HPK1 inhibitors and uses thereof

Abstract

The present invention provides a compound represented by structural formula (I) or formula (II), or a pharmaceutically acceptable salt or stereoisomer thereof, which is useful for treating a disease (such as cancer) that can be treated by inhibiting HPK1 activity. #imgabs0#

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to International Patent Application No. PCT / CN2020 / 100134 filed on July 3, 2020. The entire contents of the above application are incorporated herein by reference. Background of the Invention

[0004] Hematopoietic Progenitor Kinase (HPK1), also known as Mitogen-Activated Protein Kinase Kinase Kinase Kinase Kinase 1 (MAP4K1), is a protein kinase that acts upstream of the typical three-layer 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 cloned 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 the hematopoietic compartment, 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 six MAP4Ks including HPK1 (MAP4K1), GCK (MAP4K2), GLK (MAP4K3), HGK / NIK (MAP4K4), KHS / GCKR (MAP4K5) and MINK (MAP4K6). Collectively, these MAP4Ks are members of the approximately 26 mammalian Ste20-like serine / threonine kinases identified to date, which are homologs of the yeast sterile20 protein (Ste20p), a putative MAP4K that activates MAP3Ks in the yeast pheromone signaling pathway. These mammalian Ste20-like kinases are divided into two subfamilies based on domain structure: p21-activated kinases (PAKs) and germinal center kinases (GCKs). Several members of the GCK subfamily 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, when stimulated with anti-CD3, deficiency of LCK or ZAP70 abolishes Tyr-379 phosphorylation and the kinase activity of HPK1 (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 of HPK1 with 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 adhesion 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. HPK1KO mice are also more susceptible to EAE induction. HPK1KO T cells and B cells show enhanced cell activation and antigen receptor signaling. HPK1KO dendritic cells show higher levels of co-stimulatory molecules and proinflammatory 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, and 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 seems to be mediated mainly by a mechanism that is independent of JNK. It has been confirmed that IKK-NF-κB activation requires HPK1 kinase activation, and HPK1 is believed to operate by regulating CARMA1. CARMA1 is a transfer protein in the so-called CBM (CARMA1 / BCL10 / MALT1) complex, which promotes IKKβ activation when stimulated by anti-CD3 in Jurkat T cells. Activated IKK cleaves IκB and releases related NF-κB nuclear transcription factors. In particular, HPK1 is inducibly associated with CARMA1 and directly phosphorylates 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 an adaptor protein called SLP-76, which is transferred 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 activation, survival and proliferation of T-lymphocytes.

[0016] In this process, HPK1 directly binds to the SH2 domain of SLP-76 and acts primarily as a negative regulator of TCR signaling. For example, TCR signaling is enhanced in HPK1KO primary T cells, which show excessive proliferation and IL-2 production when connected to TCR in vitro (Shui, as mentioned above). It is believed that HPK1 can phosphorylate the SLP-76 adapter protein at Ser-376 through a negative feedback mechanism, thereby downregulating TCR signaling. When Ser-376 is phosphorylated by HPK1, SLP-76 binds to 14-3-3 through the phosphorylated Ser-376 residue, causing ubiquitination of the Lys-30 (K30) residue of SLP-76, which then becomes a target for proteasomal degradation. HPK1 also downregulates TCR signaling in other adapter proteins (including GADS) through similar mechanisms (e.g., through phosphorylation of Thr-254 of GADS and promotion of 14-3-3 interaction).

[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 different overexpression systems, HPK1-mediated inhibition of SLP-76 activation also results in inhibition of JNK activity in TCR signaling. This is consistent with the results that HPK1 knockout primary T cells showed unaffected JNK activity (Shui, supra). Similarly, HPK1 appears to regulate IKK activation by two different and opposing mechanisms - on the one hand, HPK1 activates IKK by directly phosphorylating CARMA1; on the other hand, HPK1 also negatively regulates IKK activation by inhibiting SLP-76 activation. This seems to be contrary to the dual role played by HPK1, understanding that HPK1 promotes JNK and IKK activation in the initial stage of TCR signaling, but plays an important role in hindering TCR signaling in the final stage.

[0018] HPK1 also plays a similar negative regulatory role in BCR-induced cell activation and proliferation in B cells. B cells utilize an SLP-76-like adaptor protein 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 by BLNK is 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). HPK1-deficient mice Foxp3 + Tregs are defective in suppressing TCR-induced effector T cell proliferation and, upon TCR engagement, paradoxically acquire the ability to produce IL-2 (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, HPK1 kinase activity 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 new 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 adhesion. In T cells, TCR activation also induces integrin activation, resulting in T-cell adhesion and immune synapse formation. This is achieved by SLP-76 binding to the degradation-promoting adapter protein (ADAP), which is required 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 signaling to adhesion molecules of the integrin family, thereby promoting T-cell adhesion. HPK1 negatively regulates this pathway not only by downregulating SLP-76 (as mentioned above), but also by competing with ADAP for the same SH2 binding site on SLP-76, thereby hindering the activity of Rap1, the downstream effector of ADAP (Patzak et al., Eur. J. Immunol. 40(11):3220-3225, 2010).

[0023] HPK1 also negatively regulates integrin activation and cell adhesion 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 adhesion (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 adhesion. Neutrophil trafficking, including slow rolling, tight binding, cell spreading, and diapedesis, is controlled by outside-in signaling activated by β2-integrin, inducing an interaction between actin and HIP-55 (55 kDa HPK1-interacting protein). Its high-affinity conformation that reinforces β2-integrin facilitates neutrophil adhesion (Hepper et al., J. Immunol. 188(9):4590-4601, 2012; Schymeinsky et al., Blood 114(19):4209-4220, 2009). During β2-integrin-mediated adhesion, 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 adhesion is impaired in vivo and in vitro by HPK1 deficiency or HIP-55 deficiency (Jakob, supra; Schymeinsky, supra).

[0025] Consistent with the role of HPK1 in downregulating TCR and BCR function, HPK1 negatively regulates the acquired immune response, and loss of HPK1-mediated regulatory T-cell activation and immune response may be an important mechanism in the pathogenesis of autoimmunity. In HPK1KO mice, although the development of T and B cells appears to be unaffected (Shui, supra), T cells from these animals show a substantial increase in TCR proximal signaling and downstream ERK activation when stimulated in vitro with anti-CD3, causing these cells to overproliferate (Shui, supra). T cells from immunized HPK1-deficient mice over-respond when stimulated with antigen-specific stimulation and produce significantly higher levels of proinflammatory cytokines such as IL-2, IFN-γ, and IL-4. These mice also produce higher levels of IgM and IgG isotypes, indicating enhanced function of HPK1 knockout B cells (Shui, supra).

[0026] HPK1 also negatively controls autoimmunity in mice, as HPK1KO 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 has been 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 dendritic cell (DC) maturation and activation through unknown mechanisms (Alzabin, such as the above-mentioned literature). In HPK1KO mice, bone marrow-derived dendritic cells (BMDC) show increased levels of co-stimulatory molecules CD80 / CD86, as well as increased 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 efficient (Alzabin, such as the above-mentioned literature). More importantly, tumor eradication mediated by CTL response of HPK1KO BMDC is more effective than that of wild-type BMDC (Alzabin, such as the above-mentioned literature). In addition, HPK1 can also control anti-tumor immunity through 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 efficient 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

[0030] Described herein are compounds of Formula (I), (IA), (IB), (II), (IIA) and (IIB), and pharmaceutically acceptable salts or stereoisomers thereof (collectively referred to herein as "compounds of the invention") that inhibit HPK1 activity.

[0031] Provided herein are pharmaceutical compositions comprising an effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0032] Also provided are combinations comprising a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and one or more therapeutically active co-agents.

[0033] The present invention further provides a method of inhibiting HPK1 activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, a pharmaceutically acceptable salt or stereoisomer thereof.

[0034] The present disclosure also provides a method of treating a subject having a disease or condition described herein, such as cancer (e.g., breast cancer, colorectal cancer, lung cancer, ovarian cancer, and pancreatic cancer), comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, a pharmaceutically acceptable salt or stereoisomer thereof.

[0035] Certain embodiments disclose compounds of the present disclosure, pharmaceutically acceptable salts and stereoisomers thereof, for use as medicaments, such as medicaments that act as HPK1 inhibitors.

[0036] The present disclosure also provides the use of a compound of the present disclosure, a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition comprising the same in any of the methods of the present disclosure described above. In one embodiment, a compound of the present disclosure, a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition comprising the same is provided, which is used in any of the methods of the present disclosure described herein. In another embodiment, a compound of the present disclosure, a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition comprising the same is provided for the preparation of a drug for any of the methods described in the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0038] 1. Overview

[0039] The disclosure described herein provides HPK1 / MAP4K1 inhibitors, pharmaceutically acceptable salts or stereoisomers thereof, pharmaceutical compositions thereof, and methods of using them to modulate (e.g., inhibit) HPK1 / MAP4K1 activity, the methods comprising administering the HPK1 / MAP4K1 inhibitor compounds of the present disclosure, or pharmaceutically acceptable salts thereof, to patients / subjects in need thereof. In certain embodiments, the compounds of the present disclosure, pharmaceutically acceptable salts or stereoisomers thereof are used for therapeutic administration to enhance, stimulate and / or increase immunity in the treatment of cancer.

[0040] For example, a method of treating a disease or condition associated with inhibiting HPK1 interaction may comprise administering to a patient in need thereof a therapeutically effective amount of a compound provided herein or a pharmaceutically acceptable salt or stereoisomer thereof. The compounds disclosed herein can be used alone, in combination with other agents or therapies, or as an adjuvant or neoadjuvant for the treatment of a disease or condition, including cancer.

[0041] 2. Definition

[0042] As used herein, the terms “a,” “an,” “the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0043] Compounds of any of the above formulae may exhibit one or more species of isomers (e.g., optical isomers, geometric isomers, or tautomers). Such variations are implicit in compounds of any of the above formulae because they are defined with reference to their structural features and are therefore within the scope of the present disclosure.

[0044] Compounds with one or more chiral centers can exist in various stereoisomeric forms, i.e., each chiral center can have the R or S configuration, or can be a mixture of the two. Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric and enantiomeric forms of a compound. Enantiomers are stereoisomers that are mirror images of each other. Diastereomers are stereoisomers with two or more chiral centers that are not identical and are not mirror images of each other.

[0045] "Peak 1" in the experimental section refers to the expected reaction product compound obtained from chromatographic separation / purification, which elutes earlier than the second expected reaction product compound from the same previous reaction. The second expected product compound is referred to as "Peak 2".

[0046] When a compound indicates a single enantiomer by its chemical name (e.g., "R" or "S" in the chemical name to indicate configuration) or by its structure (e.g., "wedge-shaped" bonds to indicate configuration), unless otherwise indicated, the compound is at least 60%, 70%, 80%, 90%, 99% or 99.9% optically pure (also referred to as "enantiomerically pure"). Optical purity refers to the weight of the named or described enantiomer in the mixture divided by the total weight of the two enantiomers in the mixture.

[0047] When the stereochemistry of a disclosed compound is named or depicted by a structure, and the named or depicted structure encompasses more than one stereoisomer (e.g., in a diastereomeric pair), it is understood to include one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers. It is further understood that the stereoisomeric purity of the named or depicted stereoisomer 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 stereoisomers encompassed by the name or structure in the mixture by the total weight of all stereoisomers in the mixture.

[0048] When two stereoisomers are depicted by their chemical names or structures, and the chemical names or structures are linked by "and", a mixture of the two stereoisomers is intended.

[0049] When two stereoisomers are depicted by their chemical names or structures, and the names or structures are linked by "or", it means one or the other of the two stereoisomers, but not both.

[0050] When a disclosed compound having a chiral center is depicted by a structure without showing the configuration of the chiral center, the structure is meant to encompass compounds having the S configuration at the chiral center, compounds having the R configuration at the chiral center, or compounds having a mixture of R and S configurations at the chiral center. When a disclosed compound having a chiral center is depicted by its chemical name without using "S" or "R" to indicate the configuration of the chiral center, the name is meant to encompass compounds having the S configuration at the chiral center, compounds having the R configuration at the chiral center, or compounds having a mixture of R and S configurations at the chiral center.

[0051] A racemic mixture refers to 50% of one enantiomer and 50% of the corresponding enantiomer. When a compound having one chiral center is named or depicted without indicating the stereochemistry of the chiral center, it is understood that the name or structure encompasses both possible enantiomeric forms of the compound (e.g., both enantiomerically pure, enantiomerically enriched, or racemic). When a compound having two or more chiral centers is named or depicted without indicating the stereochemistry of the chiral center, it is understood that the name or structure encompasses all possible diastereomeric forms of the compound (e.g., diastereomerically pure, diastereomerically enriched, and equimolar mixtures of one or more diastereomers (e.g., racemic mixtures)).

[0052] The term "geometric isomer" refers to compounds having at least one double bond, wherein the double bond can exist in the cis form (also known as syn or entgegen (E)) or the trans form (also known as anti or zusammen (Z)), and mixtures thereof.

[0053] When a geometric isomer is depicted by name or structure, it is understood that the named or depicted isomer is present in a greater degree than another isomer, i.e., the geometric purity of the named or depicted geometric isomer is greater than 50%, such as at least 60%, 70%, 80%, 90%, 99% or 99.9% pure by weight. Geometric purity is determined by dividing the weight of the named or depicted geometric isomer in the mixture by the total weight of all geometric isomers in the mixture.

[0054] Conventional techniques for preparing / separating individual enantiomers / diastereomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). Alternatively, the racemate (or racemic precursor) can be reacted with a suitable optically active compound, such as ethanol, or, in the case where the compound of any of the above formulas contains an acidic or basic moiety, with a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture can be separated by chromatography and / or fractional crystallization, and one or both of the diastereomers can be converted to the corresponding pure enantiomer by methods well known to the skilled person. Chiral compounds of any of the above formulae (and chiral precursors thereof) can be obtained in an enantiomerically enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon (typically heptane or hexane) containing 0 to 50% by volume of isopropanol (typically 2% to 20%) and 0 to 5% by volume of an alkylamine (typically 0.1% diethylamine). Concentration of the eluate provides an enriched mixture. Chiral chromatography with subcritical and supercritical fluids can be used. Methods of chiral chromatography that can be used for some embodiments of the present disclosure are 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). Columns can be obtained from Chiral Technologies, Inc, West Chester, Pa., USA, a subsidiary of Acquired from Chemical Industries, Ltd., Tokyo, Japan.

[0055] It must be emphasized that where a compound of any of the above formulae has been drawn in a single tautomeric form, all possible tautomeric forms are included within the scope of the present disclosure.

[0056] As used herein, the term "salt" or "salts" 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 are able to form acid and / or base salts by utilizing the presence of amino and / or carboxyl groups or groups similar thereto.

[0057] 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, chlortheophylionate, citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isothionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthoate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, stearate, succinate, subsalicylate, tartrate, toluenesulfonate and trifluoroacetate.

[0058] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like.

[0059] Organic acids that can be derived into salts 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 that can be derived into salts include, for example, ammonium salts and metals of Groups I to XII of the periodic table. In certain embodiments, salts can be derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc and copper. In certain embodiments, suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.

[0060] Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines including naturally substituted amines, cyclic amines, basic ion exchange resins and the like. Certain organic amines include isopropylamine, benzathine, cholate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

[0061] Pharmaceutically acceptable salts of the present invention can be synthesized from alkaline or acidic parts by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable 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 a suitable acid. Such 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. Lists of other suitable salts can be found in, for example, "Remington's Pharmaceutical Sciences," 20th edition, Mack Publishing Company, Easton, PA, (1985); and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0062] The terms "composition" and "formulation" are used interchangeably.

[0063] 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).

[0064] As used herein, a subject is "in need of" treatment if the subject would benefit biologically, medically, or in quality of life from such treatment.

[0065] The term "administering" refers to a method of introducing a compound of the invention or a composition thereof into or onto a subject. These methods include, but are not limited to, intraarticularly (in a joint), intravenously, intramuscularly, intratumorally, intradermally, intraperitoneally, subcutaneously, orally, topically, intrathecally, by inhalation, transdermally, rectally, and the like. Administration techniques that may be used with the agents and methods described herein may be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pennsylvania.

[0066] As used herein, the term "inhibit" refers to a reduction or suppression of a given condition, symptom, or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0067] The term "treatment" refers to reversing, slowing down or inhibiting the progress of the disease described herein. In some embodiments, treatment (i.e., therapeutic treatment) may be administered after one or more signs or symptoms of the disease have been developed or observed. In other embodiments, treatment may be administered without signs or symptoms of the disease. For example, treatment (i.e., preventive treatment) may be administered before the onset of symptoms in susceptible subjects (e.g., according to the history of symptoms and / or according to the pathogens exposed). Treatment may also be continued after symptom relief, for example, to delay or prevent recurrence.

[0068] The terms "condition," "disease," and "disorder" are used interchangeably.

[0069] Generally, the effective amount of the compounds taught herein varies depending on various factors, such as a given drug or compound, pharmaceutical formulation, route of administration, type of disease or condition, identity of the subject or host being treated, and similar factors, but can be routinely determined by one skilled in the art. The effective amount of the compounds taught herein can be readily determined by one of ordinary skill in the art by conventional methods in the art.

[0070] The term "effective amount" means an amount that, when administered to a subject, results in a beneficial or desired outcome, including a clinical outcome, e.g., inhibition, suppression or reduction of symptoms of the condition of the treated subject as compared to a control. For example, an effective amount can be administered 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).

[0071] The term "therapeutically effective amount" of a compound of the invention refers to an amount of a compound of the invention that will elicit a biological or medical response in a subject, e.g., reduction or inhibition of enzyme or protein activity, or alleviation of symptoms, alleviation of a condition, slowing or delaying disease progression, or prevention of a disease, etc. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the invention that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent and / or alleviate a condition, disorder or disease that is (i) 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 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.

[0072] All methods described herein can be carried out in any suitable order, unless otherwise indicated herein or otherwise clearly stated. Any example and other examples provided herein, or exemplary language (e.g., "such as") are used only to better illustrate the present invention, and unless otherwise stated, are not intended to limit the scope of the present invention.

[0073] The general chemical terms used in the above formulae have their general definitions.

[0074] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, agents that delay absorption, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes and the like and combinations thereof, as 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 conventional carrier is incompatible with the active ingredient, it is considered to be used in the treatment or pharmaceutical composition.

[0075] In addition, the compounds of the present invention, including their salts, may also be obtained in the form of their hydrates, or include other solvents for their crystallization. The compounds of the present invention may be inherently solvates or designed to form solvates with pharmaceutically acceptable solvents (including water); therefore, the present invention is intended to include solvates and non-solvate forms. The term "solvate" refers to a complex formed by a compound of the present invention (including its pharmaceutically acceptable salt) and one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical field, which 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.

