HPK1 inhibitors and uses thereof

Abstract

The present invention relates to compounds of structural formula (I) or (II): The present invention provides a compound represented by TIFF2023533938000069.tif8276, or a pharmaceutically acceptable salt or stereoisomer thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from International Patent Application No. PCT / CN / 2020 / 100134, filed July 3, 2020. The entire contents of the foregoing application are incorporated herein by reference. [Background technology]

[0002] Hematopoietic progenitor kinase 1 (HPK1), also known as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a protein kinase that acts upstream of the classical three-step 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, first cloned in hematopoietic progenitor cells, is primarily expressed in lymphoid organs and 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).

[0003] 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 approximately 26 mammalian Ste20-like serine / threonine kinases identified to date and are homologs of yeast sterile 20 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 their domain structure: p21-activated kinases (PAKs) and germinal center kinases (GCKs). Within the GCK subfamily, some of these kinases can activate the MAP3K kinase cascade, leading to JNK activation.

[0004] MAP4Ks are structurally very similar, with an N-terminal kinase domain (KD) followed by two to four proline-rich motifs and a C-terminal citron homology domain (CNH).

[0005] The ATP-binding site in the kinase domain of HPK1 contains Lys-46, and mutation of this residue to Met (HPK1-M46) abolishes catalytic activation of HPK1 (Hu, supra).

[0006] HPK1 contains multiple conserved Ser / Thr phosphorylation sites within its kinase domain, and a conserved Tyr phosphorylation site between the first two proline-rich motifs. Phosphorylation of Tyr379 (mouse or human Tyr381) by LCK / ZAP70 appears to be required for HPK1 activation, because LCK or ZAP70 deficiency abolishes Tyr-379 phosphorylation and kinase activity of HPK1 in anti-CD3-stimulated Jurkat T cells (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). Meanwhile, autophosphorylation of Thr-355 regulates HPK1 ubiquitination and degradation. Thr-355 is a PP4 target dephosphorylation site, and this dephosphorylation prevents CUL7 / Fbxw8-mediated ubiquitination and proteasomal degradation of activated HPK1 (Wang et al., Cancer Res. 69(3):1063-1070, 2009). Thus, HPK1 is also stabilized and activated by protein phosphatase 4 (PP4) (Zhou et al., JBC 279(47):49551-49561, 2004).

[0007] The Tyr phosphorylation site is also adjacent to a caspase cleavage site (DDVD), and 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).

[0008] Four Pro-rich motifs in HPK1 mediate the interaction of HPK1 with many SH3 domain-containing proteins (Boomer & Tan, JCB 95(1):34-44, 2005).

[0009] The CNH domain of HPK1 may be involved in HPK1-mediated lymphocyte adhesion, because the citron homology domain in another Ste20-like kinase, TNIK, binds to Rap2 and regulates the actin cytoskeleton (Taira et al., JBC 279(47):49488-49496, 2004).

[0010] MAP4Ks play an important role in the immune system, particularly in lymphocytes, by regulating cell signaling, immune cell activation, cell transformation, and cell migration. HPK1 knockout (KO) mice exhibit enhanced T cell activation, increased cytokine production, and increased antibody production after KLH immunization. HPK1 KO mice are also more susceptible to EAE induction. HPK1 KO T cells and B cells exhibit enhanced cell activation and antigen receptor signaling. HPK1 KO dendritic cells exhibit higher levels of costimulatory molecules and pro-inflammatory cytokines (Alzabin et al., J. Immunol. 182(10):6187-6194, 2009; Shui et al., Nat. Immunol. 8(1):84-91, 2007).

[0011] Overexpression in cell lines (e.g., HEK293 and COS-1 cells, as well as hematopoietic Jurkat T cells and leukemic HL-60 cells) demonstrated that HPK1 can activate the MAPK JNK (but not p38 or ERK MAP kinases) through multiple MAP3Ks (including TAK1, MEKK1, and MLK3), all of which activate the MAP2Ks MKK4 and MKK7, which in turn activate JNK.

[0012] Interestingly, the regulatory function of MAP4Ks in immune cells appears to be largely mediated by JNK-independent mechanisms. It has been demonstrated that HPK1 kinase activation is required for IKK-NF-κB activation, likely via regulation of CARMA1. CARMA1 is an adaptor protein in the so-called CBM (CARMA1 / BCL10 / MALT1) complex that promotes IKKβ activation in Jurkat T cells upon anti-CD3 stimulation. Activated IKK cleaves IκB and releases the associated NF-κB nuclear transcription factor. In particular, HPK1 inducibly associates with CARMA1 and directly phosphorylates it at Ser-551, which is required for NF-κB activation (Brenner et al., PNAS USA, 196(34):14508-14513, 2009).

[0013] In T cells, upon TCR stimulation, lymphocyte protein tyrosine kinase (Lck) phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytosolic side of the TCR / CD3 complex. Zap-70 is then recruited to the TCR / CD3 complex, where it becomes phosphorylated and activated. Activated ZAP-70 phosphorylates an adaptor protein called SLP-76, translocating it to the plasma membrane and binding to numerous proteins, including HPK1, to promote the formation of a multiprotein signalosome complex. These proteins collectively transmit TCR signaling to different effector molecules, resulting in T lymphocyte activation, survival, and proliferation.

