Each instance of is independently H or C 1-6 Alkyl, wherein R 11 or R 12 The alkyl group represented by the present invention may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6 Cycloalkyl, and 3-7 membered heterocyclic group, or

[0282] R 11 and R 12 Together with the nitrogen atom or phosphorus atom to which they are attached, they form a 3-7 membered heterocyclic group, which may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, -OH, CN, C1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, or C 1-4 Haloalkoxy;

[0283] R a Each of the examples is independently H, deuterium, halogen, OH, CN, NH 2 、NO 2 , COOH, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, -P(=O)R 11 R 12 、-S(=O) 2 R 11 , or -S(=O) 2 NR 11 R 12 , C 3-6 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R a On behalf of or by R a The alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, or heterocyclic group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 ;or

[0284] Two R's a Together with the carbon atoms to which they are attached, they form C 3-6 Cycloalkyl or 3-7 membered heterocyclic group, wherein the cycloalkyl or heterocyclic group may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, and C 1-4 Haloalkoxy;

[0285] R bEach of the examples is independently H, deuterium, halogen, OH, CN, NH 2 、NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, or NR 11 C(O)C 1-6 Alkyl, wherein R b On behalf of or by R b The alkyl, alkenyl, alkynyl, or alkoxy group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 ;

[0286] R c is phenyl, a 5-6 membered monocyclic heterocyclic group having 1 to 3 heteroatoms selected from N and O; a 5-6 membered monocyclic heteroaryl group having 1 to 3 heteroatoms selected from N and O; wherein R c The phenyl, heterocyclic or heteroaryl represented by phenyl may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、-C(O)NR 11 R 12 , and -P(O)-C 1-6 Alkyl; and

[0287] m is 0, 1, 2 or 3.

[0288] 2. The compound of paragraph 1, or a pharmaceutically acceptable salt thereof, wherein R is

[0289]

[0290] in The bond is to the pyrimidine ring, and n is 0-4.

[0291] 3. The compound of paragraph 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R a Each of the examples is independently H, halogen, OH, NH 2 ,CN,COOH,C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Hydroxyalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Hydroxyalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, NR 11 C(O)C 1-4 Alkyl, -P(=O)R 11 R 12 、-S(=O) 2 R 11 、-S(=O) 2 NR 11 R 12 , C 3-6 Cycloalkyl, or 3-7 membered heterocyclic group, or

[0292] Two R's a Together with the carbon atoms to which they are attached, they form C 3-6 Cycloalkyl or 3-7 membered heterocyclic group, wherein the cycloalkyl or heterocyclic group may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, and C 1-4 Haloalkoxy.

[0293] 4. A compound according to any one of paragraphs 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R 1 H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, wherein R 1 On behalf of or by R 1The alkyl, alkenyl, or alkoxy group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 .

[0294] 5. A compound according to any one of paragraphs 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R a Each of the examples is independently H, halo, OH, C 1-4 Alkyl, COOH, C(O)NR 11 R 12 , or –P(O)R 11 R 12 , where R 11 and R 12 Each instance of is independently H or C 1-6 alkyl.

[0295] 6. A compound according to any one of paragraphs 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R 1 H, halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 1-4 Alkoxy, or C 1-4 Haloalkoxy.

[0296] 7. A compound according to any one of paragraphs 1 to 6, or a pharmaceutically acceptable salt thereof, wherein R is

[0297] in The bond is to the pyrimidine ring, and n is 0-2.

[0298] 8. A compound according to any one of paragraphs 1 to 7, or a pharmaceutically acceptable salt thereof, wherein R is in The bond is to the pyrimidine ring, and n is 0-2.

[0299] 9. A compound according to any one of paragraphs 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R a Each of the examples is independently H, F, Cl, C 1-4 Alkyl, COOH, CONH 2 、C(O)N(CH 3 ) 2 , or –P(O)(CH3 ) 2 .

[0300] 10. A compound according to any one of paragraphs 1 to 9, or a pharmaceutically acceptable salt thereof, wherein R 1 H, Cl, or CF 3 .

[0301] 11. The compound of paragraph 1, or a pharmaceutically acceptable salt thereof, wherein R and R 1 Together with the carbon atoms to which they are attached they form the following representative ring:

[0302] in The bonds connect the pyrimidine rings.

[0303] 12. The compound of paragraph 1 or 11, or a pharmaceutically acceptable salt thereof, wherein R b Each of the examples is independently H, halogen, OH, NH 2 , C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Hydroxyalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Hydroxyalkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, or NR 11 C(O)C 1-4 Alkyl; and

[0304] R c Each of the examples is phenyl or pyridyl, each of which may be substituted with one or two substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、-C(O)NR 11 R 12 , and -P(O)-C 1-6 alkyl.

[0305] 13. A compound according to any one of paragraphs 1, 11, and 12, or a pharmaceutically acceptable salt thereof, wherein

[0306] R b Each of the examples is independently H, halogen, C1-2 Alkyl, C 1-2 Haloalkyl; and

[0307] R c Each of the examples is phenyl or pyridyl, each of which may be substituted with one or two substituents independently selected from the group consisting of the following: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, NR 11 R 12 、-C(O)NR 11 R 12 , and -P(O)-C 1-6 Alkyl, and

[0308] R 11 and R 12 Each instance of is independently H or C 1-6 alkyl.

[0309] 14. A compound according to any one of paragraphs 1 and 11 to 13, or a pharmaceutically acceptable salt thereof, wherein

[0310] R b Each instance of is independently H; and

[0311] R c Each example is phenyl or pyridyl, each of which may be substituted with one or two substituents independently selected from the group consisting of the following: 1-4 Alkyl, -C(O)N(CH 3 ) 2 , and -P(O)(CH 3 ) 2 .

[0312] 15. A compound according to any one of paragraphs 1 to 14, or a pharmaceutically acceptable salt thereof, wherein R 2 H, halogen, OH, CN, NH 2 、NO 2 , C 1-4 Alkyl, C 1-4 Alkoxy, NR 11 R 12 、-C(O)NR 11 R 12 、C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, NR 11 C(O)C 1-4 Alkyl, -P(=O)R 11 R 12 、-S(=O) 2 R11 , or -S(=O) 2 NR 11 R 12 , where R 2 On behalf of or by R 2 The alkyl or alkoxy group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 ; and m is 0 or 1.

[0313] 16. A compound according to any one of paragraphs 1 to 15, or a pharmaceutically acceptable salt thereof, wherein for

[0314] 17. A compound according to any one of paragraphs 1 to 16, or a pharmaceutically acceptable salt thereof, wherein each R 2 H, halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy; and R 3 H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, or COCH 2 NR 11 R 12 , where each case R 11 and R 12 Each is independently H or C 1-6 alkyl.

[0315] 18. A compound according to any one of paragraphs 1 to 17, or a pharmaceutically acceptable salt thereof, wherein each R 2 H, Cl, or OCH 3 ; and R 3 H, C 1-4 Alkyl, C 1-4 Hydroxyalkyl, or COCH 2 N(CH 3 ) 2 .

[0316] 19. A compound represented by structural formula (II):

[0317]

[0318] or a pharmaceutically acceptable salt thereof, wherein

[0319] A 1 CR' or N;

[0320] X is –P(=O)R 3’ R 4’ or –S(O) 2 NR 5’ R 6’ ;

[0321] R' is H, deuterium, halogen, CN, NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, wherein the alkyl, alkenyl, alkynyl, or alkoxy group represented by R' or in the group represented by R' may be substituted with one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0322] R 1’ Each of the examples is independently H, deuterium, halogen, OH, CN, NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, C 3-7 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 1’ On behalf of or by R 1’The alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, or heterocyclic group in the represented group may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0323] R 2’ Each of the examples is independently H, deuterium, halogen, OH, CN, NO 2 , C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 Alkyl, phenyl, 5-6 membered heteroaryl, C 3-7 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 2’ On behalf of or by R 2’ The alkyl, alkenyl, alkynyl, alkoxy, phenyl, heteroaryl, cycloalkyl, or heterocyclic group in the represented group may be substituted with one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;

[0324] Each R 3’ and R 4’ H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, wherein R 3’ or R 4’ The alkyl, alkenyl, or alkynyl represented by alkyl may be substituted with one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’R 12’ ;

[0325] Each R 5’ and R 6’ H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, wherein R 5’ or R 6’ The alkyl, alkenyl, or alkynyl represented by alkyl may be substituted with one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ ;or

[0326] R 5’ and R 6’ Together with the nitrogen atom to which they are attached, they form a 3-7 membered heterocyclic group, which may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, or C 1-4 Haloalkoxy;

[0327] R 11’ and R 12’ Each instance of is independently H or C 1-6 Alkyl, wherein R 11’ or R 12’ The alkyl group represented by the present invention may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 3-6 Cycloalkyl, and 3-7 membered heterocyclic group, or

[0328] R 11’ and R 12’ Together with the nitrogen atom to which they are attached, they form a 3-7 membered heterocyclic group, which may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, or C 1-4 Haloalkoxy;

[0329] m' is 0, 1, or 2; and

[0330] n' is 0, 1, or 2.

[0331] 20. The compound of paragraph 19, wherein the compound is represented by structural formula (II-1):

[0332]

[0333] or a pharmaceutically acceptable salt thereof, wherein

[0334] A 1 is CR' or N; and

[0335] R' is H, halogen, NR 11’ R 12’ , or C 1-6 Alkyl, which may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ .

[0336] 21. The compound of paragraph 19 or 20, or a pharmaceutically acceptable salt thereof, wherein X is -P(=O)R 3’ R 4’ or –S(O) 2 NR 5’ R 6’ ;

[0337] Each R 3’ and R 4’ Each is independently H or C 1-6 Alkyl, which may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11 R 12 ; and each R 5’ and R 6’ Each is independently H or C 1-6 Alkyl, or

[0338] R 5’ and R 6’ Together with the nitrogen atom to which they are attached, they form a 3-7 membered heterocyclic group, which may be substituted by one to three substituents independently selected from the group consisting of the following: halogen, -OH, CN, C 1-4Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, or C 1-4 Haloalkoxy.

[0339] 22. A compound according to any one of paragraphs 19 to 21, or a pharmaceutically acceptable salt thereof, wherein R 1’ Each of the examples is independently H, halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Cycloalkyl, or 3-7 membered heterocyclic group, wherein R 1’ The alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclic group represented by alkyl may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ .

[0340] 23. A compound according to any one of paragraphs 19 to 22, or a pharmaceutically acceptable salt thereof, wherein R 2’ Each of the examples is independently H, halogen, OH, NH 2 , C 1-6 Alkyl, C 1-6 Alkoxy, NR 11’ R 12’ 、C(O)NR 11’ R 12’ 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11’ C(O)C 1-6 alkyl, 5-6 membered heteroaryl, or 3-7 membered heterocyclic group, wherein R 2’ On behalf of or by R 2’ The alkyl, alkoxy, heteroaryl, or heterocyclic group in the group represented by may be substituted by one to three substituents independently selected from the group consisting of halogen, -OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, and NR 11’ R 12’ .

[0341] 24. The compound of any one of paragraphs 19 to 23, wherein the compound is represented by structural formula (II-2) or (II-2'):

[0342]

[0343] or a pharmaceutically acceptable salt thereof, wherein A 1 is CR' or N; and R' is H, halogen, or NH 2 .

[0344] 25. A compound according to any one of paragraphs 19 to 24, or a pharmaceutically acceptable salt thereof, wherein R 2’ Each of the examples is independently H, halogen, OH, C 1-4 Alkyl (which may be optionally substituted with halogen or OH or NR 11’ R 12’ Replace), C 1-4 Alkoxy, C(O)OC 1-6 Alkyl, C(O)NR 11’ R 12’ NR 11’ R 12’ NR 11’ C(O)C 1-6 Alkyl, 5-6 membered heterocyclic group (pyrrolidinyl or pyrrolidin-2-one), or heteroaryl ( azole).

[0345] 26. A compound according to any one of paragraphs 19 to 25, or a pharmaceutically acceptable salt thereof, wherein

[0346] X is –P(=O)R 3’ R 4’ or –S(O) 2 NR 5’ R 6’ ;

[0347] R 3’ and R 4’ Each is independent of C 1-6 Alkyl; and

[0348] R 5’ and R 6’ Each is independently H or C 1-6 alkyl.

[0349] 27. A compound according to any one of paragraphs 19 to 26, or a pharmaceutically acceptable salt thereof, wherein R 1’ Each instance of is independently H or C 1-4 alkyl.

[0350] 28. A compound according to any one of paragraphs 19 to 27, or a pharmaceutically acceptable salt thereof, wherein R 2’ Each of the examples is independently H, halogen, OH, C 1-4 Alkyl (which may be optionally substituted with halogen or OH or NR 11’ R 12’Replace), C 1-4 Alkoxy, C(O)OC 1-4 Alkyl, C(O)NR 11’ R 12’ NR 11’ R 12’ 、NHC(O)C 1-6 Alkyl, pyrrolidinyl, pyrrolidin-2-one, Azoles, where R 11’ and R 12’ Each instance of is independently H or C 1-4 alkyl.

[0351] 29. The compound of any one of paragraphs 19 to 28, or a pharmaceutically acceptable salt thereof, wherein X is -P(O)(CH 3 ) 2 or -S(O) 2 NHMe.

[0352] 30. A compound according to any one of paragraphs 19 to 29, or a pharmaceutically acceptable salt thereof, wherein R 2’ Each of the examples is independently H, F, Cl, OH, CH 3 NH 2 , or -NHCOCH 3 .

[0353] 31. The compound of any one of paragraphs 19 to 30, or a pharmaceutically acceptable salt thereof, wherein X is -P(=O)(CH 3 ) 2 .

[0354] In one embodiment, the compound or its pharmaceutically acceptable salt or stereoisomer is selected from: compounds of formula (I-0), (I-1), (I-2), (I), (II), (II-1), (II-2) or (II-2') or compounds in the examples.

[0355] In some embodiments, the HPK1 inhibitor (compound of the present invention) is selective against one or more kinases selected from the group consisting of Lck, ZAP70, JAK3, PKCθ, TBK1, and MAP4K3. In some embodiments, the HPK1 inhibitor of the present invention is selective against one or more kinases selected from the group consisting of Lck, ZAP70, and JAK3. In some embodiments, the HPK1 inhibitor of the present invention is selective against JAK3.

[0356] For example, the HPK1 inhibitors are selective against Lck such that their IC50 against Lck is at least 2-, 3-, 5-, 10-, 20-, 40-, 50-, 75-, 100-, 150-, 200-, 300-, 400-, 500-, 600-, 750-, or 1000-fold higher than their IC50 against HPK1.

[0357] In some embodiments, HPK1 is selective for ZAP70 such that its IC50 against ZAP70 is at least 2-, 3-, 5-, 10-, 20-, 40-, 50-, 75-, 100-, 150-, 200-, 300-, 400-, 500-, 600-, 750-, or 1000-fold higher than its IC50 against HPK1.

[0358] In some embodiments, HPK1 is selective for JAK3 such that its IC50 against JAK3 is at least 2-, 3-, 5-, 10-, 20-, 40-, 50-, 75-, 100-, 150-, 200-, 300-, 400-, 500-, 600-, 750-, or 1000-fold higher than its IC50 against HPK1.

[0359] In some embodiments, HPK1 is selective for PKCθ such that its IC50 against PKCθ is at least 2-, 3-, 5-, 10-, 20-, 40-, 50-, 75-, 100-, 150-, 200-, 300-, 400-, 500-, 600-, 750-, or 1000-fold higher than its IC50 against HPK1.

[0360] In some embodiments, HPK1 is selective for TBK1 such that its IC50 against TBK1 is at least 2-, 3-, 5-, 10-, 20-, 40-, 50-, 75-, 100-, 150-, 200-, 300-, 400-, 500-, 600-, 750-, or 1000-fold higher than its IC50 against HPK1.

[0361] In some embodiments, HPK1 is selective for MAP4K3 such that its IC50 against MAP4K3 is at least 2-, 3-, 5-, 10-, 20-, 40-, 50-, 75-, 100-, 150-, 200-, 300-, 400-, 500-, 600-, 750-, or 1000-fold higher than its IC50 against HPK1.

[0362] The activity against HPK1, Lck, ZAP70, PKCθ, JAK3, TBK1, and MAP4K3 can be determined by any method recognized in the relevant art (e.g., determining IC50 values), such as the procedures exemplified in the Biological Examples, such as Biological Examples 1-7 (all of which are incorporated herein by reference).

[0363] 4. Treatable diseases

[0364] HPK1 inhibitors, their pharmaceutically acceptable salts, and pharmaceutical compositions thereof can be used in a method for regulating (ie, inhibiting) HPK1 activity, which comprises administering the HPK1 inhibitor compounds of the present invention described herein, or their pharmaceutically acceptable salts, to a patient / subject in need thereof.

[0365] In particular, the present invention provides the use of the compounds of the present invention, or their stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts, particularly their pharmaceutically acceptable salts, or mixtures thereof, for treating or preventing diseases, particularly cancer (particularly hematopoietic and solid tumors) or immune response disorders or other diseases associated with abnormal MAP4K1 signaling. The pharmaceutical activity of the compounds according to the present invention can be explained at least in part by their activity as MAP4K1 inhibitors.

[0366] In certain embodiments, the compounds of the invention or their pharmaceutically acceptable salts are suitable for medical administration to a subject in need thereof to treat diseases or indications including, but not limited to, benign hyperplasia, atherosclerotic disease, sepsis, autoimmune disease, vascular disease, viral infection, neurodegenerative disease, inflammatory disease, and male fertility control disease.

[0367] In certain embodiments, the compounds of the invention or their pharmaceutically acceptable salts are suitable for therapeutic administration to enhance, stimulate and / or increase immunity in the treatment of cancer.

[0368] The HPK1 inhibitor compounds of the present invention can be used alone or in combination with other agents or medical agents, or as adjuvants or pre-adjuvants for the treatment of diseases or disorders, including cancer.

[0369] In certain embodiments, the methods of the invention can be used to treat cancers including, but not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal glands, sarcomas of soft tissue, Cancer of the urethra, cancer of the penis, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), solid tumors of children, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or urethra, carcinoma of the renal pelvis, neoplasia of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), and combinations of the foregoing cancers.

[0370] In some embodiments, cancers that can be treated using the compounds of the invention include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate cancer), breast cancer, triple-negative breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer and small cell lung cancer). In addition, refractory or recurrent malignancies whose growth can be inhibited using the compounds of the invention can also be treated.

[0371] In some embodiments, cancers that can be treated using the compounds of the present invention include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, breast cancer, lung cancer, respiratory tract cancer, brain cancer, eye cancer, thyroid and parathyroid cancer, skin cancer, head and neck cancer, reproductive organ cancer, digestive tract cancer, urinary tract cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancer (e.g., lymphoma, leukemia such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma or multiple myeloma), sarcoma, and distant metastases thereof.

[0372] In some embodiments, the diseases and indications that can be treated using the compounds of the present invention include, but are not limited to, blood cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.

[0373] Exemplary hematological cancers include lymphomas and leukemias, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma, myeloproliferative diseases (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), essential thrombocytosis (ET), myelodysplastic syndrome (MDS), T-cell acute lymphoblastic lymphoma (T-ALL), multiple myeloma, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia (Waldenstrom's Macroglubulinemia), hairy cell lymphoma, chronic myeloid lymphoma, and Burkitt's lymphoma.

[0374] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdomyosarcoma, fibromyoma, lipoma, hamartoma, and teratoma.

[0375] Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchial carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, cartilaginous hamartoma, and mesothelioma.

[0376] Exemplary gastrointestinal cancers include cancers of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (pancreatic ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibromyoma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), and colorectal cancer.

[0377] Exemplary genitourinary tract cancers include cancers of the kidney (adenocarcinoma, Wilms' tumor [Nephroblastoma]), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), and testis (spermatoblastoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibromyoma, fibroadenoma, adenoid tumor, lipoma).

[0378] Exemplary liver cancers include hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0379] Exemplary bone cancers include, for example, bone-derived sarcomas (osteosarcomas), fibrosarcomas, malignant fibrous histiocytomas, chondrosarcomas, Ewing's sarcoma, malignant lymphomas (endoplasmic reticulum cell sarcomas), multiple myeloma, malignant giant cell tumors, bone exostoses (osteocartilaginous exostoses), benign enchondromas, chondroblastomas, chondromyxofibroma, osteoid osteoma, and giant cell tumors.