[0076] The compounds of the present invention, including salts, hydrates and solvates thereof, can inherently or by design form polymorphs. On the other hand, 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 made into 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 conventional pharmaceutical operations, such as sterilization and / or can contain conventional inert diluents, lubricants or buffers, and adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.

[0077] Typically, the pharmaceutical composition is a tablet or gelatin capsule, which contains the active ingredient, together with a) diluents, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants, for example, silica, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; for tablets, there are also c) binders, for example, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if desired, d) disintegrants, for example, starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and / or e) absorbents, colorants, flavorings and sweeteners. Tablets can be film-coated or enteric-coated according to methods known in the art.

[0078] Suitable compositions for oral administration include an effective amount of the compound of the present 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 use are prepared according to any method known in the art for making pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of: sweeteners, flavoring agents, colorants, and preservatives to provide pharmaceutically beautiful and palatable preparations. Tablets may contain active ingredients mixed with non-toxic pharmaceutically acceptable excipients suitable for making tablets. Such excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating agents and disintegrants, for example, corn starch or alginic acid; binding agents, for example, starch, gelatin, or gum arabic (acacia); and lubricants, such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used. Formulations for oral use may be presented in hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or in soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

[0079] Some injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fat emulsions or suspensions. The composition may be sterilized and / or contain adjuvants, such as preservatives, tranquilizers, wetting agents or emulsifiers, dissolution promoters, salts and / or buffers for regulating osmotic pressure. In addition, it may also contain other substances of therapeutic value. The composition is prepared according to conventional mixing, granulation or coating methods, respectively, and contains about 0.1-75% or contains about 1-50% active ingredients. Suitable compositions for transdermal application include an effective amount of a 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, which includes a backing element, a reservoir containing a compound and optionally a carrier, and optionally a rate-controlling barrier, so as to deliver the compound to the host skin for a long time at a controlled and predetermined rate, and it is intended to fix the device on the skin.

[0080] Suitable compositions for topical application, e.g. to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., delivered using aerosols or the like. Such topical delivery systems will be particularly suitable for skin applications, e.g., for the treatment of skin cancer, e.g., for preventive uses in sunscreens, lotions, sprays and the like. Therefore, they are particularly suitable for surfaces, including formulations well known in the art, cosmetics. They may contain solubilizers, tranquilizers, tension enhancers, buffers and preservatives.

[0081] As used herein, topical application may also be suitable for inhalation or intranasal application. It may be conveniently delivered from a dry powder inhaler in the form of a dry powder (alone, or as a mixture, e.g., a dry blend with lactose, or mixed component particles, e.g., formed with a phospholipid), or delivered as an aerosol spray from a pressurized container, pump, spray bottle, nebulizer or sprayer with or without a suitable propellant.

[0082] The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients, since water may facilitate the degradation of certain compounds.

[0083] Anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low-moisture ingredients and 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 composition is packaged using materials known to prevent exposure to water so that it can be contained in a suitable formulation box. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0084] The present invention further provides pharmaceutical compositions and dosage forms comprising 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 "sedatives" and include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0085] 3. Compounds

[0086] In a first embodiment of the present disclosure, there is provided a compound represented by Formula I,

[0087]

[0088] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0089] In a second embodiment of the present disclosure, there is provided a compound represented by Formula IA,

[0090]

[0091] or a pharmaceutically acceptable salt.

[0092] In a third embodiment of the present disclosure, there is provided a compound represented by Formula IB,

[0093]

[0094] or a pharmaceutically acceptable salt.

[0095] In a fourth embodiment of the present disclosure, there is provided a compound represented by Formula II,

[0096]

[0097] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0098] In a fifth embodiment of the present disclosure, there is provided a compound represented by Formula IIA,

[0099] or a pharmaceutically acceptable salt.

[0100] In a sixth embodiment of the present disclosure, there is provided a compound represented by Formula IIB,

[0101]

[0102] or a pharmaceutically acceptable salt.

[0103] 4. Treatable diseases

[0104] HPK1 inhibitors, pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof can be used in a method for regulating (i.e., inhibiting) HPK1 activity, comprising administering the HPK1 inhibitor compound of the present invention described herein, or a pharmaceutically acceptable salt thereof, to a patient / subject in need thereof.

[0105] 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, in particular their pharmaceutically acceptable salts, or mixtures thereof, for treating or preventing diseases, in particular cancer (in particular hematopoietic and solid tumors) or conditions with dysregulated immune responses or other conditions 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.

[0106] In certain embodiments, the compounds of the invention, or pharmaceutically acceptable salts thereof, are used for therapeutic administration to a subject in need thereof to treat diseases or indications including, but not limited to, benign hyperplasia, atherosclerotic disorders, sepsis, autoimmune disorders, cardiovascular disorders, viral infections, neurodegenerative disorders, inflammatory disorders, and male fertility control disorders.

[0107] In certain embodiments, the compounds of the invention or pharmaceutically acceptable salts thereof are used for therapeutic administration to enhance, stimulate and / or increase immunity in the treatment of cancer.

[0108] The HPK1 inhibitor compounds of the invention may be used alone or in combination with other agents or therapies, or as adjuvants or neoadjuvants for the treatment of diseases or conditions, including cancer.

[0109] 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, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or urethra cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor neovascularization, spinal axis tumors (spinal axis tumors), tumor), 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.

[0110] 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 adenocarcinoma), 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.

[0111] 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, 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.), blood 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 lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin lymphoma or multiple myeloma), sarcoma, and distant metastases thereof.

[0112] In some embodiments, diseases and indications that can be treated using the compounds of the present invention include, but are not limited to, hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, cancers of the nervous system, gynecological cancers, and skin cancers.

[0113] 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 lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin lymphoma, myeloproliferative disorders (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), essential thrombocythemia (ET), myelodysplastic syndrome (MDS), T-cell acute lymphoblastic lymphoma (T-ALL), multiple myeloma, cutaneous T-cell lymphoma, Waldenstrom's Macroglubulinemia, hairy cell lymphoma, chronic myeloid lymphoma, and Burkitt's lymphoma.

[0114] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdomyosarcoma, fibroma, lipoma, hamartoma, and teratoma.

[0115] Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, cartilaginous hamartoma, and mesothelioma.

[0116] Exemplary gastrointestinal cancers include cancers of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), and colorectal cancer.

[0117] 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 (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroma, adenoid, lipoma).

[0118] Exemplary liver cancers include hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0119] Exemplary bone cancers include, e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostosis), benign enchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor of bone.

[0120] Exemplary nervous system cancers include cancers of the skull (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meninges (meningiomas, meningiosarcomas, gliomas), brain (astrocytomas, meduoblastomas, gliomas, ependymomas, blastomas (pinealomas), glioblastomas, glioblastomas multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), and spinal cord (neurofibromas, meningiomas, gliomas, sarcomas), as well as neuroblastoma and dysplastic gangliocytoma (Lhermitte-Duclos disease).

[0121] Exemplary gynecological cancers include cancers of the uterus (endometrial cancer), cervix (cervical cancer, preneoplastic cervical dysplasia), ovary (ovarian cancer (serous cystic adenocarcinoma, mucinous cystic adenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, 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).

[0122] 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 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 cancer.

[0123] 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.

[0124] 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). Overexpressed Cox-2 has been detected in many types of tumors, such as colorectal, breast, pancreatic and lung cancers, and is associated with poor prognosis. Overexpression of COX-2 has been reported in blood cancer models (such as RAJI (Burkitt's lymphoma) and U937 (acute pro-monocytic leukemia)) and in patients' embryonic cells. CD73 is upregulated in a variety of different human cancers, including those of the colon, lung, pancreas and ovary. Higher expression levels of CD73 have been associated with tumor angiogenesis, invasiveness and metastasis, and are associated with shorter survival of breast cancer patients.

[0125] Examples of treatable breast cancers include, but are not limited to, triple negative breast cancer, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

[0126] Examples of respiratory tract cancers include, but are not limited to, small cell and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma.

[0127] Examples of brain cancers that may be treated include, but are not limited to, brainstem and hypothalamic gliomas, cerebellar and cerebral astrocytomas, glioblastomas, medulloblastomas, ependymomas, and neuroectodermal and pineal tumors.

[0128] Male reproductive organ tumors that may be treated include, but are not limited to, prostate and testicular cancers.

[0129] Tumors of the female reproductive organs that may be treated include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer, as well as uterine sarcoma.

[0130] Ovarian cancers that may be treated include, but are not limited to, serous tumors, endometrioid tumors, mucinous cystadenocarcinomas, granulosa cell tumors, Sertoli-Leydig cell tumors, and arrhenoblastoma.

[0131] 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.

[0132] Treatable digestive tract tumors include, but are not limited to, cancers of the anus, colon, colorectum, esophagus, bladder, stomach, pancreas, rectum, small intestine, and salivary glands.

[0133] 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.

[0134] Gastric cancers that may be treated include, but are not limited to, intestinal and diffuse gastric adenocarcinomas.

[0135] Pancreatic cancers that may be treated include, but are not limited to, pancreatic ductal adenocarcinoma, adenosquamous carcinoma, and pancreatic endocrine tumors.

[0136] Treatable urinary tract tumors include, but are not limited to, bladder, penis, kidney, renal pelvis, ureter, urethra, and human papillary renal cancer.

[0137] 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.

[0138] Bladder cancers that may be treated include, but are not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, and small cell carcinoma.

[0139] Treatable eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

[0140] 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.

[0141] 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.

[0142] Head and neck cancers that may be treated include, but are not limited to, head and neck squamous cell carcinoma, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, salivary gland cancer, lip and oral cavity cancer, and squamous cell.

[0143] 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.

[0144] Treatable sarcomas include, but are not limited to, sarcomas of soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

[0145] Treatable leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

[0146] In certain embodiments, the compounds of the invention can be used to treat various other diseases involving MAP4K1, such as cardiovascular and pulmonary diseases.

[0147] In certain embodiments, the compounds of the present invention can be used as medicaments for the treatment and / or prevention of cardiovascular, inflammatory and fibrotic disorders, renal disorders, in particular acute and chronic renal insufficiency, and acute and chronic renal failure.

[0148] As used herein, the term "renal insufficiency" includes both acute and chronic clinical manifestations of renal insufficiency, and underlying or associated renal disorders, such as diabetic and non-diabetic nephropathy, hypertensive nephropathy, ischemic renal disorder, renal hypoperfusion, intradialytic hypotension, obstructive uropathy, renal stenosis, glomerulopathy, glomerulonephritis (such as, for example, primary glomerulonephritis; minimal change glomerulonephritis (lipid nephropathy); 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, amyloid deposition disease, Goodpasture syndrome, Wegener's granulomatosis 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, renal disorders such as primary and congenital or acquired renal disorders, Alport syndrome syndrome), nephritis, immune renal disorders such as renal transplant rejection and immune complex-induced renal disorders, toxic substance-induced nephropathy, contrast agent-induced nephropathy, diabetic and non-diabetic nephropathy, renal cysts, nephrosclerosis, hypertensive nephrosclerosis and nephrotic syndrome, which can be characterized by diagnosis, for example, by abnormal decreases in creatinine and / or water output, abnormal increases in blood concentrations of urea, nitrogen, potassium and / or creatinine, changes in renal enzyme activity (e.g., glutamyl 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.

[0149] In certain embodiments, the compounds of the invention can be used to treat and / or prevent the sequelae of renal insufficiency, for example, pulmonary edema, heart failure, uremia, anemia, electrolyte disturbances (e.g., hypercalcemia, hyponatremia) and disorders of bone and carbohydrate metabolism.

[0150] In certain embodiments, the compounds of the invention can be used to treat and / or prevent the sequelae of renal insufficiency, for example, pulmonary edema, heart failure, uremia, anemia, electrolyte disturbances (e.g., hypercalcemia, hyponatremia) and disorders of bone and carbohydrate metabolism.

[0151] In certain embodiments, the compounds of the present invention are further useful for the treatment and / or prevention of polycystic kidney disease (PCKD) and syndrome of inappropriate ADH secretion (SIADH).

[0152] In certain embodiments, the compounds of the present invention are also useful for treating and / or preventing metabolic syndrome, hypertension, resistant hypertension, acute and chronic heart failure, coronary heart disease, stable and unstable angina, peripheral and cardiovascular disorders, 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, ventricular Tachycardia, torsade de pointes, atrial and ventricular extrasystole, AV-junctional extrasystole, sick sinus syndrome, syncope, AV-nodal cyclotron tachycardia, Wolff-Parkinson-White syndrome, acute coronary syndrome (ACS), autoimmune heart diseases (pericarditis, endocarditis, valvulitis, aortitis, cardiomyopathy), shock (e.g., cardiogenic shock, septic shock, and anaphylactic shock), aneurysm, boxer cardiomyopathy (boxe r-cardiomyopathy) (premature ventricular contraction (PVC)); for the treatment and / or prevention of thromboembolic disorders and ischemic disorders such as myocardial ischemia, myocardial infarction, stroke, cardiac hypertrophy, transient and ischemic attacks, preeclampsia, inflammatory cardiovascular disorders, coronary and peripheral artery spasm, edema formation (e.g., pulmonary edema, cerebral edema, renal edema or edema caused by heart failure, peripheral circulatory disturbances, reperfusion injury, arterial and venous thrombosis), myocardial insufficiency, endothelial dysfunction; to prevent restenosis (e.g., after thrombolytic therapy, percutaneous transluminal angioplasty (PTA), transluminal coronary angioplasty (PTCA), heart transplantation and bypass surgery), as well as microvascular and macrovascular damage (vasculitis), increased fibrinogen and low-density lipoprotein (LDL) levels and increased plasminogen activator inhibitor 1 (PAI-1) concentrations, and also for the treatment and / or prevention of erectile dysfunction and female sexual dysfunction.

[0153] In certain embodiments, the compounds of the invention are also useful for treating and / or preventing asthmatic conditions, pulmonary arterial hypertension (PAH), and other forms of pulmonary hypertension (PH), including left 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 induced by cigarette smoke), and cystic fibrosis (CF).

[0154] In certain embodiments, the compounds of the invention are also effective for controlling central nervous system disorders characterized by disturbances in the NO / cGMP system. They are particularly useful for improving perception, attention, learning or memory after cognitive impairment, such as those occurring in association with conditions / diseases / syndromes such as mild cognitive impairment, age-related learning and memory impairment, age-related memory loss, vascular dementia, brain trauma, stroke, dementia occurring after a stroke (post-stroke dementia), post-traumatic brain trauma, general concentration impairment, childhood concentration impairment with learning and memory problems, Alzheimer's disease, Lewy body dementia, frontal lobe degeneration dementia, including Pick's syndrome, Parkinson's disease, progressive dementia with cortical basal degeneration, amyotrophic lateral sclerosis (ALS), Huntington's disease, demyelination, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacob dementia, HIV dementia, schizophrenia with dementia or Korsakoff's psychosis.

[0155] In certain embodiments, the compounds of the invention are also useful in 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 for controlling pathological disorders in the intake of food, stimulants and addictive substances.

[0156] In certain embodiments, the compounds of the present invention are further useful for controlling cerebral blood flow and thus represent effective agents for controlling migraine headaches.

[0157] In certain embodiments, the compounds of the present invention are also suitable for preventing and controlling the sequelae of cerebral infarction (stroke), such as stroke, cerebral ischemia and brain trauma. The compounds according to the present invention are also useful for controlling pain and tinnitus.

[0158] 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 disorders of the kidney, chronic intestinal inflammation (IBD, Crohn's disease, UC), pancreatitis, peritonitis, rheumatoid disorders, inflammatory skin disorders and inflammatory eye disorders.

[0159] In certain embodiments, the compounds of the present invention may also be used to treat and / or prevent autoimmune diseases.

[0160] In certain embodiments, the compounds of the invention are also useful for treating and / or preventing fibrotic disorders of internal organs, for example, lung, heart, kidney, bone marrow and especially liver, as well as skin fibrosis and fibrotic eye disorders.

[0161] As used herein, the term "fibrotic disorder" specifically includes the following: liver fibrosis, cirrhosis, pulmonary fibrosis, myocardial fibrosis, kidney disease, glomerulonephritis, interstitial renal fibrosis, fibrotic lesions 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).

[0162] In certain embodiments, the compounds of the invention are also useful for controlling post-surgical scarring, for example, scarring resulting from glaucoma surgery.

[0163] In certain embodiments, the compounds of the present invention may also be used for the cosmetic treatment of aged and keratinized skin.

[0164] In certain embodiments, the compounds of the present invention are useful for treating and / or preventing hepatitis, neoplasms, osteoporosis, glaucoma, and gastroparesis.

[0165] In certain embodiments, the compounds of the invention are useful 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., restenosis and hypertrophy).

[0166] In another embodiment, 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 in a woman's life, and their growth depends on the female sex hormones estradiol and progesterone. Therefore, the peak incidence of uterine fibroids in about 70% and >80% of white and African-American women is between the ages of 35 and menopause, respectively, and then shrinks due to the reduction in hormone levels. About 30% and 45% of white and African-American women, respectively, have 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 80mL during the menstrual cycle. The submucosal location of uterine fibroids (e.g., those located directly beneath the endometrium) appears to have an even more severe effect on uterine bleeding, potentially causing anemia in affected women and, along with the symptoms of uterine fibroids, significantly affecting the quality of life of affected women.

[0167] In certain embodiments, the compounds of the present invention are useful for treating and / or preventing chronic renal disorders, acute and chronic renal insufficiency, diabetes, inflammatory or hypertensive kidney disease, fibrotic disorders, cardiac insufficiency, angina pectoris, hypertension, pulmonary hypertension, ischemia, vascular disorders, thromboembolic disorders, arteriosclerosis, sickle cell anemia, erectile dysfunction, benign prostatic hyperplasia, dysuria associated with benign prostatic hyperplasia, Huntington's disease, dementia, Alzheimer's disease, and Creutzfeldt-Jakob disease.

[0168] The present invention provides methods for treating and / or preventing chronic kidney disorders, acute and chronic renal insufficiency, diabetes, inflammatory or hypertensive kidney disease, fibrotic disorders, cardiac insufficiency, angina, 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.

[0169] The present invention further provides the use of the compounds according to the invention for the treatment and / or prophylaxis of disorders, in particular the disorders mentioned above.

[0170] The present invention further provides a method for treating and / or preventing disorders, in particular the disorders mentioned above, using an effective amount of at least one of the compounds according to the invention.

[0171] Therefore, the compounds of the present invention can be used to inhibit, block, reduce, or decrease MAP4K1 activation through 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 condition.