[0014] During this process, HPK1 directly binds to the SH2 domain of SLP-76 and primarily functions as a negative regulator of TCR signaling. For example, TCR signaling is elevated in HPK1 KO primary T cells, i.e., they exhibit hyperproliferation and IL-2 production upon in vitro TCR ligation (Shui, supra). HPK1 is thought to down-regulate TCR signaling through negative feedback by phosphorylating the SLP-76 adaptor protein at Ser-376. Upon HPK1-mediated Ser-376 phosphorylation, SLP-76 binds to 14-3-3 via the phosphorylated Ser-376 residue, resulting in ubiquitination of SLP-76 at the Lys-30 (K30) residue, which subsequently targets it for proteasomal degradation. HPK1 also down-regulates TCR signaling through similar mechanisms in other adaptor proteins, including GAD (e.g., by phosphorylating Thr-254 of GADS to promote 14-3-3 interaction).

[0015] Thus, HPK1 plays dual and opposing roles in JNK activation and TCR signaling. HPK1 has been demonstrated to directly activate the JNK pathway in different overexpression systems via the MAP3K-MAP2K-MAPK pathway, whereas HPK1-mediated inhibition of SLP-76 activation also results in the inhibition of JNK activity in TCR signaling. This is consistent with the observation that HPK1 knockout primary T cells exhibit unaffected JNK activity (Shui, supra). Similarly, HPK1 appears to regulate IKK activation through two distinct and contrasting mechanisms: on the one hand, HPK1 directly phosphorylates CARMA1 to activate IKK; and on the other hand, HPK1 also inhibits SLP-76 activation, negatively regulating IKK activation. This seemingly contrasting dual role of HPK1 is best understood as HPK1 promoting JNK and IKK activation in the early stages of TCR signaling but playing a key role in attenuating TCR signaling in the later stages.

[0016] HPK1 also plays a similar negative role in BCR-induced cell activation and B cell proliferation in B cells. B cells use an SLP-76-like adaptor protein called BLNK to transduce BCR signaling, including activation of JNK and IKK. In B cells, the Tyr kinases Syk and Lyn promote Tyr phosphorylation and activation of HPK1, and the resulting pY379 of HPK1 mediates HPK1-BLNK binding. The negative feedback of BLNK by HPK1 is via Thr-152 of BLNK. pY379 binding by 14-3-3 leads to BLNK ubiquitination at multiple Lys residues, resulting in the proteasomal degradation of BLNK (thus attenuating BCR signaling).

[0017] Interestingly, HPK1 is involved in the regulation of regulatory T cells (T reg ) appears to be a positive regulator of the inhibitory function of Foxp3 (Sawasdikosol et al., J Immunol. 188(Suppl. 1):163, 2012). + Tregs from mice are defective in suppressing TCR-induced effector T cell proliferation and, paradoxically, gain the ability to produce IL-2 after TCR engagement (Sawasdikosol, supra). Thus, HPK1 is a critical regulator of Treg function and peripheral self-tolerance.

[0018] HPK1 also inhibits CD4 + HPK1 kinase activity is also involved in PGE2-mediated inhibition of T cell activation (Ikegami et al., J Immunol. 166(7):4689-4696, 2001). US2007 / 008798 reports that HPK1 kinase activity is involved in the inhibition of CD4 T cell activation via PGE2-induced PKA activation. + We show that HPK1 expression increased in T cells exposed to physiological concentrations of PGE2. The proliferation of HPK1-deficient T cells was resistant to the inhibitory effects of PGE2 (US2007 / 0087988). Thus, PGE2-mediated activation of HPK1 may represent a novel regulatory pathway that modulates immune responses.

[0019] In addition to TCR and BCR, HPK1 also transmits signals downstream of TGF-R (transforming growth factor receptor) (Wang et al., JBC 272(36):22771-22775, 1997) or G-coupled PGE2 receptors (EP2 and EP4) (Ikegami et al., J Immunol. 166(7):4689-4696, 2001).

[0020] 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 of the degranulation-promoting adaptor protein (ADAP), which is required for TCR-induced integrin activation (Wang et al., J. Exp. Med. 200(8):1063-1074, 2004). However, the constitutively associated SKAP55 protein, which targets the activated small GTPase Rap1 to the plasma membrane, results 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 (see above), but also by competing with ADAP for the same SH2 binding site on SLP-76, and then attenuating the activity of the ADAP downstream effector Rap1 (Patzak et al., Eur. J. Immunol. 40(11):3220-3225, 2010).

[0021] HPK1 also negatively regulates integrin activation and cell adhesion in B cells, where it associates with a SKAP55 homolog called SKAP-HOM (Königsberger 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). HPK1 is thought to induce negative phosphorylation sites in SKAP-HOM, which in turn suppresses Rap1 activation.

[0022] However, in neutrophils, HPK1 positively regulates adhesion. Neutrophil trafficking, including slow rolling, tight binding, cell spreading, and extravasation, is regulated by outside-in signaling of β2 integrin activation, which induces the interaction of actin with HIP-55 (a 55-kDa HPK1-interacting protein). This reinforces the high-affinity conformation of β2 integrin and contributes to neutrophil adhesion (Hepper et al., J. Immunol. 188(9):4590-4601, 2012; Schymeinsky et al., Blood 114(19):4209-4220, 2009). HPK1 colocalizes with HIP-55 and actin to the lamellipodia of neutrophils upon β2 integrin-mediated adhesion (Jakob et al., Blood 121(20):4184-4194, 2013). CXCL1-mediated neutrophil adhesion is abolished by either HPK1 deficiency or HIP-55 deficiency in vitro and in vivo (Jakob, supra; Schymeinsky, supra).