[0380] Exemplary cancers of the nervous system include cancers of the skull (osteomas, hemangiomas, granulomas, xantholipomas, osteitis deformans), meninges (meningiomas, meningiosarcomas, gliomatosis), brain (astrocytomas, medulloblastomas, gliomas, ependymomas, blastomas (pineal adenomas), glioblastomas, glioblastomas multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), and spine (neurofibromas, meningiomas, gliomas, sarcomas), as well as neuroblastoma and dysplastic gangliocytoma (Lhermitte-Duclos disease).

[0381] Exemplary gynecological cancers include cancers of the uterus (endometrial carcinoma), cervix (cervical carcinoma, preneoplastic cervical dysplasia), ovary (ovarian carcinoma (serous cystic adenocarcinoma, mucinous cystic adenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), and fallopian tube (carcinoma).

[0382] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, Merkel cell skin cancer, dysplastic nevus, lipoma, hemangioma, cutaneous fibromyoma, and keloid. In some embodiments, diseases and indications that can be treated using the compounds of the present invention include, but are not limited to, sickle cell disease (e.g., sickle cell anemia), triple negative breast cancer (TNBC), myelodysplastic syndrome, testicular cancer, biliary tract cancer, esophageal cancer, and urothelial carcinoma.

[0383] Exemplary head and neck cancers include glioblastoma, melanoma, rhabdomyosarcoma, lymphosarcoma, osteosarcoma, squamous cell carcinoma, adenocarcinoma, oral cancer, laryngeal cancer, nasopharyngeal cancer, nasal and paranasal cancers, and thyroid and parathyroid cancers.

[0384] In some embodiments, the HPK1 inhibitor can be used to treat tumors that produce PGE2 (e.g., tumors that overexpress Cox-2) and / or adenosine (tumors that overexpress CD73 and CD39). Overexpression of Cox-2 has been detected in many tumors, such as colorectal, breast, pancreatic and lung cancers, and is associated with a poor prognosis. Overexpression of COX-2 has been reported in hematological cancer models such as RAJI (Burkey's lymphoma) and U937 (acute promonocytic leukemia) and in blasts of patients. CD73 is upregulated in a variety of human cancers, including those of the colon, lung, pancreas and ovary. Increased expression of CD73 has been associated with tumor angiogenesis, invasiveness, and metastasis, and has been associated with shortened survival in breast cancer patients.

[0385] Examples of breast cancers that may be treated include, but are not limited to, triple negative breast cancer, invasive ductal carcinoma, invasive follicular carcinoma, ductal carcinoma in situ, and follicular carcinoma in situ.

[0386] Examples of respiratory tract cancers include, but are not limited to, small cell and non-small cell lung cancers, as well as bronchial adenomas and pleuropulmonary blastomas.

[0387] Examples of brain cancers that may be treated include, but are not limited to, brainstem and hypothalamic gliomas, cerebellar and cerebral astrocytomas, glioblastomas, medullary blastomas, ependymomas, and neuroectodermal and pineal tumors.

[0388] Examples of male reproductive organ tumors that may be treated include, but are not limited to, cancers of the prostate and testicles.

[0389] Tumors of the female reproductive organs that may be treated include, but are not limited to, cancers of the endometrium, cervix, ovary, vagina, and vulva, as well as uterine sarcomas.

[0390] Ovarian cancers that may be treated include, but are not limited to, serous tumors, endometrioid tumors, mucinous cystic adenocarcinomas, granulosa cell tumors, Sertoli-Leydig cell tumors, and androgenic tumors.

[0391] Cervical cancers that may be treated include, but are not limited to, squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, neuroendocrine tumors, glassy cell carcinoma, and papillary cervical adenocarcinoma.

[0392] Digestive tract tumors that may be treated include, but are not limited to, cancers of the anus, colon, colorectum, esophagus, bladder, stomach, pancreas, rectum, small intestine, and salivary glands.

[0393] Esophageal cancers that may be treated include, but are not limited to, esophageal cell carcinoma and adenocarcinoma, as well as squamous cell carcinoma, leiomyosarcoma, malignant melanoma, rhabdomyosarcoma, and lymphoma.

[0394] Gastric cancers that may be treated include, but are not limited to, intestinal and diffuse gastric adenocarcinomas.

[0395] Pancreatic cancers that may be treated include, but are not limited to, pancreatic ductal adenocarcinoma, adenosquamous carcinoma, and pancreatic endocrine tumors.

[0396] Urological tumors that may be treated include, but are not limited to, bladder, penis, kidney, renal pelvis, ureter, urethra, and human papillary renal cancers.

[0397] Kidney cancers that may be treated include, but are not limited to, renal cell carcinoma, urothelial carcinoma, juxtaglomerular cell tumor (renninoma), angiomyolipoma, renal parotid gland eosinophilic granulocytoma, Bellini duct carcinoma, clear cell sarcoma of the kidney, mesodermal nephroma, and Wilms' tumor.

[0398] Bladder cancers that may be treated include, but are not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, and small cell carcinoma.

[0399] Treatable eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

[0400] Liver cancers that may be treated include, but are not limited to, hepatocellular carcinoma (liver cell carcinoma with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.

[0401] Skin cancers that may be treated include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

[0402] Head and neck cancers that may be treated include, but are not limited to, squamous cell carcinoma of the head and neck, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, salivary gland cancer, lip and oral cancer, and squamous cell.

[0403] Lymphomas that may be treated include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt's lymphoma, Hodgkin's disease, and central nervous system lymphoma.

[0404] Treatable sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

[0405] Treatable leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

[0406] In certain embodiments, the compounds of the invention can be used to treat various other diseases involving MAP4K1, such as cardiovascular and pulmonary diseases.

[0407] In certain embodiments, the compounds of the invention can be used as medicines for the treatment and / or prevention of cardiovascular, inflammatory and fibrotic diseases, renal diseases, in particular acute and chronic renal insufficiency, and acute and chronic renal failure.

[0408] As used herein, the term "renal insufficiency" includes both acute and chronic symptoms of renal insufficiency, and primary or associated renal diseases, such as diabetic and non-diabetic nephropathy, hypertensive nephropathy, ischemic renal disease, renal hypoperfusion, intradialytic hypotension, obstructive uropathy, renal vascular stenosis, glomerular disease, glomerulonephritis (e.g., primary glomerulonephritis; minimal change glomerulonephritis (steatohepatitis); membranous glomerulonephritis; focal segmental glomerulosclerosis (FSGS); membranoproliferative glomerulonephritis; rapidly progressive glomerulonephritis; inter-annular membranoproliferative glomerulonephritis (IgA nephritis, Berger's disease); post-infectious glomerulonephritis; secondary glomerulonephritis), diabetes, systemic lupus erythematosus, amyloidosis, Goodpasture's syndrome (Goodpasture's syndrome), syndrome), Wegener's granulomatosis, Henoch-Schonlein purpura, microscopic polyangiitis, acute glomerulonephritis, pyelonephritis (e.g., due to urolithiasis, benign prostatic hypertrophy, diabetes, malformations, analgesic abuse, Crohn's disease), glomerulosclerosis, arteriolonecrose of the kidney, tubulointerstitial disease, nephrotic disorders such as primary and congenital or acquired renal disease, Alport syndrome renal syndrome, nephritis, immune renal diseases such as renal transplant rejection and immune complex-induced renal disease, toxic substance-induced nephropathy, contrast agent-induced nephropathy, diabetic and non-diabetic nephropathy, renal cysts, nephrosclerosis, hypertensive nephrosclerosis and renal syndromes with diagnostic features, such as abnormally decreased creatinine and / or water output, abnormally increased blood concentrations of urea, nitrogen, potassium and / or creatinine, changes in renal enzyme activity (e.g., glutamate synthetase), changes in urine osmolality or urine volume, increased microalbuminuria, macroalbuminuria, glomerular and arteriolar damage, renal tubular dilatation, hyperphosphatemia and / or the need for dialysis.

[0409] In certain embodiments, the compounds of the invention can be used to treat and / or prevent sequelae of renal insufficiency, such as pulmonary edema, heart failure, uremia, anemia, electrolyte imbalance (eg, hypercalcemia, hyponatremia), and bone and carbohydrate metabolism disorders.

[0410] In certain embodiments, the compounds of the invention can be used to treat and / or prevent sequelae of renal insufficiency, such as pulmonary edema, heart failure, uremia, anemia, electrolyte imbalance (eg, hypercalcemia, hyponatremia), and bone and carbohydrate metabolism disorders.

[0411] In certain embodiments, the compounds of the present invention are further suitable for treating and / or preventing polycystic kidney disease (PCKD) and syndrome of inappropriate ADH secretion (SIADH).

[0412] In certain embodiments, the compounds of the present invention are also suitable for the treatment and / or prevention of metabolic syndrome, hypertension, resistant hypertension, acute and chronic heart failure, coronary heart disease, stable and unstable angina, peripheral and cardiovascular diseases, arrhythmias, atrial and ventricular arrhythmias and impaired conduction function, for example, atrioventricular block of the first to third degree (AB block 1-111), supraventricular tachycardia, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular flutter, heart failure ... ventricular tachycardia, torsades de pointes, atrial and ventricular extrasystoles, AV-junctional extrasystoles, sick sinus syndrome, syncope, AV-nodal cyclotron tachycardia, Wolff-Parkinson-White syndrome, acute coronary syndrome (ACS), autoimmune heart disease (pericarditis, endocarditis, valvulitis, aortitis, cardiomyopathy), shock (e.g., cardiogenic shock, septic shock, for the treatment and / or prevention of thromboembolic disorders and ischemic diseases such as myocardial ischemia, myocardial infarction, stroke, cardiac hypertrophy, transient and ischemic attacks, preeclampsia, inflammatory cardiovascular diseases, coronary and peripheral arterial spasm, edema formation (e.g., pulmonary edema, cerebral edema, renal edema or edema caused by heart failure, poor peripheral circulation, reperfusion injury, arterial and venous thrombosis), myocardial insufficiency for the prevention of postoperative restenosis (e.g., after thrombolytic therapy, percutaneous transluminal angioplasty (PTA), transluminal coronary angioplasty (PTCA), heart transplantation and bypass surgery), and microvascular and macrovascular damage (vasculitis), increase in the concentrations of fibrinogen and low-density lipoprotein (LDL) and increase in the concentration of plasminogen activator inhibitor 1 (PAI-1), and for the treatment and / or prevention of erectile dysfunction and female sexual dysfunction.

[0413] In certain embodiments, the compounds of the invention are also suitable for the treatment and / or prevention of asthmatic disorders, pulmonary arterial hypertension (PAH) and other forms of pulmonary hypertension (PH), including left sided heart disease, HIV, sickle cell anemia, thromboembolism (CTEPH), sarcoidosis, pulmonary hypertension associated with COPD or pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), acute lung injury (ALI), alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, emphysema (e.g., emphysema caused by smoking) and cystic fibrosis (CF).

[0414] In certain embodiments, the compounds of the invention are also effective in controlling central nervous system disorders characterized by dysregulation of the NO / cGMP system. They are particularly suitable for improving perception, attention, learning or memory after cognitive impairment, such as those cognitive impairments that occur in particular in association with conditions / diseases / syndromes such as mild cognitive impairment, aging-related learning and memory impairment, aging-related memory loss, vascular dementia, brain trauma, stroke, dementia occurring after stroke (post-stroke dementia), post-traumatic brain injury, general attention impairment, attention impairment in children with learning and memory problems, Alzheimer's disease, Lewy body dementia, dementia with anterior temporal lobe degeneration, including Pick's syndrome, Parkinson's disease, progressive dementia with cortical basal degeneration, amyotrophic lateral sclerosis (ALS), Huntington's disease, Disease), demyelination, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacob dementia, HIV dementia, schizophrenia with dementia, or Korsakoff's psychosis.

[0415] In certain embodiments, the compounds of the invention are also suitable for the treatment and / or prevention of central nervous system disorders, such as anxiety, stress and depressive states, CNS-related sexual dysfunction and sleep disorders, and pathological disturbances in the control of the intake of food, stimulants, and addictive substances.

[0416] In certain embodiments, the compounds of the present invention are further suitable for controlling cerebral blood flow and thus represent effective agents for controlling migraine headaches.

[0417] In certain embodiments, the compounds of the present invention are also suitable for preventing and controlling the sequelae of cerebral infarction (such as stroke, cerebral ischemia and brain trauma). The compounds of the present invention are also useful for controlling pain and tinnitus.

[0418] In certain embodiments, the compounds of the present invention have anti-inflammatory effects and can therefore be used as anti-inflammatory agents for the treatment and / or prevention of sepsis (SIRS), multiple organ failure (MODS, MOF), inflammatory diseases of the kidney, chronic intestinal inflammation (IBD, Crohn's disease, UC), pancreatitis, peritonitis, rheumatoid diseases, inflammatory skin diseases and inflammatory diseases of the eye.

[0419] In certain embodiments, the compounds of the present invention can also be used to treat and / or prevent autoimmune diseases.

[0420] In certain embodiments, the compounds of the invention are also suitable for the treatment and / or prevention of fibrotic disorders of internal organs, such as the lung, heart, kidney, bone marrow and in particular the liver, as well as skin fibrosis and ocular fibrotic disorders.

[0421] The term "fibrotic disorder" as used herein specifically includes the following: hepatic fibrosis, cirrhosis, pulmonary fibrosis, myocardial fibrosis, nephropathy, glomerulonephritis, interstitial renal fibrosis, fibrotic damage due to diabetes, myelofibrosis and similar fibrotic disorders, scleroderma, morphea, keloids, hypertrophic scars (also following surgical procedures), nevi, diabetic retinopathy, proliferative vitreoretinopathy, and connective tissue disorders (e.g., sarcoidosis).

[0422] In certain embodiments, the compounds of the invention are also suitable for controlling post-surgical scarring, for example, resulting from glaucoma surgery.

[0423] In certain embodiments, the compounds of the present invention can also be used for the cosmetic treatment of aged and keratinized skin.

[0424] In certain embodiments, the compounds of the invention are suitable for treating and / or preventing hepatitis, neoplasia, osteoporosis, glaucoma and gastroparesis.

[0425] In certain embodiments, the compounds of the invention are suitable for treating and / or preventing viral infections (e.g., HIV and Kaposi's sarcoma); inflammatory and autoimmune diseases (e.g., colitis, arthritis, Alzheimer's disease, glomerulonephritis and wound healing); bacterial, fungal, and / or parasitic infections; skin diseases (e.g., psoriasis); hyperproliferative-based diseases characterized by an increase in the number of cells (e.g., fibroblasts, hepatocytes, bone and bone marrow cells, cartilage or smooth muscle cells or epithelial cells) (e.g., endometrial hyperplasia); bone diseases and cardiovascular diseases (e.g., postoperative restenosis and hypertrophy).

[0426] In another specific example, the compounds of the present invention can also be used to treat or prevent uterine fibroids (uterine leiomyoma or uterine myoma) in women. Uterine fibroids are benign tumors of the uterine myometrium (the smooth muscle layer of the uterus). Uterine fibroids grow slowly during a woman's life, and their growth is dependent on the female sex hormones estradiol and progesterone. Therefore, about 70% and >80% of white and African-American women have a peak incidence of uterine fibroids between the ages of 35 and menopause, respectively, and then shrink due to reduced hormone levels. About 30% and 45% of white and African-American women, respectively, experience clinically relevant symptoms due to their fibroids, which are heavy menstrual bleeding and pain associated with the menstrual cycle (David et al., Eur J Obstet Gynecol Reprod Biol. 199: 137-140, 2016). The heavy menstrual bleeding at this time is defined as bleeding more than 80 mL during the menstrual cycle. The submucosal location of uterine fibroids (i.e., they are located directly beneath the endometrium) appears to have an even more serious effect on uterine bleeding, possibly causing anemia in women, and thus severely affecting the quality of life of women with symptoms of uterine fibroids.

[0427] In certain embodiments, the compounds of the invention are useful for the treatment and / or prevention of chronic renal disease, acute and chronic renal insufficiency, diabetes, inflammatory or hypertensive nephropathy, fibrotic disorders, cardiac insufficiency, angina pectoris, hypertension, pulmonary hypertension, ischemia, vascular disorders, thromboembolic disorders, arteriosclerosis, sickle cell anemia, erectile dysfunction, benign prostatic hypertrophy, dysuria associated with benign prostatic hypertrophy, Huntington's disease, dementia, Alzheimer's disease and Creutzfeldt-Jakob disease.

[0428] The present invention provides a method for treating and / or preventing chronic kidney disease, acute and chronic renal insufficiency, diabetes, inflammatory or hypertensive nephropathy, fibrotic diseases, cardiac insufficiency, angina pectoris, hypertension, pulmonary hypertension, ischemia, vascular diseases, thromboembolic diseases, arteriosclerosis, sickle cell anemia, erectile dysfunction, benign prostatic hypertrophy, dysuria associated with benign prostatic hypertrophy, Huntington's disease, dementia, Alzheimer's disease and Creutzfeldt-Jakob disease.

[0429] The present invention further provides the use of the compounds according to the invention for the treatment and / or prevention of disorders, in particular the disorders mentioned above.

[0430] The present invention further provides a method for treating and / or preventing disorders, in particular the disorders mentioned above, by using an effective amount of at least one compound according to the invention.

[0431] Therefore, the compounds of the present invention can be used to inhibit, block, reduce, or decrease MAP4K1 activation by exogenous and / or endogenous ligands, thereby reducing tumor growth and dysregulated immune responses, for example, in the context of cancer and cancer immunotherapy, for blocking immunosuppression and increasing immune cell activation and infiltration. This method includes administering to a mammal (including a human) in need thereof an amount of a compound of the present invention or a pharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrate, solvate or ester thereof to treat the disease.

[0432] The present invention also provides a method for treating various other diseases involving MAP4K1, such as (but not limited to) diseases with dysregulated immune responses, inflammation, vaccination of infections and cancers, viral infections, obesity, and diet-induced obesity, obesity, metabolic diseases, hepatic steatosis, and uterine fibroids. These diseases are well characterized in humans, but also have similar causes in other mammals and can be treated by administering the pharmaceutical compositions of the present invention.

[0433] 5. Combination therapy

[0434] The compounds of the invention may be used in combination therapy with one or more additional / secondary medical agents suitable for treating the disease or indication for which the compounds are useful.

[0435] Thus, in certain embodiments, for example, the methods of the invention using the compounds of the invention may include administering to a subject in need thereof an additional therapeutic agent. The additional therapeutic agent may be: (i) an immunomodulator that blocks or inhibits an immune system checkpoint, which may or may not be a component of the NF-κB pathway; and / or (ii) an agent that directly stimulates an immune effector response, such as a cytokine, or a tumor-specific donor-transferred T cell population, or an antibody directed against a protein expressed by a tumor cell; and / or (iii) a composition comprising a tumor antigen or an immunogenic fragment thereof; and / or (iv) a chemotherapeutic agent.

[0436] In some embodiments, the second medical agent includes an inhibitor of the PI3K-AKT-mTOR pathway, an inhibitor of the Raf-MAPK pathway, an inhibitor of the JAK-STAT pathway, an inhibitor of the β-catenin pathway, an inhibitor of the notch pathway, an inhibitor of the hedgehog signaling pathway, an inhibitor of the Pim kinase, and / or an inhibitor of chaperone proteins and cell cycle progression. In some embodiments, the combination therapy of the present invention can reduce the possibility of drug resistance in cell populations and / or reduce the toxicity of treatment.

[0437] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βPv, PKA, PKG, PKC, CaM-kinase, phosphorylation kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, CSFIR, KIT, FLK-II, KDR / FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR / Flt2, Flt4, EphAl, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK, and B-Raf.