[0172] The present invention also provides methods of treating various other disorders involving MAP4K1, such as, but not limited to, disorders with dysregulated immune responses, inflammation, vaccination against infection and cancer, viral infections, obesity and diet-induced obesity, lipogenesis, metabolic disorders, hepatic steatosis, and uterine fibroids. These disorders have been well characterized in humans, but also have similar etiologies in other mammals and can be treated by administering the pharmaceutical compositions of the present invention.

[0173] 5. Combination therapy

[0174] The compounds of the present invention may be used in combination therapy with one or more additional / second therapeutic agents suitable for the treatment of the diseases or indications treatable using the subject compounds.

[0175] Thus, in certain embodiments, for example, the methods of the invention using the compounds of the invention may include administering a further therapeutic agent to a subject in need thereof. The further 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 adoptively transferred T cell population, or a specific 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.

[0176] In certain embodiments, the second therapeutic 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 Pim kinases, and / or an inhibitor of chaperone proteins and cell cycle progression. In certain embodiments, the combination therapy of the present invention can reduce the likelihood of drug resistance in a cell population and / or reduce the toxicity of treatment.

[0177] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be combined with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βPv, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, CSFIR, KIT, FLK-1 I, KDR / FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR / Flt 2. Flt4, EphAl, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf.

[0178] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be combined with one or more of the following inhibitors for the treatment of cancer, including: FGFR inhibitors (FGFR1, FGFR2, FGFR3 or FGFR4, for example, AZD4547, BAY1187982, 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 (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 (e.g., PI3K-γ selective inhibitors), CSF1R inhibitors (e.g., PLX3397 and LY3022855), TAM receptors tyrosine kinases (Tyro-3, Axl and Mer), aryl hydrocarbon receptor (AhR) modulators (such as laquinimod, aminoflavonoids, CB7993113, CH223191, 6,2',4'-trimethoxyflavone (TMF), GNF351 (N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine), aminoflavonoids, NKI150460, indole-3-methanol, β-naphthylidene flavonoids and their dimers, diindolylmethane (DIM), 4-hydroxytamoxifen, leflunomide, raloxifene (

[00136] The invention relates to angiotensin-converting agents, such as raloxifene, tranilast, flutamide, mexiletine, nimodiphine, omeprazole, sulindac, tranilast and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), angiogenesis inhibitors, interleukin receptor inhibitors, bromodomain inhibitors or BET inhibitors (e.g., bromodomain inhibitors or BET inhibitors, such as OTX015, CPI-0610, INCB54329 and INCB57643), and adenosine receptor antagonists, or a combination thereof.

[0179] In certain embodiments, the HPK1 inhibitor compounds of the present invention may be combined with HDAC inhibitors, such as panobinostat and vorinostat.

[0180] In certain embodiments, the HPK1 inhibitor compounds of the invention may be combined with inhibitors of c-Met, such as onartumzumab, tivantnib, and capmatinib (INC-280).

[0181] In certain embodiments, the HPK1 inhibitor compounds of the invention may be combined with an inhibitor of BTK, such as ibrutinib.

[0182] In certain embodiments, the HPK1 inhibitor compounds of the invention may be combined with inhibitors of mTOR, for example, rapamycin, sirolimus, temsirolimus, and everolimus.

[0183] In certain embodiments, the HPK1 inhibitor compounds of the present invention may be combined with inhibitors of MEK, such as trametinib, selumetinib, and GDC-0973.

[0184] In certain embodiments, the HPK1 inhibitor compounds of the present invention can be combined with inhibitors of Hsp90 (e.g., tanespimycin), inhibitors of cyclin-dependent kinases (e.g., palbociclib), inhibitors of PARP (e.g., olaparib), and inhibitors of Pim kinases (LGH447, INCB053914, and SGI-1776).

[0185] In certain embodiments, the HPK1 inhibitor compounds of the present invention may be combined with agonists of the DNA sensor (c-GAS) and / or its downstream adaptor protein STING.

[0186] 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 triggers 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 STING and triggering the activation of the transcription factor IRF3. Activated IRF3 causes the transcription of type 1 IFN-β, which initiates a variety of biological responses in 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 strengthen anti-tumor immunity.

[0187] In certain embodiments, the HPK1 inhibitor compounds of the present invention may be combined with one or more immune checkpoint inhibitors.

[0188] Effector T cell activation is usually triggered by antigenic peptides recognized by TCR presented by MHC complexes. The type and degree of activation achieved are then determined by the balance between the stimulating signal and the signal that inhibits the effector T cell response. As used herein, "immune system checkpoint" refers 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. Such interactions may be direct, such as the interaction between a ligand and a cell surface receptor that transmits an inhibitory signal to the effector T cell. Or it may be indirect, such as blocking or inhibiting the interaction between a ligand and a cell surface receptor that transmits an activation signal to the effector T cell, or the interaction promotes the suppression of inhibitory molecules or cells, or is eliminated by enzymes of metabolites required by effector T cells, or any combination thereof.

[0189] Examples of immune system checkpoints include: a) interactions between indoleamine 2,3-dioxygenase (IDO1) and its substrates; b) interactions between PD1 and PD-L1 and / or PD1 and PD-L2; c) interactions between CTLA-4 and CD86 and / or CTLA-4 and CD80; d) interactions between B7-H3 and / or B7-H4 and their respective ligands; e) interactions between HVEM and BTLA; f) interactions between GAL9 and TIM3; g) interactions between MHC class I or II and LAG 3; and h) interactions between MHC class I or II 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).

[0190] Exemplary immune checkpoint inhibitors therefore 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.

[0191] 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 targets of the methods of the present invention.

[0192] 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 targeted by the methods of the present invention.

[0193] In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137.

[0194] 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, NOX2, PD-1, TIΜ3, SIGLEC7, SIGLEC9 and VISTA, and its binding partners (such as PD-L1 and PD-L2).

[0195] In some embodiments, the compounds provided herein can be used in combination with one or more agents selected from the group consisting of a KIR inhibitor, a TIGIT inhibitor, a LAIR1 inhibitor, a CD160 inhibitor, a 2B4 inhibitor, and a TGFRβ inhibitor.

[0196] 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 certain embodiments, inhibitors of IDO1 include Epacadostat (INCB24360), Indoximod, GDC-0919 (NLG919) and F001287. Other inhibitors of IDO1 include 1-methyltryptophan (1MT).

[0197] In some embodiments, inhibitors of immune checkpoint molecules are also referred to as "immunomodulators," 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.

[0198] 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 blockade or inhibition. Therefore, the agent is generally referred to as an inhibitor of the checkpoint.

[0199] 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 fragments, Fab' fragments, 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.

[0200] In certain embodiments, the immunomodulator used in the HPK1 inhibitor of the present invention is an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody or an anti-CTLA-4 antibody.

[0201] 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 certain embodiments, the inhibitor of PD-1 is AMP-224 (PD-L2F that binds to PD-1). c fusion protein) or AUNP-12 (anti-PD-1 peptide).

[0202] 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 certain embodiments, the anti-PD-L1 antibodies include atezolizumab, avelumab, durvalumab or MEDI-4736, and MPDL3280A.

[0203] 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 certain embodiments, the antibody is ipilimumab.

[0204] In certain embodiments, the inhibitor of the immune checkpoint molecule is a combination of two or more regulators 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.

[0205] In some embodiments, inhibitors of immune checkpoint molecules are antibodies that block or inhibit the interaction between 4-1BB and its ligands, including utomilumab.

[0206] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of CSF1R, for example, an anti-CSF1R antibody. In some embodiments, the anti-CSF1R antibody is IMC-CS4 or RG7155.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] In some embodiments, the compounds of the present invention can be used in combination with bispecific antibodies. In some embodiments, one of the domains of the bispecific antibody targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3, CD137, ICOS, CD3 or TGFβ receptor.

[0214] In some embodiments, the compounds of the invention can be used in combination with one or more agents for treating a disease, such as 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 mustard, chlormethine, cyclophosphamide, and succinimidyl. TM ), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, 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).

[0215] The compounds of the present invention can be further used in combination with other methods for treating cancer, for example, 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.

[0216] The compounds of the present invention may be administered in combination with one or more anticancer drugs, such as chemotherapeutic agents. Examples of chemotherapeutic agents include any of the following: abarelix, abiraterone, afatinib, aflibercept, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, axitinib, azacitidine, bevacizumab, bexarotene, baricitinib, bicalutamide, bleomycin, bortezombi, bortezomib, ezomib, 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, temozolomide omide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, triptorelin, uracil mustard, valrubicin, vandetanib, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, and zoledronate.

[0217] Other anticancer agents include antibody therapy, such as trastuzumab (Herceptin), antibodies to co-stimulatory 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 present invention for the treatment of cancer or infection (e.g., viral, bacterial, fungal and parasitic infections) include, but are not limited to, nivolumab, pembrolizumab, MPDL3280A, MEDI-4736 and SHR-1210.

[0218] Other anticancer agents include inhibitors of kinases associated with cell proliferative disorders. These 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.

[0219] Other anticancer agents also include those that block immune cell migration, such as antagonists of chemokine receptors, including CCR2 and CCR4. The compounds of the present disclosure may be further used in combination with one or more anti-inflammatory agents, steroids, immunosuppressants, or therapeutic antibodies.

[0220] 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 adoptively transferred tumor-specific T cell populations, or antibodies specific for proteins expressed by tumor cells.

[0221] As used herein, "agent that directly stimulates an immune effector response" means any suitable agent, but typically refers to a cytokine or chemokine (or an agent that stimulates the production of one of them), a tumor-specific adoptively transferred T cell population, or an antibody specific for a protein expressed by tumor cells.

[0222] Cytokines may be interferons selected from the following: IFNα, IPNβ, IFNγ and IFNA, or interleukins, such as IL-2. Chemokines may be inflammatory mediators, for example, selected from CXCL9, 10 and 11, which attract T cells expressing CXCR3. The agent 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 regimen of BCG for bladder cancer is once a week for 6 weeks, but for the sake of 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, particularly for colon cancer. Such BCG usage is also encompassed within the present invention. Tumor-specific adoptive transfer of T cell populations directly increases the number of tumor-specific T cell populations in a subject 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.

[0223] Antibodies specific for proteins expressed by tumor cells usually stimulate immune activity by binding to tumor cells and promote cell lysis via 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.

[0224] Therefore, in certain exemplary embodiments, the compounds of the present invention can be used in combination with calcineurin inhibitors, for example, cyclosporine A or FK 506; mTOR inhibitors, for example, rapamycin, 40-0-(2-hydroxyethyl)-rapamycin, biolimus-7 or biolimus-9; ascomycins with immunosuppressive properties, for example, ABT-281, ASM981; corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic acid or salts; mycophenolate mofetil; IL-1β inhibitors.

[0225] In another embodiment, the compounds of the invention are combined with a concomitant agent that is a PI3 kinase inhibitor.

[0226] In another embodiment, the compounds of the invention are combined with a concomitant agent that affects BTK (Bruton's tyrosine kinase).

[0227] The compounds of the present invention for treating neoplastic diseases can 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.

[0228] In some embodiments, the compounds of the invention (including salts thereof) can be combined 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 that have been transfected to express the cytokine GM-CSF.

[0229] In some embodiments, the compounds of the present invention or their salts can also be used in combination with vaccination processes 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 heat shock proteins isolated from tumor tissue itself. In some embodiments, the compounds of the present invention or their salts can be combined with dendritic cell immunization to activate a strong anti-tumor response.

[0230] In some embodiments, the compounds of the invention can 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 can also be combined with macrocyclic peptides that activate the host immune response.

[0231] In some embodiments, the compounds of the present invention can be used in combination with bone marrow transplantation to treat various tumors of hematopoietic origin.

[0232] Suitable antiviral agents intended for use in combination with the compounds of the present 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 (adefovir 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.

[0233] It is understood that many of the other therapeutic agents used in the methods of the present invention may be biologics that require intravenous, intraperitoneal, or depot administration. In yet another embodiment, the compound of the present invention is administered orally, while the other therapeutic agent is administered parenterally, for example, intravenously, intraperitoneally, or as a depot.

[0234] 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).

[0235] In one embodiment, the present invention provides a product comprising a compound of the present invention (such as a principal compound or any subfamily thereof) and at least one other therapeutic 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 subfamily thereof and other therapeutic agents (groups) in the same pharmaceutical composition, or compositions comprising a principal compound or any subfamily thereof and other therapeutic agents (groups) in separate form, for example, in kit form.

[0236] In one embodiment, the invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which comprises a principal compound, and another comprising a second therapeutic agent as discussed herein. In one embodiment, the kit comprises means for separately housing the composition, such as a container, a separate bottle, or a separate foil package. Examples of such kits are blister packs, which are commonly used for packaging tablets, capsules, and the like. The kit of the present invention can be used to administer different dosage forms at different administration intervals, for example, oral and parenteral, administering separate compositions, or adjusting dosages from separate compositions to each other. In order to promote adaptability, the kit of the present invention typically comprises instructions for administration.

[0237] 6. Compound screening / assay methods

[0238] The compounds of the invention inhibit the kinase activity of HPK1, which can be directly measured 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 effect of the compound can also be evaluated using bioassays to determine the effect of the compound on cytokine secretion by T cells after TCR and CD28 stimulation.

[0239] For example, Example 5 describes such a functional assay for determining the effect of HPK1 inhibitors on IL-2 & IFN-γ release from stimulated Pan T cells. The secreted IL-2 & IFN-γ can be measured / quantified using a standard ELISA assay. Briefly, pan T cells are isolated from peripheral blood (PB) mononuclear cells (MNC), or PBMC using a commercially available kit, such as the MACS (Miltenyl Biotec) Pan T Isolation Kit (Cat. No. 130-096-535). Primary human pan-T cells include CD4 and CD8 T cells and some γ / δ T cell subsets. Pan-T cells can be isolated using a negative immunomagnetic separation technique without the use of a column.

[0240] Isolated pan-T cells were plated at 100,000 cells / well in 96-well plates and stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies or with PMA / Ionomycin as a positive control (or medium as a negative control). Test compounds were added to the cells at different concentrations to assess the effect of the compounds on cytokine secretion after TCR-stimulation / CD28 co-stimulation. After the stimulated cells were incubated for an additional 2 days, the supernatant from each well (containing cytokines secreted by pan-T cells) was collected for ELISA assays and quantification of IL-2 and IFN-γ.

[0241] Other assays can also be used to assess the ability of any HPK1 inhibitor to inhibit HPK1, or to screen for compounds having HPK1 inhibitory activity.

[0242] For example, in one assay, the inhibition of HPK1 kinase activity can be determined using the Treg assay (regulatory T-cell proliferation assay) described below. Specifically, primary CD4 Tregs are isolated from human donated peripheral blood mononuclear cells (PBMCs) using a suitable kit, such as one from ThermoFisher Scientific (Cat. No. 11363D). + / CD25 - T-cells and CD4 + / CD25 + Regulatory T cells were isolated from CD4 + / CD25 - T-cells were labeled with CFSE (Thermo Fisher Scientific, C34554). + / CD25 + Regulatory T cells were expressed at 1×10 6The cells were resuspended in RPMI-1640 medium at a concentration of 10 cells / mL. 100 μL of CFSE-labeled T cells were incubated with or without 50 μL of CD4 + / CD25 + Regulatory T-cells were mixed and treated with 5 μL anti-CD3 / CD28 beads (Thermo Fisher 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% CO2). On day 5, the proliferation of CFSE-labeled T-cells was analyzed using the FITC approach using a BD LSRFortessa X-20. The lead compound was expected to inhibit HPK1 to enhance Treg function and inhibit the proliferation of CFSE-labeled primary CD4 + / CD25 - Proliferation of T-cells.

[0243] In another example, inhibition of HPK1 kinase activity can be determined 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 serves as a scaffold on which key signaling complexes can be established 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 assess the phosphorylation status of proteins in a no-wash assay format.

[0244] 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 with cell culture medium to obtain 40-fold dilutions (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, diluted 1:3 starting from 3 μM) for 1 hour at 37°C and 5% CO2, followed by treatment with anti-CD3 (5 μg / ml, OKT3 clone) for 30 min to activate TCR and HPK1. A 1:25 dilution of 100× blocking reagent (from p-SLP76ser376HTRF 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. Cell lysate (16 μL) was added to a Greiner white plate, treated with p-SLP76Ser376HTRF 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 a curve of percent inhibition versus inhibitor concentration using GraphPad Prism 5.0 software.

[0245] Any of the above assays can be scaled up or scaled up for high throughput screening (HTS). Any of the above assays can be used to determine the IC50 of a lead compound.

[0246] 7. Pharmaceutical Compositions

[0247] The present invention provides pharmaceutical compositions comprising any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0248] "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 the 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 can 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 will appreciate other pharmaceutical carriers and excipients suitable for use with the compounds of the present invention.

[0249] These compositions optionally further comprise one or more additional therapeutic agents. Alternatively, the compounds of the invention may be administered to patients in need thereof in combination with one or more other therapeutic regimens (e.g., Gleevec or other kinase inhibitors, interferon, bone marrow transplant, farnesyl transferase inhibitors, bisphosphonates, thalidomide, cancer vaccines, hormone therapy, antibodies, radiation, etc.). For example, the additional therapeutic agent for co-administration with the compounds of the invention or included in the pharmaceutical composition may be another one or more anticancer agents.

[0250] As described herein, the compositions of the present invention comprise a compound 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 particular dosage form. Remington's Pharmaceutical Sciences, 15th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1975) discloses known techniques for preparing various different carriers and methods for preparing pharmaceutical compositions. Unless any known carrier medium is incompatible with the compounds of the present invention, such as producing any undesirable biological effects or interacting in a harmful manner with any other component (group) in the pharmaceutical composition, 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 included in the composition.

[0251] 8. Formulations

[0252] The present invention also includes a class of compositions comprising an active compound of the present invention in association with one or more pharmaceutically acceptable carriers and / or diluents and / or adjuvants (collectively referred to herein as "carrier" materials) and, if desired, other active ingredients.

[0253] 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.

[0254] 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.

[0255] The compounds of the present invention can be administered by any suitable route, preferably in the form of pharmaceutical compositions compatible with such routes and in doses that are effective for treatment. The compounds and compositions of the present invention can be administered, for example, orally, mucosally, topically, rectally, pulmonary (such as inhalation spray) or parenterally (including via blood vessels, intravenously, intraperitoneally, subcutaneously, intramuscularly, transdermally, intraorbitally, intrathecally, intraventricularly, intratumorally, intranasally, intracranially, implanted, inhaled, and by infusion techniques, in dosage unit formulations containing pharmaceutically acceptable carriers, adjuvants and vehicles.

[0256] Typically, the pharmaceutical composition is a tablet or gelatin capsule comprising the active ingredient, together with 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; for tablets, c) a binder, for example, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if desired, 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 may be film-coated or enteric-coated according to techniques known in the art.