[0023] Consistent with its role in downregulating TCR and BCR function, HPK1 negatively regulates adaptive immune responses, and loss of HPK1-mediated control of T cell activation and immune responses may be an important mechanism for autoimmune pathogenesis. Although T and B cell development appears unaffected in HPK1 KO mice (Shui, supra), T cells from these animals exhibit dramatically increased activation of TCR-proximal signaling and downstream ERK, leading to overgrowth of these cells in vitro upon anti-CD3 stimulation (Shui, supra). T cells from immunized HPK1-deficient mice are hyperresponsive to antigen-specific stimulation and produce significantly higher levels of inflammatory cytokines, such as IL-2, IFN-γ, and IL-4. These mice also produce significantly higher levels of IgM and IgG isoforms, suggesting enhanced function of HPK1 knockout B cells (Shui, supra).

[0024] HPK1 also negatively regulates autoimmunity in mice, as HPK1 KO mice are more susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) (Shui, supra). Attenuation of HPK1 also contributes to abnormal T and B cell activation and autoimmunity in human patients. HPK1 downregulates peripheral blood mononuclear cells in patients with psoriatic arthritis or T cells in patients with systemic lupus erythematosus (SLE).

[0025] The physiological function of HPK1 is not limited to lymphocytes; HPK1 also negatively regulates the maturation and activation of dendritic cells (DCs) through an unknown mechanism (Alzabin, supra). In HPK1 KO mice, bone marrow-derived dendritic cells (BMDCs) exhibit elevated levels of the costimulatory molecules CD80 / CD86 and increased production of pro-inflammatory cytokines (Alzabin, supra). Thus, the antigen-presenting activity of dendritic cells is more efficient in HPK1 KO mice (Alzabin, supra). More importantly, tumor elimination by HPK1 KO BMDC-mediated CTL responses is more effective than that by wild-type BMDCs (Alzabin, supra). Furthermore, HPK1 also regulates antitumor immunity through T- and B-lymphocyte-dependent mechanisms. Adoptive transfer of HPK1-deficient T cells has been shown to control tumor growth and metastasis more effectively than wild-type T cells (Alzabin et al., Cancer Immunol Immunother 59(3):419-429, 2010). Similarly, BMDCs from HPK1-knockout mice were more efficient at mounting T cell responses that eradicated Lewis lung carcinoma compared with wild-type BMDCs (Alzabin et al., J Immunol. 182(10):6187-6194, 2009).

[0026] Therefore, there is a need for HPK1 inhibitory compounds to treat diseases or disorders through modulation of HPK1 activity. Summary of the Invention [Means for solving the problem]

[0027] Described herein are compounds of formula (I), (IA), (IB), (II), (IIA), and (IIB), and pharmaceutically acceptable salts or stereoisomers thereof (collectively referred to as "compounds of the invention"), which inhibit the activity of HPK1.

[0028] Provided herein is a pharmaceutical composition comprising an effective amount of a compound of the present disclosure, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and a pharmaceutically acceptable carrier. Also provided is a combination 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.

[0029] The present disclosure 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.

[0030] The present disclosure further provides a method of treating a subject having a disease or condition described herein, e.g., 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.

[0031] Certain embodiments disclose the compounds of the present disclosure, pharmaceutically acceptable salts or stereoisomers thereof, for use as pharmaceuticals, eg, to act as HPK1 inhibitors.

[0032] 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, provided is a compound of the present disclosure, a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition comprising the same, for use in any of the methods of the present disclosure described herein. In another embodiment, provided is the use of a compound of the present disclosure, a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition comprising the same, for the manufacture of a medicament in any of the methods of the present disclosure described. DETAILED DESCRIPTION OF THE INVENTION

[0033] 1. Overview The disclosure described herein provides HPK1 / MAP4K1 inhibitors, pharmaceutically acceptable salts thereof, and methods of using them to modulate (e.g., inhibit) HPK1 / MAP4K1 activity, comprising administering a disclosed HPK1 / MAP4K1 inhibitor compound or a pharmaceutically acceptable salt thereof to a patient / subject in need thereof. In certain embodiments, the disclosed compounds, pharmaceutically acceptable salts thereof, or stereoisomers thereof are useful for therapeutic administration to enhance, stimulate, and / or increase immunity in the treatment of cancer.

[0034] For example, a method for treating a disease or disorder associated with inhibition of HPK1 interaction can include administering a therapeutically effective amount of a compound provided herein, a pharmaceutically acceptable salt or stereoisomer thereof to a patient in need thereof. The compounds of the present disclosure can be used alone, in combination with other drugs or therapies, or as adjuvants or neoadjuvants for the treatment of diseases or disorders, including cancer. 2.Definition As used herein, the terms "a," "an," "the," and similar terms as used in the context of the present invention (especially in the context of the claims) are to be construed as including both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0035] Compounds of any one of the above formulas may exhibit one or more types of isomerism (e.g., optical, geometric, or tautomeric isomerism). Such variants are implicit in compounds of any one of the above formulas that are defined as such by reference to their structural features, and are therefore implicitly included within the scope of the present disclosure.

[0036] Compounds with one or more chiral centers can exhibit various stereoisomeric forms, i.e., each chiral center can have an R or S configuration, or a mixture of both. 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.

[0037] "Peak 1" in the experimental section refers to a target reaction product compound obtained from chromatographic separation / purification that elutes faster than a second target reaction product compound from the same preceding reaction. The second target reaction product compound is designated "Peak 2."