[0438] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with one or more of the following inhibitors for treating cancer, including: FGFR inhibitors (FGFR1, FGFR2, FGFR3 or FGFR4, for example, AZD4547, BAY 1187982, ARQ087, BGJ398, BIBF1120, TKI258, lucitanib, dovitinib, TAS-120, JJ-42756493, Debiol347, INCB54828, INCB62079, and INCB63904), JAK inhibitors (JAK1 and / or JAK2, for example, ruxolitinib, baricitinib, or itacitinib (INCB39110)), IDO inhibitors inhibitors (e.g., epacadostat and NLG919), LSD1 inhibitors (e.g., GSK2979552, INCB59872, and INCB60003), TDO inhibitors, PI3K-δ inhibitors (e.g., INCB50797 and INCB50465), PI3K-γ inhibitors (such as PI3K-γ selective inhibitors), CSF1R inhibitors (e.g., PLX3397 and LY3022855), TAM receptor tyrosine kinases (Tyro-3, Axl, and Mer), aryl hydrocarbon receptor (AhR) modulators (e.g., laquinimod), mod), aminoflavonoids, CB7993113, CH223191, 6,2',4'-trimethoxyflavone (TMF), GNF351 (N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purine-6-amine), aminoflavonoids, NKI150460, indole-3-methanol, β-naphthoflavonoids and their dimers, diindolylmethane (DIM), 4-hydroxytamoxifen, leflunomide, raloxifene, tranilast, Flutamide, mexiletine, nimodiphine, omeprazole, sulindac, tranilast, and TCDD (2,3,7,8-tetrachlorodiphenyl-p-dioxin)), angiogenesis inhibitors, interleukin receptor inhibitors, bromodomain and extra-terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors, such as OTX015, CPI-0610, INCB54329, and INCB57643), and adenosine receptor antagonists or a combination thereof.

[0439] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with HDAC inhibitors, such as panobinostat and vorinostat.

[0440] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with c-Met inhibitors, such as onartumzumab, tivantnib, and capmatinib (INC-280).

[0441] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with BTK inhibitors, such as ibrutinib.

[0442] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with mTOR inhibitors, such as rapamycin, sirolimus, temsirolimus, and everolimus.

[0443] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with MEK inhibitors, such as trametinib, selumetinib, and GDC-0973.

[0444] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with Hsp90 inhibitors (e.g., tanespimycin), cyclin-dependent kinase inhibitors (e.g., palbociclib), PARP inhibitors (e.g., olaparib), and Pim kinase inhibitors (LGH447, INCB053914, and SGI-1776).

[0445] In certain embodiments, the HPK1 inhibitor compounds of the present invention may be used in combination with agonists of the DNA sensor (c-GAS) and / or its downstream adaptor protein STING.

[0446] The cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway is a component of the innate immune system that functions to detect the presence of cytosolic DNA, and the response activates the expression of inflammatory genes, causing aging or activating defense mechanisms. The movement of DNA from its normal nuclear location to the cytosol is associated with tumorigenesis or viral infection. cGAS has been found in the cytosol, and when directly bound to cytosolic DNA, cGAS forms a dimer that catalyzes the production of 2'3'-cGAMP from ATP and GTP. The resulting cGAMP then acts as a second messenger, binding to STING and initiating the activation of the transcription factor IRF3. Activated IRF3 causes the transcription of type 1 IFN-β, which initiates a variety of biological responses from many downstream target genes, such as viral responses, tumor detection, autoimmunity, and cellular senescence. In many tumor cells, a constitutively active DNA damage response causes the accumulation of cytoplasmic DNA and activation of the cGAS / STING pathway. The NKG2D ligand Rae1 has been shown to be upregulated in lymphoma cells in a STING / IRF3-dependent manner, thus contributing to NK-mediated tumor clearance. Activation of the c-GAS-STING pathway in antigen-presenting cells such as dendritic cells has been shown to enhance their function and augment anti-tumor immunity.

[0447] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be used in combination with one or more immune checkpoint inhibitors.

[0448] Effector T cell activation is usually initiated by antigenic peptides recognized by TCR presented by MHC complexes. The type and degree of activation achieved is then determined by the balance between stimulatory signals and signals that inhibit the effector T cell response. As used herein, "immune system checkpoints" refer to any molecular interaction that can change the equilibrium state and tend to inhibit the effector T cell response. That is, when the molecular interaction occurs, the activation of effector T cells is negatively regulated. These interactions may be direct, such as the interaction between ligands and cell surface receptors that transmit inhibitory signals to effector T cells. Or they may be indirect, such as blocking or inhibiting the interaction between ligands and cell surface receptors that transmit activation signals to effector T cells, or the interaction promotes the deregulation of inhibitory molecules or cells, or is consumed by enzymes of metabolites required by effector T cells, or any combination thereof.

[0449] Examples of immune system checkpoints include: a) interaction between indoleamine 2,3-dioxygenase (IDO1) and its substrate; b) interaction between PD1 and PD-L1 and / or between PD1 and PD-L2; c) interaction between CTLA-4 and CD86 and / or between CTLA-4 and CD80; d) interaction between B7-H3 and / or between B7-H4 and their respective ligands; e) interaction between HVEM and BTLA; f) interaction between GAL9 and TIM3; g) interaction between class I or II MHC and LAG 3; and h) interactions between class I or II MHC and KIR; i) interactions between OX40 (CD134) and OX40L (CD252); j) interactions between CD40 and CD40L (CD154); k) interactions between 4-1BB (CD137) and ligands (including 4-1BBL); l) interactions between GITR and ligands (including GITRL).

[0450] Thus, examples of immune checkpoint inhibitors include inhibitors against the following immune checkpoint molecules, such as CD20, CD27, CD28, CD39, CD40, CD 122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, PD-1, PD-L1, and PD-L2.

[0451] A representative checkpoint for the purposes of the present invention is checkpoint (b), which is the interaction between PD1 and either of its ligands, PD-L1 and PD-L2. PD1 is expressed on effector T cells. The signal generated by engagement with either of its ligands downregulates activation. Some tumors express these ligands. Many solid tumors, including melanomas, specifically express PD-L1. These tumors therefore downregulate immune-mediated anti-tumor effects through activation of the inhibitory PD-1 receptor of T cells. By blocking the interaction between PD1 and one or both of its ligands, the checkpoint of the immune response can be removed, resulting in an increased anti-tumor T cell response. Therefore, PD1 and its ligands are examples of immune system checkpoint components that may be targeted by the methods of the present invention.

[0452] Another checkpoint for the purposes of the present invention is checkpoint (c), which is the interaction between the T cell receptor CTLA-4 and its ligands, the B7 proteins (B7-1 and B7-2). CTLA-4 is usually upregulated on the surface of T cells after initial activation, and when bound to the ligand, further / continued activation is inhibited. CTLA-4 competes with the receptor CD28, which is also expressed on the surface of T cells, for binding to the B7 protein, but upregulates activation. Therefore, by blocking the interaction of CTLA-4 with the B7 protein, but not blocking the interaction of CD28 with the B7 protein, one of the normal checkpoints of the immune response can be removed, resulting in an increased anti-tumor T cell response. Therefore, CTLA-4 and its ligands are examples of immune system checkpoint components that may be targets of the methods of the present invention.

[0453] In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137.

[0454] In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, HΜ3, and VISTA.

[0455] In some embodiments, the compounds provided herein can be used in combination with one or more agents selected from the group consisting of KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGFRβ inhibitors.

[0456] In some embodiments, the compounds provided herein can be used in combination with immune checkpoint inhibitors, which are generally small molecule inhibitors (SMIs) of small organic molecules. For example, in some embodiments, inhibitors of IDO1 include Epacadostat (INCB24360), Indoximod, GDC-0919 (NLG919) and F001287. Other inhibitors of IDO1 include 1-methyltryptophan (1MT).

[0457] In some embodiments, inhibitors of immune checkpoint molecules are also referred to as "immunomodulatory agents," which include any agent that, when administered to a subject, blocks or inhibits the action of an immune system checkpoint, resulting in upregulation of an immune effector response, typically a T cell effector response, which may include an anti-tumor T cell effector response, in the subject.

[0458] The immunomodulator used in the method of the present invention can block or inhibit any of the above-mentioned immune system checkpoints. The agent may be an antibody or any other suitable agent that can cause the blocking or inhibitory effect. Therefore, the agent is generally referred to as an inhibitor of the checkpoint.

[0459] As used herein, "antibody" includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. Antibodies may be polyclonal or monoclonal and may be produced by any suitable method. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include Fab fragments, F(ab') 2 fragment, Fab' fragment, F d Fragments, Fv fragments, dAb fragments, and isolated complementarity determining regions (CDRs). Single-chain antibodies (such as scFv) and heavy chain antibodies (such as VHH and camelid antibodies) are also intended to be encompassed within the term "antigen-binding portion" of an antibody.

[0460] In some embodiments, the immunomodulator used in the HPK1 inhibitor of the present invention is an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0461] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of PD-1, for example, an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab (MDX-1106), pembrolizumab (Merck 3475 or Lambrolizumab), pidilizumab (CT-011), tislelizumab (BGB-A317), camrelizumab (SHR-1210), spartalizumab (PDR001), or AMP-514 (MEDI0680). In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is camrelizumab (SHR-1210). In some embodiments, the PD-1 inhibitor is AMP-224 (PD-L2 F that binds to PD-1). c fusion protein) or AUNP-12 (anti-PD-1 peptide).

[0462] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of PD-L1, for example, an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is BMS-935559, BMS-936559 (MDX-1105), MEDI-4736 (durvalumab), MPDL3280A (also known as RG7446), YW243.55.S70 (HPAB-0381-WJ), or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A or MEDI-4736. In some embodiments, the anti-PD-L1 antibody includes atezolizumab, avelumab, durvalumab or MEDI-4736, and MPDL3280A.

[0463] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of CTLA-4, for example, an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab, or any antibody disclosed in WO2014 / 207063 (incorporated herein by reference). Other molecules include polypeptides, or soluble mutant CD86 polypeptides. In some embodiments, the antibody is ipilimumab.

[0464] In some embodiments, the inhibitor of the immune checkpoint molecule is a combination of two or more of the modulators described herein, such as a combination targeting two or more different targets (e.g., PD-1, PD-L1, and PD-L2). Exemplary combinations include: α-PD-1 and α-PD-L1; α-CTLA-4, α-PD-L1, and α-CD20, etc.

[0465] In some embodiments, the inhibitor of the immune checkpoint molecule is an antibody that blocks or inhibits the interaction between 4-1BB and its ligand, including utomilumab.

[0466] In some embodiments, the inhibitor of immune checkpoint molecules is an inhibitor of CSF1R, for example, an anti-CSF1R antibody. In some embodiments, the anti-CSF1R antibody is IMC-CS4 or RG7155.

[0467] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of LAG3, for example, an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016, LAG525, IMP321 or GSK2831781.

[0468] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of GITR, for example, an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518, MK-4166, MK1248, BMS-986156, MEDI1873, or GWN323.

[0469] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of OX40, for example, an anti-OX40 antibody or an OX40L fusion protein. In some embodiments, the anti-OX40 antibody is MEDI0562, MEDI6469, MOXR0916, PF-04518600, or GSK3174998. In some embodiments, the OX40L fusion protein is MEDI6383.

[0470] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of TIM3, for example, an anti-TIM3 antibody. In some embodiments, the anti-TIM3 antibody is MBG-453.

[0471] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of CD20, for example, an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is obinutuzumab or rituximab.

[0472] In some embodiments, the compounds of the present invention can be used in combination with one or more metabolic enzyme inhibitors. In some embodiments, the metabolic enzyme inhibitor is an inhibitor of IDO 1, TDO, or arginase. Examples of IDO1 inhibitors include epacadostat and NGL919. An example of an arginase inhibitor is CB-1158.

[0473] In some embodiments, the compounds of the present invention can be used in combination with bispecific antibodies. In some embodiments, one of the functional domains of the bispecific antibody targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3, CD137, ICOS, CD3 or TGFβ receptor.

[0474] In some embodiments, the compounds of the invention may be used in combination with one or more agents for treating a disease (e.g., cancer). In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulator. Examples of alkylating agents include bendamustine, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitroureas and triazenes, uracil nitrogen mustard, chlormethine, cyclophosphamide, and succinimidyl. TM), ifosfamide, melphalan, chlorambucil, pipobroman, triethyl-melamine, triethylthiophosphoramide, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulator is lenalidomide (LEN) or pomalidomide (POM).

[0475] The compounds of the present invention can be further used in combination with other methods for treating cancer, such as chemotherapy, radiotherapy, tumor targeted therapy, chemotherapy pre-therapy, immunotherapy or surgery. Examples of immunotherapy include cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccines, monoclonal antibodies, adoptive T cell transfer, oncolytic virus therapy and immunomodulatory small molecules, including thalidomide or JAK1 / 2 inhibitors and analogs.

[0476] The compounds of the invention may be administered in combination with one or more anticancer drugs, such as chemotherapeutic agents. Examples of chemotherapeutic agents include any of abarelix, abiraterone, afatinib, aflibercept, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, axitinib, azacitidine, bevacizumab, bexarotene, baricitinib, bicalutamide, bleomycin, bortezombi, bortezomib, zomib, brivanib, buparlisib, busulfan (intravenous), busulfan (oral), calusterone, capecitabine, carboplatin, carmustine, cediranib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dacomitinib, dactinomycin, dalteparin sodium), dasatinib, dactinomycin, daunorubicin, decitabine, degarelix, denileukin, denileukindiftitox, deoxycoformycin, dexrazoxane, docetaxel, doxorubicin, droloxafine, dromostanolone propionatepropionate, eculizumab, enzalutamide, epidophyllotoxin, epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, flutamide, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate acetate), ibritumomab tiuxetan, idarubicin, idelalisib, ifosfamide, imatinib mesylate, interferon α2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mithramycin, mitomycinC), mitotane, mitoxantrone, nandrolone phenpropionate, navelbene, necitumumab, nelarabine, neratinib, nilotinib, nilutamide, nofetumomab, oserelin, oxaliplatin, paclitaxel, pamidronate, panitumumab, pazopanib, pegaspargase, pegfilgrastim, pemetrexeddisodium, pentostatin, pilaralisib, pipobroman, plicamycin, ponatinib, prednisone, procarbazine, quinacrine, rasburicase, regorafenib, reloxafine, rituximab, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, tegafur, temozolo mide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, triptorelin, uracil mustard, valrubicin, vandetanib, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, and zoledronate.

[0477] Other anticancer agents include antibody therapy, such as trastuzumab (Herceptin), antibodies to costimulatory molecules such as CTLA-4 (e.g., ipilimumab or tremelimumab), 4-1BB, antibodies to PD-1 and PD-L1, or antibodies to cytokines (IL-10, TGF-β, etc.). Examples of antibodies to PD-1 and / or PD-L1 that can be used in combination with the compounds of the invention for the treatment of cancer or infection (such as viral, bacterial, fungal, and parasitic infections) include, but are not limited to, nivolumab, pembrolizumab, MPDL3280A, MEDI-4736, and SHR-1210.

[0478] Other anticancer agents include inhibitors of kinases associated with cell proliferative disorders. Such kinases include, but are not limited to, Aurora-A, CDK1, CDK2, CDK3, CDK5, CDK7, CDK8, CDK9, ephrin receptor kinases, CHK1, CHK2, SRC, Yes, Fyn, Lck, Fer, Fes, Syk, Itk, Bmx, GSK3, JNK, PAK1, PAK2, PAK3, PAK4, PDK1, PKA, PKC, Rsk, and SGK.

[0479] Other anticancer agents also include those that block the migration of immune cells, such as antagonists of chemokine receptors (including CCR2 and CCR4). The compounds of the present invention may be further used in combination with one or more anti-inflammatory agents, steroids, immunosuppressants, or therapeutic antibodies.

[0480] In some embodiments, the compounds of the invention may be used in combination with other therapeutic agents that directly stimulate immune effector responses, such as cytokines, or antibodies directed against tumors that transfer T cell populations or against proteins expressed by tumor cells.

[0481] As used herein, "agents that directly stimulate immune effector responses" refers to any suitable agent, but typically refers to cytokines or chemokines (or agents that stimulate the production of one), tumor-specific adoptively transferred T cell populations, or antibodies directed against proteins expressed by tumor cells.

[0482] The cytokine may be an interferon selected from the following: IFNα, IPNβ, IFNγ and IFNA, or an interleukin, such as IL-2. Chemokines may be inflammatory mediators, for example, selected from CXCL9, 10, and 11, which attract T cells expressing CXCR3. The preparation that stimulates the production of cytokines or chemokines may be an adjuvant suitable for administration to humans. An example is Bacille Calmette-Guerin (BCG), which is usually administered into the bladder (i.e., the urethra) for the treatment of bladder cancer. The typical dosage course of BCG for bladder cancer is once a week for a total of 6 weeks, but for the long-term safety history, administration can also be maintained indefinitely. It is known that BCG can stimulate an immune response to bladder cancer. BCG has also been used as an adjuvant in combination with a composition comprising a tumor antigen (i.e., using a cancer vaccine), usually when administered intradermally, especially for colon cancer. These BCG uses are also included in the present invention. Tumor-specific adoptive transfer of T cell populations directly increases the number of tumor-specific T cell populations in an individual and can be produced by any suitable means. However, the process generally involves isolating tumor-specific T cells from a tumor sample from a patient, selectively culturing those cells, and then returning the expanded tumor-specific T cell population to the patient. Alternatively, genetic engineering of the T cell receptor locus can be used, followed by amplification of the altered cells to produce tumor-specific T cell populations.

[0483] Antibodies specific for proteins expressed by tumor cells usually stimulate immune activity by binding to tumor cells and promoting cell lysis through antibody-dependent cell-mediated cytotoxicity (ADCC). Examples of such antibodies include anti-CD20 antibodies such as ofatumumab or rituximab, and anti-CD52 antibodies such as alemtuzumab.

[0484] Thus, in certain exemplary embodiments, the compounds of the invention may be used in combination with a calcineurin inhibitor, e.g., cyclosporine A or FK 506; an mTOR inhibitor, e.g., rapamycin, 40-0-(2-hydroxyethyl)-rapamycin, biolimus-7 or biolimus-9; an ascomycin with immunosuppressive properties, e.g., ABT-281, ASM981; a corticosteroid; cyclophosphamide; azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic acid or a salt; mycophenolate mofetil; or an IL-1β inhibitor.

[0485] In another embodiment, the compounds of the invention are used in combination with an adjunct agent that is a PI3 kinase inhibitor.

[0486] In another embodiment, the compounds of the invention are used in combination with an agent that affects BTK (Bruton's tyrosine kinase).

[0487] The compounds of the present invention for treating neoplastic diseases may be used in combination with B-cell modulators, for example, rituximab, BTK or Syk inhibitors; inhibitors of PKC, PI3 kinase, PDK, PIM, JAK and mTOR and BH3 mimetics.

[0488] In some embodiments, the compounds of the present invention (including salts thereof) can be used in combination with another immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines. Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MARTI and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

[0489] In some embodiments, the compounds of the present invention or their salts can also be used in combination with a vaccination process for treating cancer. In some embodiments, the tumor cells are transduced to express GM-CSF. In some embodiments, tumor vaccines include proteins from viruses involved in human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV) and Kaposi's herpes sarcoma virus (KHSV). In some embodiments, the compounds of the present invention can be used in combination with tumor-specific antigens, such as cardiac shock proteins isolated from tumor tissue itself. In some embodiments, the compounds of the present invention or their salts can be used in combination with dendritic cell immunization to activate a strong anti-tumor response.

[0490] In some embodiments, the compounds of the invention may be used in combination with bispecific macrocyclic peptides that target effector cells expressing Feα or Feγ receptors to tumor cells. The compounds of the invention may also be used in combination with macrocyclic peptides that activate host immune responses.

[0491] In some embodiments, the compounds of the present invention can be used in combination with bone marrow transplants to treat various tumors of hematopoietic origin.

[0492] Suitable antiviral agents contemplated for use in combination with the compounds of the invention include nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and other antiviral drugs. Examples of suitable NRTIs include zidovudine (AZT); didanosine (ddl); zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir (1592U89); adefovir dipivoxil (d4T); adefovir sirox (d4T); dapoxetine ... dipivoxil (bis(POM)-PMEA); lobucavir (BMS-180194); BCH-10652; emitricitabine ((-)-FTC); β-L-FD4 (also known as β-L-D4C, designated β-L-2',3'-dideoxy-5-fluoro-cytidine); DAPD ((-)-β-D-2,6-diamino-purine dioxolane); and dideoxyadenosine (lodenosine) (FddA). Typical suitable NNRTIs include nevirapine (BI-RG-587); delaviradine (BHAP, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442 (l-(ethoxy-methyl)-5-(l-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione); and (+)-calanolide A (NSC-675451) and B. Typical suitable protease inhibitors include saquinavir (Ro 31-8959); ritonavir (ABT-538); indinavir (MK-639); nelfnavir (AG-1343); amprenavir (141W94); lasinavir (BMS-234475); DMP-450; BMS-2322623; ABT-378; and AG-1549. Other antiviral agents include hydroxyurea, ribavirin, IL-2, IL-12, pentafuside, and Yissum Project No. 11607.