[0257] Suitable compositions for oral administration include an effective amount of a compound of the present 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 use are prepared according to any method known in the art for making pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, colorants, and preservatives to provide pharmaceutically aesthetic and palatable preparations. Tablets may contain active ingredients mixed with non-toxic pharmaceutically acceptable excipients suitable for making tablets. Such excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating agents and disintegrants such as corn starch, or alginic acid; binding agents such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained action over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used. Formulations for oral use may be presented in hard gelatin capsules where the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate or kaolin), or in soft gelatin capsules where the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil).

[0258] Some injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fat emulsions or suspensions. The composition may be sterilized and / or contain adjuvants, such as preservatives, tranquilizers, wetting agents or emulsifiers, dissolution promoters, salts and / or buffers for adjusting osmotic pressure. In addition, it may also contain other substances of therapeutic value. The composition is prepared according to conventional mixing, granulation or coating methods, respectively, and contains about 0.1-75% or about 1-50% of active ingredients. Suitable compositions for transdermal application include an effective amount of a 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, which includes a backing element, a reservoir containing a compound and optionally a carrier, and optionally a rate-controlling barrier, so as to deliver the compound to the host skin for a long time at a controlled and predetermined rate, and includes a means for fixing the device on the skin.

[0259] Suitable compositions for topical application, e.g. to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., delivered using aerosols or the like. Such topical delivery systems will be particularly suitable for skin applications, e.g., for the treatment of skin cancer, e.g., for preventive uses in sunscreens, lotions, sprays and the like. Therefore, they are particularly suitable for surfaces, including formulations well known in the art, cosmetics. They may contain solubilizers, tranquilizers, tension enhancers, buffers and preservatives.

[0260] As used herein, topical application may also be suitable for inhalation or intranasal application. It may be conveniently delivered from a dry powder inhaler in the form of a dry powder (alone, or as a mixture, e.g., a dry blend with lactose, or mixed component particles, e.g., formed with a phospholipid), or delivered as an aerosol spray from a pressurized container, pump, spray bottle, nebulizer or sprayer with or without a suitable propellant.

[0261] 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.

[0262] Anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low-moisture ingredients and 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 composition is packaged using materials known to prevent exposure to water so that it can be contained in a suitable formulation box. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0263] The present invention further provides pharmaceutical compositions and dosage forms comprising 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 "sedatives" and include, but are not limited to, antioxidants such as ascorbic acid, pH buffers or salt buffers, and the like.

[0264] The pharmaceutically active compounds of the present invention can be formulated into pharmaceutical preparations for administration to patients (including humans and other mammals) according to known pharmaceutical methods.

[0265] The amount of compound used and the dosage regimen for treating disease conditions using the compounds and / or compositions according to the present invention are determined according to various factors, including the age, weight, sex and medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the specific compound used. Therefore, the dosage regimen may vary greatly, but is routinely determined by standard methods. As previously described, the daily dose can be administered once or can be divided into 2, 3, 4 or more administrations.

[0266] Based on the purpose of treatment, the active compound of the present invention is usually combined with one or more adjuvants, excipients or carriers suitable for the specified route of administration. If administered orally, the compound can be mixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talcum, stearic acid, magnesium stearate, magnesium oxide, sodium salts and calcium salts of phosphoric acid or sulfuric acid, gelatin, gum arabic, sodium alginate, polyvinyl pyrrolidone, and / or polyvinyl alcohol, and then tableted or encapsulated for easy administration. Such capsules or tablets may include controlled release formulations because the active compound can be dispersed in hydroxypropyl methylcellulose.

[0267] Taking skin conditions as an 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 application 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 no more than 5% w / w, and more preferably 0.1% to 1%.

[0268] The compounds of the present invention can also be applied by transdermal devices. Preferably, transdermal application is achieved by using a patch, which is a reservoir and porous membrane type or a solid matrix variable type. The activating agent in both cases is continuously delivered from the reservoir or microcapsule through the membrane to the activating agent permeable adhesive in contact with the skin or mucous membrane of the recipient. If the activating agent is absorbed through the skin, the activating agent is applied to the recipient at a controlled and predetermined flow rate. Taking microcapsules as an example, 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.

[0269] Although the phase may contain only an emulsifier, it may contain a mixture of at least one emulsifier and a fat or oil or both a fat and an oil. Preferably, a hydrophilic emulsifier and a lipophilic emulsifier are included together as a stabilizer. 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 matrix 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, distearic acid glyceryl alone or in combination with wax, or other materials known in the art.

[0270] According to the cosmetic properties to be expected to be achieved, select the oils or fats suitable for formulation, because the solubility of the active compound in most oils that may be used in the pharmaceutical emulsion formulation is extremely low. Therefore, the cream should preferably be a non-greasy, non-sticky and washable product with a suitable firmness to avoid leakage from a pipe or other container. The mono- or dibasic alkyl esters of straight or branched chains such as di-isoadipate, isocetyl stearate, propylene glycol esters of coconut fatty acids, 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.

[0271] Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils may be used.

[0272] 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.

[0273] Preferred concentrations of the active ingredient in such formulations are from 0.5 to 20%, advantageously from 0.5 to 10%, especially about 1.5% w / w.

[0274] 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 dispersants 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 pharmaceutical field. The active ingredient may also be formed into a composition using a suitable carrier (including saline, dextrose, or water, or using cyclodextrin (i.e. Captisol), a co-solvent dissolution method (i.e. propylene glycol) or a micellar dissolution method (i.e. Tween 80)) for injection.

[0275] 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 can 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 can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids (such as oleic acid) can be used in injection preparations.

[0276] Pharmaceutical compositions for pulmonary administration may be administered in the form of an aerosol or using an inhaler containing a dry powder aerosol.

[0277] 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 glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

[0278] The pharmaceutical composition may be subjected to conventional pharmaceutical operations, such as sterilization, and / or contain commonly used adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, and the like. Tablets and pills may be prepared using an enteric coating 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 formula described herein, or a pharmaceutically acceptable salt thereof; an additional agent selected from the following: a kinase inhibitor (small molecule, polypeptide, antibody, etc.), an immunosuppressant, an anticancer agent, an antiviral agent, an anti-inflammatory agent, an antifungal agent, an antibiotic, or an anti-vascular hyperproliferation compound; and any pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0279] 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. Such compositions optionally comprise one or more additional therapeutic agents, including, for example, kinase inhibitors (small molecules, polypeptides, antibodies, etc.), immunosuppressants, anticancer agents, antiviral agents, anti-inflammatory agents, antifungal agents, antibiotics, or anti-vascular hyperproliferative compounds.

[0280] 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 when administered at a dose sufficient to deliver a therapeutic amount of the compound, will not destroy its pharmacological activity and is non-toxic. 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). delivery systems) (SEDDS) (e.g., d-tocopherol 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene glycol-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. It is also advantageous 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 delivery of the compounds of the formula described herein.

[0281] The pharmaceutical composition can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions 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 aqueous suspensions and / or emulsions are administered orally, the active ingredient can be suspended or dissolved in the oil phase and combined with an emulsifier and / or suspending agent.

[0282] If desired, certain sweeteners, flavorings and / or coloring agents may be added. Pharmaceutical compositions may include formulations utilizing liposome or microencapsulation technology. Various different examples thereof are known in the art.

[0283] 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 prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents, examples of which are also known in the art.

[0284] 9. Treatment kit

[0285] One aspect of the present invention relates to a kit for conveniently and effectively carrying out the method and use according to the present invention. Typically, a pharmaceutical package or kit comprises one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. Such kits are particularly suitable for delivering oral solid dosage forms, such as tablets or capsules. Such kits preferably include many unit doses, and may also include cards to arrange the doses in the order in which they are intended to be used. If necessary, a way to help memory can be provided, for example, in the form of numbers, letters or other marks, or inserted into a calendar, indicating the date on which the dose can be applied in the treatment plan. Optionally associated with such containers may be a form of notification prescribed by a government agency that manages the manufacture, usage or sale of drugs, which reflects that the agency has approved the manufacture, usage or sale for human use.

[0286] The following representative examples contain important supplementary information, exemplification and guidance that may be applicable to various embodiments of the present invention and its effects. 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 art will understand various modifications of the present invention and many other embodiments thereof upon review of the present invention, including the following examples and references to the scientific and patent literature cited herein.

[0287] The contents of the cited references are incorporated herein by reference to help illustrate the state of the art.

[0288] Furthermore, for purposes of the present invention, chemical elements are identified 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.

[0289] 10. Synthesis Scheme

[0290] The compounds of the present invention can be prepared by those of ordinary skill in the art according to techniques and methods recognized in the art. More specifically, the compounds of the present invention can be prepared as shown in the reaction schemes, methods and examples below. Those skilled in the art will appreciate 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. Example

[0291] The following are the abbreviations used in this manual and their meanings:

[0292] Ac: Acetyl

[0293] Boc: tert-Butyloxycarbonyl

[0294] EtOAc: Ethyl acetate

[0295] DCM: dichloromethane

[0296] ACN: Acetonitrile

[0297] THF: Tetrahydrofuran

[0298] DMSO: dimethyl sulfoxide

[0299] MeOH: Methanol

[0300] EtOH: ethanol

[0301] DMAP:4-(dimethylamino)pyridine

[0302] DIPEA: N,N-isopropylethylamine

[0303] NMR: Nuclear Magnetic Resonance

[0304] LC-MS: Liquid chromatography-mass spectrometry

[0305] TLC: Thin layer chromatography

[0306] TCR: T cell receptor

[0307] BCR: B cell receptor

[0308] mM: millimole

[0309] μM: micromolar

[0310] mL: milliliters

[0311] ng: nanogram

[0312] nM: nanomolar

[0313] nm: nanometer

[0314] IC50: half maximal inhibitory concentration

[0315] OD: Optical density

[0316] A. Biological Examples

[0317] Biological Example 1 HPK1 biochemical assay

[0318] This example uses ADP-GLO TM Kinase assay to measure the effects of potential HPK1 inhibitor compounds on HPK1 kinase activity.

[0319] ADP-GLO TM The 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.

[0320] Materials and instruments

[0321] 1. Reagents:

[0322]

[0323] 2. Instruments & Suppliers:

[0324]

[0325]

[0326] 3. Board Setup

[0327] Serial 3-fold dilutions of compounds from 10 μM (highest concentration) to 0.508 nM (lowest concentration). Positive control is 10 μM reference + enzyme + substrate. Negative control is 1% DMSO + enzyme + substrate.

[0328] 4. Regulations

[0329] 1. Buffer Preparation

[0330] 40mM Tris pH7.5; 20mM MgCl2, 0.1mg / ml BSA, 50μM DTT

[0331] Buffer stock

[0332] 1M Tris, pH 7.5, 121.14 g / mol <![CDATA[Add 6.057 g to 50 mL of H2O and adjust the pH to 7.5]]>

[0333] <![CDATA[1M MgCl2,95.21g / mol]]> <![CDATA[Add 4.7605 g to 50 mL of H2O]]>

[0334] Add 20 mL 1 M Tris and 10 mL 1 M MgCl2 to 470 mL ddH2O to make a buffer stock solution and store at room temperature.

[0335] 2. Prepare fresh 1* assay buffer

[0336] 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 3

[0337] 3. Compound Preparation

[0338] 1) Dilute compounds to 1 mM by mixing 10 μL of a 10 mM stock solution of each compound with 90 μL DMSO.

[0339] 2) The compound was then diluted 3-fold by BRAVO into 10 doses (from 5 μL to 10 μL). The highest compound concentration was 1 mM (100×), and the DMSO concentration was 100%.

[0340] 3) Transfer 100 nL of each diluted compound sample to a 384-well plate (Corning-4512) by ECHO

[0341] 4) Centrifuge the plate at 1,500 rpm for 1 minute.

[0342] 4. Prepare 2× ATP-MBP mixture:

[0343] 20μM ATP, 0.2μg / μL MBP in kinase buffer (final concentration: 10μM ATP, 0.1μg / μL MBP)

[0344]

[0345] 5. Prepare 2× HPK1 working solution using assay buffer.

[0346] The final concentration of HPK1 was 0.6 ng / μL. The more potent compounds were treated with lower concentrations of HPK1 (0.26 ng / μL-0.065 ng / μL).

[0347] 6. Add 5 μL / well of 2×HPK1 working solution and centrifuge at 1,500 rpm for 1 minute.

[0348] 7. Add 5 μL / well of 2×ATP-substrate and centrifuge at 1,500 rpm for 1 minute.

[0349] 8. Incubate at 25°C for 1 hour (or 6 hours for high potency compounds).

[0350] 9. Add 5 μL / well ADP-GLO TMThe reagent stops the kinase reaction and depletes unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0351] 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.

[0352] 11. Record the luminescent signal on Envision plate reader (384-CTG).

[0353] 5. Data Analysis

[0354] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the high and low controls contained in each assay plate. The high control wells were taken as 0% inhibition, and the low control wells, which contained no compound but only DMSO (final concentration = 0.5%), were taken as 100% inhibition. The concentration of the test compound and the % inhibition values ​​were plotted and the concentration of compound required for 50% inhibition (IC50) was determined using a three-parameter logistic dose response equation. The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a quality control measurement. The experiment was considered acceptable if the endpoint value was within 3 times of the expected value.

[0355] Biological Example 2 PKC-theta biochemical assay

[0356] This embodiment uses ADP-GLO TM Kinase assays were used to measure the effects of potential HPK1 inhibitor compounds on PKC-θ kinase activity. An exemplary experimental setup is described below, but minor modifications may be required for individual assays.

[0357] Materials and instruments

[0358] 1. Reagents:

[0359]

[0360] 2. Instruments & Suppliers:

[0361]

[0362]

[0363] 3. Board Setup

[0364] Serial 3-fold dilutions of compounds from 10 μM (highest concentration) to 0.508 nM (lowest concentration). Positive control is 10 μM reference + enzyme + substrate. Negative control is 1% DMSO + enzyme + substrate.

[0365] 4. Regulations

[0366] 1. Buffer Preparation

[0367] 40mM Tris pH7.5; 20mM MgCl2, 0.1mg / ml BSA, 50μM DTT

[0368] Buffer stock

[0369] 1M Tris, pH 7.5, 121.14 g / mol <![CDATA[Add 6.057 g to 50 mL of H2O and adjust the pH to 7.5]]>

[0370] <![CDATA[1M MgCl2,95.21g / mol]]> <![CDATA[Add 4.7605 g to 50 mL of H2O]]>

[0371] Add 20 mL 1 M Tris and 10 mL 1 M MgCl2 to 470 mL ddH2O to make a buffer stock solution and store at room temperature.

[0372] 2. Prepare fresh 1* assay buffer

[0373]

[0374] 3. Compound Preparation

[0375] 1) Dilute compounds to 1 mM by mixing 10 μL of a 10 mM stock solution of each compound with 90 μL DMSO.

[0376] 2) The compound was then diluted 3-fold by BRAVO into 10 doses (from 5 μL to 10 μL). The highest compound concentration was 1 mM (100×), and the DMSO concentration was 100%.

[0377] 3) By ECHO, 50 nL of each diluted compound sample was transferred to a 384-well plate (Corning-4512).

[0378] 4) Centrifuge the plate at 1,500 rpm for 1 minute.

[0379] 4. Prepare 2× enzyme working solution in kinase buffer:

[0380]

[0381] 5. Prepare 2× ATP-mix:

[0382]

[0383] 6. Add 2.5 μL / well of 2× enzyme working solution and centrifuge at 1,500 rpm for 1 minute.

[0384] 7. Add 2.5 μL / well of 2×ATP-substrate and centrifuge at 1,500 rpm for 1 minute.

[0385] 8. Incubate at 25°C for 60 min.

[0386] 9. Add 5 μL / well of ADP-GLO TM The reagent stops the kinase reaction and depletes unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0387] 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.

[0388] 11. Record the luminescent signal on Envision plate reader (384-USL).

[0389] 5. Data Analysis

[0390] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the high and low controls contained in each assay plate. The high control wells were taken as 0% inhibition, and the low control wells, which did not contain any compound but only DMSO (final concentration = 0.5%), were taken as 100% inhibition. The concentration of the test compound was plotted against the % inhibition value, and the concentration of the compound required for 50% inhibition (IC50) was determined using a three-parameter logistic dose response equation. The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a quality control measurement. If the endpoint value was within 3 times of the expected value, the experiment was considered acceptable.

[0391] Biological Example 3 TBK1 biochemical assay

[0392] This embodiment uses ADP-GLO TM Kinase assays are used to 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.

[0393] Materials and instruments

[0394] 1. Reagents:

[0395]

[0396] 2. Instruments & Suppliers:

[0397] instrument source Catalog Number 96-V Bottom Hole Plate Greiner 651201 384-well plate Corning 4512 LDV Board labcyte LP0200 Bravo Agilent - Envision PerkinElmer - Water System Millipore Milli-Q Reference System - Bravo 10μL Tip Axygen VT-384-10uL-R Bravo 30μL Tip Axygen VT-384-31uL-R Manual single channel pipette RAININ - Multichannel electronic pipette Thermo / RAININ -

[0398] 3. Board Setup

[0399] See above.

[0400] 4. Regulations

[0401] 1. Buffer Preparation

[0402] 40mM Tris pH7.5; 20mM MgCl2, 0.1mg / ml BSA, 50μM DTT

[0403] Buffer stock

[0404] 1M Tris, pH 7.5, 121.14 g / mol <![CDATA[Add 6.057 g to 50 mL of H2O and adjust the pH to 7.5]]>

[0405] <![CDATA[1M MgCl2,95.21g / mol]]> <![CDATA[Add 4.7605 g to 50 mL of H2O]]>

[0406] Add 20 mL 1 M Tris and 10 mL 1 M MgCl2 to 470 mL ddH2O to make a buffer stock solution and store at RT (room temperature).

[0407] 2. Prepare fresh 1* assay buffer

[0408]

[0409] 3. Compound Preparation

[0410] 1) Dilute compounds to 1 mM by mixing 10 μL of a 10 mM stock solution of each compound with 90 μL DMSO.

[0411] 2) The compound was then diluted 3-fold by BRAVO into 10 doses (from 5 μL to 10 μL). The highest compound concentration was 1 mM (100×), and the DMSO concentration was 100%.

[0412] 3) 50 nL of each diluted compound sample was transferred to a 384-well plate (Corning-4512) by ECHO.

[0413] 4) Centrifuge the plate at 1,500 rpm for 1 minute.

[0414] 4. Prepare 2× enzyme working solution in kinase buffer:

[0415]

[0416] 5. Prepare 2× ATP-mix:

[0417]

[0418] 6. Add 2.5 μL / well of 2× enzyme working solution and centrifuge at 1,500 rpm for 1 minute.

[0419] 7. Add 2.5 μL / well of 2×ATP-substrate and centrifuge at 1,500 rpm for 1 minute.