[0038] When a compound is designated by its chemical name (e.g., when the configuration is indicated by an "R" or "S" chemical name) or its structure (e.g., when the configuration is indicated by a "wedge" bond) that indicates a single enantiomer, 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 is the weight of the named or depicted enantiomer in a mixture divided by the total weight of both enantiomers in the mixture.

[0039] When the stereochemistry of a disclosed compound is named or depicted in a structure, and the named or depicted structure encompasses more than one stereoisomer (e.g., as in a diastereomeric pair), it is understood that one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers is included. 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, stereoisomeric purity is determined by dividing the total weight of the stereoisomer encompassed by that name or structure in the mixture by the total weight of all stereoisomers in the mixture.

[0040] When two stereoisomers are depicted by their chemical names or structures and the chemical names or structures are connected by "and," a mixture of the two stereoisomers is intended. When two stereoisomers are depicted by their chemical names or structures and the chemical names or structures are connected by "or," either one or the other of the two stereoisomers is intended, but not both.

[0041] When a disclosed compound having a chiral center is depicted by a structure without indicating the configuration at that chiral center, the structure is meant to encompass compounds having the S configuration at that chiral center, compounds having the R configuration at that chiral center, or compounds having a mixture of R and S configurations at that chiral center. When a disclosed compound having a chiral center is depicted by its chemical name without indicating the configuration at that chiral center with "S" or "R," the name is meant to encompass compounds having the S configuration at that chiral center, compounds having the R configuration at that chiral center, or compounds having a mixture of R and S configurations at that chiral center.

[0042] A racemic mixture means 50% of one enantiomer and 50% of the corresponding enantiomer. When a compound with one chiral center is named or depicted without indicating the stereochemistry of the chiral center, the name or structure is understood to encompass both possible enantiomeric forms of the compound (e.g., enantiomerically pure, enantiomerically enriched, or racemic). When a compound with two or more chiral centers is named or depicted without indicating the stereochemistry of the chiral centers, the name or structure is understood to encompass 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)).

[0043] The term "geometric isomer" refers to a compound containing at least one double bond, where the double bond can exist in cis (also called syn or entgegen (E)) or trans (also called anti or zusammen (Z)) form, as well as mixtures thereof.

[0044] When a geometric isomer is designated by name or structure, it is understood that the named or depicted isomer is present to a greater degree than another isomer, i.e., the geometric isomeric purity of the named or depicted geometric isomer is greater than 50% by weight, e.g., at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. Geometric isomeric 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.

[0045] Conventional techniques for the preparation / isolation of individual enantiomers / diastereomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or a salt or derivative of the racemate) using, for example, chiral high-pressure liquid chromatography (HPLC). Alternatively, the racemate (or racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, if a compound of any one of the above formulas contains an acidic or basic moiety, 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 diastereoisomers converted to the corresponding pure enantiomer by methods well known to those skilled in the art. Chiral compounds of any one of the above formulas (and their chiral precursors) can be obtained in enantiomerically enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing 0-50% by volume, typically 2% to 20% by volume, of isopropanol, and 0-5% by volume of an alkylamine, typically 0.1% diethylamine. The concentration of the eluent will result in an enriched mixture. Chiral chromatography using subcritical and supercritical fluids may also be used. Methods of chiral chromatography useful in some embodiments of the present disclosure are known in the art (see, e.g., 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 Daicel® Chemical Industries, Ltd., Tokyo, Japan.

[0046] It must be emphasized that although compounds of any one of the above formulas are depicted herein in a single tautomeric form, all possible tautomeric forms are included within the scope of the present disclosure.

[0047] As used herein, the singular or plural terms "salt" refer to acid addition salts or base addition salts of the compounds of the present invention. "Salt" specifically includes "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, and typically are not biologically or otherwise undesirable; in many cases, the compounds of the present invention are capable of forming acids and / or bases by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0048] Pharmaceutically acceptable acid addition salts, such as acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorotheophylionate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydrogen iodide / iodide, isothionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methyl sulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate The phosphate / dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, subsalicylate, tartrate, tosylate and trifluoroacetate salts can be formed with inorganic and organic acids.

[0049] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I-XII of the periodic table; in certain embodiments, salts are 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.

[0050] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Particular organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0051] The pharmaceutically acceptable salts of the present invention can be synthesized from basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of an appropriate base (e.g., hydroxide, carbonate, bicarbonate, etc., Na, Ca, Mg, or K, etc.), or by reacting the free base form of these compounds with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water or an organic solvent, or a mixture of the two. Generally, the use of non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable, where feasible. Additional lists of suitable salts can be found, for example, in "Remington's Pharmaceutical Sciences," 20th Edition, Mack Publishing Company, Easton, PA, (1985); and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0052] The terms "composition" and "formulation" are used interchangeably. A "subject" is a mammal, preferably a human, but may also be an animal in need of veterinary treatment, such as companion animals (e.g., dogs, cats, etc.), livestock (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0053] As used herein, a subject is "in need of" a treatment if such subject would receive biological, medical, or quality of life benefit from such treatment. The terms "administer," "administering," or "administration" refer to a method of introducing a compound of the present invention, or a composition thereof, into or onto a subject. These methods include, but are not limited to, intraarticular (intra-articular), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, subcutaneous, oral, topical, intrathecal, by inhalation, transdermal, rectal, and the like. Administration techniques that can be used with the agents and methods described herein can 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.

[0054] As used herein, the terms "inhibit," "inhibition," or "inhibiting" refer to the 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.