[0493] It is understood that many other medical agents used in the method of the present invention may be biological agents that require intravenous, intraperitoneal, or depot administration. In another embodiment, the compounds of the present invention are administered orally, while the other medical agent is administered parenterally, for example, intravenously, intraperitoneally, or as a depot.

[0494] In any combination therapy described herein, when more than one pharmaceutical agent is administered to a patient, they may be administered simultaneously, separately, sequentially, or in combination (eg, when there are more than two agents).

[0495] In one embodiment, the present invention provides a product comprising a compound of the present invention (e.g., a principal compound or any subgroup thereof) and at least one other medical agent as a combined preparation for simultaneous, separate, or sequential use in therapy. Products provided as combined preparations include compositions comprising a compound of the present invention or any subgroup thereof and other medical agent(s) in the same pharmaceutical composition, or compositions comprising the principal compound or any subgroup thereof and other medical agent(s) in separate forms, for example, in the form of a kit.

[0496] In one embodiment, the present invention provides a set comprising two or more separate pharmaceutical compositions, at least one of which comprises a main compound, and another comprises a second medical agent discussed herein. In one embodiment, the set comprises means for separately accommodating the composition, such as a container, a separate bottle, or a separate foil package. Such set examples are blister packs, which are commonly used for packaging tablets, capsules, and the like. The set of the present invention can be used to administer different dosage forms at different dosing intervals, for example, oral and parenteral, administering separate compositions, or adjusting dosages by separate compositions. In order to promote adaptability, the set of the present invention generally comprises instructions for administration.

[0497] 6. Compound Screening / Analysis

[0498] The compounds of the invention inhibit the kinase activity of HPK1, which can be directly analyzed using a variety of biochemical assays, such as the assay described in Example 1. The IC50 value of any compound can be determined based on a range of inhibitor concentrations. In addition, the inhibitory potency of the compound can also be evaluated using bioassays to determine the effect of the compound on cytokine secretion after T cell stimulation with TCR and CD28.

[0499] For example, Example 5 illustrates such functional assays for determining the effects of HPK1 inhibitors on IL-2 & IFN-γ release from stimulated Pan T cells. Standard ELISA assays can be used to measure / quantify secreted IL-2 & IFN-γ. Briefly, pan T cells are isolated from peripheral blood (PB) mononuclear cells (MNCs), or PBMCs using a commercially available kit, such as the MACS (Miltenyl Biotec) Pan T Isolation Kit (Catalog No. 130-096-535). Native human pan-T cells include CD4 and CD8 T cells and some γ / δ T cell subsets. Pan-T cells can be isolated using negative immunomagnetic separation techniques without the use of columns.

[0500] Isolated pan-T cells are plated at 100,000 cells / well in a 96-well plate and stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies or with PMA / Ionomycin as a positive control (or culture medium as a negative control). Test compounds are added to the cells at different concentrations and analyzed for their effects on cytokine secretion after TCR-stimulation / CD28 co-stimulation. After the stimulated cells are cultured for an additional 2 days, the supernatant from each well (containing cytokines secreted by pan-T cells) is collected for ELISA analysis and quantification of IL-2 and IFN-γ.

[0501] Other assays may also be used to assay the ability of any HPK1 inhibitor to inhibit HPK1, or to screen for compounds having HPK1 inhibitory activity.

[0502] For example, in one assay, the inhibition of HPK1 kinase activity can be analyzed using the Treg assay (regulatory T-cell proliferation assay) described below. Specifically, naive CD4 T cells are isolated from human donated peripheral blood mononuclear cells (PBMC) using a suitable kit, such as one from Thermo Fisher Scientific (Cat. No. 11363D). + / CD25 - T-cells and CD4 + / CD25 + Regulatory T cells. + / CD25 - T-cells were labeled with CFSE (Thermo Fisher Scientific, C34554). + / CD25 + Regulatory T cells were 1×10 6Resuspend in RPMI-1640 medium at a concentration of 10 cells / mL. Take 100 μL of CFSE-labeled T-cells with or without 50 μL CD4 + / CD25 + Regulatory T cells were mixed and treated with 5 μL anti-CD3 / CD28 beads (ThermoFisher Scientific, 11132D) and various concentrations of compounds diluted in 50 μL RPMI-1640 medium. The mixed cell populations were cultured for 5 days (37°C, 5% CO 2 ), and on day 5, the proliferation of CFSE-labeled T-cells was analyzed using the FITC channel using a BD LSRFortessa X-20. The lead compound is expected to inhibit HPK1 to enhance Treg function and inhibit CFSE-labeled native CD4 + / CD25 - T-cell proliferation.

[0503] In another example, inhibition of HPK1 kinase activity can be analyzed using the p-SLP-76S376 HTRF assay (Cisbio) described below. This HTRF cell-based assay allows for rapid quantitative detection of phosphorylation of SLP-76 at Serine 376 by HPK1. Phospho-SLP-76 forms a scaffold upon which key signaling complexes can be built and serves as a marker for T-lymphocyte activation. According to the manufacturer, the phospho-SLP-76 (Ser376) assay uses two labeled antibodies: one with a donor fluorophore and the other with an acceptor. The first antibody is specific for binding to the phosphorylated S376 motif on SLP-76, and the second for its ability to recognize SLP-76 independent of its phosphorylation state. Protein phosphorylation can form an immune-complex involving the two labeled antibodies and bring the donor fluorophore into close proximity with the acceptor, thereby generating a FRET signal. Its intensity is positively correlated with the concentration of phosphorylated protein contained in the sample and provides a means to analyze the phosphorylation status of proteins in a no-wash assay format.

[0504] Briefly, Jurkat cells (cultured in RPMI1640 medium containing 10% FBS) were collected and centrifuged, and then 3×10 6Cells / mL were suspended in appropriate culture medium. Jurkat cells (35 μL) were then distributed to each well of a 384-well plate. Test compounds were diluted 40-fold using cell culture medium (39 μL of cell culture medium was added to 1 μL of compound). Jurkat cells in multiwell plates were treated with various concentrations of test compounds (5 mL of diluted compound was added to 35 μL of Jurkat cells, starting from 3 μM and diluted 1:3) and incubated at 37°C and 5% CO 2 For 1 hour, anti-CD3 (5 μg / ml, OKT3 clone) was then used for 30 min to activate TCR and HPK1. A 1:25 dilution of 100× blocking reagent (from p-SLP76ser376 HTRF kit) was prepared using 4× lysis buffer (LB), and 15 μL of 4× LB buffer containing blocking reagent was added to each well and incubated for 45 mins at room temperature with gentle shaking. Lysate (16 μL) was added to Greiner white plates, treated with p-SLP76 Ser376 HTRF reagent (2 μL donor, 2 μL acceptor), and incubated overnight at 4°C. The next day, homogeneous time-lapse fluorescence (HTRF) was measured on a PHERAstar plate reader. IC50 was determined by fitting the percentage of inhibition to the curve against inhibitor concentration using GraphPad Prism 5.0 software.

[0505] Any of the above assays can be scaled up or scaled up for high throughput screening (HTS). IC50s of lead compounds can be determined using any of the above assays.

[0506] 7. Pharmaceutical compositions

[0507] The present invention provides a pharmaceutical composition comprising any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0508] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that assist in the formulation and / or administration of an active agent to a subject and / or absorption by a subject and may be included in the compositions of the present invention without causing significant adverse toxic side effects to a subject. Non-limiting examples of pharmaceutically acceptable carriers and excipients include water, NaCl, physiological saline solutions, lactated Ringer's solutions, physiological sucrose solutions, physiological glucose solutions, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, salt solutions (such as Ringer's solutions), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and colorants and the like. Such preparations may be sterilized and, if necessary, mixed with adjuvants that do not adversely react or interfere with the activity of the compounds provided herein, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants and / or aromatic substances and the like. Those skilled in the art are aware of other pharmaceutical carriers and excipients suitable for use with the compounds of the present invention.

[0509] These compositions may further comprise one or more additional medical agents as needed. Alternatively, the compounds of the present invention may be administered to patients in need in combination with one or more other medical treatments (e.g., Gleevec or other kinase inhibitors, interferon, bone marrow transplantation, farnesyl transferase inhibitors, bisphosphonates, thalidomide, cancer vaccines, hormone therapy, antibodies, radiation, etc.). For example, the additional medical agent co-administered with the compounds of the present invention or included in the pharmaceutical composition may be another one or more anticancer agents.

[0510] As described herein, the compositions of the present invention include the compounds of the present invention and a pharmaceutically acceptable carrier, as used herein, including any and all solvents, diluents, or other vehicles, dispersants or suspending aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants and the like suitable for the desired specific dosage form. Remington's Pharmaceutical Sciences, 15th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1975) discloses the known techniques for preparing various different carriers and methods for preparing pharmaceutical compositions. Unless any conventional carrier medium is incompatible with the compounds of the present invention, such as producing any undesirable biological effects or interacting with any other component (group) in the pharmaceutical composition in a harmful manner, its use is intended to be included within the scope of the present invention. Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars (such as lactose, glucose and sucrose); starches (such as corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate); tragacanth powder; malt; gelatin; talc; excipients (such as cocoa butter and suppository wax); oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be contained in the composition.

[0511] 8. Preparation

[0512] The present invention also includes a composition comprising the active compound of the present invention and one or more pharmaceutically acceptable carriers and / or diluents and / or adjuvants (collectively referred to herein as "carrier" materials), and if necessary, in combination with other active ingredients.

[0513] In certain embodiments, the present invention provides a pharmaceutical formulation for treating cancer, particularly a cancer described herein, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0514] In certain embodiments, the present invention provides a pharmaceutical formulation for treating a cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancer, ovarian cancer, and pancreatic cancer, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0515] The compounds of the present invention may be administered by any suitable route, preferably in the form of pharmaceutical compositions compatible with these routes and in doses effective for treatment. The compounds and compositions of the present invention may be administered, for example, orally, mucosally, topically, rectally, pulmonary (such as inhalation spray) or parenterally (including intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular, transdermal, intraorbital, intrathecal, intraventricular, intratumoral, intranasal, intracranial, implanted, inhaled, and administered by infusion techniques in dosage unit formulations containing pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0516] Typically, the pharmaceutical composition is a tablet or gelatin capsule, which contains the active ingredient and a) a diluent, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) a lubricant, for example, silica, talc, stearic acid, its magnesium or calcium salt, and / or polyethylene glycol; c) a binder for tablets, for example, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone; d) a disintegrant, for example, starch, sugar, alginic acid or its sodium salt, or a foaming mixture, if necessary; and / or e) an adsorbent, a colorant, a flavoring agent, and a sweetener. The tablet can be further coated with a film coating or an enteric coating according to techniques known in the art.

[0517] Compositions suitable for oral administration include an effective amount of the compound of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral administration are prepared according to any method known in the art for making pharmaceutical compositions, and these compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, colorants and preservatives to produce pharmaceutically aesthetic and palatable preparations. Tablets may contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for making tablets. These excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch, or alginic acid; binders such as starch, gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a long period of time. For example, sustained-release materials such as glyceryl monostearate or glyceryl distearate may be used. Oral formulations may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate or kaolin), or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil).

[0518] Some injection compositions are aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fat emulsions or suspensions. Such compositions may be sterilized and / or contain adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, dissolution promoters, salts for adjusting osmotic pressure, and / or buffers. In addition, they may also contain other substances of medical value. The composition is prepared according to the commonly used mixing, granulation, or coating methods, respectively, and contains about 0.1-75% or about 1-50% of active ingredients. Compositions suitable for transdermal administration include an effective amount of the compound of the present invention and a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmaceutically acceptable solvents to promote passage through the host skin. For example, the transdermal device is in the form of a bandage, which includes a backing element, a reservoir containing the compound and an optional carrier, an optional rate-controlling barrier, so as to deliver the compound to the host skin for a long time at a controlled and predetermined rate, and a means for fixing the device on the skin.

[0519] Compositions suitable for topical application (e.g., administration to the skin and eyes) include aqueous solutions, suspensions, ointments, creams, gels, or spray formulations, e.g., delivered by aerosol or the like. Such topical delivery systems are particularly suitable for skin application, e.g., for the treatment of skin cancer, e.g., for prophylactic use in sunscreens, lotions, sprays, and the like. They are therefore particularly suitable for topical, including cosmetic formulations known in the art. These may contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0520] As used herein, topical administration also relates to inhalation or intranasal administration, which are suitably delivered in the form of dry powders (alone, in a mixture, for example, with lactose, or with, for example, phospholipids to form mixed component particles) from a dry powder inhaler, or delivered as an aerosol spray from a pressurized container, pump, spray bottle, nebulizer or aerosol dispenser, with or without a suitable propellant.

[0521] The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients, since water may promote the degradation of certain compounds.

[0522] The anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low moisture content ingredients and under low moisture and low humidity conditions. Anhydrous pharmaceutical compositions can be prepared and stored to maintain their anhydrous nature. Therefore, anhydrous compositions are packaged using materials known to prevent exposure to moisture so that they can be contained in a suitable formulation kit. Examples of suitable packaging include, but are not limited to, airtight foil packages, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0523] The present invention further provides a pharmaceutical composition and dosage form, which comprises one or more agents that can reduce the degradation rate of the compounds of the present invention as active ingredients. Such agents are referred to herein as "stabilizers" and include (but are not limited to) antioxidants such as ascorbic acid, pH buffers, or salt buffers, etc.

[0524] The pharmaceutically active compounds of the present invention can be prepared into pharmaceutical preparations for administration to patients (including humans and other mammals) according to conventional pharmaceutical methods.

[0525] The dosage and dosage course of the compound used to treat the disease condition according to the compound and / or composition of the present invention are determined according to various factors, including the age, weight, sex and medical condition of the individual, the disease pattern, the severity of the disease, the route and frequency of administration, and the specific compound used. Therefore, the dosage course may vary greatly, but is determined by standard methods as usual. As mentioned above, the daily dose can be administered once or can be divided into 2, 3, 4 or more administrations.

[0526] For medical purposes, the active compounds of the present invention are usually combined with one or more adjuvants, excipients or carriers suitable for the specified route of administration. If the drug is administered orally, the compound can be mixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid or sulfuric acid, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then compressed or encapsulated to facilitate administration. Such capsules or tablets may contain a controlled release formulation because the active compound can be dispersed in hydroxypropyl methylcellulose.

[0527] For skin conditions, for example, it is preferred to apply a topical formulation of the compounds of the invention to the affected area 2 to 4 times a day. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration of the skin (e.g., liniments, lotions, ointments, creams, or pastes), and drops suitable for administration to the eyes, ears, or nose. The active ingredient for topical administration may comprise 0.001% to 10% w / w of the formulation, e.g., 1% to 2% by weight, but it may comprise up to 10% w / w of the formulation, but preferably not more than 5% w / w, and more preferably 0.1% to 1%.

[0528] The compounds of the present invention may also be administered by transdermal devices. Preferably, transdermal administration is achieved by using a patch, which is in the form of a reservoir and porous membrane or a solid matrix variation. In both cases, the active agent is continuously delivered from the reservoir or microcapsule through the membrane to the active agent permeable adhesive in contact with the skin or mucous membrane of the recipient. If the active agent is absorbed through the skin, the active agent is administered to the recipient at a controlled and predetermined flow rate. In the case of microcapsules, the encapsulating agent also has the function of a membrane. The oil phase of the emulsion of the present invention can be composed of known ingredients in a known manner.

[0529] Although the phase may contain only an emulsifier, it may contain a mixture of at least one emulsifier and a fat or oil or a fat and an oil. Preferably, a hydrophilic emulsifier and a lipophilic emulsifier are included together as stabilizers. It is also preferred to include both oil and fat. In summary, the emulsifier (group) with or without the stabilizer (group) constitutes the so-called emulsifying wax, and the wax together with the oil and fat constitutes the so-called emulsified ointment base to form the oil dispersed phase of the cream formulation. The emulsifiers and emulsion stabilizers of the present invention suitable for the formulation of the present invention include Tween 60, Span 80, cetearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone or in combination with wax, or other materials known in the relevant art.

[0530] The oil or fat suitable for the formulation is selected according to the desired cosmetic properties, because the solubility of the active compound in most oils that may be used in the formulation of pharmaceutical emulsions is extremely low. Therefore, the cream should preferably be a non-greasy, non-sticky and washable product with suitable solidity to avoid leakage from tubes or other containers. Straight or branched monobasic- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol ester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a mixture of branched chain esters can be used. These can be used alone or in combination according to the desired properties.

[0531] Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils may be used.

[0532] Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.

[0533] The preferred concentration of the active ingredient in such formulations is 0.5 to 20%, preferably 0.5 to 10%, especially about 1.5% w / w.

[0534] Preparations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. Such solutions and suspensions may be prepared from sterile powders or granules using one or more of the above-mentioned carriers or diluents for preparing oral administration, or using other suitable homogenizers or wetting agents and suspending agents. The compound may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum, and / or various buffers. Other adjuvants and modes of administration are widely known in the medical arts. The active ingredient may also be formed into a composition using a suitable carrier (including physiological saline, dextrose, or water, or using cyclodextrin (i.e. Captisol), co-solvent dissolution (i.e. propylene glycol) or micellar dissolution (i.e. Tween 80)) for injection administration.

[0535] Sterile injections may also be sterile injection solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils are often used as solvents or suspension media. Any mild non-volatile oils may be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in injection preparations.

[0536] Pharmaceutical compositions for pulmonary administration may be administered in the form of an aerosol or using an inhaler containing a dry powder aerosol.

[0537] Suppositories for rectal administration of the drug are prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycol, which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

[0538] The pharmaceutical composition may be subjected to general pharmaceutical operations, such as sterilization, and / or contain commonly used adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Tablets and pills may be prepared using enteric coatings in addition. Such compositions may also contain adjuvants, such as wetting agents, sweeteners, flavoring agents, and fragrances. The pharmaceutical composition of the present invention comprises a compound of the chemical formula described herein. Or a pharmaceutically acceptable salt thereof; additional agents selected from the following: kinase inhibitors (small molecules, polypeptides, antibodies, etc.), immunosuppressants, anticancer agents, antiviral agents, anti-inflammatory agents, antifungal agents, antibiotics, or anti-angiogenic compounds; and any pharmaceutically acceptable carrier, adjuvant or vehicle.

[0539] Another composition of the invention comprises a compound of the formula described herein. Or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle. These compositions may optionally contain one or more additional medical agents, including, for example, kinase inhibitors (small molecules, polypeptides, antibodies, etc.), immunosuppressants, anticancer agents, antiviral agents, anti-inflammatory agents, antifungal agents, antibiotics, or anti-angiogenic compounds.

[0540] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be co-administered with the compound of the present invention to a patient and that does not destroy the pharmacological activity and is non-toxic when administered in an amount sufficient to deliver a therapeutic amount of the compound. Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical composition of the present invention include (but are not limited to) ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (self emulsifying drug delivery systems), and the like. systems) (SEDDS) (e.g., d-tocopheryl polyethylene glycol 1000 succinate), surfactants for pharmaceutical dosage forms (e.g., Tweens or other similar polymeric delivery matrices), serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene glycol-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. It is also convenient to use cyclodextrins, such as u-, p-, and y-cyclodextrins, or chemically modified derivatives thereof, such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-cyclodextrins, or other soluble derivatives, to enhance the delivery of the compounds of the formulae described herein.

[0541] The pharmaceutical composition can be orally administered in any orally acceptable dosage form, including (but not limited to) capsules, tablets, emulsions and aqueous suspensions, homogenates and solutions. Taking oral tablets as an example, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. When administered orally in capsule form, suitable diluents include lactose and dry corn starch. When an aqueous suspension and / or emulsion is administered orally, the active ingredient can be suspended or dissolved in an oil phase and combined with an emulsifier and / or suspending agent.

[0542] If necessary, certain sweeteners, flavoring agents and / or coloring agents may be added. The pharmaceutical composition may include formulations utilizing liposome or microencapsulation technology. Various different examples thereof are known in the art.

[0543] The pharmaceutical composition can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques known in the art of pharmaceutical formulation and can be dissolved in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or homogenizing agents, examples of which are also known in the relevant art.