[0420] 8. Incubate at 25°C for 60 min.

[0421] 9. Add 5 μL / well of ADP-GLO TM The reagent stops the kinase reaction and depletes unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0422] 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.

[0423] 11. Record the luminescent signal on Envision plate reader (384-USL).

[0424] 5. Data Analysis

[0425] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the high and low controls contained in each assay plate. The high control wells were taken as 0% inhibition, and the low control wells, which did not contain any compound but only DMSO (final concentration = 0.5%), were taken as 100% inhibition. The concentration of the test compound was plotted against the % inhibition value, and the concentration of the compound required for 50% inhibition (IC50) was determined using a three-parameter logistic dose response equation. The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a quality control measurement. If the endpoint value was within 3 times of the expected value, the experiment was considered acceptable.

[0426] Biological Example 4 JAK3 biochemical assay

[0427] This embodiment uses ADP-GLO TM Kinase assay to measure the effects of potential HPK1 inhibitor compounds on JAK3 kinase activity. An exemplary experimental setup is described below, but minor modifications may be required for individual assays.

[0428] Materials and instruments

[0429] 1. Reagents:

[0430]

[0431] 2. Instruments & Suppliers:

[0432]

[0433]

[0434] 3. Regulations

[0435] 1. Preparation of JAK3 Kinase Buffer

[0436] DTT and BSA were freshly added to the buffer (final concentrations: 40 mM Tris pH 7.5; 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT).

[0437]

[0438] 2. Compound Preparation

[0439] Test compounds: Compounds were diluted to 1 mM by mixing 5 μL of 10 mM compound stock solution with 45 μL DMSO. Compound solutions were then serially diluted 3-fold to make 10 doses. The highest compound concentration was 1 mM (100×) and the DMSO concentration was 100%.

[0440] 50 nL of each of the 10 doses of diluted compound was added to a 384-well assay plate (Corning #4512) by ECHO.

[0441] Positive Control: 50 nL of 1 mM reference compound was added to a 384-well assay plate (Corning #4512) by ECHO.

[0442] Negative Control: Transfer 50 nL of DMSO to a 384-well assay plate as a negative control.

[0443] The assay plate was centrifuged at 1,500 rpm for 1 minute.

[0444] 3. Transfer compounds using ECHO according to the following procedure:

[0445] Reference compounds and test compounds 1-15: serial 3-fold dilutions from 10 μM to 0.508 nM (10 doses); negative control was 0.78 ng JAK3, 4 μM ATP & 0.2 μg / μL poly, 1% DMSO; positive control was the highest dose of reference compound, 0.78 ng JAK3, 0.2 μg / μL Poly (E4Y1), 4 μM ATP, 1% DMSO.

[0446] 4. Use a pipette (Thermo, 30 μL multichannel) to add 2.5 μL / well of 2×JAK3 working solution and centrifuge at 1,500 rpm for 1 minute.

[0447] 5. Use a pipette (Thermo, 30 μL multichannel) to add 2.5 μL / well of 2×ATP-Poly (E4Y1) working mixture and centrifuge at 1,500 rpm for 1 minute.

[0448] 6. Incubate the assay plate at 25°C for 60 min.

[0449] 7. Use BRAVO to add 5 μL / well of ADP-GLO TM The reagent stops the kinase reaction and depletes unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0450] 8. Add 10 μL of kinase detection reagent using BRAVO to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0451] 9. Record the luminescent signal on Envision plate reader (384-USL).

[0452] 10. Process the data using XL-fit. Inhibition % = [1-(test well-negative control) / (positive control-negative control)]*100%.

[0453] 5. Data Analysis

[0454] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the negative and positive controls contained in each assay plate. Negative control wells were taken as 0% inhibition, and positive control wells were taken as 100% inhibition. The concentration of the test compound was plotted against the % inhibition value, and the concentration of the compound required for 50% inhibition (IC50) was determined using a three-parameter logistic dose response equation. The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a quality control measurement. If the endpoint value was within 3 times of the expected value, the experiment was considered acceptable.

[0455] Biological Example 5 ZAP70 biochemical assay

[0456] This embodiment uses ADP-GLO TM Kinase assay to measure the effects of potential HPK1 inhibitor compounds on ZAP70 kinase activity. An exemplary experimental setup is described below, but minor modifications may be required for individual assays.

[0457] Materials and instruments

[0458] 1. Reagents:

[0459]

[0460]

[0461] 2. Instruments & Suppliers:

[0462]

[0463] 3. Regulations

[0464] 1. Preparation of ZAP70 kinase buffer:

[0465] DTT and BSA were freshly added to the buffer (final concentrations: 40 mM Tris pH 7.5; 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT, 2 mM MnCl2).

[0466]

[0467]

[0468] 2. Compound preparation:

[0469] Test compounds: Compounds were first diluted to 1 mM by mixing 5 μL of 10 mM compound stock with 45 μL DMSO. These compounds were then diluted 3-fold to make 10 doses. The highest compound concentration was 1 mM (100×) and the DMSO concentration was 100%.

[0470] 50 nL of compound solution was transferred to a 384-well assay plate (Corning #4512) by ECHO.

[0471] Positive Control: By ECHO, transfer 50 nL of 1 mM staurosporine to a 384-well assay plate as a positive control.

[0472] Negative control: Transfer 50 nL DMSO to a 384-well assay plate as a negative control. Centrifuge the plate at 1,500 rpm for 1 minute.

[0473] 3. Transfer compounds using ECHO according to the following procedure:

[0474] Staurosporine with test compounds 1-15: serial 3-fold dilutions from 10 μM to 0.508 nM (10 doses); negative control was 6.25 ng ZAP70, 10 μM ATP & 0.4 μg / μL poly, 1% DMSO; positive control was 10 μM staurosporine, 6.25 ng ZAP70, 10 μM ATP & 0.4 μg / μL poly, 1% DMSO.

[0475] 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).

[0476] 5. Prepare 2× ZAP70 working solution: (2.5 ng / μL) in kinase buffer (final concentration is 1.25 ng / μL).

[0477] 6. Use a pipette (Thermo, 30 μL multichannel) to add 2.5 μL / well of 2× ZAP70 working solution and centrifuge at 1,500 rpm for 1 minute.

[0478] 7. Use a pipette (Thermo, 30 μL multichannel) to add 2.5 μL / well of 2×ATP-Poly (E4Y1) working mixture and centrifuge at 1,500 rpm for 1 minute.

[0479] 8. Incubate the assay plate at 25°C for 60 min.

[0480] 9. Add 5 μL / well of ADP-GLO using BRAVO TM The reagent stops the kinase reaction and depletes unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0481] 10. Add 10 μL of kinase detection reagent using BRAVO to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0482] 11. Record the luminescent signal on Envision plate reader (384-USL).

[0483] 12. Process the data using XL-fit. Inhibition % = [1-(test well-negative control) / (positive control-negative control)]*100%.

[0484] 5. Data Analysis

[0485] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the negative and positive controls contained in each assay plate. Negative control wells were taken as 0% inhibition, and positive control wells were taken as 100% inhibition. The concentration of the test compound was plotted against the % inhibition value, and the concentration of the compound required for 50% inhibition (IC50) was determined using a three-parameter logistic dose response equation. The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a quality control measurement. If the endpoint value was within 3 times of the expected value, the experiment was considered acceptable.

[0486] Biological Example 6 LCK biochemical assay protocol

[0487] This embodiment uses ADP-GLO TM Kinase assay to measure the effects of potential HPK1 inhibitor compounds on Lck kinase activity. An exemplary experimental setup is described below, but minor modifications may be made in individual assays.

[0488] Materials and instruments

[0489] 1. Reagents:

[0490]

[0491] 2. Instruments & Suppliers:

[0492]

[0493]

[0494] 3. Regulations

[0495] 1. Preparation of LCK kinase buffer:

[0496] DTT and BSA were freshly added to the buffer (final concentrations: 40 mM Tris pH 7.5; 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT, 2 mM MnCl2).

[0497]

[0498] 2. Compound preparation:

[0499] Test compounds: Compounds were first diluted to 1 mM by mixing 5 μL of 10 mM compound stock solution with 45 μL DMSO. Compound solutions were then serially diluted 3-fold to make 10 doses. The highest compound concentration was 1 mM (100×) and the DMSO concentration was 100%.

[0500] Positive control: dilute to 300 μM by mixing 3 μL of 10 mM staurosporine stock solution with 97 μL DMSO.

[0501] Using BRAVO, 50 nL of compound solution and 300 μM staurosporine were transferred to a 384-well assay plate (Corning #4512).

[0502] Negative control: 50 nL DMSO was transferred to a 384-well assay plate as a negative control. The assay plate was centrifuged at 1,500 rpm for 1 minute.

[0503] 3. Transfer compounds using ECHO according to the following procedure:

[0504] Staurosporine with test compounds 1-15: serial 3-fold dilutions from 10 μM to 0.508 nM (10 doses); negative control was 7 ng LCK, 20 μM ATP & 0.4 μg / μL poly, 1% DMSO; positive control was 3 μM staurosporine, 7 ng LCK, 20 μM ATP & 0.4 μg / μL poly, 1% DMSO.

[0505] 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)).

[0506] 5. Prepare 2× LCK working solution: (2.8 ng / μL) in kinase buffer (final concentration is 1.4 ng / μL).

[0507] 6. Use a pipette (Thermo, 30 μL multichannel) to add 2.5 μL / well of 2× LCK working solution and centrifuge at 1,000 rpm for 1 minute.

[0508] 7. Use a pipette (Thermo, 30 μL multichannel) to add 2.5 μL / well of 2×ATP-Poly (E4Y1) working mixture and centrifuge at 1,000 rpm for 1 minute.

[0509] 8. Incubate the assay plate at 25°C for 60 min.

[0510] 9. Add 5 μL / well of ADP-GLO using BRAVO TM The reagent stops the kinase reaction and depletes unconsumed ATP after 1 hour. Centrifuge at 1,000 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0511] 10. Add 10 μL of kinase detection reagent using BRAVO to convert ADP to ATP. Centrifuge at 1,000 rpm for 1 minute. Incubate at 25°C for 30 minutes.

[0512] 11. Record the luminescent signal on Envision plate reader (384-USL).

[0513] 12. Use XL-fit to process the data. Inhibition % = (negative control - test well) / (negative control - positive control) × 100%.

[0514] 4. Data Analysis

[0515] The percent inhibition (%) for each compound concentration was calculated based on and relative to the signal in the negative and positive controls contained in each assay plate. Negative control wells were taken as 0% inhibition, and positive control wells were taken as 100% inhibition. The concentration of the test compound was plotted against the % inhibition value, and the concentration of the compound required for 50% inhibition (IC50) was determined using a three-parameter logistic dose response equation. The endpoint value (IC50) of the reference peptide / compound in each experiment was evaluated as a treatment control measurement. The experiment was considered acceptable if the endpoint value was within 3-fold of the expected value.

[0516] Biological Example 7 MAP4K3 protein kinase assay

[0517] This example provides an assay protocol for measuring phosphorylation of a peptide substrate by the protein kinase MAP4K3.

[0518] Briefly, MAP4K3, its substrates and cofactors (ATP and Mg) were 2+ ) were combined in the wells of a microtiter plate and incubated at 25° C. for 5 hours. At the end of the incubation, the reaction was terminated by adding a buffer containing EDTA. The separation and electrophoretic quantification of substrates and products were performed using the microfluidics-based LabChip 3000 Drug Discovery System from Caliper LifeSciences.

[0519] 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%.

[0520] Typical assay setups and conditions are provided below.

[0521] 1. Add 5 μL of 2X enzyme buffer (or control) to the wells of a 384-well plate.

[0522] 2. Add 100 nL 100X compound. If desired, enzyme and compound can be pre-incubated at this point.

[0523] 3. Add 5 μL 2× substrate buffer.

[0524] 4. Incubate the plate at 25°C for 5 hours.

[0525] 5. Add 40 μL of 1.25× Stop Buffer to stop the reaction.

[0526] 6. Use the values ​​provided in the table below to 3000 Drug Discovery System operations.

[0527] Separation conditions of 12 aspiration chips

[0528] Initial delayed aspiration time 50sec Sample buffer post-aspiration time 40sec Dye buffer post-absorption 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

[0529] 7. Load the plate and start electrophoresis, using a blue laser (480 nm) as excitation light and a green CCD (520 nm) for detection (CCD2).

[0530] The assay was run with the following reaction conditions: 5 hours total; at 25°C in the presence of 20 mM 100% inhibitor EDTA.

[0531] 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.

[0532] It should be noted that the specific activity of MAP4K3 varies between batches and the enzyme concentration may need to be adjusted to give a yield of approximately 10-20% conversion of substrate to product.

[0533] The substrate and product peptides present in each sample were separated by electrophoresis using a LabChip 3000 capillary electrophoresis instrument. Two fluorescence peaks were observed when the substrate and product peptides were separated. The relative fluorescence intensity changes of the measured substrate and product peaks were used as parameters to reflect the enzyme activity. The capillary electropherograms (RDA acquisition archives) 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.

[0534] Enzyme activity was measured at various concentrations (12 compound concentrations, 3× dilution intervals) of each compound. 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 for each compound at each concentration.

[0535] The inhibition percentage (P inh ):

[0536] P inh =(PSR 0％ -PSR inh ) / (PSR 0％-PSR 100 %)*100, where PSR inh is the total ratio in the presence of inhibitor, PSR 0％ is the average total product ratio in the absence of inhibitors, and PSR 100％ is 100% - the average total product ratio of the inhibition control samples.

[0537] The inhibition curves (P inh The IC50 value of the inhibitor was determined relative to the inhibitor concentration.

[0538] Certain materials and buffers used in this assay are listed in the table below for reference.

[0539]

[0540]

[0541] Biological Example 8 Study on the efficacy of compounds releasing IL2 & IFN-γ from human Pan T cells

[0542] This example is an assay that can be used to determine the effect of a compound on the release of IL2 & IFN-γ using pan T cells and an ELISA assay format.

[0543] Materials and instruments

[0544] 1. Reagents:

[0545]

[0546]

[0547] 2. Instruments & Suppliers:

[0548]

[0549] 3. Regulations

[0550] Protocol for Pan T Isolation and Reagent Preparation (Day 0):

[0551] 1. Cell growth medium:

[0552] RPMI1640: ATCC, catalog number 30-2001

[0553] 10% FBS: Gibco, catalog number 10099141

[0554] 1% Pen-Strep: Gibco, catalog number 15140122

[0555] 1% non-essential amino: Gibco, catalog number 11140050

[0556] β-Mercaptoethanol: Gibco, catalog number 21985023

[0557] 2. Preparation of Pan T separation buffer:

[0558] By using The flushing solution will Prepare a solution containing phosphate buffered saline (PBS) (pH 7.2), 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting BSA stock solution (#130-091-376) 20 times. Store the buffer at 4°C. Degas the buffer before use, as air bubbles will block the column.

[0559] 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

[0560] 3. Thawing of Frozen PBMCs

[0561] 1) Preheat the culture medium in a 37°C water bath.

[0562] 2) Thaw the cells quickly in a 37°C water bath.

[0563] 3) Add pre-warmed culture medium to a 15 mL test tube and transfer the cells to the tube.

[0564] 4) Centrifuge at 300×g for 8 min. (0 for increase and 0 for decrease).

[0565] 5) Wash PBMCs with wash buffer.

[0566] 6) PBMCs were centrifuged at 300×g for 8 min and washed twice (9 for centrifugation up and 1 for centrifugation down).

[0567] 7) The cells were resuspended in Pan T cell isolation buffer and the cell number was counted.

[0568] 4.Pan T cell isolation:

[0569] Stain cells using a bead cocktail

[0570] 1) Prepare cells and determine cell number. Filter cells using a 70 μm cell strainer.

[0571] 2) Resuspend the cell pellet and 7 total number of cells in 40 μL of buffer.

[0572] 3) Every 10 7Add 10 μL of PanT cell biotin-antibody mixture per total cell number.

[0573] 4) Mix the wells and incubate in refrigerator (ice) for 10 minutes.

[0574] 5) Every 10 7 Add 30 μL of buffer per total number of cells.

[0575] 6) Every 10 7 Add 20 μL of Pan T cell microbead mixture per total cell number.

[0576] 7) Mix the wells and incubate in refrigerator (ice) for 15 minutes.

[0577] 8) Perform subsequent magnetic cell separation.

[0578] 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.

[0579] Subsequent manual cell separation:

[0580] 1) Place the LS column in the magnetic field of a suitable MACS separator. Details can be found in the data sheet of each MACS column.

[0581] 2) Prepare the column by flushing with 3 mL of buffer.

[0582] 3) Apply the cell suspension to the column and collect the flow-through, which represents the enriched T cells.

[0583] 4) Wash the column with 5 mL of buffer and collect the unlabeled cells that have passed through, which represent the enriched T cells.

[0584] Reminder: Be sure to wait until the column reservoir is empty before proceeding to the next step.

[0585] 5. Pan T cell FACS:

[0586] 1) Take out 50 μL of PBMC and Pan T cells respectively into FACS tubes.

[0587] 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 unstained controls, the cells were incubated with FACS staining buffer.

[0588] 3) Wash twice with cold staining buffer (PBS containing 0.2% BSA and 1 mM EDTA).

[0589] 4) Run FACS. Gate CD3 + 、CD3 + CD4 + and CD3 + CD8 + Group, used for % analysis.

[0590] 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.

[0591] 6) Dispense the cell suspension into sterile disposable containers for future use.

[0592] Protocol for preparation of compounds and anti-human CD3 / CD28 (Day 0):

[0593] 1. Compound Preparation

[0594] Compound serial dilutions (1000× from source plate)

[0595] Compounds were dissolved in 100% DMSO to a concentration of 10 mM and then serially diluted 3-fold to 8 dose points.

[0596] 4× Compound Dose Preparation (Middle Panel: Corning-3599):

[0597] Prepare 4X compound solutions in culture medium. Pipette up and down. For ZPE controls, prepare 0.4% DMSO in culture medium (4X). For HPE controls, prepare 0.4 μM RGT003-026 in culture medium (4X).

[0598] 2. Anti-human CD3 (stock concentration 6.76 mg / ml): Store at 4°C.

[0599] 1) Dilute anti-human CD3 with PBS to a final concentration of 0.5 μg / mL.

[0600] 2) 50 μL / well of CD3 was added to each well except for the positive and negative control wells. The positive and negative control wells were not stimulated with CD3 / CD28.

[0601] 3) Incubate in a 37°C 5% CO2 incubator for 2 hours.

[0602] 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.

[0603] 3. Preparation of Anti-human CD28 (4×)

[0604] Antibodies were diluted in culture medium to a stock concentration of 11.07 mg / mL to 2 μg / mL (4×).