[0055] The terms "treatment," "treat," and "treating" refer to reversing, alleviating, or inhibiting the progression of a disease as described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of disease have developed or been observed (i.e., therapeutic treatment). In other embodiments, treatment may be administered in the absence of signs or symptoms of disease. For example, treatment may be administered to a suspected subject prior to the onset of symptoms (i.e., prophylactic treatment) (e.g., in light of a history of symptoms and / or exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0056] The terms "condition," "disease," and "disorder" are used interchangeably. Generally, the effective amount of a compound taught herein will vary depending on a variety of factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, etc., but can, in any event, be determined by one of ordinary skill in the art using routine methods. An effective amount of a compound of the present teachings can be readily determined by one of ordinary skill in the art using routine methods known in the art.

[0057] The term "effective amount" means an amount that, when administered to a subject, produces beneficial or desired results, including clinical results, e.g., inhibits, suppresses, or reduces the symptoms of the condition being treated in the subject compared to a control. For example, an effective amount may be provided in a unit dosage form (e.g., 1 mg to about 50 g per day, e.g., 1 mg to about 5 grams per day).

[0058] The term "therapeutically effective amount" of a compound of the present invention refers to an amount of a compound of the present invention that induces a biological or medical response in a subject, such as reducing or inhibiting enzyme or protein activity, or improving symptoms, alleviating a condition, slowing or delaying disease progression, or preventing a disease. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present invention that, when administered to a subject, (1) at least partially alleviates, inhibits, prevents, and / or improves a condition, disorder, or disease (i) mediated by HPK1, (ii) associated with HPK1 activity, or (iii) characterized by HPK1 activity (normal or abnormal); or (2) reduces or inhibits HPK1 activity; or (3) reduces or inhibits HPK1 expression; or (4) alters HPK1 protein levels. In another non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present invention that, when administered to a cell, tissue, or non-cellular biological material, or medium, at least partially reduces or inhibits HPK1 activity; or partially or completely reduces or inhibits HPK1 expression.

[0059] All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") herein is intended merely to better clarify the invention and does not pose a limitation on the scope of the invention as otherwise claimed.

[0060] General chemical terms used in the above formula have their usual meanings. 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), isotonicity agents, absorption delaying agents, 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). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0061] Furthermore, the compounds of the present invention, including their salts, may also be obtained in the form of their hydrates, or may include other solvents used for their crystallization. The compounds of the present invention inherently or by design form solvates with pharmaceutically acceptable solvents (including water), and therefore, the present invention is intended to encompass both solvated and unsolvated forms. The term "solvate" refers to a molecular complex of the compounds of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical field that are known to be harmless to the recipient, such as water, ethanol, etc. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0062] The compounds of the present invention may naturally or by design form polymorphs, including salts, hydrates, and solvates thereof. In another aspect, the present invention provides pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions may be formulated for a particular route of administration, such as oral, parenteral, or rectal administration. Furthermore, the pharmaceutical compositions of the present invention may be made in solid form (including, without limitation, capsules, tablets, pills, granules, powders, or suppositories) or in liquid form (including, without limitation, solutions, suspensions, or emulsions). The pharmaceutical compositions may be subjected to conventional pharmaceutical operations, such as sterilization, and / or may contain inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers.

[0063] Typically, pharmaceutical compositions are tablets or gelatin capsules containing active ingredients together with a) diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants such as silica, talc, stearic acid, its magnesium or calcium salt, and / or polyethylene glycol; for tablets, also c) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if desired, d) disintegrants such as starch, agar, alginic acid or its sodium salt, or effervescent mixture; and / or e) absorbents, coloring agents, flavoring agents and sweeteners.Tablets can be film-coated or enteric-coated by methods known in the art.

[0064] Compositions suitable for oral administration include an effective amount of the compounds 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 can be prepared by any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer-lasting effect.For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used.The preparation for oral use can be presented as a hard gelatin capsule, in which active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as a soft gelatin capsule, in which active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

[0065] Certain injectable compositions are isotonic aqueous solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and / or contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solubility enhancers, salts for adjusting osmotic pressure, and / or buffers. Furthermore, the compositions may also contain other therapeutically valuable substances. The compositions are prepared by conventional mixing, granulating, or coating methods, respectively, and contain about 0.1-75% or about 1-50% of the active ingredient. Compositions suitable for transdermal application include an effective amount of the compound of the present invention with a suitable carrier. Suitable carriers for transdermal delivery include absorbable pharmacologically acceptable solvents that assist passage through the host's skin. For example, transdermal devices are in the form of a bandage comprising a backing material, optionally a reservoir containing the carrier, a rate-controlling barrier to the host's skin for delivering the compound at a controlled, predetermined rate over an extended period of time, and a means for securing the device to the skin.

[0066] For example, compositions suitable for topical application to skin and eyes include aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations, such as for aerosol delivery.Such topical delivery systems are particularly suitable for transdermal application, for example, for the treatment of skin cancer, and for preventative use, such as sun creams, lotions, sprays, etc.Therefore, they are particularly suitable for use in topical formulations, including cosmetic formulations, well known in the art.Such formulations may contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0067] As used herein, topical application can also refer to inhalation or nasal application. They can be conveniently delivered in the form of a dry powder from a powder inhaler (either alone, as a mixture, for example, as a dry blend with lactose, or as mixed component particles, for example, with phospholipids), or in the form of an aerosol spray from a pressurized container, pump, spray, atomizer, or nebulizer, with or without the use of a suitable propellant.