[0544] 9. Treatment Kit

[0545] One aspect of the present invention is related to a convenient and effective set for carrying out the method and use according to the present invention. Typically, a pharmaceutical package or set comprises one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. Such sets are particularly suitable for delivering oral solid dosage forms, such as tablets or capsules. Such sets preferably include many unit doses, and may also include a card to arrange the doses in the order of their planned use. If necessary, a way to help memory can be provided, for example, with numbers, letters, or other marks or inserting a calendar to indicate the date of administration of the dose according to the treatment plan. Optionally, these containers may be added with instructions in the form of instructions prescribed by a government agency that manages the manufacture, usage, or sale of pharmaceutical products, which reflect that the agency has approved the manufacture, usage, or sale of drugs for human administration.

[0546] The following representative examples contain important additional information, examples and guidance for cooperating with the present invention in various embodiments and their equivalents. These examples are intended to help illustrate the present invention and are not intended to and should not be construed as limiting the present invention. Indeed, in addition to those shown and described herein, those skilled in the relevant art will understand various modifications of the present invention and many other embodiments thereof upon review of this case, including the following examples and references to the scientific and patent literature cited herein.

[0547] The contents of the cited references have been incorporated herein by reference to help illustrate the relevant art.

[0548] Furthermore, for purposes of the present invention, chemical elements are described according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition, inside pages. In addition, general organic chemistry principles and specific functional group moieties and reactivities are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "Organic Chemistry," Morrison & Boyd (3rd edition), both of which are incorporated herein by reference in their entirety.

[0549] 10. Synthesis reaction scheme

[0550] The compounds of the present invention can be prepared by those skilled in the art according to techniques and processes recognized by the art. More specifically, the compounds of the present invention can be prepared according to the reaction schemes, methods and examples shown below. It is understood by those skilled in the art that the individual steps of the following reaction schemes can be varied to produce the compounds of the present invention. Reagents and starting materials are readily available to those skilled in the art. Unless otherwise expressly stated, all substituents are as defined above.

[0551] Examples

[0552] The following are the abbreviations and their definitions used in this specification:

[0553] EtOAc: ethyl acetate

[0554] DCM: dichloromethane

[0555] ACN: Acetonitrile

[0556] THF: Tetrahydrofuran

[0557] DMSO: dimethyl sulfoxide

[0558] MeOH: Methanol

[0559] EtOH: ethanol

[0560] DMF: N,N-dimethylformamide

[0561] DMA: N,N-dimethylacetamide

[0562] DMF DMA: N,N-dimethylformamide dimethyl acetal

[0563] NCS: N-chlorosuccinimide

[0564] NBS: N-bromosuccinimide

[0565] NIS: N-iodosuccinimide

[0566] Pd-C: Palladium on Carbon

[0567] LDA: lithium diisopropylamide

[0568] TFA: trifluoroacetic acid

[0569] PTSA: p-Toluenesulfonic acid

[0570] DIBAL-H: Diisobutylaluminum hydride

[0571] LAH: Lithium Aluminum Hydride

[0572] Py: pyridine

[0573] DPPA: diphenylphosphoryl azide

[0574] CDI: 1,1-Carbonyldiimidazole

[0575] TEA: triethylamine

[0576] DIPEA: N,N-isopropylethylamine

[0577] DMAP: 4-(dimethylamino)pyridine

[0578] EDCI: N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride

[0579] HATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0580] HOBT: 1-Hydroxybenzotriazole

[0581] TfOH: trifluoromethanesulfonic acid

[0582] dppf: 1,1-diphenylphosphine-1,1-difluorocenyl-bis(diphenylphosphine)

[0583] DAST: (diethylamino)sulfur trifluoride

[0584] Pd2(dba)3: Di(benzylideneacetone)dipalladium(0)

[0585] Boc: tert-butoxycarbonyl

[0586] Ac:Acetyl

[0587] TMSI: trimethylsilyl iodide

[0588] TBAI: Tetrabutylammonium iodide

[0589] PPh 3 :Triphenylphosphine

[0590] dba: dibenzylidene acetone

[0591] BINAP: 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl

[0592] MsCl: methanesulfonyl chloride

[0593] TsCl: Tosyl chloride

[0594] DMAP: 4-dimethylaminopyridine

[0595] LiHMDS: Lithium bis(trimethylsilyl)amide

[0596] NaHMDS: Sodium bis(trimethylsilyl)amide

[0597] DMS: dimethyl sulfide

[0598] DME: dimethoxyethane

[0599] DCE: dichloroethane

[0600] NMR: Nuclear Magnetic Resonance

[0601] LC-MS: Liquid chromatography-mass spectrometry

[0602] ESI-MS: Electrospray ionization-mass spectrometry

[0603] GCMS: Gas chromatography-mass spectrometry

[0604] TLC: Thin layer chromatography

[0605] TCR: T cell receptor

[0606] BCR: B cell receptor

[0607] CARD: caspase activation and recruitment domain

[0608] mM: millimolar concentration

[0609] μM: micromolar concentration

[0610] mL: milliliters

[0611] ng: Nike

[0612] nM: nanomolar concentration

[0613] nm: nanometer

[0614] IC 50 : Half maximal inhibitory concentration

[0615] OD: Optical density

[0616] A. Biological Examples

[0617] Biological Example 1 HPK1 Biochemical Analysis

[0618] This example uses ADP-GLO TM The kinase assay measures the effect of potential HPK1 inhibitor compounds on HPK1 kinase activity.

[0619] ADP-GLO TMThe kinase assay (Promega Corp., Madison, WI) measures the ADP formed by the kinase reaction. According to the manufacturer, the ADP formed in the kinase assay is first converted to ATP, which is then used to generate light in the luciferase reaction. Luminescence is correlated with kinase activity. An exemplary experimental setup is described below, but slight adjustments may be made in individual assays.

[0620] Materials and instruments

[0621] 1. Reagents:

[0622]

[0623] 2. Instruments & Suppliers:

[0624]

[0625] 3. Disk settings

[0626] The compound was serially diluted 3 times from 10 μM (highest concentration) to 0.508 nM (lowest concentration). The positive control group was 10 μM reference + enzyme + substrate. The negative control group was 1% DMSO + enzyme + substrate.

[0627] 4. Process

[0628] 1. Buffer Preparation

[0629] 40mM Tris pH7.5; 20mM MgCl 2 , 0.1 mg / ml BSA, 50 μM DTT

[0630] Buffer stock solutions

[0631]

[0632]

[0633] <![CDATA[1M MgCl 2 ,95.21g / mol]]> <![CDATA[Add 4.7605 g to 50 mL H 2 O]]>

[0634] Add 20 mL 1M Tris and 10 mL 1M MgCl 2 To 470mL ddH 2 O to obtain a buffer stock solution and store at room temperature.

[0635] 2. Prepare fresh 1* assay buffer

[0636] Reagents [Reservoir] [final] multiple Add (mL) DTT(mM) 10 0.05 200 0.015 BSA (mg / mL) 100 0.1 1000 0.003 Buffer stock solutions 3

[0637] 3. Compound Preparation

[0638] 1) Take 10 μL of 10 mM stock solution of each compound and mix with 90 μL DMSO to dilute the compound to 1 mM.

[0639] 2) Then, the compound was diluted 3-fold using BRAVO to prepare 10 doses (from 5 μL to 10 μL). The highest compound concentration was 1 mM (100×), and the DMSO concentration was 100%.

[0640] 3) Using ECHO, transfer 100 nL of each diluted compound sample to a 384-well plate (Corning-4512)

[0641] 4) Centrifuge the assay plate at 1,500 rpm for 1 minute.

[0642] 4. Prepare 2× ATP-MBP mixture: 20 μM ATP, 0.2 μg / μL MBP in kinase buffer (final concentration: 10 μM ATP, 0.1 μg / μL MBP)

[0643]

[0644] 5. Prepare 2×HPK1 working solution using assay buffer. The final concentration of HPK1 is 0.6 ng / μL. For highly potent compounds, use a lower concentration of HPK1 (0.26 ng / μL-0.065 ng / μL).

[0645] 6. Add 5 μL / well of 2×HPK1 working solution and centrifuge at 1,500 rpm for 1 minute.

[0646] 7. Add 5 μL / well of 2×ATP-substrate and centrifuge at 1,500 rpm for 1 minute.

[0647] 8. Incubate at 25°C for 1 hour (or 6 hours for high potency compounds).

[0648] 9. Add 5 μL / well ADP-GLO TM The reagent stops the kinase reaction and consumes the unused ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0649] 10. Add 10 μL of kinase assay reagent to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0650] 11. Record the luminescent signal on the Envision reader (384-CTG).

[0651] 5. Data Analysis

[0652] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the high and low control groups contained in each assay plate. The high control wells were considered 0% inhibition, and the low control wells, which did not contain any compound but only DMSO (final concentration = 0.5%), were considered 100% inhibition. The concentration of the test compound and the % inhibition value were plotted and the three-parameter logarithmic dose-response equation was used to determine the compound concentration required to achieve 50% inhibition (IC50). The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a quality control measure. The experiment was considered acceptable if the endpoint value was within 3 times of the expected value.

[0653] Biological Example 2 PKC-θ Biochemical Assay

[0654] This example uses ADP-GLO TM The kinase assay measures the effect of potential HPK1 inhibitor compounds on PKC-θ kinase activity. An exemplary experimental setup is described below, but minor modifications may be made in individual assays.

[0655] Materials and instruments

[0656] 1. Reagents:

[0657]

[0658] 2. Instruments & Suppliers:

[0659] instrument source Catalog Number 96-hole V-type chassis Greiner 651201 384-well plate Corning 4512 LDV disk Labcyte LP0200 Bravo Agilent - Envision PerkinElmer - Pure water system Millipore Milli-Q Reference System - Bravo 10μL Dropper Tip Axygen VT-384-10uL-R Bravo 30μL Dropper Tip Axygen VT-384-31uL-R Manual single tube straw RAININ - Multi-tube electronic pipette Thermo / RAININ -

[0660] 3. Disk settings

[0661] The compound was serially diluted 3 times from 10 μM (highest concentration) to 0.508 nM (lowest concentration). The positive control group was 10 μM reference + enzyme + substrate. The negative control group was 1% DMSO + enzyme + substrate.

[0662] 4. Process

[0663] 1. Buffer Preparation

[0664] 40mM Tris pH7.5; 20mM MgCl 2 , 0.1 mg / ml BSA, 50 μM DTT

[0665] Buffer stock solutions

[0666] 1M Tris, pH 7.5, 121.14 g / mol <![CDATA[Add 6.057 g to 50 mL of H 2 O and adjust the pH to 7.5]]>

[0667] <![CDATA[1M MgCl 2 ,95.21g / mol]]> <![CDATA[Add 4.7605 g to 50 mL H 2 O]]>

[0668] Add 20 mL 1M Tris and 10 mL 1M MgCl2 To 470mL ddH 2 O to obtain a buffer stock solution and store at room temperature.

[0669] 2. Prepare fresh 1* assay buffer

[0670]

[0671] 3. Compound Preparation

[0672] 1) Take 10 μL of 10 mM stock solution of each compound and mix with 90 μL DMSO to dilute the compound to 1 mM.

[0673] 2) Then, the compound was diluted 3-fold using BRAVO to prepare 10 doses (from 5 μL to 10 μL). The highest compound concentration was 1 mM (100×), and the DMSO concentration was 100%.

[0674] 3) Using ECHO, 50 nL of each diluted compound sample was transferred to a 384-well plate (Corning-4512).

[0675] 4) The assay plate was centrifuged at 1,500 rpm for 1 minute.

[0676] 4. Prepare 2× enzyme working solution in kinase buffer:

[0677]

[0678] 5. Prepare 2× ATP-mix:

[0679]

[0680] 6. Add 2.5 μL / well of 2× enzyme operating solution and centrifuge at 1,500 rpm for 1 minute.

[0681] 7. Add 2.5 μL / well of 2×ATP-substrate and centrifuge at 1,500 rpm for 1 minute.

[0682] 8. Incubate at 25°C for 60 min.

[0683] 9. Add 5 μL / well of ADP-GLO TM The reagent stops the kinase reaction and consumes the unused ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0684] 10. Add 10 μL of kinase assay reagent to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0685] 11. Record the luminescent signal on the Envision reader (384-USL).

[0686] 5. Data Analysis

[0687] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the high and low control groups contained in each assay plate. The high control wells were considered 0% inhibition, and the low control wells, which did not contain any compound but only DMSO (final concentration = 0.5%), were considered 100% inhibition. The concentration of the test compound was plotted against the % inhibition value, and the three-parameter logarithmic dose-response equation was used to determine the compound concentration required to achieve 50% inhibition (IC50). The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a quality control measure. The experiment was considered acceptable if the endpoint value was within 3 times of the expected value.

[0688] Biological Example 3 TBK1 biochemical analysis

[0689] This example uses ADP-GLO TM Kinase assays measure the effects of potential HPK1 inhibitor compounds on TBK1 kinase activity. An exemplary experimental setup is described below, but minor modifications may be required for individual assays.

[0690] Materials and instruments

[0691] 1. Reagents:

[0692]

[0693] 2. Instruments & Suppliers:

[0694] instrument source Catalog Number 96-hole V-type chassis Greiner 651201 384-well plate Corning 4512 LDV disk labcyte LP0200 Bravo Agilent - Envision PerkinElmer - Pure water system Millipore Milli-Q Reference System - Bravo 10μL Dropper Tip Axygen VT-384-10uL-R Bravo 30μL Dropper Tip Axygen VT-384-31uL-R Manual single tube straw RAININ - Multi-tube electronic pipette Thermo / RAININ -

[0695] 3. Disk settings

[0696] See above.

[0697] 4. Process

[0698] 1. Buffer Preparation

[0699] 40mM Tris pH7.5; 20mM MgCl 2 , 0.1 mg / ml BSA, 50 μM DTT

[0700] Buffer stock solutions

[0701] 1M Tris, pH 7.5, 121.14 g / mol <![CDATA[Add 6.057 g to 50 mL of H 2 O and adjust the pH to 7.5]]>

[0702] <![CDATA[1M MgCl 2 ,95.21g / mol]]> <![CDATA[Add 4.7605 g to 50 mL H 2 O]]>

[0703] Add 20 mL 1M Tris and 10 mL 1M MgCl 2 To 470mL ddH 2 O to make a buffer stock solution and store at RT (room temperature).

[0704] 2. Prepare fresh 1* assay buffer

[0705]

[0706] 3. Compound Preparation

[0707] 1) Take 10 μL of 10 mM stock solution of each compound and mix with 90 μL DMSO to dilute the compound to 1 mM.

[0708] 2) Then, the compound was diluted 3-fold using BRAVO to prepare 10 doses (from 5 μL to 10 μL). The highest compound concentration was 1 mM (100×), and the DMSO concentration was 100%.

[0709] 3) Using ECHO, 50 nL of each diluted compound sample was transferred to a 384-well plate (Corning-4512).

[0710] 4) Centrifuge the assay plate at 1,500 rpm for 1 minute.

[0711] 4. Prepare 2× enzyme working solution in kinase buffer:

[0712]

[0713] 5. Prepare 2× ATP-mix:

[0714]

[0715] 6. Add 2.5 μL / well of 2× enzyme operating solution and centrifuge at 1,500 rpm for 1 minute.

[0716] 7. Add 2.5 μL / well of 2×ATP-substrate and centrifuge at 1,500 rpm for 1 minute.

[0717] 8. Incubate at 25°C for 60 min.

[0718] 9. Add 5 μL / well of ADP-GLO TM The reagent stops the kinase reaction and consumes the unused ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0719] 10. Add 10 μL of kinase assay reagent to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0720] 11. Record the luminescent signal on the Envision reader (384-USL).

[0721] 5. Data Analysis

[0722] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the high and low control groups contained in each assay plate. The high control wells were considered 0% inhibition, and the low control wells, which did not contain any compound but only DMSO (final concentration = 0.5%), were considered 100% inhibition. The concentration of the test compound was plotted against the % inhibition value, and the three-parameter logarithmic dose-response equation was used to determine the compound concentration required to achieve 50% inhibition (IC50). The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a quality control measure. The experiment was considered acceptable if the endpoint value was within 3 times of the expected value.

[0723] Biological Example 4: JAK3 biochemical analysis

[0724] This example uses the ADP-GLO kinase assay to measure the effects of potential HPK1 inhibitor compounds on JAK3 kinase activity. An exemplary experimental setup is described below, but there may be slight adjustments in individual assays.

[0725] Materials and instruments

[0726] 1. Reagents:

[0727]

[0728] 2. Instruments & Suppliers:

[0729]

[0730] 3. Process

[0731] 1. Preparation of JAK3 Kinase Buffer

[0732] DTT and BSA were freshly added to the buffer (final concentration: 40 mM Tris pH 7.5; 20 mM MgCl 2 , 0.1 mg / ml BSA, 50 μM DTT).

[0733]

[0734]

[0735] 2. Compound Preparation

[0736] Test compound: Take 5 μL of 10 mM compound stock solution and mix it in 45 μL DMSO to dilute the compound to 1 mM. The compound solution is then serially diluted 3 times to make 10 doses. The highest compound concentration is 1 mM (100×), and the DMSO concentration is 100%.

[0737] Using ECHO, 50 nL of each of the 10 doses of diluted compound was added to a 384-well assay plate (Corning #4512).

[0738] Positive control group: Using ECHO, 50 nL of 1 mM reference compound was added to a 384-well assay plate (Corning #4512).

[0739] Negative control group: 50 nL DMSO was transferred to a 384-well assay plate as a negative control group.

[0740] The assay plate was centrifuged at 1,500 rpm for 1 minute.

[0741] 3. Transfer compounds using ECHO according to the following procedure:

[0742] Reference compound and test compound 1-15: serially diluted 3-fold from 10 μM to 0.508 nM (10 doses); negative control group was 0.78 ng JAK3, 4 μM ATP & 0.2 μg / μL poly, 1% DMSO; positive control group was the highest dose of reference compound, 0.78 ng JAK3, 0.2 μg / μL Poly (E4Y1), 4 μM ATP, 1% DMSO.

[0743] 4. Use a pipette (Thermo, 30 μL multitube) to add 2.5 μL / well of 2×JAK3 working solution and centrifuge at 1,500 rpm for 1 minute.

[0744] 5. Use a pipette (Thermo, 30 μL multitube) to add 2.5 μL / well of 2×ATP-Poly (E4Y1) working mixture and centrifuge at 1,500 rpm for 1 minute.

[0745] 6. Incubate the assay plate at 25°C for 60 min.

[0746] 7. Use BRAVO to add 5 μL / well of ADP-GLO TM The reagent stops the kinase reaction and consumes the unused ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0747] 8. Add 10 μL of kinase assay reagent using BRAVO to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0748] 9. Record the luminescent signal on the Envision reader (384-USL).

[0749] 10. Use XL-fit to process the data. Inhibition % = [1-(test well-negative control group) / (positive control group-negative control group)]*100%.

[0750] 5. Data Analysis

[0751] Calculate the percent inhibition (%) for each compound concentration based on and relative to the signal in the negative and positive control groups contained in each assay plate. Negative control wells are considered 0% inhibition, and positive control wells are considered 100% inhibition. Plot the concentration of test compound against the % inhibition value and use a three-parameter logarithmic dose-response equation to determine the compound concentration required to achieve 50% inhibition (IC50). Evaluate the endpoint value (IC50) of the reference peptide / compound in each experiment as a quality control measure. The experiment is considered acceptable if the endpoint value is within 3 times of the expected value.

[0752] Biological Example 5 ZAP70 Biochemical Analysis

[0753] This example uses ADP-GLO TM Kinase assays measure the effects of potential HPK1 inhibitor compounds on ZAP70 kinase activity. An exemplary experimental setup is described below, but minor modifications may be made in individual assays.

[0754] Materials and instruments

[0755] 1. Reagents:

[0756]

[0757] 2. Instruments & Suppliers:

[0758]

[0759] 3. Process

[0760] 1. Preparation of ZAP70 kinase buffer:

[0761] DTT and BSA were freshly added to the buffer (final concentration: 40 mM Tris pH 7.5; 20 mM MgCl 2 , 0.1mg / ml BSA, 50μM DTT, 2mM MnCl 2 ).

[0762]

[0763] 2. Compound preparation:

[0764] Test compound: Take 5 μL of 10 mM compound stock solution and mix it in 45 μL DMSO to dilute the compound to 1 mM. The compound solution is then diluted 3 times to make 10 doses. The highest compound concentration is 1 mM (100×) and the DMSO concentration is 100%.

[0765] Using ECHO, 50 nL of compound solution was transferred to a 384-well assay plate (Corning #4512).