[0605] 4.PMA / Ionomycin Preparation (4×)

[0606] 1) Dilute PMA to 400 ng / mL (8×) in culture medium.

[0607] 2) Dilute ionomycin to 8 μM (8×) in culture medium.

[0608] 3) Mix equal volumes of PMA and ionomycin to obtain a 4× mixture.

[0609] Protocol for stimulating cells (Day 0)

[0610] 1. Place 1×10 5 The cell suspension of 10 cells / well (100 μL / well) was transferred to a 96-well plate (cell plate: Greiner-655180).

[0611] 2. For test compounds and ZPE / HPE controls, add 50 μL / well of anti-human CD28. For positive or negative controls, add 50 μL / well of 4x PMA / ionomycin solution or culture medium, respectively.

[0612] 3. Add 50 μL / well of compound to the cell plate according to the plate plot shown below. For ZPE / HPE controls, add 50 μL / well of 0.4% DMSO solution or 0.4 μM RGT003-026 (4×), respectively. For positive or negative controls, add 50 μL / well of culture medium.

[0613] 4. Incubate the plates for 48 hours.

[0614] Protocol for IL-2 & IFN-γ ELISA:

[0615] Day 1: Coating plates:

[0616] 1) Coat microtiter wells with 100 μL per well of IL-2 & IFN-γ capture antibody diluted in coating buffer. See lot-specific instructions / certificate of analysis for recommended antibody coating dilutions. Seal plate and incubate overnight at 4°C.

[0617] Day 2: Sample Collection:

[0618] 1) After incubation for 48 hours at 37°C and 5% CO2 incubator, centrifuge the cell plate at 1,000 rpm for 10 minutes. Collect 100 μL / well of supernatant and then perform IL-2 and IFN-γ ELISA assays. Store the supernatant at -80°C and perform IL-2 and IFN-γ ELISA assays the next day. The supernatant may need to be diluted 30 to 40 times to ensure that the assay does not exceed the linear range of the IL-2 and IFN-γ standard curves.

[0619] 2) Add new culture medium (containing anti-CD28, P / I and compound) to the plate, 100 μL / well.

[0620] 3) Plate drawing:

[0621] Day 3-4: IL-2 & IFN-γ ELISA:

[0622] 1) Aspirate the wells and wash 3 times with ≥300 μL / well of wash buffer. After the final wash, invert the plate and blot on absorbent paper to remove any residual buffer.

[0623] 2) Block the plate with ≥ 200 μL / well Assay Diluent. Incubate at room temperature for 1 hour.

[0624] 3) Aspirate / wash as in step 2.

[0625] 4) Prepare standards in assay diluent.

[0626] Preparation of IL-2 standard stock solution:

[0627] 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 portions and freeze at -80°C.

[0628] Preparation of IFN-γ standard stock solution:

[0629] Add 1 mL of deionized water to the vial (145 ng / vial) to obtain a stock concentration of 145 ng / mL. Aliquot the standard stock solution into 10 μL / vial portions and freeze at -80°C.

[0630] Preparation of IL-2 / IFNγ standard curve:

[0631] The standard samples were diluted to a maximum concentration of 500 pg / mL. Then a 2-fold serial dilution was run to 10 dose points (including a blank control). The standards of different concentrations were transferred to the ELISA plate, 100 μL / well.

[0632] 1) Pipette 100 μL of each standard, sample, and control into appropriate wells. Seal the plate and incubate at room temperature for 2 hours.

[0633] 2) Aspirate / wash as in step 2, but wash a total of 5 times.

[0634] 3) Add 100 μL of the operating detection reagent (detection antibody + SAv-HRP reagent) to each well. Seal the plate and incubate at room temperature for 1 hour.

[0635] 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.

[0636] 5) Add 100 μL of substrate solution to each well. Incubate the plate (without plate seal cover) at room temperature in the dark for 30 minutes.

[0637] 6) Add 50 μL of stop reaction solution to each well.

[0638] 7) Within 30 minutes of stopping the reaction, read the absorbance at 450 nm. Normalize OD450nm to the OD value at 570nm.

[0639] 4. Data Analysis

[0640] In the biochemical assays, the percent inhibition (%) of each compound concentration was calculated based on and relative to the signal from the HPE and ZPE wells contained in each assay plate. HPE wells were considered 100% effective, and ZPE wells containing no compound but DMSO (final concentration = 0.1%) were considered 0% effective.

[0641] In the cell assay, the response of IL-2 or IFN-γ was calculated at each compound concentration relative to the DMSO control (data not shown). EC2x represents the compound concentration that produces a 200% response (2-fold) and EC50 was calculated using GraphPad Prism.

[0642] Biological Example 9 Kinetic Solubility Determination

[0643] This protocol aims to measure the kinetic solubility of the test sample in the assay buffer. The study will be conducted in accordance with the international bioethical standards "World Medical Association Declaration of Helsinki".

[0644] 1. Reagents

[0645] Comparison:

[0646] 10 mM Propranolol in DMSO

[0647] 10 mM Ketoconazole in DMSO

[0648] 10 mM Tamoxifen in DMSO

[0649] Deionized 18.2M-cm MQ Ultrapure water (UltraPure water)

[0650] Phosphoric acid, 85% (H3PO4)

[0651] Dimethyl sulfoxide

[0652] Assay buffer

[0653] Phosphate buffer, pH 7.4

[0654] 2. Regulations

[0655] Calibration curve preparation:

[0656] 300 μM compound solution: add 6 μL of compound stock solution to 192 μL of MeOH / H2O (1:1).

[0657] Prepare working solution in MeOH / H2O (1:1)

[0658]

[0659]

[0660] 1) Prepare stock solutions of test compounds at 10 mM concentration in DMSO. Dilute the stock solution in triplicate into 100 mM phosphate buffer to a target concentration of 100 μM in 1.5 mL Eppendorf tubes with a final DMSO concentration of 1%.

[0661] 2) Add 4 μL of 10 mM DMSO stock solution to 396 μL of assay buffer.

[0662] 3) Keep the sample tube shaking (1000 rpm) at room temperature for 1 hour.

[0663] 4) Centrifuge the sample tube at 12000 rpm (Eppendrof-5424, about 13500 g) for 10 minutes to precipitate undissolved particles.

[0664] 5) Transfer the supernatant to new tubes at different dilutions (undiluted, 10-fold diluted, 100-fold diluted, or other dilution multiples).

[0665] 6) Add 5 μL of the working solution and diluted supernatant to 95 μL of ACN (containing IS).

[0666] Samples were analyzed by LC-MS-MS against a calibration curve.

[0667] 3. Data Analysis

[0668] A standard calibration curve was prepared in methanol / H2O (1:1). After 1 hour of incubation, the test compound concentration in the supernatant was determined from a calibration curve constructed by plotting peak area versus nominal concentration over a dynamic range of 0.02 μM to 60 μM and used to assess the aqueous solubility of the test compounds.

[0669] Biological Example 10 MDCK Permeability Assay

[0670] This assay is only used to generate screening data and estimate the intestinal absorption potential of candidate drugs. The study will be conducted in accordance with the international bioethical standards "World Medical Association Declaration of Helsinki".

[0671] instrument

[0672] ◆24-well cell culture plate (PET membrane): Millipore

[0673] ◆24-well feeder tray:Millipore

[0674] ◆Pipette and tube (Eppendorf)

[0675] Millicell ERS system

[0676] ◆96-well U-shaped plate (Greiner)

[0677] ◆96-well microplate (Greiner)

[0678] ◆37℃CO2 incubator

[0679] ◆FlexStation 3(Molecular Devices)

[0680] Reagents

[0681] Cell lines:

[0682] MDCK(ATCC) or MDCK MDR1(NKI).

[0683] Cell culture growth medium (MEM + 10% FBS + 1% NEAA).

[0684] Prepare growth medium by adding 50 mL FBS and 5 mL NEAA to 445 mL of MEM, or adjust the final volume as needed.

[0685] Trypsin-EDTA (Invitrogen, Cat# 25200-072).

[0686] Assay and dosing solution buffers.

[0687] Hanks Balanced Salt Solution (HBSS, Invitrogen), containing 25 mM HEPES, pH 7.4

[0688] Hanks Balanced Salt Solution (HBSS, Invitrogen).

[0689]

[0690] Prepare donor buffer:

[0691] For direction A to B:

[0692] √HBSS buffer containing 0.3% DMSO and 5 μM Lucifer Yellow (LY): Add 150 μL DMSO and 125 μL LY (2 mM) to 50 mL HBSS buffer (pH 7.4).

[0693] √HBSS buffer containing 0.1% DMSO and 5 μM Lucifer Yellow (LY): Add 50 μL DMSO and 125 μL LY (2 mM) to 50 mL HBSS buffer (pH 7.4).

[0694] √HBSS buffer containing 5μM Lucifer Yellow (LY): Add 125μL LY (2mM) to 50mL HBSS buffer (pH 7.4).

[0695] For direction B to A:

[0696] √HBSS buffer containing 0.3% DMSO: Add 150 μL DMSO to 50 mL HBSS buffer (pH 7.4).

[0697] √HBSS buffer containing 0.1% DMSO: Add 50 μL DMSO to 50 mL HBSS buffer (pH 7.4).

[0698] √ HBSS buffer without DMSO.

[0699] Prepare receiver buffer:

[0700] For A-to-B direction: Prepare HBSS buffer containing 0.4% DMSO: add 200 μL DMSO to 50 mL HBSS buffer (pH 7.4).

[0701] For B-to-A direction: Prepare HBSS buffer containing 0.4% DMSO and 5 μM LY: add 200 μL DMSO and 125 μL LY (2 mM) to 50 mL HBSS buffer (pH 7.4).

[0702] Comparison:

[0703] Prepare 10 mM stock concentrations of compound in DMSO and Lucifer Yellow in assay buffer using the following formula:

[0704] (actual weight / molecular weight) / mL solvent = 10 mM

[0705] Reference compounds: Erythromycin, Metoprolol, Atenolol

[0706]

[0707] The diluted solution was centrifuged at 4000 rpm for 5 minutes and the supernatant was collected for compound dosing.

[0708] Prepare compound solutions for standard curve (3 μM / 1 μM / 0.2 μM / 0.04 μM / 0.01 μM / 0.005 μM):

[0709] 20× solution:

[0710] 15μL (10mM)+485μL (MeOH:H2O=1:1)—500μL (300μM)

[0711] 200μL(300μM)+800μL(MeOH:H2O=1:1)---1000μL(60μM)

[0712] 200μL(60μM)+400μL(MeOH:H2O=1:1)---600μL(20μM)

[0713] 200μL(20μM)+800μL(MeOH:H2O=1:1)---1000μL(4μM)

[0714] 200μL (4μM)+800μL (MeOH:H2O=1:1)---1000μL (0.8μM)

[0715] 200μL (0.8μM)+600μL (MeOH:H2O=1:1)---800μL (0.2μM)

[0716] 200μL (0.2μM)+200μL (MeOH:H2O=1:1)---400μL (0.1μM)

[0717] 1× solution: 3 μL of 20× solution (0.1–60 μM) + 57 μL 0.4% DMSO HBSS + 60 μL ACN containing IS (200 ng / mL Osalmid)

[0718] Study Design

[0719] ◆Test concentration: determined by the initiator

[0720] ◆Incubation temperature and time point: 37°C, 0 and 60 minutes

[0721] ◆Sample specifications: 1-3

[0722] Regulations

[0723] Routine culture and maintenance

[0724] Stock cultures were maintained in MEM + 10% FBS + 1% NEAA at 75 cm 2 The cells were grown in tissue culture treated flasks and split (passaged) twice a week to maintain the desired confluence.

[0725] For maintenance passaging: split the trypsinized cells into new flasks at the standard passaging ratio of 1:20.

[0726] Subculture plan:

[0727] √ Remove and discard the culture medium.

[0728] √ Rinse the cell layer twice with 0.25% (w / v) trypsin-0.53 mM EDTA solution to remove all traces of serum containing trypsin inhibitor.

[0729] √ Add 2.0 to 3.0 mL of Trypsin-EDTA solution to the flask and observe the cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 15 minutes).

[0730] (Note: To avoid clumping, do not agitate the cells by tapping or shaking the flask while waiting for them to detach. Cells that are difficult to detach can be placed at 37°C to facilitate dispersion).

[0731] √ Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gentle pipetting.

[0732] √ Add an appropriate aliquot of the cell suspension to a new culture vessel.

[0733] √ Incubate the culture at 37°C.

[0734] Inoculation of assay plates

[0735] MDCK assay plates were seeded 3-4 days before running the assay. 24-well plates were seeded at 0.88 × 10 5 The cells were seeded at a density of 2.2 × 10 / well in a 400 μL top chamber volume. 5 / mL), and the 24-well basal chamber was provided with a volume of 25 mL of growth medium. The assay plate was usually provided with a change of growth medium 24 hours before the assay.

[0736] Assay plate preparation and transepithelial electrical resistance (TEER) measurement

[0737] The MDCK assay plate was rinsed with HBSS+ buffer before running the assay. After rinsing, fresh HBSS+ was added to the assay plate at 400 μL apical chamber volume and 0.8 mL HBSS+ basal chamber volume. The resistance across the monolayer was measured using a Millicell ERS system ohmmeter. (If TEER is above 100 ohms*cm 2 , then the cell will be used).

[0738] Preparation of standard curve

[0739] Preparation of cell plates:

[0740] ◆ Remove buffer from apical and basolateral sides. Add 600 μL of donor solution (for A-to-B) or 500 μL of receiver solution (for B-to-A) to the top wells according to the plate mapping.

[0741] ◆ Prepare a new substrate plate by adding 800 μL of Receiver Solution (for A-to-B) or 900 μL of Donor Solution (B-to-A) to the wells of a new 24-well plate.

[0742] ◆Put the top plate and base plate into a 37℃ incubator.

[0743] Preparation of assay plates:

[0744] ◆After 5 minutes, transfer 100 μL of sample from all donors (for A-to-B and B-to-A) to the appropriate wells of the D0 sample plate. And transfer 100 μL of sample from all top chambers (donors for A-to-B and recipients for B-to-A) to the appropriate wells of the Lucifer Yellow D0 (D0LY) microplate.

[0745] ◆Lay the top board onto the base board and start the transfer process.

[0746] ◆At 90 minutes, separate the top and bottom plates and transfer 100 μL of sample from all donors (for both A-to-B and B-to-A) to the appropriate wells of a new D90 sample plate and 200 μL of sample from all receivers to the appropriate wells of an R90 sample plate. Transfer 100 μL of sample from all bottom chambers (receivers for A-to-B and donors for B-to-A) to the appropriate wells of a new Lucifer Yellow R90 (R90LY) microplate.

[0747] ◆ Using a fluorescence plate reader, determine LY permeability by reading D0LY and R90LY at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0748] ◆Sample preparation.

[0749] ◆For receiver solution: 60 μL sample + 60 μL ACN containing IS (200 ng / mL Osalmid).

[0750] ◆For donor solution: 6 μL sample + 54 μL 0.4% DMSO / HBSS + 60 μL ACN containing IS (200 ng / mL Osalmid).

[0751] calculate

[0752] Transepithelial electrical resistance (TEER) = (resistance 样品 -resistance 空白 )×effective membrane area

[0753] Fluorescent Yellow Penetration

[0754] P app =(V A / (area × time)) × ([RFU] 接收者 –[RFU] 空白 ) / (([RFU] 初始，供体 –[RFU] 空白 )×dilution factor)×100

[0755] 7.1 Prepare the drug transport assay using the following equation:

[0756] Transepithelial electrical resistance (TEER) = (resistance 样品-resistance 空白 )×effective membrane area

[0757] Fluorescent Yellow Penetration

[0758] P app =(V R / (area × time)) × ([RFU] 接收者 –[RFU] 空白 ) / (([RFU] 初始，供体 –[RFU] 空白 ) × dilution factor)

[0759] Drug permeability

[0760] P app =(V R / (area×time))×([drug] 接收者 / (([drug] 初始，供体 ) × dilution factor)

[0761] Where V R is the volume in the receiver well (0.8 mL for A to B and 0.4 mL for B to A), and area is the surface area of ​​the membrane (0.7 cm for Millipore-24 cell culture plates). 2 ), and time is the total transfer time in seconds.

[0762] Percent recovery = 100 x (total compound in 90 min donor x dilution factor + total compound in 90 min recipient) / (total compound in 0 min donor x dilution factor).

[0763] Data analysis

[0764] The apparent permeability coefficient (P) of the test compound was calculated using the following equation: app ):

[0765] P app =(V R / (area×time))×([drug] 接收者 / (([drug] 初始，供体 ) × dilution factor)

[0766] Where V R is the volume in the receptor well (0.8 mL for A to B and 0.4 mL for B to A), and area is the surface area of ​​the membrane (0.7 cm for Millipore-24 cell culture plates). 2 ), and time is the total transfer time in seconds. The test compound concentrations on the receiver and donor sides were determined from standard calibration curves constructed by plotting peak areas versus nominal concentrations.

[0767] According to the top to base (AB) P app Data and Base to Top (BA)P app The average value of the data is used to calculate the efflux ratio: efflux ratio = P app(B-A) / P app(A-B) .

[0768] Transepithelial electrical resistance (TEER) was measured to determine the formation of tight junctions between cells, and if the TEER value was greater than 100 Ω cm 2 , then the cell monolayer will be used. To assess the integrity of the cell monolayer, 5 μM Lucifer Yellow was co-treated with the test compound in the donor compartment. app Greater than 5×10 -6 cm / sec, the data point will be excluded from the calculation.

[0769] Biological Example 11 Metabolic Stability in Liver Microsome Assay

[0770] This assay is used to determine the stability of the test compound in liver microsomes. This study will be conducted in accordance with the international bioethical standards "World Medical Association Declaration of.

[0771] Liver microsome samples

[0772] Human, Sprague Dawley rat, CD-1 mouse, Beagle dog, and Cynomolgus monkey liver microsome samples were purchased from BD Gentest or RILD.

[0773] instrument

[0774] Incubator (37°C)

[0775] Centrifuge (Eppendorf 5810)

[0776] Eppendorf pipettes and tubes

[0777] 96-well plate (Greiner)

[0778] Reagents

[0779] Compound Stock Solutions:

[0780] Test compounds were 10 mM in DMSO and stored at -80°C.

[0781] Assay buffer

[0782] 0.1 M potassium phosphate buffer, pH 7.4:

[0783] Buffer A: 1.0 L of 0.1 M potassium dihydrogen phosphate buffer containing 1.0 mM EDTA

[0784] Buffer B: 1.0 L of 0.1 M potassium phosphate buffer containing 1.0 mM EDTA

[0785] Buffer C (K-phosphate buffer): 0.1 M potassium phosphate buffer, 1.0 mM EDTA, pH 7.4, titrated with 700 mL of buffer B against buffer A while monitoring the pH meter.