[0068] The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising compounds of the invention as active ingredients, since water can facilitate the degradation of certain compounds. The anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low-moisture ingredients and low-moisture or low-humidity conditions.The anhydrous pharmaceutical compositions can be prepared and stored so that their anhydrous nature is maintained.Therefore, the anhydrous compositions are packaged using known materials that prevent exposure to water so that they can be included in suitable prescription kits.Suitable packaging examples include, but are not limited to, hermetically sealed foils, plastics, unit-dose containers (e.g., vials), blister packs and strip packs.

[0069] The present invention further provides pharmaceutical compositions and dosage forms that comprise one or more agents that reduce the rate at which a compound of the invention as an active ingredient will decompose. Such agents, referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers. 3.Compound In a first embodiment of the present disclosure, provided is a compound of formula I

[0070] [ka]

[0071] or a pharmaceutically acceptable salt or stereoisomer thereof. In a second embodiment of the present disclosure, there is provided a compound of formula IA

[0072] [ka]

[0073] or a pharmaceutically acceptable salt thereof. In a third embodiment of the present disclosure, there is provided a compound of formula IB

[0074] [ka]

[0075] or a pharmaceutically acceptable salt thereof. In a fourth embodiment of the present disclosure, there is provided a compound of formula II

[0076] [ka]

[0077] or a pharmaceutically acceptable salt or stereoisomer thereof. In a fifth embodiment of the present disclosure, there is provided a compound of formula IIA

[0078] [ka]

[0079] or a pharmaceutically acceptable salt thereof. In a sixth embodiment of the present disclosure, there is provided a compound of formula IIB

[0080] [ka]

[0081] or a pharmaceutically acceptable salt thereof. 4. Treatable diseases The HPK1 inhibitors, pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof can be used in methods of modulating (i.e., inhibiting) HPK1 activity, the methods comprising administering an HPK1 inhibitor compound of the present invention, or a pharmaceutically acceptable salt thereof, to a patient / subject in need thereof, as described herein.

[0082] In particular, the present invention provides the use of a compound of the present invention, or a stereoisomer, tautomer, N-oxide, hydrate, solvate, or salt thereof, in particular a pharmaceutically acceptable salt thereof, or a mixture thereof, for use in the treatment or prevention of a disease, in particular cancer (especially hematopoietic and solid tumors), or a condition of immune response dysregulation, or other disorder associated with aberrant 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.

[0083] In certain embodiments, the compounds of the invention, or pharmaceutically acceptable salts thereof, are useful 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, vascular disorders, viral infections, neurodegenerative disorders, inflammatory disorders, and disorders of male fertility control.

[0084] In certain embodiments, the compounds of the invention, or pharmaceutically acceptable salts thereof, are useful for therapeutic administration to enhance, stimulate and / or increase immunity in the treatment of cancer.

[0085] The HPK1 inhibitor compounds of the present invention can be used alone, in combination with other agents or therapies, or as adjuvants or neoadjuvants for the treatment of diseases or disorders, including cancer.

[0086] In certain embodiments, the methods of the present invention are directed to the treatment of cancers including, but not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, cancer of the endometrium, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, and the like. The compounds may be used to treat cancers including cancer of the bladder, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or urethra, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis 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 above cancers.

[0087] In some embodiments, cancers treatable with the compounds of the present invention include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate cancer), breast cancer, triple-negative breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer and small cell lung cancer). In addition, refractory or recurrent malignant tumors whose growth can be inhibited using the compounds of the present invention can also be treated.

[0088] In some embodiments, cancers treatable 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 cancer, brain cancer, eye cancer, thyroid and parathyroid cancer, skin cancer, head and neck cancer, reproductive cancer, gastrointestinal cancer, urinary cancer, glioblastoma, sarcoma, bladder cancer, etc.), blood cancers (e.g., lymphoma, leukemia, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma, or multiple myeloma), sarcoma and distant metastases thereof.

[0089] In some embodiments, diseases and indications treatable using the compounds of the present invention include, but are not limited to, hematological cancers, sarcomas, lung cancer, gastrointestinal cancers, genitourinary cancers, liver cancer, bone cancer, nervous system cancers, gynecological cancers, and skin cancers.

[0090] Exemplary hematological cancers include lymphomas and leukemias, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma, myeloproliferative disorders (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), essential thrombocytosis (ET), myelodysplastic syndromes (MDS), T-cell acute lymphoma (T-ALL), multiple myeloma, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, hairy cell lymphoma, chronic myeloid lymphoma, and Burkitt's lymphoma.

[0091] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdomyosarcoma, fibroma, lipoma, hamartoma, and teratoma.

[0092] Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic lung carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, chondroid hamartoma, and mesothelioma.

[0093] Exemplary gastrointestinal cancers include cancer of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (tubular adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), and colorectal cancer.

[0094] Exemplary genitourinary cancers include cancer 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, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma).

[0095] Exemplary liver cancers include hepatocellular carcinoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma. Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondral exostosis), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor.

[0096] Exemplary nervous system cancers include cancers of the skull (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), meninges (meningioma, meningeal sarcoma, glioma), brain (astrocytoma, meduoblastoma, glioma, ependymoma, germ cell tumor (pinealoma), glioblastoma, glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors) and spinal cord (neurofibroma, meningioma, glioma, sarcoma), as well as neuroblastoma and Lhermitte-Duclos disease.

[0097] Exemplary gynecological cancers include cancer of the uterus (endometrial carcinoma), cervix (cervical carcinoma, preneoplastic cervical dysplasia), ovary (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid rhabdomyosarcoma (embryonic rhabdomyosarcoma)), and fallopian tube (carcinoma).