[0766] Positive control group: Using ECHO, 50 nL of 1 mM staurosporine was transferred to a 384-well assay plate as a positive control group.

[0767] Negative control group: Take 50nL DMSO and transfer it to a 384-well assay plate as the negative control group. Centrifuge the assay plate at 1,500rpm for 1 minute.

[0768] 3. Transfer compounds using ECHO according to the following procedure:

[0769] Staurosporine and test compounds 1-15: serially diluted 3-fold from 10 μM to 0.508 nM (10 doses); negative control group was 6.25 ng ZAP70, 10 μM ATP & 0.4 μg / μL poly, 1% DMSO; positive control group was 10 μM staurosporine, 6.25 ng ZAP70, 10 μM ATP & 0.4 μg / μL poly, 1% DMSO.

[0770] 4. Prepare 2×ATP-Poly(E4Y1) mixture: 20 μM ATP, 0.8 μg / μL poly(E4Y1) in kinase buffer (final concentration: 10 μM ATP, 0.4 μg / μL poly(E4Y1).

[0771] 5. Prepare 2× ZAP70 working solution: (2.5 ng / μL) in kinase buffer (final concentration 1.25 ng / μL).

[0772] 6. Use a pipette (Thermo, 30 μL multitube) to add 2.5 μL / well of 2×ZAP70 working solution and centrifuge at 1,500 rpm for 1 minute.

[0773] 7. Use a pipette (Thermo, 30 μL multitube) to add 2.5 μL / well of 2×ATP-Poly (E4Y1) working mixture and centrifuge at 1,500 rpm for 1 minute.

[0774] 8. Incubate the assay plate at 25°C for 60 min.

[0775] 9. Use BRAVO to add 5 μL / well of ADP-GLO TM The reagent stops the kinase reaction and consumes the unused ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0776] 10. Add 10 μL of kinase assay reagent using BRAVO to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0777] 11. Record the luminescent signal on the Envision reader (384-USL).

[0778] 12. Use XL-fit to process the data. Inhibition % = [1-(test well-negative control group) / (positive control group-negative control group)]*100%.

[0779] 5. Data Analysis

[0780] Calculate the percent inhibition (%) for each compound concentration based on and relative to the signal in the negative and positive control groups contained in each assay plate. Negative control wells are considered 0% inhibition, and positive control wells are considered 100% inhibition. Plot the concentration of test compound against the % inhibition value and use a three-parameter logarithmic dose-response equation to determine the compound concentration required to achieve 50% inhibition (IC50). Evaluate the endpoint value (IC50) of the reference peptide / compound in each experiment as a quality control measure. The experiment is considered acceptable if the endpoint value is within 3 times of the expected value.

[0781] Biological Example 6 LCK biochemical analysis

[0782] This example uses the ADP-GLO kinase assay to measure the effects of potential HPK1 inhibitor compounds on Lck kinase activity. An exemplary experimental setup is described below, but there may be slight adjustments in individual assays.

[0783] Materials and instruments

[0784] 1. Reagents:

[0785]

[0786] 2. Instruments & Suppliers:

[0787]

[0788] 3. Process

[0789] 1. Preparation of LCK kinase buffer:

[0790] DTT and BSA were freshly added to the buffer (final concentration: 40 mM Tris pH 7.5; 20 mM MgCl 2 , 0.1mg / ml BSA, 50μM DTT, 2mM MnCl 2 ).

[0791]

[0792] 2. Compound preparation:

[0793] Test compound: Take 5 μL of 10 mM compound stock solution and mix it in 45 μL of DMSO, dilute the compound to 1 mM first. The compound solution is then diluted 3 times in series to make 10 doses. The highest compound concentration is 1 mM (100×), and the DMSO concentration is 100%.

[0794] Positive control group: 3 μL of 10 mM staurosporine stock solution was mixed with 97 μL DMSO and diluted to 300 μM.

[0795] Using BRAVO, 50 nL of compound solution and 300 μM staurosporine were transferred to a 384-well assay plate (Corning #4512).

[0796] Negative control group: 50 nL of DMSO was transferred to a 384-well assay plate as a negative control group. The assay plate was centrifuged at 1,500 rpm for 1 minute.

[0797] 3. Transfer compounds using ECHO according to the following procedure:

[0798] Staurosporine and test compounds 1-15: serial dilution 3-fold from 10 μM to 0.508 nM (10 doses); negative control group was 7 ng LCK, 20 μM ATP & 0.4 μg / μL poly, 1% DMSO; positive control group was 3 μM staurosporine, 7 ng LCK, 20 μM ATP & 0.4 μg / μL poly, 1% DMSO.

[0799] 4. Prepare 2×ATP-Poly(E4Y1) mixture: 40 μM ATP, 0.8 μg / μL poly(E4Y1) in kinase buffer (final concentration: 20 μM ATP, 0.4 μg / μL poly(E4Y1)).

[0800] 5. Prepare 2× LCK working solution: (2.8 ng / μL) in kinase buffer (final concentration is 1.4 ng / μL).

[0801] 6. Use a pipette (Thermo, 30 μL multitube) to add 2.5 μL / well of 2×LCK working solution and centrifuge at 1,000 rpm for 1 minute.

[0802] 7. Use a pipette (Thermo, 30 μL multitube) to add 2.5 μL / well of 2×ATP-Poly (E4Y1) working mixture and centrifuge at 1,000 rpm for 1 minute.

[0803] 8. Incubate the assay plate at 25°C for 60 min.

[0804] 9. Use BRAVO to add 5 μL / well of ADP-GLO TM The reagent stops the kinase reaction and consumes the unused ATP after 1 hour. Centrifuge at 1,000 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0805] 10. Add 10 μL of kinase assay reagent using BRAVO to convert ADP to ATP. Centrifuge at 1,000 rpm for 1 minute. Incubate at 25°C for 30 minutes.

[0806] 11. Record the luminescent signal on the Envision reader (384-USL).

[0807] 12. Use XL-fit to process the data. Inhibition % = (negative control group - test well) / (negative control group - positive control group) × 100%.

[0808] 4. Data Analysis

[0809] Calculate the percent inhibition (%) for each compound concentration based on and relative to the signal in the negative and positive control groups contained in each assay plate. Negative control wells are considered 0% inhibition, and positive control wells are considered 100% inhibition. Plot the concentration of test compound against the % inhibition value and use a three-parameter logarithmic dose-response equation to determine the compound concentration required to achieve 50% inhibition (IC50). Evaluate the endpoint value (IC50) of the reference peptide / compound in each experiment as a quality control measure. The experiment is considered acceptable if the endpoint value is within 3 times of the expected value.

[0810] Biological Example 7 MAP4K3 Protein Kinase Assay

[0811] This example provides an assay to measure phosphorylation of a peptide substrate by the protein kinase MAP4K3.

[0812] Briefly, MAP4K3, its substrate, and cofactors (ATP and Mg 2+ ) were combined in the wells of a microtiter plate and incubated at 25°C for 5 hours. At the end of the incubation period, the reaction was terminated by adding a buffer containing EDTA. The substrate and product were separated and quantified by electrophoresis using the microfluidics-based LabChip 3000 Drug Discovery System from Caliper Life Sciences.

[0813] For this assay, the MAP4K3 substrate is FAM-GAGRLGRDKYKTLRQIRQ-NH2 (FAM is carboxyfluorescein). The purity of the peptide substrate by capillary electrophoresis is preferably >98%.

[0814] Typical assay setup and conditions are provided below.

[0815] 1. Add 5 μL of 2× enzyme buffer (or control) to each well of a 384-well plate.

[0816] 2. Add 100 nL of 100× compound. If necessary, enzyme and compound can be pre-incubated at this time.

[0817] 3. Add 5 μL of 2× substrate buffer.

[0818] 4. Incubate the plate at 25°C for 5 hours.

[0819] 5. Add 40 μL of 1.25× Stop Buffer to stop the reaction.

[0820] 6. Use the values ​​provided in the table below to 3000 Drug Discovery System operations.

[0821] 12 separation conditions of liquid crystal absorption film

[0822] Initial delayed aspiration time 50sec Sample buffer post-pipetting time 40sec Post-stain buffer aspiration time 40sec Sample aspiration time 0.2sec Final delayed aspiration time 120sec Dye absorption time 0.2sec pressure -2psi Downstream voltage -3000 volts Upstream voltage -800 volts

[0823] 7. Set up the plate and start electrophoresis, using blue laser (480nm) as excitation light and green CCD (520nm) for detection (CCD2).

[0824] The assay was run under the following reaction conditions: 5 hours total; at 25°C in the presence of 20 mM 100% inhibitor EDTA.

[0825] The final assay reaction mixture was: 100 mM HEPES, pH 7.5; 0.1% BSA; 0.01% Triton X-100; 1 mM DTT; 5 mM MgCl2 , 10 μM sodium orthovanadate, 10 μM β-glycerophosphate, 20 μM ATP; 1% DMSO (from compound); 0.5 μM FAM-GAGRLGRDKYKTLRQIRQ-NH2; 0.5 nM MAP4K3 enzyme.

[0826] It should be noted that the specific activity of MAP4K3 varies with each batch and the enzyme concentration may have to be adjusted to give a substrate to product conversion yield of -10-20%.

[0827] The LabChip 3000 capillary electrophoresis instrument was used to perform electrophoresis to separate the substrate and product peptides present in each sample. Two fluorescence peaks were observed when the substrate and product peptides were separated. The relative fluorescence intensity changes of the substrate and product peaks were used as parameters to reflect the enzyme activity. The capillary electropherograms (RDA acquired files) were analyzed using HTS WELL Analyzer software (Caliper Life Sciences). The kinase activity in each sample was determined by the total ratio (PSR): P / (S+P), where P is the peak height of the product peptide and S is the peak height of the substrate peptide.

[0828] Each compound was tested for enzyme activity at various concentrations (12 compound concentrations, 3× dilution intervals). Four replicates of negative control samples (0% - inhibition without inhibitor) and positive control samples (100% - inhibition in the presence of 20 mM EDTA) were collected and used to calculate the % inhibition value of each compound at each concentration.

[0829] The percentage of inhibition (P inh ):

[0830] P inh =(PSR 0％ -PSR inh ) / (PSR 0％ -PSR 100 %)*100, where PSR inh is the total ratio in the presence of inhibitors, PSR 0％ is the average product-to-total ratio in the absence of inhibitors, and PSR 100％ The average product-to-total ratio is 100% - the inhibition control sample.

[0831] The 4-parameter sigmoid dose-response model was used, and the inhibition curve (P inh The IC50 value of the inhibitor was determined relative to the inhibitor concentration.

[0832] Some of the materials and buffers used in this analysis are listed in the table below for reference.

[0833]

[0834]

[0835] Biological Example 8 Study on the efficacy of compounds on IL2 & IFN-γ released by human Pan T cells

[0836] This example is an analytical method using pan T cells and ELISA analysis format to determine the effect of compounds on the release of IL2 & IFN-γ.

[0837] Materials and instruments

[0838] 1. Reagents:

[0839]

[0840]

[0841] 2. Instruments & Suppliers:

[0842]

[0843]

[0844] 3. Process

[0845] Pan T Isolation and Reagent Preparation Process (Day 0):

[0846] 1. Cell growth medium:

[0847] RPMI1640: ATCC, catalog number 30-2001

[0848] 10% FBS: Gibco, catalog number 10099141

[0849] 1% Pen-Strep: Gibco, catalog number 15140122

[0850] 1% non-essential amine: Gibco, catalog number 11140050

[0851] β-Mercaptoethanol: Gibco, catalog number 21985023

[0852] 2. Preparation of Pan T Isolation Buffer:

[0853] use Dilution of rinse solution Prepare a solution containing phosphate buffered saline (PBS) (pH 7.2), 0.5% bovine serum albumin (BSA), and 2 mM EDTA using BSA stock solution (#130-091-376) 20 times. Keep the buffer at 4°C. Degas the buffer before use because bubbles will block the column.

[0854] Contents [Reservoir] [Final Concentration] multiple Volume (mL) BSA 10％ 0.5％ 20 5mL EDTA 0.5M 2mM 250 0.4mL PBS, pH 7.2 1× 1× 1 94.6mL total 100mL

[0855] 3. Thawing of Frozen PBMCs

[0856] 1) Preheat the culture medium in a 37°C water bath.

[0857] 2) Thaw the cells quickly in a 37°C water bath.

[0858] 3) Add pre-warmed culture medium to a 15 mL test tube and transfer the cells to the tube.

[0859] 4) Centrifuge at 300×g for 8 min. (0 for increase and 0 for decrease).

[0860] 5) Wash PBMCs using wash buffer.

[0861] 6) PBMCs were centrifuged at 300×g for 8 min and washed twice (9 for centrifugation up and 1 for centrifugation down).

[0862] 7) The cells were resuspended in Pan T cell isolation buffer and the cell number was counted.

[0863] 4.Pan T cell isolation method:

[0864] Staining cells with a bead cocktail

[0865] 1) Prepare cells and determine cell number. Filter cells using a 70 μm cell strainer.

[0866] 2) The cell aggregates were resuspended and the 7 Total cells were contained in 40 μL of buffer.

[0867] 3) Every 10 7 Add 10 μL of Pan T cell biotin-antibody mixture per total cell number.

[0868] 4) Mix wells and incubate in refrigerator (ice) for 10 minutes.

[0869] 5) Every 10 7 Add 30 μL of buffer per total number of cells.

[0870] 6) Every 10 7 Add 20 μL of Pan T cell microbead mixture per total cell number.

[0871] 7) Mix wells and incubate in refrigerator (ice) for 15 minutes.

[0872] 8) Then, magnetic cell separation was performed.

[0873] Note: a. Move quickly, keep cells cold, and use pre-cooled solutions (2-8°C). b. Aim to achieve 10 7 Magnetic labeling volume for total cell number. When working with fewer cells, use the same volumes as indicated. c. When working with higher cell numbers, scale up all reagent volumes and total volume accordingly. d. For optimal performance, it is important to obtain a single cell suspension prior to magnetic labeling.

[0874] Subsequent manual cell separation:

[0875] 1) Place the LS column in the magnetic field of a suitable MACS separator. For details, refer to the data sheet of each MACS column.

[0876] 2) Prepare the column by rinsing with 3 mL of buffer.

[0877] 3) Load the cell suspension onto the column and collect the flow-through, which represents the enriched T cells.

[0878] 4) Wash the column with 5 mL of buffer and collect the unlabeled cells that have passed through, which represent the enriched T cells.

[0879] Reminder: Be sure to wait until the tubing tank is empty before proceeding to the next step.

[0880] 5. Pan T cell FACS:

[0881] 1) Take out 50 μL of PBMC and Pan T cells respectively and add them into FACS tubes.

[0882] 2) The cells were incubated with anti-human CD3 / CD4 / CD8 antibodies (1 μL / 1 μL / 1 μL / tube) at 4° C. for 20 min. For the unstained control group, the cells were incubated with FACS staining buffer.

[0883] 3) Wash twice with cold staining buffer (PBS containing 0.2% BSA and 1 mM EDTA).

[0884] 4) Operate FACS. Block CD3 + 、CD3 + CD4 + , and CD3 + CD8 + Ethnic groups, analyze their percentages.

[0885] 5) If the purity of Pan T cells is higher than 90%, dilute the cell suspension to 1 million cells / mL using an appropriate volume of cell culture medium.

[0886] 6) Dispense the cell suspension into a sterile disposable storage tank for later use.

[0887] Process for preparation of compounds and anti-human CD3 / CD28 (Day 0):

[0888] Compound preparation

[0889] Compound serial dilution (starting assay plate 1000×)

[0890] The compound was dissolved in 100% DMSO to a concentration of 10 mM and then serially diluted 3-fold to prepare 8 dose points.

[0891] Preparation of 4× compound doses (intermediate assay plate: Corning-3599):

[0892] Prepare 4× compound solutions in culture medium. Pipette up and down. For the ZPE control group, prepare 0.4% DMSO in culture medium (4×). For the HPE control group, prepare 0.4 μM RGT003-026 in culture medium (4×).

[0893] 2. Anti-human CD3 (stock concentration 6.76 mg / ml): stored at 4°C.

[0894] 1) Dilute anti-human CD3 with PBS to a final concentration of 0.5 μg / mL.

[0895] 2) 50 μL / well of CD3 was added to each well except the positive and negative control wells. The positive and negative control wells did not have CD3 / CD28 stimulation.

[0896] 3) 5% CO at 37°C 2 Incubate in an incubator for 2 hours.

[0897] 4) Remove 50 μL of antibody solution from the cell culture plate and rinse each well twice with 200 μL of sterile PBS each time.

[0898] 3. Preparation of Anti-Human CD28 (4×)

[0899] The antibody was diluted in culture medium from a stock concentration of 11.07 mg / mL to 2 μg / mL (4×).

[0900] 4.PMA / Ionomycin Preparation (4×)

[0901] 1) Dilute PMA to 400 ng / mL (8×) in culture medium.

[0902] 2) Dilute ionomycin to 8 μM (8×) in culture medium.

[0903] 3) Mix equal volumes of PMA and ionomycin to obtain a 4× mixture.

[0904] Stimulation Cell Process (Day 0)

[0905] 1. Take 1×10 5 The cell suspension of 100 cells / well (100 μL / well) was transferred to a 96-well plate (cell analysis plate: Greiner-655180).

[0906] 2. The test compound group and ZPE / HPE control group were added with 50 μL / well of anti-human CD28. The positive or negative control group was added with 50 μL / well of 4x PMA / ionomycin solution or culture medium, respectively.

[0907] 3. According to the analysis plate map shown below, add 50 μL / well of the compound to the cell analysis plate. ZPE / HPE control group is added with 50 μL / well of 0.4% DMSO solution or 0.4 μM RGT003-026 (4×). Positive or negative control group, add 50 μL / well of culture medium.

[0908] 4. Incubate the assay plate for 48 hours.

[0909] IL-2&IFN-γ ELISA process:

[0910] Day 1: Coating dishes:

[0911] 1) Coat the microtiter wells with 100 μL of IL-2 & IFN-γ capture antibody (diluted in coating buffer) per well. Refer to the batch specific instructions / certificate of analysis for recommended antibody coating dilutions. Seal the assay plate and incubate overnight at 4°C.

[0912] Day 2: Collect samples:

[0913] 1) At 37°C and 5% CO 2 After 48 hours of incubation in the incubator, the cell plate was centrifuged at 1,000 rpm for 10 minutes. 100 μL / well of supernatant was collected and analyzed by IL-2 and IFN-γ ELISA. The supernatant was stored at -80°C and analyzed by IL-2 and IFN-γ ELISA the next day. The supernatant may need to be diluted 30 to 40 times to ensure that the analysis does not exceed the linear range of the IL-2 and IFN-γ standard curves.

[0914] 2) Add new culture medium (containing anti-CD28, P / I and compound) to the assay plate, 100 μL / well.

[0915] 3) Disk map:

[0916] Day 3-4: IL-2 & IFN-γ ELISA:

[0917] 1) Aspirate wells and wash 3 times with ≥300 μL / well of wash buffer. After the final wash, invert the assay plate and place on blotting paper to remove any residual buffer.

[0918] 2) Block the assay plate with ≥ 200 μL / well of assay diluent and incubate at room temperature for 1 hour.

[0919] 3) Aspirate / wash as in step 2.

[0920] 4) Prepare standards in assay diluent.

[0921] Preparation of IL-2 standard stock solution:

[0922] Add 1 mL of deionized water to the vial (235 ng / vial) to obtain a stock concentration of 235 ng / mL. Aliquot the standard stock solution into 10 μL / vial and freeze at -80°C.

[0923] Preparation of IFN-γ standard stock solution:

[0924] Add 1 mL of deionized water to the vial (145 ng / vial), the stock concentration is 145 ng / mL. Aliquot the standard stock solution into 10 μL / vial and freeze at -80°C

[0925] Preparation of IL-2 / IFNγ standard curve:

[0926] Dilute the standard sample to a maximum concentration of 500 pg / mL. Then dilute it 2-fold to make 10 doses (including a blank control group). Transfer the standard samples of different concentrations to the ELISA plate, 100 μL / well.

[0927] 1) Pipette 100 μL of each standard, sample, and control into appropriate wells. Seal the assay plate and incubate at room temperature for 2 hours.

[0928] 2) Aspirate / wash as in step 2, but wash a total of 5 times.

[0929] 3) Add 100 μL of the detection reagent (detection antibody + SAv-HRP reagent) to each well. Seal the assay plate and incubate at room temperature for 1 hour.