[0786] Spiking Solution

[0787] Prepare 3× test compound or positive control solution:

[0788] 500 μM spiking solution: add 10 μL of 10 mM DMSO stock solution to 190 μL ACN.

[0789] 1.5 μM spiking solution in microsomes (0.75 mg / mL): 1.5 μL of 500 μM spiking solution and 18.75 μL of 20 mg / mL liver microsomes were added to 479.75 μL of K-phosphate buffer.

[0790] Other solutions

[0791] 6 mM NADPH in 0.1 M K-phosphate buffer.

[0792] Dissolve 25.1 mg of NADPH tetrasodium salt in 5 mL of K-phosphate buffer.

[0793] Regulations

[0794] Add 30 μL of a 1.5 μM spiking solution containing 0.75 mg / ml microsomal solution to wells designated 45 min, 30 min, 15 min, 5 min, and 0 min. Pre-incubate the plate at 37°C for 5 min.

[0795] 15 μL of NADPH stock solution (6 mM) was added to all wells and the timer was started. Immediately, 135 μL of ACN containing IS was added to the well designated as time 0.

[0796] At 5 min, 15 min, 30 min and 45 min, 135 μL of ACN containing IS was added to the wells.

[0797] At the end of the incubation, 15 μL of NADPH stock solution (6 mM) was added to the wells designated as time 0.

[0798] After quenching, the reaction mixture was centrifuged at 3220×g for 10 minutes.

[0799] 50 μL of supernatant from each well was transferred to a 96-well sample plate containing 50 μL of ultrapure water (Millipore) for LC / MS analysis.

[0800] Table 11-1 Concentrations of components in the final incubation mixture

[0801]

[0802] calculate

[0803] Results are expressed as % of test compound remaining at each time point:

[0804] Remaining % = Peak area ratio t x / peak area ratio t0, where

[0805] t0 = 0 minutes of incubation

[0806] t x = any given incubation time.

[0807] In vitro half-lives are reported in minutes and are calculated as follows:

[0808] T 1 / 2 =-(0.693 / slope)

[0809] When y-intercept = 100% at x = 0 minutes, slope = In (% remaining) vs. incubation time.

[0810] According to T 1 / 2 Calculation of in vitro intrinsic clearance Cl′ int .

[0811] Cl′ int =(0.693 / T 1 / 2 )×(1 / (microsomal protein concentration (0.5 mg / mL))×scaling factor

[0812] The scaling factors used for each species are listed in Table 11-2.

[0813] The predicted hepatic clearance for each species was calculated based on the half-life using the fully stirred model.

[0814] CL hep =(Q H ×Cl′ int ×f ub ) / (Q H +Cl′ int ×f ub ),in

[0815] Q His the hepatic blood flow (mL / min / kg) (Table 11-2).

[0816] f ub is the unbound drug fragment in plasma, and

[0817] Cl′ int is the in vitro intrinsic clearance.

[0818] Table 11-2 Proportional factors for the prediction of intrinsic clearance in mouse, rat, dog, monkey and human microsomes

[0819]

[0820] a Scaling factor = (microsomal protein per gram of liver) × (liver weight per kilogram of body weight)

[0821] Data analysis

[0822] By comparing with the peak area ratio (compound peak area / internal standard peak area) at T0 (incubation 0 minute), calculate the parent remaining % at each incubation time point. The ln value of the peak area ratio is plotted against time and the gradient of the line is determined. The half-life and metabolism of the compound are calculated according to the natural logarithm linear regression of relative peak area vs time. Use the following formula to calculate the half-life and intrinsic clearance rate (Cl in microsomes) int ).

[0823] In vitro T 1 / 2 (min) = 0.693 / clearance rate constant (k)

[0824] Cl int (μL / min / mg of microsomal protein) = (0.693 / in vitro T 1 / 2 )(incubation volume / mg microsomes).

[0825] The predicted clearance of the test compound for each species will be determined based on the calculated half-life using the well-stirred model.

[0826] A positive control will also be included in the assay. If the positive control is not within the specified limits, the compound's value will be rejected.

[0827] Biological Example 12 Comparison of the Disclosed Compounds and Analogs

[0828] According to the scheme provided by the present disclosure, the biological properties of the disclosed compounds and various compound analogs were tested. The test results are summarized in the following Tables 12-1 and 12-2. According to Table 1, the compounds of the present disclosure (Examples 1, 2, 4 and 5) show excellent HPK1 inhibitory activity (less than 1nM). They also show good efficacy in inducing the production of IL-2. In addition, Table 12-2 shows that these compounds have good overall properties when considering solubility, permeability and metabolic stability, which have unexpectedly better overall properties than close analogs (Compounds 7-10).

[0829] Table 12-1 IC of HPK1 50 and efficacy

[0830]

[0831]

[0832] Table 12-2 Kinetic solubility, permeability and metabolic stability

[0833]

[0834]

[0835]

[0836] B. Synthesis Examples

[0837] Instrument Description

[0838] 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 specified in the experimental methods for each compound. Chemical shifts are expressed in parts per million (ppm, delta units). Coupling constants are expressed in Hertz units (Hz). Splitting patterns describe the apparent diversity and are designated as s (singlet), d (doublet), t (triplet), q (quadruplet), quint (pentaplast), m (multiplet), br (broad peak).

[0839] 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:

[0840] Time (min) Acetonitrile (%) 0.1% ammonium acetate aqueous solution (%) 0.50 5 95 4.50 95 5 6.00 95 5

[0841] Experimental procedures: All reactions were performed under anhydrous nitrogen atmosphere unless otherwise stated. TLC plates were visually detected with UV light. Flash chromatography refers to column chromatography on silica gel using glass columns (40-60 μm). Alternatively, automated chromatography was performed using a Biotage SP1 or Biotage Isolera system with UV detection at 220 or 254 nm and Biotage normal or reverse phase silica cartridges. Details can be found in the relevant experimental methods.

[0842] General Methods and Preparation

[0843] The compounds and intermediates disclosed herein are prepared by the synthetic methods described herein. During the experiments, except for the acids, bases, reagents, coupling reagents, solvents, etc. explicitly mentioned, modifications to reaction conditions such as temperature, solution concentration, solvent volume, microwave conditions applied, reaction duration, or combinations thereof are intended to be part of the present invention, and alternative suitable acids, bases, reagents, coupling reagents, solvents, etc. may be used, and are all included in the scope of the present disclosure. All possible geometric isomers, stereoisomers, and salt forms are intended to fall within the scope of the present disclosure.

[0844] Intermediates

[0845] Int.0

[0846]

[0847] 1-(tert-butyl)3-methylindoline-1,3-dicarboxylate

[0848]

[0849] Step 1: Sodium hydride (50.77 mg, 1.27 mmol, 60% in mineral oil) was added to a solution of indoline-1,3-dicarboxylic acid-1-(tert-butyl) 3-methyl ester (320 mg, 1.15 mmol) and iodomethane (491.36 mg, 3.46 mmol) in DMF (8 mL) at 25 ° C. The mixture was stirred at 25 ° C for 2 hours, diluted with EA (200 mL), and then washed with water (20 mL×3) and brine (30 mL). The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (EA / PE: 15%) to provide 3-methylindoline-1,3-dicarboxylic acid-1-(tert-butyl) 3-methyl ester as a light yellow oil (244 mg, 70% yield). LC-MS (ESI) m / z: 236 [M+H-56] + .

[0850] Int.1

[0851]

[0852] Methyl 2-(3-methylindolin-3-yl)acetate

[0853]

[0854] Step 1: To a solution of 3-methylindoline-1,3-dicarboxylic acid-1-(tert-butyl) 3-methyl ester (730 mg, 2.51 mmol) in THF (20 mL) was added an aqueous solution (1.41 mL) of sodium hydroxide (3.01 g, 75.17 mmol). The mixture was stirred at room temperature for 16 hours, acidified with 1M HCl aqueous solution, and extracted with EtOAc (100 mL x 3). The organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to provide crude 1-tert-butyloxycarbonyl-3-methylindoline-3-carboxylic acid as a yellow oil (620 mg, 85% yield). LC-MS (ESI) m / z: 222 [M+H-56] + .

[0855] Step 2: To a mixture of 1-tert-butyloxycarbonyl-3-methylindoline-3-carboxylic acid (630 mg, 2.27 mmol) in DCM (15 mL) was slowly added oxalyl dichloride (865.1 mg, 6.82 mmol) and N,N-dimethylformamide (8.3 mg, 0.114 mmol) at 0°C. The mixture was stirred at room temperature for 16 hours and concentrated in vacuo. The residue (670 mg, 1.13 mmol) was dissolved in THF (10 mL) and MeCN (5.00 mL), and diazomethyl(trimethyl)silane (258.75 mg, 2.27 mmol) (2M solution in diethyl ether) was slowly added to the above solution. The reaction mixture was stirred under N2 for 2 hours, quenched with 10% citric acid (10 mL), and partitioned between DCM (50 mL) and water (50 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography on silica gel (EA / PE=0-10%) to afford tert-butyl 3-(2-diazoacetyl)-3-methylindoline-1-carboxylate as a yellow oil (200 mg, 56% yield).

[0856] Step 3: To a solution of tert-butyl 3-(2-diazoacetyl)-3-methylindoline-1-carboxylate (200 mg, 0.664 mmol) in methanol (5 mL) was added silver benzoate (76.0 mg, 0.332 mmol). The reaction mixture was stirred at room temperature and N2 for 1.5 hours and diluted with DCM (50 mL) and water (50 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (EA / PE=0-15%) to provide tert-butyl 3-(2-methoxy-2-oxoethyl)-3-methylindoline-1-carboxylate as a colorless oil (100 mg, 42% yield). LC-MS (ESI) m / z: 250 [M+H-56] + .

[0857] Step 4: A mixture of tert-butyl 3-(2-methoxy-2-oxoethyl)-3-methylindoline-1-carboxylate (100 mg, 0.278 mmol) and HCl / EA (4M, 1.5 mL) in DCM (3 mL) was stirred at room temperature for 2 h and concentrated in vacuo to afford crude methyl 2-(3-methylindolin-3-yl)acetate without further purification. LC-MS (ESI) m / z: 206 [M+H] + .

[0858] Alternative synthesis methods:

[0859]

[0860] Step 1: NaH (6.52 g, 163.09 mmol, 60% suspension in mineral oil) was added to a solution of 3-methylindolin-2-one (20 g, 135.89 mmol) in THF (200 mL) at 0 ° C. within 30 minutes. The reaction mixture was stirred at 0 ° C. for 30 minutes, and then a solution of di-tert-butyl carbonate (29.07 g, 133.18 mmol) in THF (50 mL) was added dropwise. The reaction mixture was stirred for 1 hour, diluted with saturated NH4Cl aqueous solution (50 mL), and extracted with CH2Cl2 (100 mL×2). The combined organic phases were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (PE / EA=9 / 1) to provide tert-butyl 3-methyl-2-oxoindolin-1-carboxylate (30 g, 74% yield) as a yellow oil. LC-MS (ESI) m / z: 192 [M+H] + .

[0861] Step 2: To a solution of tert-butyl 3-methyl-2-oxoindoline-1-carboxylate (30 g, 103.12 mmol) in THF (300 mL) was added NaH (4.95 g, 123.74 mmol, 60% in mineral oil) at 0°C over 30 minutes. The reaction mixture was stirred at 0°C for 30 minutes, and then methyl 2-bromoacetate (18.93 g, 123.74 mmol) was added dropwise. The reaction mixture was stirred for 1 hour, quenched with saturated aqueous NH4Cl solution (100 mL), and extracted with CH2Cl2 (200 mL x 2). The combined organic phases were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (PE / EA=8 / 2) to provide tert-butyl 3-(2-methoxy-2-oxoethyl)-3-methyl-2-oxoindoline-1-carboxylate (28 g, 83% yield) as a white solid. LC-MS (ESI) m / z: 264 [M+H-56] + .

[0862] Step 3: A mixture of 4M HCl in 1,4-dioxane (35 mL) and tert-butyl 3-(2-methoxy-2-oxoethyl)-3-methyl-2-oxoindoline-1-carboxylate (28 g, 87.68 mmol) in CH2Cl2 (200 mL) was stirred at room temperature for 2 hours and concentrated in vacuo. The residue was partitioned between CH2Cl2 (200 mL) and saturated aqueous NaHCO3 solution (50 mL). The separated aqueous layer was extracted with CH2Cl2 (50 mL×3), and the combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo to provide methyl 2-(3-methyl-2-oxoindoline-3-yl) acetate (17.6 g, 82% yield). LC-MS (ESI) m / z: 220 [M+H] + .

[0863] Step 4: A mixture of methyl 2-(3-methyl-2-oxoindol-3-yl)acetate (15.6 g, 71.16 mmol) and Lawesson's reagent (14.71 g, 36.38 mmol) in toluene (220 mL) was stirred at 130° C. for 1.5 hours and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (PE / EA=80:20) to provide methyl 2-(3-methyl-2-thioindol-3-yl)acetate (15.5 g, 74% yield) as a white solid. LC-MS (ESI) m / z: 236 [M+H] + .

[0864] Step 5: To a mixture of 2-(3-methyl-2-thioindolin-3-yl)acetic acid methyl ester (9.5 g, 40.37 mmol) and nickel chloride (10.46 g, 80.75 mmol) in mixed THF (70 mL) and methanol (70 mL) at 0°C over 1 hour, NaBH4 (9.16 g, 242.24 mmol) was added in batches. The resulting mixture was stirred for 10 minutes and filtered through a Celite pad. The solid cake was washed with MeOH (100 mL), and the filtrate was concentrated in vacuo. The residue was dissolved in EA (200 mL) and washed with water (60 mL×3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (CH2Cl2: methanol = 95: 5) to provide 2-(3-methylindolin-3-yl)acetic acid methyl ester (6.5 g, 75% yield) as a yellow oil. LC-MS (ESI) m / z: 206 [M+H] + .

[0865] Int.2

[0866]

[0867] 6-Methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[0868]

[0869] 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 50% aqueous NaOH (17.44 g, 218 mmol) dropwise at 0°C. The resulting mixture was stirred at room temperature overnight and filtered. The filtrate was concentrated to yield bis(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)methane as a white solid (23 g, 100% yield).

[0870] 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 6-methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride as a white solid, which was suspended in DCM (300 mL). Saturated aqueous NaHCO3 solution (500 mL) was added to the mixture, 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 dried over anhydrous Na2SO4, filtered, and concentrated to provide 6-methoxy-1,2,3,4-tetrahydroisoquinoline as a yellow oil (10 g, 75% yield). LC-MS (ESI) m / z: 164 [M+H] + .

[0871] 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, to which NaBH4 (1.13 g, 29.96 mmol) was added in portions. 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 flash column chromatography on silica gel (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] + .

[0872] 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 subsequently added 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 dried over anhydrous Na2SO4, filtered, and concentrated. 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] + .

[0873] Step 5: Fe powder (5.5 g, 93.99 mmol) and ammonium chloride (793.96 mg, 13.08 mmol) were added to a solution of EA (32 mL), H2O (16 mL) and EtOH (144 mL) containing 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (4.2 g, 14.40 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 flash column chromatography on silica gel (DCM / MeOH=20 / 1 to 10 / 1 v / v) to provide 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine hydrochloride as a yellow solid (4.5 g, 100% yield). LC-MS (ESI) m / z: 193 [M+H] + .

[0874] Alternative synthesis methods:

[0875]

[0876] Step 1: At 0°C, BOC2O (15.88 g, 72.75 mmol, 16.70 mL) was slowly added 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). 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), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1) to provide tert-butyl (3-methoxyphenethyl)carbamate (14.6 g, 79.06% yield, 90% purity) as a colorless liquid. LC-MS (ESI) m / z: 196 [M+H-56] + .

[0877] Step 2: To a solution of tert-butyl (3-methoxyphenethyl)carbamate (7 g, 27.85 mmol) and 2-chloropyridine (4.74 g, 41.78 mmol, 3.92 mL) in CH2Cl2 (50 mL) at -78 °C was added a solution of Tf2O (8.64 g, 30.64 mmol, 5.15 mL) in CH2Cl2 (5 mL). After 20 minutes, BH3.Et2O (19.77 g, 139.26 mmol) was added dropwise to the above solution. The reaction mixture was then warmed to room temperature, stirred for 2 hours, and carefully quenched with saturated NaHCO3 solution. The resulting mixture was extracted with CH2Cl2 (3 x 100 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, 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] + .

[0878] Step 3: At -20°C, HNO3 (2.16 g, 22.29 mmol, 65% purity) was added dropwise to a solution of 6-methoxy-3,4-dihydroisoquinolin-1(2H)-one (3.8 g, 21.44 mmol, 9.62 mL) in concentrated H2SO4 (50 mL). 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 (3x200 mL). The combined organic layers were concentrated in vacuo, and the residue was purified by flash column chromatography (SiO2, 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] + .

[0879] Step 4: 1M BH3 / THF (45.46mmol, 45.5mL) was added to a solution of 6-methoxy-7-nitro-3,4-dihydroisoquinolin-1(2H)-one (2.02g, 9.09mmol) in THF (200mL). The mixture was stirred at reflux for 20 hours and the reaction was carefully quenched with methanol (30mL). The resulting solution was concentrated in vacuo. The residue was heated at 80°C for 3 hours in 2N HCl (50mL), cooled, basified with aqueous ammonium hydroxide, and extracted with dichloromethane (3x100mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to provide 6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (1.89g, 100.00% yield). LC-MS (ESI) m / z: 209 [M+H] + .

[0880] Step 5: Formaldehyde (1.64 g, 54.46 mmol, 1.51 mL) was added 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. The mixture was stirred at room temperature for 15 minutes and cooled to 0°C. NaBH4 (1.03 g, 27.23 mmol) was then added to the above mixture in portions. 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 flash column chromatography on silica gel (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] + .

[0881] Step 6: Fe (2.19 g, 39.19 mmol) and ammonium chloride (284.74 mg, 5.32 mmol) were added to a solution of 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (1.3 g, 5.85 mmol) in EtOAc (2 mL), H2O (1 mL) and EtOH (10 mL). The mixture was stirred at 60 ° C for 16 hours, cooled to room temperature, and filtered. The 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-tetrahydroisoquinoline-7-amine hydrochloride as a yellow solid (0.85 g, 75.6% yield). LC-MS (ESI) m / z: 193 [M+H] + .