[0098] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, Merkel cell skin cancer, lentigineous dysplastic nevi, lipoma, hemangioma, dermatofibroma, and keloids. In some embodiments, diseases and indications treatable using the compounds of the present invention include, but are not limited to, sickle cell disease (e.g., sickle cell anemia), triple-negative breast cancer (TNBC), myelodysplastic syndrome, testicular cancer, bile duct cancer, esophageal cancer, and urothelial carcinoma.

[0099] Exemplary head and neck cancers include glioblastoma, melanoma, rhabdomyosarcoma, lymphosarcoma, osteosarcoma, squamous cell carcinoma, adenocarcinoma, oral cavity cancer, laryngeal cancer, nasopharyngeal cancer, nasal cavity and paranasal sinus cancer, and thyroid and parathyroid cancer.

[0100] In some embodiments, the subject HPK1 inhibitors can be used to treat tumors that produce PGE2 (e.g., Cox-2-overexpressing tumors) and / or adenosine (CD73 and CD39-overexpressing tumors). Cox-2 overexpression has been detected in many tumors, such as colorectal, breast, pancreatic, and lung cancer, and is correlated with poor prognosis. COX-2 overexpression has been reported in blood cancer models such as RAJI (Burkitt's lymphoma) and U937 (acute promonocytic leukemia), as well as in patient blast cells. CD73 is upregulated in various human cancers, including colon, lung, pancreatic, and ovarian. Higher CD73 expression levels are associated with tumor angiogenesis, invasiveness, and metastasis, as well as shorter patient survival times in breast cancer.

[0101] Examples of breast cancers that can be treated 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.

[0102] Examples of cancers of the respiratory tract include, but are not limited to, small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of treatable brain cancers include, but are not limited to, brainstem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, glioblastoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumor.

[0103] Treatable tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Treatable tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.

[0104] Treatable ovarian cancers include, but are not limited to, serous tumors, endometriotic tumors, mucinous cystadenocarcinoma, granulosa cell tumors, Sertoli-Leydig cell tumors, and male germ cell tumors.

[0105] Treatable cervical cancers include, but are not limited to, squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, neuroendocrine tumors, hyaloid cell carcinoma, and chorioadenoma carcinoma.

[0106] Treatable tumors of the digestive tract include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers. Treatable esophageal cancers include, but are not limited to, esophageal cell carcinoma and adenocarcinoma, as well as squamous cell carcinoma, leiomyosarcoma, malignant melanoma, rhabdomyosarcoma, and lymphoma.

[0107] Treatable gastric cancers include, but are not limited to, intestinal-type and diffuse-type gastric adenocarcinoma. Treatable pancreatic cancers include, but are not limited to, tubular adenocarcinoma, adenosquamous carcinoma, and pancreatic endocrine tumors.

[0108] Treatable tumors of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, urethral, ​​and human papillary renal carcinoma. Treatable kidney cancers include, but are not limited to, renal cell carcinoma, urothelial cell carcinoma, juxtaglomerular cell carcinoma (nephroma), angiomyolipoma, oncocytoma of the kidney, Bellini duct carcinoma, clear cell sarcoma of the kidney, mesoblastic nephroma, and Wilms' tumor.

[0109] Bladder cancers that can be treated include, but are not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, and small cell carcinoma. Treatable eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

[0110] Treatable liver cancers include, but are not limited to, hepatocellular carcinoma (liver cell carcinoma with or without the fibrolamellar variant), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular-cholangiocarcinoma.

[0111] Treatable skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell carcinoma, and non-melanoma skin cancer. Treatable head and neck cancers include, but are not limited to, squamous cell carcinoma of the head and neck, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, salivary gland cancer, lip and oral cavity cancer, and squamous cell.

[0112] Lymphomas that can be treated include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.

[0113] Treatable sarcomas include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Treatable leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

[0114] In certain embodiments, the compounds of the present invention may be used to treat a variety of other disorders in which MAP4K1 is involved, such as cardiovascular and pulmonary diseases. In certain embodiments, the compounds of the present invention may be used as pharmaceuticals for the treatment and / or prevention of cardiovascular, inflammatory and fibrotic disorders, renal disorders, particularly acute and chronic renal insufficiency, and also acute and chronic renal failure.

[0115] As used herein, the term "renal insufficiency" includes both acute and chronic conditions of renal insufficiency, as well as underlying or associated renal disorders, such as diabetic and non-diabetic nephropathy, hypertensive nephropathy, ischemic nephropathy, renal hypoperfusion, dialysis-induced hypotension, obstructive uropathy, renal stenosis, glomerulopathy, glomerulonephritis (e.g., primary glomerulonephritis; minimal change glomerulonephritis (lipoid nephrosis); membranous glomerulonephritis; focal segmental glomerulosclerosis (FSGS); membranous hyperplasia), and the like. Proliferative glomerulonephritis; crescentic glomerulonephritis; mesangial proliferative glomerulonephritis (IgA nephritis, Berger's disease); post-infectious glomerulonephritis; secondary glomerulonephritis), diabetes mellitus, lupus erythematosus, amyloidosis, Goodpasture's syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, microscopic polyangiitis, acute glomerulonephritis, pyelonephritis (e.g., urinary tract stones, benign prostatic hyperplasia, diabetes mellitus, dysplasia, painkiller abuse, Crohn's disease) nephropathy), glomerulosclerosis, renal arteriolar necrosis, tubulointerstitial disease, kidney damage, e.g., primary and congenital or acquired kidney damage, Alport syndrome, nephritis, immune-mediated kidney damage, e.g., renal transplant rejection and immune complex-induced kidney damage, toxic substance-induced nephropathy, contrast agent-induced nephropathy, diabetic and non-diabetic nephropathy, renal cysts, nephrosclerosis, hypertensive nephrosclerosis and nephrotic syndrome, which may be characterized diagnostically by, e.g., abnormally decreased creatinine and / or water excretion, abnormally elevated blood levels of urea, nitrogen, potassium and / or creatinine, altered activity of kidney enzymes, e.g., glutamyl synthetase, altered urine osmolality or urine volume, hypermicroalbuminuria, macroalbuminuria, glomerular and arteriolar lesions, ureteral dilation, hyperphosphatemia and / or the need for dialysis.