[0930] 4) Aspirate / wash as in step 2, but wash a total of 7 times. Note: In this final wash step, soak the wells in wash buffer for 30 seconds to 1 minute per wash.

[0931] 5) Add 100 μL of substrate solution to each well. Incubate the plate (without the plate seal cover) in the dark at room temperature for 30 minutes.

[0932] 6) Add 50 μL of stop reaction solution to each well.

[0933] 7) Within 30 minutes of stopping the reaction, read the absorbance at 450 nm. OD450 nm was calibrated to the OD value at 570 nm.

[0934] 4. Data Analysis

[0935] In the biochemical assay, the percent inhibition (%) of each compound concentration was calculated based on and relative to the signal contained in the HPE and ZPE wells in each assay plate. HPE wells were considered 100% efficacy, and ZPE wells containing no compound but DMSO (final concentration = 0.1%) were considered 0% efficacy.

[0936] In the cell-based assay, the response of IL-2 or IFN-γ was calculated at each compound concentration relative to the DMSO control group (data not shown). See Table 2. EC2x represents the compound concentration that produces a 200% response (2-fold), and EC50 was calculated using GraphPad Prism.

[0937] The biochemical and cellular data obtained from the example compounds are listed in Tables 1 and 2 below.

[0938]

[0939]

[0940]

[0941]

[0942]

[0943]

[0944]

[0945]

[0946]

[0947]

[0948]

[0949]

[0950]

[0951]

[0952]

[0953]

[0954]

[0955]

[0956]

[0957]

[0958]

[0959]

[0960]

[0961]

[0962]

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969]

[0970]

[0971]

[0972]

[0973]

[0974] B. Synthesis Examples

[0975] Instrument Description

[0976] NMR spectroscopy was performed using a Varian Mercury spectrometer at 400 MHz ( 1 H), 376MHz( 19 F) or 75MHz( 13 C). The solvents used for the samples are clearly stated in the experimental procedures for each compound. Chemical shifts are expressed in parts per million (ppm, delta units). Coupling constants are expressed in Hertz units (Hz). Apparent multimodality is described by split peak pattern and is expressed as s (singlet), d (doublet), t (triplet), q (quadruplet), quint (quintuplet), m (multiplet), br (broad peak).

[0977] The LCMS system used was: Agilent 6120 (Binary Gradient Module pump), Xbridge analytical column C 18 , 5 μm, 4.6×50 mm, 25° C., 5 μL injection volume, 2 mL / min, using a gradient of acetonitrile in 0.1% ammonium acetate in water according to the following schedule:

[0978]

[0979] Experimental procedures: All reactions were performed under anhydrous nitrogen blanket unless otherwise stated. TLC plates were visually detected by UV light. Flash chromatography refers to silica gel column chromatography (40-60 μm) using glass columns. Alternatively, automated chromatography was performed using a Biotage SP1 or Biotage Isolera system, with UV detection at 220 or 254 nm, and Biotage normal phase or reverse phase silica cartridges. Details can be found in the relevant experimental procedures.

[0980] General methods and preparation

[0981] The compounds, intermediates and specific examples of the general formula (I-0), (I-1), (I-2), (I), (II), (II-1), (II-2), or (II-2') are prepared by the synthetic methods described herein. In the experimental process, in addition to the acids, bases, reagents, coupling reagents, solvents, etc. explicitly mentioned, modifications of reaction conditions such as temperature, solution concentration, solvent volume, microwave conditions applied, reaction duration or a combination thereof are all intended to be part of the present invention, and alternative suitable acids, bases, reagents, coupling reagents, solvents, etc. can be used and are all included in the scope of the present invention. All possible geometric isomers, stereoisomers, and salt forms are intended to be included in the scope of the present invention.

[0982] General reaction diagram and synthesis process of pyrimidine compounds:

[0983]

[0984] Condition A1: Sodium hydride (60% dispersed in mineral oil, 1.2 eq.) was added to a DMF solution containing Int.E (or a reagent obtained from a commercial product) (1.0 eq.) at 0°C, and the mixture was stirred at 0°C for 30 minutes. 5-R 1 The resulting mixture was then stirred at room temperature for 1 hour, cooled to 0°C, and heated to room temperature using ice-H 2 The reaction was terminated by purifying the crude product to produce 4-R and 5-R 1 Substituted 2-Cl-pyrimidines.

[0985] Condition A2: In the presence of 5-R 1 Potassium carbonate (2.0 eq.) and Int.E (or a commercially available reagent) (1.0 eq.) were added to a DMF solution of substituted 2,4-dichloropyrimidine (1.0-1.5 eq.). The mixture was stirred at 80°C overnight, cooled to room temperature, and H 2 O and extracted with EA. The crude product was purified to yield 4-R and 5-R 1 Substituted 2-Cl-pyrimidines.

[0986] Condition A3: Add 5-R 1 Substituted 2,4-dichloropyrimidine (1.0-1.5 eq.) and DIPEA (1.0-2.0 eq.). The reaction mixture was stirred at 100°C for 18 hours, diluted with water, and extracted with EA. The crude product was purified to provide 4-R and 5-R 1 Substituted 2-Cl-pyrimidines.

[0987] Condition B1: Add 4-R and 5-R to a 2-methoxyethanol solution containing Int.W (1.0-1.4 eq.) 1 Substituted 2-Cl-pyrimidine (1.0 eq.) and HCl in EtOH (3.0 eq.). The resulting mixture was stirred at 120°C for 20 hours and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel and / or preparative-HPLC.

[0988] Condition B2: In the presence of 4-R and 5-R 1 To a solution of substituted 2-Cl-pyrimidine (1.0 eq.) and Int. W (1-1.3 eq.) in isopropanol was added trifluoroacetic acid (1.0-3.0 eq.). The mixture was heated in N 2 and stirred at 100 °C for 20 h, cooled to room temperature, and saturated NaHCO 3The aqueous solution was basified to pH 8 and extracted with DCM. The organic phase was washed with brine and washed with anhydrous Na 2 SO 4 The filtrate was concentrated in vacuo and the residue was purified by silica gel flash column chromatography and / or preparative HPLC.

[0989] Condition B3: In the presence of 4-R and 5-R 1 To a solution of substituted 2-Cl-pyrimidine (1.0 eq.) and Int.W (1-1.3 eq.) in isopropanol was added TsOH. 2 The mixture was stirred at 100°C overnight, cooled to room temperature, and saturated NaHCO 3 The aqueous solution was basified to pH 8 and extracted with DCM. The organic phase was washed with brine and washed with anhydrous Na 2 SO 4 The filtrate was concentrated in vacuo and the residue was purified by silica gel flash column chromatography and / or preparative HPLC.

[0990] Condition B4: In the presence of 4-R and 5-R 1 Substituted 2-Cl-pyrimidine (1.0 eq.) bis Int.W (1.0-1.2 eq.), Pd 2 (dba) 3 (0.2 eq.), Xantphos (0.2 eq.), and potassium carbonate (2.0 eq.). The mixture was heated at 100 °C and N 2 The mixture was stirred at 4 °C for 12 h, cooled to room temperature and filtered. The residue was purified by flash column chromatography on silica gel and / or preparative HPLC.

[0991] Depending on the specific example, 4-R and 5-R 1 Substituted 2-Cl-pyrimidines are additionally transformed between conditions A and B, and further transformed after condition B.

[0992] Intermediate

[0993] Int.W1

[0994]

[0995] 6-Methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[0996]

[0997] Step 1: A mixture of 2-(3-methoxyphenyl)ethaneamine (20.0 g, 132.27 mmol, 19.23 mL) in aqueous HCl (1 N, 192 mL, 192 mmol) was added with aqueous formaldehyde (37% wt, 41.64 g, 529.08 mmol). The mixture was stirred at 60°C for 1 hour, cooled to 0°C, and alkalized by adding dropwise 0°C 50% aqueous NaOH (17.44 g, 218 mmol). The resulting mixture was stirred at room temperature overnight and filtered. The filtrate was concentrated to give bis(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)methane as a white solid (23 g, 100% yield).

[0998] Step 2: Concentrated HCl (15.20 g, 182.01 mmol, 15.3 mL) was added dropwise to a suspension of bis(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)methane (28 g, 82.73 mmol) in i-PrOH (200 mL) at 0°C, and the mixture was stirred at room temperature for 18 hours. MTBE (70 mL) was added to the above mixture, and the suspension was stirred at room temperature for another 4 hours. After filtration, the filter cake was washed with MTBE / i-PrOH mixture (100 mL, 1 / 1 v / v) and dried to provide a white solid of 6-methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, which was suspended in DCM (300 mL). Saturated NaHCO was added to the mixture. 3 Aqueous solution (500 mL) was added, and the mixture was stirred at room temperature for 2 hours. After separation, the aqueous layer was extracted with DCM (50 mL x 4). The combined organic layers were washed with anhydrous Na 2 SO 4 Dehydration, filtration, and concentration provided 6-methoxy-1,2,3,4-tetrahydroisoquinoline as a yellow oil (10 g, 75% yield). LC-MS (ESI) m / z: 164 [M+H] + .

[0999] Step 3: Aqueous formaldehyde solution (37% w / w, 4.8 g, 59.92 mmol, 1.67 mL) was added to a solution of 6-methoxy-1,2,3,4-tetrahydroisoquinoline (1.63 g, 9.99 mmol) in MeOH (30 mL) at room temperature. The mixture was stirred at room temperature for 15 minutes and cooled to 0°C. NaBH was added in portions. 4(1.13 g, 29.96 mmol). The mixture was stirred at room temperature for 3 hours, quenched with ice-water (10 mL), and extracted with DCM (20 mL x 5). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (DCM / MeOH=10 / 1 v / v) to provide 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline as a yellow oil (1.7 g, 96% yield). LC-MS (ESI) m / z: 178 [M+H] + .

[1000] Step 4: 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline (2 g, 11.28 mmol) and guanidine nitrate (1.17 g, 9.59 mmol) were added sequentially to a pre-cooled (0°C) solution of concentrated sulfuric acid (4 mL) at 0°C. The resulting mixture was stirred at 0°C for 30 minutes, quenched with ice-water (20 mL), basified to pH 10-11 with aqueous NaOH (4N), and extracted with DCM (50 mL x 4). The combined organic layers were washed with anhydrous Na 2 SO 4 Dehydrate, filter, and concentrate. The residue was purified by silica gel flash column chromatography (PE 100% v / v, PE / EA = 1 / 1 v / v, EA 100% v / v, and then DCM / MeOH = 20 / 1, v / v) to provide 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline as a yellow solid (0.9 g, 36% yield). LC-MS (ESI) m / z: 223 [M+H] + .

[1001] Step 5: In the presence of 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (4.2 g, 14.40 mmol) in EA (32 mL), H 2 O (16 mL), and EtOH (144 mL) solution were added with Fe powder (5.5 g, 93.99 mmol) and ammonium chloride (793.96 mg, 13.08 mmol). The mixture was stirred at 60 ° C for 48 hours, cooled to room temperature, and filtered. The filter cake was washed with methanol (30 mL x 3), and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (DCM / MeOH=20 / 1 to 10 / 1 v / v) to provide 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine hydrochloride as a yellow solid (4.5 g, 100% yield). LC-MS (ESI) m / z: 193 [M+H] + .

[1002] Another synthesis method:

[1003]

[1004] Step 1: At 0°C, slowly add BOC to a solution of triethylamine (13.38 g, 132.27 mmol, 18.44 mL), 2-(3-methoxyphenyl)ethaneamine (10 g, 66.13 mmol) and DMAP (807.96 mg, 6.61 mmol) in DCM (100 mL). 2 O (15.88 g, 72.75 mmol, 16.70 mL). The mixture was then stirred at room temperature for 16 hours, diluted with ice-water (200 mL) and extracted with EtOAc (3 x 200 mL). The combined organic phases were washed with brine (100 mL) and washed with anhydrous Na 2 SO 4 The residue was purified by flash chromatography (SiO 2 , petroleum ether / ethyl acetate = 10 / 1) to provide (3-methoxyphenethyl)carbamic acid tert-butyl ester (14.6 g, 79.06% yield, 90% purity) as a colorless liquid. LC-MS (ESI) m / z: 196 [M+H-56] + .

[1005] Step 2: At -78 °C, in a CH 2 Cl 2 Tf was added to the solution (50 mL) 2 O (8.64 g, 30.64 mmol, 5.15 mL) 2 Cl 2 (5 mL) solution. After 20 minutes, BH 3 .Et 2 O (19.77 g, 139.26 mmol) was added to the above solution. The reaction mixture was then warmed to room temperature, stirred for 2 hours, and carefully quenched with saturated NaHCO 3 The resulting mixture was quenched with CH 2 Cl 2 Extraction (3 x 100 mL). The combined organic layers were washed with brine (50 mL) and purified by anhydrous Na 2 SO 4 The residue was purified by flash chromatography (SiO 2, petroleum ether / ethyl acetate / methanol = 5 / 1 / 0 to 10 / 10 / 1) to provide 6-methoxy-3,4-dihydroisoquinolin-1(2H)-one (3.4 g, 68.89% yield) as an off-white solid. LC-MS (ESI) m / z: 178 [M+H] + .

[1006] Step 3: At -20°C, in a concentrated H 2 SO 4 Add HNO (50 mL) to the solution 3 (2.16 g, 22.29 mmol, 65% purity). The mixture was stirred at -20°C to -25°C for 3 hours and poured into ice water (300 mL). The aqueous layer was extracted with dichloromethane (3 x 200 mL). The combined organic layers were concentrated in vacuo and the residue was purified by flash column chromatography (SiO 2 , petroleum ether / ethyl acetate / methanol = 10 / 1 / 0 to 10 / 10 / 1) to provide 6-methoxy-7-nitro-3,4-dihydroisoquinolin-1(2H)-one (2.02 g, 40.78% yield). LC-MS (ESI) m / z: 223 [M+H] + .

[1007] Step 4: To a solution of 6-methoxy-7-nitro-3,4-dihydroisoquinolin-1(2H)-one (2.02 g, 9.09 mmol) in THF (200 mL) was added 1 M BH 3 / THF (45.46 mmol, 45.5 mL). The mixture was stirred at reflux for 20 hours, and the reaction was carefully quenched with methanol (30 mL). The resulting solution was concentrated in vacuo. The residue was heated at 80°C for 3 hours in 2N HCl (50 mL), cooled, basified with aqueous ammonium hydroxide solution, and extracted with dichloromethane (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to provide 6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (1.89 g, 100.00% yield). LC-MS (ESI) m / z: 209 [M+H] + .

[1008] Step 5: Add formaldehyde (1.64 g, 54.46 mmol, 1.51 mL) to a solution of 6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (1.89 g, 9.08 mmol, 9.62 mL) in methanol (30 mL) at room temperature. Stir the mixture at room temperature for 15 minutes and cool to 0°C. Then add NaBH 4(1.03 g, 27.23 mmol) was added to the above mixture. The resulting mixture was stirred at room temperature for 3 hours, quenched with ice-water (10 mL), and extracted with DCM (20 mL x 5). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (DCM / MeOH=10 / 1, v / v) to provide 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (1.4 g, 69.40% yield) as a yellow oil. LC-MS (ESI) m / z: 223 [M+H] + .

[1009] Step 6: In the presence of 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (1.3 g, 5.85 mmol) in EtOAc (2 mL), H 2 O (1 mL), and EtOH (10 mL) solution, Fe (2.19 g, 39.19 mmol) and ammonium chloride (284.74 mg, 5.32 mmol) were added. The mixture was stirred at 60 ° C for 16 hours, cooled to room temperature, and filtered. The filter cake was washed with methanol (30 mL x 3), and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (DCM / MeOH=20 / 1 to 10 / 1 v / v) to provide 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine hydrochloride as a yellow solid (0.85 g, 75.6% yield). LC-MS (ESI) m / z: 193 [M+H] + .

[1010] Int.W2

[1011]

[1012] 2-Methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[1013]

[1014] Step 1: Potassium nitrate (4.01 g, 47.21 mmol) was added portionwise to an ice-cold solution of 1,2,3,4-tetrahydroisoquinoline (4.8 g, 36.04 mmol) in concentrated sulfuric acid (20 mL), keeping the temperature below 5°C. The reaction mixture was stirred at room temperature overnight, poured into ice (100 g), and NH 3 .H 2 O to adjust pH to 9, and use CH 2 Cl 2Extraction (50 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was crystallized in methanol to provide 7-nitro-1,2,3,4-tetrahydroisoquinoline hydrochloride as a yellow solid (4.2 g, 54% yield). LC-MS (ESI) m / : 179 [M+H] + .

[1015] Step 2: To a stirred mixture of 7-nitro-1,2,3,4-tetrahydroisoquinoline hydrochloride (5.0 g, 23.29 mmol) in anhydrous 1,2-dichloroethane (200 mL) was added formalin (37% aqueous formaldehyde solution, 2.0 mL, 25.86 mmol) and then triacetoxyborohydride (19.81 g, 104.82 mmol). The reaction mixture was stirred vigorously at room temperature for 4 hours and concentrated. The residue was diluted with EtOAc (400 mL) and slowly treated with saturated NaHCO 3 The reaction was quenched with aqueous solution (400 mL) and stirred for 30 minutes. The mixture was extracted with EA (100 mL x 4). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to provide 2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline as a brown solid (3.8 g, 85% yield). LC-MS (ESI) m / : 193 [M+H] + .

[1016] Step 3: Take 2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (3.8 g, 19.77 mmol), Fe (5.52 g, 98.85 mmol), NH 4 Cl (15.86 g, 296.54 mmol) in EA (10 mL), EtOH (100 mL), and H 2 O (20 mL) suspension was vigorously stirred at 70 ° C for 24 hours. After filtration, the filter cake was washed with EA / EtOH mixed solvent (100 mL, 10 / 1, v / v), and the filtrate was concentrated. The residue was purified by silica gel flash column chromatography (MeOH in DCM, 0% to 25% v / v) to provide 2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine as a yellow solid (1.84 g, 57% yield). LC-MS (ESI) m / z: 163 [M+H] + .

[1017] Int.W3

[1018]

[1019] 6-Chloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[1020]

[1021] Step 1: At room temperature, MeI (5.21 g, 36.67 mmol) was added to a solution of 6-chloroisoquinoline (2.0 g, 12.22 mmol) in MeCN (15 mL). The mixture was stirred in a sealed tube at 50°C for 2 days. After cooling to room temperature, the solid was collected by filtration and dried to provide 6-chloro-2-methylisoquinolin-2-ium iodide as a yellow solid (3.2 g, 86% yield). LC-MS (ESI) m / z: 178 [M] + .

[1022] Step 2: NaBH was added portionwise to a solution of 6-chloro-2-methylisoquinolin-2-ium iodide (200 mg, 0.65 mmol) in MeOH (8 mL) at 0°C. 4 (127.06 mg, 3.36 mmol). The mixture was stirred at room temperature for 3 hours and saturated with NaHCO 3 The aqueous solution was adjusted to pH ~ 9 and extracted with DCM (30 mL x 3). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by preparative-TLC (DCM / MeOH=20 / 1, v / v) to provide 6-chloro-2-methyl-1,2,3,4-tetrahydroisoquinoline as a yellow solid (90 mg, 44% yield). LC-MS (ESI) m / z: 182 [M+H] + .

[1023] Step 3 & 4: Follow the same process as Int.W1 Step 4 & 5, using 6-chloro-2-methyl-1,2,3,4-tetrahydroisoquinoline instead of 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline. LC-MS (ESI) m / z: 197 [M+H] + .

[1024] Int.W4

[1025]

[1026] 2-Methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine

[1027]

[1028] Step 1: Take 1-methyl-3,5-dinitro-pyridin-2-one (4.5 g, 22.60 mmol) and 1-methylpiperidin-4-one (2.81 g, 24.86 mmol) in NH 3 / MeOH solution (7N, 50 mL) was placed in a sealed tube and stirred at 50°C overnight. After cooling to room temperature, the mixture was concentrated, and the residue was dissolved in DCM (100 mL) and saturated NaHCO3 The layers were separated and the aqueous layer was extracted with DCM (50 mL x 2). The combined organic layers were washed with anhydrous Na 2 SO 4 Dehydration, filtration, and concentration provided 6-methyl-3-nitro-5,6,7,8-tetrahydro-1,6-naphthyridine as a red solid (2.84 g, 65% yield). LC-MS (ESI) m / z: 194 [M+H] + .