[0882] Int.3

[0883]

[0884] 6-Fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[0885]

[0886] Step 1: A mixture of 6-fluoro-3,4-dihydro-2H-isoquinolin-1-one (20.0 g, 121.09 mmol) in H2SO4 (160 mL) was cooled to -5 °C and potassium nitrate (12.85 g, 127.15 mmol) was added portionwise. The resulting mixture was stirred at the same temperature for 4 hours and poured into ice water. The solid was collected by filtration, washed with water and dried in vacuo to give 6-fluoro-7-nitro-3,4-dihydro-2H-isoquinolin-1-one (25.2 g, 119.91 mmol, 99.0% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ8.46(d,J=7.9Hz,1H),8.33(d,J=27.5Hz,1H),7.63(d,J=11.8Hz,1H),3.43(td,J=6.6,2.8Hz,2H),3.04(t,J=6.5Hz,2H).LCMS(ESI + )[(M+H) + ]:211.

[0887] Step 2: 1M borane tetrahydrofuran (599.54mmol, 600mL) was added to 6-fluoro-7-nitro-3,4-dihydro-2H-isoquinolin-1-one (25.2g, 119.91mmol). The mixture was stirred at reflux for 20 hours, cooled to room temperature and carefully quenched with methanol (150mL). The resulting solution was concentrated in vacuo. The residue was stirred in 2N HCl aqueous solution (500mL) at 80°C for 3 hours, cooled to room temperature, basified to pH 8-9 with ammonium hydroxide solution, and extracted with dichloromethane (500mL×3). The combined organic layers were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo to provide 6-fluoro-7-nitro-1,2,3,4-tetrahydroisoquinoline (22.3g, 113.67mmol, 94.8% yield) as a yellow solid. LCMS (ESI + )[(M+H) + ]:197.

[0888] Step 3: To a solution of 6-fluoro-7-nitro-1,2,3,4-tetrahydroisoquinoline (22.3 g, 113.67 mmol) in DCM (500 mL) was added formaldehyde (20.48 g, 682.03 mmol) at room temperature. The mixture was stirred at room temperature for 30 minutes and cooled to 0°C. Sodium triacetoxyborohydride (96.37 grams, 454.69 mmol) was added to the mixture in portions. The resulting mixture was stirred at room temperature for 36 hours, quenched with ice water (400 mL) and extracted with DCM (600 mL×3). The combined organic layers were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (DCM / MeOH=10 / 1 v / v) to afford 6-fluoro-2-methyl-7-nitro-3,4-dihydro-1H-isoquinoline (21.3 g, 101.33 mmol, 89.1% yield) as a yellow oil. LCMS (ESI + )[(M+H) + ]:211.

[0889] Step 4: To a solution of 6-fluoro-2-methyl-7-nitro-3,4-dihydro-1H-isoquinoline (21.3 g, 101.33 mmol) in EtOH (213 mL) and H2O (40 mL) was added ammonium chloride (37.22 g, 709.31 mmol) and iron powder (56.59 grams, 1.01 mol, 7.20 mL). The mixture was stirred at 60 ° C for 3 hours, cooled to room temperature and filtered. The solid filter cake was washed with methanol (100 mL×2), and the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (DCM / MeOH=20 / 1 to 10 / 1 v / v) to provide 6-fluoro-2-methyl-3,4-dihydro-1H-isoquinoline-7-amine (17.3 g, 95.99 mmol, 94.7% yield) as a yellow solid. LCMS (ESI + )[(M+H) + ]:181.

[0890] Example 1

[0891]

[0892] 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid

[0893] Step 1: To a mixture of Int.1 (67 mg, 0.248 mmol) in n-BuOH (3 mL) was slowly added 2,4,5-trichloropyrimidine (45.5 mg, 0.248 mmol) and DIPEA (96.19 mg, 0.744 mmol). The mixture was stirred at 100 ° C for 16 hours, quenched with ice water (20 mL) and extracted with EA (30 mL×3). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (DCM / PE=0-100%) to give methyl 2-(1-(2,5-dichloropyrimidine-4-yl)-3-methylindolin-3-yl) acetate (80 mg, 83.3% yield) as a yellow solid. LC-MS (ESI) m / z: 352 [M+H] + .

[0894] Step 2: To a mixture of methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate (80 mg, 0.227 mmol) in i-PrOH (5 mL) was slowly added Int.2 (51.5 mg, 0.227 mmol) and TsOH H2O (43.2 mg, 0.227 mmol). The mixture was stirred at 100 ° C for 16 hours, quenched with saturated NaHCO3 aqueous solution (10 mL) and extracted with DCM / MeOH (v / v=10 / 1, 30 mL×3). The organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (DCM / MeOH=0-15%) to give methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)3-methylindolin-3-yl)acetate (70 mg, 42.5% yield) as a yellow solid. LC-MS (ESI) m / z: 508 [M+H] + . 1 H NMR(500MHz,DMSO-d6)δ8.26(s,1H),7.98(s,1H),7.59(s,1H),7.30(t,J=8 .8Hz,2H),7.12(s,1H),6.98(s,1H),6.74(s,1H),4.38(d,J=10.7Hz,1H),4. 05(d,J=10.7Hz,1H),3.78(s,3H),3.50(s,3H),3.27(s,2H),2.78(dd,J=10. 3, 4.8Hz, 3H), 2.68 (s, 1H), 2.55 (t, J = 5.7Hz, 2H), 2.30 (s, 3H), 1.36 (s, 3H).

[0895] Step 3: A mixture of methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate (70 mg, 0.096 mmol) and aqueous LiOH solution (2N, 1 mL) in THF (5 mL) was stirred at room temperature for 16 hours, adjusted to pH ~5 with formic acid, and extracted with DCM (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo and the residue was purified by preparative HPLC (MeCN / 10 mM NH4HCO3, 0.025% NH3.H2O) to give 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid (23.0 mg, 46.2% yield) as a white solid. LC-MS (ESI) m / z: 494 [M+H] + . 1 H NMR(500MHz,DMSO-d6)δ8.25(s,1H),7.99(s,1H),7.57(s,1H),7.29(dd,J=17 .2,7.6Hz,2H),7.11(t,J=7.3Hz,1H),6.98(d,J=7.3Hz,1H),6.75(s,1H),4.4 2(d,J=10.7Hz,1H),4.02(d,J=10.6Hz,1H),3.78(s,3H),3.30(s,2H),2.78(s ,2H),2.69(d,J=15.4Hz,1H),2.58(d,J=6.1Hz,3H),2.32(s,3H),1.34(s,3H).

[0896] Examples 2 and 3

[0897]

[0898] (R)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid and (S)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid

[0899] Method 1:

[0900] Step 1: SFC chiral separation of methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate (157 mg) to provide methyl (R)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate and methyl (S)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate (67 mg, 63 mg), respectively. The stereochemistry was not absolutely determined.

[0901] Component corresponding to Peak 1: (R)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate or (S)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate.

[0902] LC-MS (ESI) m / z: 508 [M+H] + Chiral-HPLC retention time: 1.829 minutes; ee value: 98.3%.

[0903] The component corresponding to Peak 2: (S)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate or (R)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate.

[0904] LC-MS (ESI) m / z: 508 [M+H] + . Chiral-HPLC retention time: 3.427 min; ee value: >99%.

[0905] SFC separation conditions:

[0906] Instrument: SFC-80 (Thar, Waters)

[0907] Column: AD-H 20*250mm, 10μm (Daicel)

[0908] Column temperature: 35°C

[0909] Mobile phase: CO2 / EtOH (0.2% methanol / ammonia) = 50 / 50

[0910] Flow rate: 80g / min

[0911] Back pressure: 100bar

[0912] Detection wavelength: 214nm

[0913] Cycle time: 6.9min

[0914] Step 2: Under the same conditions as in Step 3 of Example 1, (R)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate and (S)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate were hydrolyzed. To the mixture were added methyl esters of the mixture to give (R)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid and (S)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid (17.6 mg, 33.8 mg, respectively).

[0915] Example 2: Methyl ((R)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate or (S)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate corresponds to the product derived from Peak 1 separated by SFC in Step 1 of Method 1.

[0916] LC-MS (ESI) m / z: 494 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.25(s,1H),8.00(s,1H),7.57(s,1H),7.30(dd,J=13.7,7.7H z,2H),7.11(t,J=7.6Hz,1H),6.97(t,J=7.3Hz,1H),6.75(s,1H),4.42(d,J=10.7Hz,1 H), 4.02 (d, J = 10.8 Hz, 1H), 3.78 (s, 3H), 3.30 (s, 2H), 2.78 (s, 2H), 2.69 (d, J = 15.4 Hz, 1H), 2.62-2.54 (m, 3H), 2.32 (s, 3H), 1.34 (s, 3H). Chiral-HPLC retention time: 1.384 min; ee value: >99%.

[0917] Example 3: Methyl ((S)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate or (R)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate corresponds to the product derived from Peak 2 separated by SFC in Step 1 of Method 1.

[0918] LC-MS (ESI) m / z: 494 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ8.25(s,1H),8.00(s,1H),7.56(s,1H),7.29(dd,J=13.6,7.4Hz,2H), 7.10(t,J=7.1Hz,1H),6.97(t,J=7.4Hz,1H),6.75(s,1H),4.41(d,J=10.8Hz,1H),4.02(d,J=1 0.8Hz,1H),3.77(s,3H),3.29(s,2H),2.77(t,J=5.6Hz,2H),2.69(d,J=15.4Hz,1H),2.57(dd,J=13.1,7.1Hz,3H),2.30(d,J=11.1Hz,3H),1.34(s,2H). Chiral-HPLC retention time: 1.132min; ee value:>99%.

[0919] Method 2:

[0920] Step 1: SFC chiral separation of methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate to provide methyl (R)-2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate and methyl (S)-2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate.

[0921] Component corresponding to peak 1: LC-MS (ESI) m / z: 352 [M+H] + Chiral-HPLC retention time: 1.191 min (peak 1); ee value: >99%.

[0922] Component corresponding to peak 2: LC-MS (ESI) m / z: 352 [M+H] + Chiral-HPLC retention time: 1.519 min (peak 2); ee value: >99%.

[0923] SFC separation conditions:

[0924] Instrument: SFC-80 (Thar, Waters)

[0925] Column: AD-H 20*250mm, 10μm (Daicel)

[0926] Column temperature: 35°C

[0927] Mobile phase: CO2 / isopropanol (0.2% methanol / ammonia) = 87 / 13

[0928] Flow rate: 80g / min

[0929] Back pressure: 100bar

[0930] Detection wavelength: 214nm

[0931] Cycle time: 2.8min

[0932] An additional large batch SFC chiral separation of methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate was performed under similar or identical conditions to provide methyl (R)-2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate and methyl (S)-2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate.

[0933] (One stereoisomer as a representative example of steps 2 & 3)

[0934] Step 2: A mixture of Int.2 (1.36 g, 5.96 mmol), (S)-2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid methyl ester or (R)-2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid methyl ester (2.1 g, 5.96 mmol, derived from the peak 2 of SFC separation of step 1 of method 2), and TsOH H2O (1.13 g, 5.96 mmol) in tert-butyl alcohol (25 mL) was stirred at 100°C for 12 hours, cooled to room temperature, poured into saturated aqueous NaHCO3 solution (10 mL) and extracted with EA (30 mL×3). The organic phase was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (DCM / MeOH=20:1) to provide (S)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate or (R)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate (1.8 g, yield 56.3%) as a light yellow solid. LC-MS (ESI) m / z: 508 [M+H] + .

[0935] Step 3: A mixture of a solution of lithium hydroxide hydrate (264 mg, 6.3 mmol) in water and (S)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate or (R)-methyl 2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate (1.6 g, 3.15 mmol) in MeOH (5 mL) was stirred at room temperature for 16 hours and acidified to pH about 3 with acetic acid. The resulting mixture was purified by preparative HPLC (MeCN / 10 mM NH4HCO3) to provide Example 3: ((S)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid or (R)-2-(1-(5-chloro-2-((6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid) (839.3 mg, 53.9% yield) as a light yellow solid derived from peak 2 of the SFC separation of step 1 of method 2. LC-MS (ESI) m / z: 494 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.30(s,1H),8.02(s,1H),7.63(s,1H),7.37(d,J=8.0Hz,1H),7. 33(d,J=8.0Hz,1H),7.16(t,J=8.0Hz,1H),7.02(t,J=8.0Hz,1H),6.80(s,1H),4.48(d,J= 10.8Hz,1H),4.08(d,J=10.8Hz,1H),3.83(s,3H),2.83(t,J=5.6Hz,2H),2.74(d,J=15.2Hz,1H),2.66–2.59(m,3H),2.37(s,3H),1.40(s,3H). Chiral-HPLC retention time: 1.132min; ee value:>99%.

[0936] Example 4

[0937]

[0938] 2-(1-(5-chloro-2-((6-fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid

[0939] Step 1: A solution of methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate (195.4 mg, 0.555 mmol), Int.3 (100 mg, 0.555 mmol) and TsOH.H2O (105.5 mg, 0.555 mmol) in i-PrOH (4 mL) was stirred at 120 ° C for 16 hours, cooled to room temperature, and neutralized to pH about 8 using saturated NaHCO3 aqueous solution. The mixture was extracted with DCM / MeOH (v / v, 10 / 1, 20 mL x3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (DCM / MeOH=10 / 1) to give a mixture of methyl 2-(1-(5-chloro-2-((6-fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate and isopropyl 2-(1-(5-chloro-2-((6-fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate (440 mg) as a pale yellow solid, which was used in the next step without further purification. LC-MS (ESI) m / z: 496 (methyl ester), 524 (isopropyl ester) [M+H] + .

[0940] Step 3: A mixture of methyl 2-(1-(5-chloro-2-((6-fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate and isopropyl 2-(1-(5-chloro-2-((6-fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetate (220 mg) in THF (4 mL) and aqueous LiOH (2N, 4 mL) was stirred at room temperature for 5 hours and at 50° C. for 16 hours, adjusted to pH about 5 with formic acid, and extracted with DCM / MeOH (10 / 1, 20 mL×3). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo and the residue was purified by preparative HPLC (MeCN / 10 mM NH4HCO3) to give 2-(1-(5-chloro-2-((6-fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)-3-methylindolin-3-yl)acetic acid (26.5 mg, 12.4% yield) as a white solid. LC-MS (ESI) m / z: 482 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.93(s,1H),8.22(s,1H),7.35-7.24(m,3H),7.07-6.92(m,3H),4.41(d,J=10.7Hz,1H),4.03(d, J=10.8Hz,1H),3.36(s,2H),2.78(d,J=5.4Hz,2H),2.68(d,J=15.5Hz,1H),2.60-2.54(m,3H),2.32(s,3H),1.33(s,3H).

[0941] Examples 5 and 6

[0942]

[0943] Method 2 used for Examples 2 and 3 was followed.

[0944] Step 1: To the first eluate of (Peak 1) ((R)-2-[1-(2,5-dichloropyrimidin-4-yl)-3-methyl-indolin-3-yl]acetic acid methyl ester or (S)-2-[1-(2,5-dichloropyrimidin-4-yl)-3-methyl-indolin-3-yl]acetic acid methyl ester) (27.8 g, 78.93 mmol) described in Method 2 of Examples 2 and 3 in n-butanol (anhydrous, 99.9%, 600 mL) was added 6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-amine (Int. 3, 17.07 g, 94.71 mmol) and 4-methylbenzenesulfonic acid (14.95 g, 86.82 mmol). The mixture was stirred at 120° C. for 20 hours. After the reaction was completed as judged by LCMS, the reaction mixture was cooled to room temperature, quenched with saturated aqueous NaHCO3 (100 mL), and extracted with DCM (600 mL×3). The organic phase was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography on silica gel (DCM / MeOH=50 / 1 to 10 / 1 v / v) to provide (R)-2-[1-[5-chloro-2-[(6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl)amino]pyrimidin-4-yl]-3-methyl-indolin-3-yl]acetic acid methyl ester or (S)-2-[1-[5-chloro-2-[(6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl)amino]pyrimidin-4-yl]-3-methyl-indolin-3-yl]acetic acid methyl ester (22.6 g, 45.57 mmol, yield 57.7%, mixed with butyl ester) as a yellow solid. LCMS (ESI + )[(M+H) + ]:496.

[0945] Step 2: A mixture of (R)-2-[1-[5-chloro-2-[(6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl)amino]pyrimidin-4-yl]-3-methyl-indolin-3-yl]acetic acid methyl ester or (S)-2-[1-[5-chloro-2-[(6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl)amino]pyrimidin-4-yl]-3-methyl-indolin-3-yl]acetic acid methyl ester (22.6 g, 45.57 mmol) and aqueous lithium hydroxide solution (5.46 g, 227.83 mmol) (1N in H2O) in THF (200 mL) and MeOH (100 mL) was stirred at 25°C for 6 hours until the reaction was complete as indicated by LCMS. The pH was adjusted to 5-6 with FA at 0°C and extracted with 10% MeOH in DCM (500 mL x 3). The organic phase was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue (20 g, 80-90% purity) was purified by preparative HPLC (NH4HCO3) to give Example 5 ((R)-2-[1-[5-chloro-2-[(6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl)amino]pyrimidin-4-yl]-3-methyl-indolin-3-yl]acetic acid or (S)-2-[1-[5-chloro-2-[(6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl)amino]pyrimidin-4-yl]-3-methyl-indolin-3-yl]acetic acid) (9.6 g, 19.92 mmol, 43.7% yield) as a light yellow solid. LCMS (ESI + )[(M+H) + ]:482. Optical rotation: -7.24 (Solvent: 1% DEA in CAN: water = 50:50; Concentration: 0.3 g / 100 mL; Temperature: 25°C). Chiral-HPLC retention time: 13.962 min (Column conditions: Column IG (4.6×250 mm 5 μm); Mobile phase: n-hexane (0.1% DEA): EtOH (0.1% DEA) = 80:20; Wavelength: 225 nm; Flow rate: 1.0 mL / min; Temperature: 40°C). Example 5 corresponds to the product derived from the peak 1 separated by SFC in step 1 of method 2 of Examples 2 and 3, which results in a more potent product.

Claims

1. A compound represented by formula I, or a pharmaceutically acceptable salt thereof.

2. A compound represented by formula IA, or a pharmaceutically acceptable salt thereof.

3. A compound represented by formula IB, or a pharmaceutically acceptable salt thereof.

4. A compound represented by formula II, or a pharmaceutically acceptable salt thereof.

5. A compound represented by formula IIB, or a pharmaceutically acceptable salt thereof.

6. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 2, 3 and 5, wherein the compound has an optical purity of 60% or more.

7. The compound or a pharmaceutically acceptable salt thereof according to claim 5, wherein the compound has an optical purity of 90% or more.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the compound has an optical purity of 99% or greater.

9. A pharmaceutical composition, which consists of an effective amount of the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

10. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and one or more therapeutically active concomitant agents.

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