[1029] Step 2: 10% Pd / C (817.20 mg) was added to a solution of 6-methyl-3-nitro-7,8-dihydro-5H-1,6-naphthyridine (1.3 g, 6.73 mmol) in ethanol (35 mL) and THF (35 mL). 2 The mixture was stirred at 1 atm for 2 hours. After filtration, the filter cake was washed with EtOH (5 mL x 2), and the filtrate was concentrated to provide 6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine as a yellow solid (1.1 g, 100% yield). LC-MS (ESI) m / z: 164 [M+H] + .

[1030] Step 3: Bromine (1.17 g, 7.35 mmol) was added to a solution of 6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine (1.2 g, 7.35 mmol) and AcONa (1.21 g, 14.70 mmol) in acetic acid (25 mL) at room temperature, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted in DCM (100 mL) and water (30 mL) and NaHCO 3 Slowly adjust the pH to 9. The organic layer is washed with anhydrous Na 2 SO 4 Dehydrate, filter, and concentrate. The residue was purified by silica gel flash column chromatography (DCM / MeOH=20 / 1, v / v) to provide 2-bromo-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine as a yellow solid (570 mg, 32%). LC-MS (ESI) m / z: 242 [M+H].

[1031] Step 4: To a mixture of 2-bromo-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine (250 mg, 1.03 mmol), cesium carbonate (672.86 mg, 2.07 mmol) and MeONa (83.67 mg, 1.55 mmol) in MeOH (2 mL) were added CuI (19.67 mg, 103.26 umol) and 1,10-phenanthroline monohydrate (40.94 mg, 206.51 umol). The mixture was heated to N2 Under microwave conditions, the mixture was stirred at 120°C for 2 hours. After cooling to room temperature, the residue was purified by preparative-TLC (DCM / MeOH=15 / 1, v / v) to provide 2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine as a yellow solid (74 mg, 37% yield). LC-MS (ESI) m / z: 194 [M+H] + .

[1032] Int.W5

[1033]

[1034] 2-Isopropyl-6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-amine

[1035]

[1036] Step 1: Acetone (725.93 mg, 12.50 mmol, 917.74 uL) was added dropwise to a mixture of 6-methoxy-1,2,3,4-tetrahydroisoquinoline (340 mg, 2.08 mmol) in MeOH (10 mL) at room temperature, and the mixture was stirred at room temperature for 30 minutes. Then, NaBH(AcO) was added in portions at 0°C. 3 (6.25 mmol) was added to the above mixture. The resulting mixture was stirred at room temperature for 3 hours and then treated with saturated NaHCO 3 The reaction was quenched with aqueous solution (10 mL) and concentrated. The residue was extracted with DCM (50 mL x 2), and the combined organic layers were washed with brine (20 mL x 2) and washed with anhydrous Na 2 SO 4 Dehydrate, filter and concentrate. The residue was purified by silica gel flash column chromatography (DCM / MeOH=20 / 1, v / v) to provide 2-isopropyl-6-methoxy-1,2,3,4-tetrahydroisoquinoline as a white solid (150 mg, 35% yield). LC-MS (ESI) m / z: 206 [M+H] + .

[1037] Step 2: Follow the same process as Int.W1 step 4, using 2-isopropyl-6-methoxy-1,2,3,4-tetrahydroisoquinoline instead of 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline. LC-MS (ESI) m / z: 251 [M+H] + .

[1038] Step 3: A mixture of 2-isopropyl-6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (200 mg, 799.06 umol) and 10% palladium on carbon (30 mg, 799.06 umol) in EA (6 mL) was heated at room temperature under H 2 The mixture was stirred at 40 °C for 16 h and filtered. The filtrate was concentrated and the residue was purified by preparative-TLC (DCM / MeOH=10 / 1, v / v) to provide 2-isopropyl-6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-amine as a white solid (50 mg, 28% yield). LC-MS (ESI) m / z: 221 [M+H] + .

[1039] Int.W6

[1040]

[1041] 7-Amino-6-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester

[1042]

[1043] Step 1: At -10 °C, in a solution of concentrated H 2 SO 4 6-methoxy-1,2,3,4-tetrahydroisoquinoline (1.0 g, 1.84 mmol) was added to a cold solution of 4-nitro-1,2,3,4-tetrahydroisoquinoline (5 mL). Guanidine nitrate (758 mg, 6.13 mmol) was added to the above mixture in portions at -10 °C. The mixture was stirred at -10 °C for 30 minutes, poured into cold water (20 mL), adjusted to pH 8 with aqueous NaOH (4 N), and extracted with EA (40 mL x 4). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (DCM 100% v / v, and then DCM / MeOH = 10 / 1, v / v) to provide 6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline as a yellow solid (200 mg, 16% yield). LC-MS (ESI) m / z: 209 [M+H] + .

[1044] Step 2: Take 6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (100 mg, 0.48 mmol), Boc 2 O (157 mg, 0.72 mmol), and Et 3A mixture of N (97 mg, 0.96 mmol) and DCM (5 mL) was stirred at room temperature for 4 hours and concentrated. The residue was purified by silica gel flash column chromatography (DCM 100% v / v, and then DCM / MeOH = 10 / 1, v / v) to provide 6-methoxy-7-nitro-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester as a yellow oil (150 mg, 100% yield). LC-MS (ESI) m / z: 209 [M-100+1] + .

[1045] Step 3: A mixture of 6-methoxy-7-nitro-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester (150 mg, 0.48 mmol) and 10% Pd / C (50 mg) in EtOH (5 mL) was added to H 2 (1 atm) and stirred at room temperature for 16 hours, and filtered. The filtrate was concentrated to provide 7-amino-6-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester as a yellow solid (110 mg, 81% yield). LC-MS (ESI) m / z: 223 [M-55] + .

[1046] Int.W7

[1047]

[1048] 6-Fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[1049]

[1050] Step 1: At 0°C, in a solution of 6-fluoro-3,4-dihydro-2H-isoquinolin-1-one (900 mg, 5.45 mmol) 2 SO 4 Potassium nitrate (578.47 mg, 5.72 mmol) was added to the solution (9 mL) in portions. The resulting mixture was stirred at 0°C for 2 hours and poured into ice-water. A white solid precipitated, which was collected by filtration and dried in vacuo to provide 6-fluoro-7-nitro-3,4-dihydro-2H-isoquinolin-1-one (1.04 g, 86% yield). LC-MS (ESI) m / z: 211 [M+H] + .

[1051] Steps 2, 3, and 4: Follow the same process as Steps 4, 5, and 6 of another synthesis method of Int. W1, using 6-fluoro-7-nitro-3, 4-dihydro-2H-isoquinolin-1-one instead of 6-methoxy-7-nitro-3, 4-dihydro-2H-isoquinolin-1-one. LC-MS (ESI) m / z: 181 [M+H]+ .

[1052] Int.W8

[1053]

[1054] 2-Ethyl-6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-amine

[1055]

[1056] Step 1: In the presence of 6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (300 mg, 1.44 mmol) 3 To the CN (10 mL) solution, iodoethane (224.72 mg, 1.44 mmol, 115.83 uL) and K 2 CO 3 (796.52 mg, 5.76 mmol). The reaction mixture was stirred at 20°C for 16 hours, and partitioned between ethyl acetate (100 mL) and water (100 mL). The organic layer was separated, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (0-20% methanol in dichloromethane, containing aqueous ammonium hydroxide) to provide 2-ethyl-6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (136 mg, 40% yield). LC-MS (ESI) m / z: 237 [M+H] + .

[1057] Step 2: Follow the same process as step 6 of another synthesis method of Int. W1, using 2-ethyl-6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline instead of 2-methyl-6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline. LC-MS (ESI) m / z: 207 [M+H] + .

[1058] Int.W9

[1059]

[1060] 6-Methoxy-2-methylisoindolin-5-amine

[1061]

[1062] Step 1: Follow the same process as Int. W7 step 1, using 5-methoxyisoindolin-1-one instead of 6-fluoro-3,4-dihydroisoquinolin-1(2H)-one. LC-MS (ESI) m / z: 209 [M+H] + .

[1063] Steps 2, 3, and 4: Follow the same process as Steps 4, 5, and 6 of another synthesis method of Int. W1, using 5-methoxy-6-nitroisoindolin-1-one instead of 6-methoxy-7-nitro-3,4-dihydro-2H-isoquinolin-1-one. LC-MS (ESI) m / z: 179 [M+H] + .

[1064] Int.W10

[1065]

[1066] 6-Isopropoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[1067]

[1068] Step 1: 1M hydrogen bromide (1 mL) was added to a solution of 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (50 mg, 224.98 umol) in water (5 mL). The mixture was stirred at reflux for 16 hours, basified to pH 8 with saturated aqueous sodium bicarbonate solution, and extracted with dichloromethane / methanol (v / v, 10 / 1, 20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo to provide the crude product 2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinolin-6-ol (46.84 mg, 90% yield). LC-MS (ESI) m / z: 209 [M+H] + .

[1069] Step 2: Potassium carbonate (229.00 mg, 1.66 mmol) was added to a DMF (2 mL) solution containing 2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinolin-6-ol (230 mg, 1.10 mmol) and 2-iodopropane (187.78 mg, 1.10 mmol, 110.46 uL). The mixture was stirred at room temperature for 16 hours and diluted with brine (10 mL). The aqueous phase was separated and extracted with ethyl acetate (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel flash column chromatography (EA / PE: 10-50%) to provide 6-isopropoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (240 mg, 87% yield). LC-MS (ESI) m / z: 251 [M+H] + .

[1070] Step 3: Follow the same process as step 6 of another synthesis method of Int. W1, using 6-isopropoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline instead of 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline 5-methoxy-6-nitroisoindolin-1-one. LC-MS (ESI) m / z: 221 [M+H] + .

[1071] Int.W11

[1072]

[1073] 1-(7-amino-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethane-1-one

[1074]

[1075] Step 1: At 0°C, triethylamine (838.43 mg, 8.29 mmol, 1.15 mL) was added to a solution of 6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (908 mg, 4.14 mmol) in dichloromethane (15 mL), followed by trifluoroacetic anhydride (1.04 g, 4.97 mmol, 700.78 uL). The reaction mixture was stirred at room temperature for 3 hours and saturated NaHCO 3 The aqueous solution was basified to pH 8 and extracted with dichloromethane (10 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo to provide the crude product 2,2,2-trifluoro-1-(6-methoxy-7-nitro-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one as a yellow solid (1.26 g, 95% yield). LC-MS (ESI) m / z: 305 [M+H] + .

[1076] Step 2: A mixture of 2,2,2-trifluoro-1-(6-methoxy-7-nitro-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one (1.33 g, 4.14 mmol) and 10% palladium on carbon (400 mg, 3.76 mmol) in methanol (30 mL) was stirred at 25 °C with H 2 The mixture was stirred under vacuum for 16 hours and filtered through celite. The filter cake was washed with methanol (30 mL x 3) and the filtrate was concentrated in vacuo to provide 1-(7-amino-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)-2,2,2-trifluoroethane-1-one as a light yellow solid (1.14 g, 95% yield). LC-MS (ESI) m / z: 275 [M+H] + .

[1077] Int.E1

[1078]

[1079] (1H-Indol-3-yl)dimethylphosphine oxide

[1080]

[1081] Step 1: To a mixture of 1H-indole (2.34 g, 19.97 mmol) and KOH (1.34 g, 23.97 mmol) in DMF (40 mL) was added dropwise 1H-indole (2.34 g, 19.97 mmol) at 0°C. 2 (5.08 g, 19.97 mmol) in DMF (10 mL). The resulting mixture was stirred at 0°C for 30 minutes, diluted with cold water (200 mL), and stirred for another 30 minutes. The solid was collected by filtration and purified by HPLC. 2 O (30 mL x 3), and dried in vacuo to provide 3-iodo-1H-indole as a yellow solid (2.58 g, 53% yield).

[1082] Step 2: Triethylamine (3.00 g, 4.13 mL, 29.62 mmol), BocO (4.31 g, 19.75 mmol), and DMAP (120.64 mg, 0.99 mmol) were added to a solution of 3-iodo-1H-indole (2.4 g, 9.87 mmol) in DCM (15 mL). The mixture was stirred at room temperature for 1 hour and concentrated in vacuo. The residue was purified by silica gel flash column chromatography (EA / PE=20 / 1, v / v) to provide 3-iodo-1H-indole-1-carboxylic acid tert-butyl ester as a brown oil (4.6 g, 100% yield).

[1083] Step 3: Take 3-iodoindole-1-carboxylic acid tert-butyl ester (1.0 g, 2.91 mmol), dimethylphosphine oxide (250.19 mg, 3.21 mmol), Pd(OAc) 2 (65.42mg, 0.29mmol), Xantphos (168.61mg, 0.29mmol), and K 3 PO 4 A mixture of (680.40 mg, 3.21 mmol) and DMF (8 mL) was stirred at N 2Under microwave conditions, the mixture was stirred at 80°C for 5 hours. After cooling to room temperature, the mixture was concentrated, and the residue was purified by silica gel flash column chromatography (DCM 100% v / v, and then DCM / MeOH=20 / 1 v / v) to provide 3-(dimethylphosphoryl)-1H-indole-1-carboxylic acid tributyl ester as a brown oil (1 g, 100% yield). LC-MS (ESI) m / z: 294 [M+H] + .

[1084] Step 4: TFA (3 mL) was added to a solution of tert-butyl 3-(dimethylphosphoryl)-1H-indole-1-carboxylate (1.0 g, 3.41 mmol) in DCM (6 mL) at 0°C. The mixture was stirred at room temperature overnight and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (DCM / MeOH=20 / 1, v / v) to provide (1H-indol-3-yl)dimethylphosphine oxide as a white solid (460 mg, 70% yield). LC-MS (ESI) m / z: 194 [M+H] + .

[1085] Int.E2

[1086]

[1087] N,N-Dimethyl-1H-indole-3-carboxamide

[1088]

[1089] Triethylamine (2.51 g, 3.46 mL, 24.82 mmol) was added dropwise to a DMF (15 mL) solution containing 1H-indole-3-carboxylic acid (1.0 g, 6.21 mmol), HATU (4.72 g, 12.41 mmol), and dimethylamine hydrochloride (1.01 g, 12.41 mmol) at 0°C. The mixture was stirred at room temperature for 14 hours and then heated to room temperature with H 2 The mixture was diluted with 4% paraformaldehyde (100 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic layers were washed with brine (100 mL x 5), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated...

Claims

1. A compound or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is represented by the structural formula (I-2): in A is CR2 or N; R is in The bond system connects the pyrimidine ring, R a Each instance of is independently H, F or Cl; R a1 Independent for H, C 1-4 Alkyl or C 1-4 Hydroxyalkyl; R a1’ Independently -(CHR aa ) k OH, -(CHR aa ) k CN, -(CHR aa ) k C(O)OR 11 , -(CHR aa ) k C(O)NR 11 R 12 , -(CHR aa ) k C(O)NR 11 OR 12 , -(CHR aa ) k C(O)NR 11 S(=O)2R 12 , -(CHR aa ) k S(=O)2R 11 ,-(CHR aa ) k S(=O)2NR 11 R 12 ,-(CHR aa ) k NR 11 S(=O)2R 12 , -(CHR aa ) k -5-6 membered heteroaryl or -(CHR aa ) k P(=O)R 11 R 12 ; R aa is independently H or optionally halogen-substituted C 1-3 alkyl; R1 is H, deuterium, halogen, OH, CN, NH2, NO2, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, phenyl, 5-6 membered heteroaryl, C 3-6 Cycloalkyl or 3-7 membered heterocyclic group, wherein the alkyl, alkenyl, alkynyl, alkoxy, phenyl, heteroaryl, cycloalkyl or heterocyclic group represented by R1 or in the group represented by R1 may be substituted by one to three substituents independently selected from the group consisting of halogen, OH, CN, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Haloalkyl, C 1-4 Haloalkoxy and NR 11 R 12 ;or Each instance of R2 is independently H, deuterium, halogen, OH, CN, NH2, NO2, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, P(=O)R 11 R 12 、S(=O)2R 11 or S(=O)2NR 11 R 12 , wherein the alkyl, alkenyl, alkynyl or alkoxy group represented by R2 or in the group represented by R2 may be optionally substituted by one to three independently selected from halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy and NR 11 R 12 substituted by a substituent of the group formed; R3 is C 1-6 Alkyl, C 1-6 Hydroxyalkyl or COCH2NR 11 R 12 ; R 11 and R 12 Each instance of is independently H or C 1-4 alkyl; m is 0, 1, 2, or 3; and k is 0 or 1.

2. The compound according to claim 1 or its pharmaceutically acceptable salt or stereoisomer, wherein R1 is H, halogen, CN, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-6 Alkyl, C(O)OC 1-6 Alkyl, NR 11 C(O)C 1-6 Alkyl, wherein the alkyl, alkenyl or alkoxy represented by R1 or in the group represented by R1 may be optionally substituted by one to three independently selected from halogen, OH, CN, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Haloalkyl, C 1-4 Haloalkoxy and NR 11 R 12 The group consisting of the substituents is substituted.

3. The compound according to claim 2, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each instance of R2 is independently H, halogen, OH, CN, NH2, NO2, C 1-4 Alkyl, C 1-4 Alkoxy, NR 11 R 12 、C(O)NR 11 R 12 、C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, NR 11 C(O)C 1-4 Alkyl, P(=O)R 11 R 12 、S(=O)2R 11 or S(=O)2NR 11 R 12 , wherein the alkyl or alkoxy group represented by R2 or in the group represented by R2 may be optionally substituted by one to three independently selected from halogen, OH, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy and NR 11 R 12 The group consisting of the substituents is substituted.

4. The compound according to claim 3 or its pharmaceutically acceptable salt or stereoisomer, wherein R1 is H, halogen, CN, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 1-4 Alkoxy or C 1-4 Haloalkoxy.

5. The compound according to claim 4, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is H, F, Cl, CN or CF3.

6. The compound according to claim 1 or its pharmaceutically acceptable salt or stereoisomer, wherein R a1 Each is independently H, CH3 or CH2OH; R a1’ Each is independently -(CHR aa ) k OH, -(CHR aa ) k CN, -(CHR aa ) k C(O)OR 11 、-(CHR aa ) k C(O)NR 11 R 12 、-(CHR aa ) k C(O)NR 11 OR 12 、-(CHR aa ) k C(O)NR 11 S(=O)2R 12 、-(CHR aa ) k S(=O)2R 11 、-(CHR aa ) k S(=O)2NR 11 R 12 、-(CHR aa ) k NR 11 S(=O)2R 12 、-(CHR aa ) k -tetrazolyl or -(CHR aa ) k P(=O)R 11 R 12 ; R aa Each is independently H, CH3 or CF3; R 11 and R 12 Each is independently H or C 1-2 Alkyl; and k is 0 or 1.

7. The compound according to claim 6 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R a Each instance of is independently H or F.

8. The compound according to claim 7 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is Cl.

9. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein m is 0 or 1.

10. The compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein for 11. The compound according to claim 10 or its pharmaceutically acceptable salt or stereoisomer, wherein each of R2 is H, halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy.

12. The compound according to claim 11, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of R2 is H, F, Cl or OCH3; and R3 is C 1-4 alkyl.

13. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

14. A combination comprising a therapeutically effective amount of a compound as claimed in any one of claims 1 to 12 or a pharmaceutically acceptable salt or stereoisomer thereof, and one or more therapeutically active adjunct agents.

15. Use of a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt or stereoisomer thereof, in the preparation of a medicament for treating a subject suffering from cancer.

16. The use according to claim 15, wherein the cancer is selected from breast cancer, colorectal cancer, lung cancer, ovarian cancer and pancreatic cancer.

17. Use according to claim 16, wherein the cancer is selected from acute lymphoblastic leukemia (ALL), gastric cancer, genitourinary tract cancer, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck.

18. Use of a compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt or stereoisomer thereof in the preparation of a medicament for inhibiting HPK1 activity in a subject in need thereof.

19. The use of claim 18, wherein the subject suffers from cancer, and wherein the cancer is treated.

20. The use of claim 19, wherein the cancer is selected from breast cancer, colorectal cancer, lung cancer, ovarian cancer and pancreatic cancer.

21. The use according to claim 20, wherein the cancer is selected from acute lymphoblastic leukemia (ALL), gastric cancer, genitourinary tract cancer, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck.

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