[0116] In certain embodiments, the compounds of the present invention may be used to treat and / or prevent sequelae of renal insufficiency, such as pulmonary edema, heart failure, uremia, anemia, electrolyte abnormalities (e.g., hypercalemia, hyponatremia), and disturbances of bone and glucose metabolism.

[0117] In certain embodiments, the compounds of the present invention may be used to treat and / or prevent sequelae of renal insufficiency, such as pulmonary edema, heart failure, uremia, anemia, electrolyte abnormalities (e.g., hyperkalemia, hyponatremia), and disturbances of bone and glucose metabolism.

[0118] In certain embodiments, the compounds of the present invention are further suitable for the treatment and / or prevention of polycystic kidney disease (PCKD) and syndrome of inappropriate ADH secretion (SIADH). In certain embodiments, the compounds of the present invention are also useful in treating 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 conduction disorders, such as atrioventricular block grades 1-3 (AB block grades 1-3), supraventricular tachyarrhythmias, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular flutter, ventricular tachyarrhythmias, polymorphic ventricular tachycardia, atrial and ventricular premature contractions, AV junctional premature contractions, sick sinus syndrome, collapse, AV nodal reentrant tachycardia, Wolff-Parkinson-White syndrome, acute coronary syndrome (ACS), autoimmune heart disorders (pericarditis, endocarditis, valvulitis, aortitis, cardiomyopathies), shock, such as cardiogenic shock, septic shock, and anaphylactic shock, aneurysms, boxer cardiomyopathy (ventricular and / or prevention of premature extravascular contractions (PVCs), thromboembolic disorders and ischemia, such as myocardial ischemia, myocardial infarction, stroke, cardiac hypertrophy, transient ischemic attacks, inflammatory cardiovascular disorders, spasm of coronary and peripheral arteries, edema formation, such as pulmonary edema, cerebral edema, renal edema or edema caused by heart failure, peripheral circulatory disorders, reperfusion injury, arterial and venous thrombosis, myocardial failure, endothelial dysfunction, such as restenosis after thrombolytic therapy, percutaneous transluminal coronary angioplasty (PTA), percutaneous transluminal coronary angioplasty (PTCA), heart transplantation and bypass surgery, and also for the prevention of micro- and macrovascular damage (vasculitis), high levels of fibrinogen and low-density lipoprotein (LDL) and high concentrations of plasminogen activator inhibitor 1 (PAI-1), and also for the treatment and / or prevention of erectile dysfunction and female sexual dysfunction.

[0119] In certain embodiments, the compounds of the present invention are also suitable for the treatment and / or prevention of asthma disorders, pulmonary arterial hypertension (PAH) and left heart disease, HIV, sickle cell anemia, thromboembolism (CTEPH), sarcoidosis, other forms of pulmonary hypertension (PH) including COPD or pulmonary fibrosis-associated pulmonary hypertension, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), acute lung injury (ALI), alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, emphysema (e.g., cigarette smoke-induced emphysema) and cystic fibrosis (CF).

[0120] In certain embodiments, the compounds of the present invention are also effective in controlling central nervous system disorders characterized by disruptions of the NO / cGMP system. The compounds of the invention are particularly suitable for improving perception, concentration, learning or memory following cognitive impairment such as that occurring in connection with environmental / disease / syndrome, for example, mild cognitive impairment, age-related learning and memory impairment, age-related memory loss, vascular dementia, craniocerebral trauma, stroke, dementia occurring after stroke (post-stroke dementia), post-traumatic craniocerebral injury, general concentration loss, childhood concentration loss with learning and memory problems, Alzheimer's disease, Lewy body disease, Pick's syndrome, dementia due to frontal lobe degeneration including Parkinson's disease, corticobasal degeneration, amyotrophic lateral sclerosis (ALS), Huntington's disease, demyelination, multiple sclerosis, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia or progressive dementia due to Korsakoff's psychosis.

[0121] In certain embodiments, the compounds of the present invention are also suitable for the treatment and / or prevention of central nervous system disorders, such as states of anxiety, tension and depression, CNS-related dysfunction and sleep disorders, and for controlling pathological disturbances in the intake of food, stimulants and addictive substances.

[0122] In certain embodiments, the compounds of the invention are also suitable for regulating cerebral blood flow and therefore represent effective agents for controlling migraine headaches. In certain embodiments, the compounds of the present invention are also suitable for the prevention and control of the sequelae of cerebral infarction (stroke), such as stroke, cerebral ischemia, and craniocerebral trauma. The compounds according to the present invention can also be used to control pain and tinnitus conditions.

[0123] In certain embodiments, the compounds of the present invention have anti-inflammatory properties and may 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, rheumatic disorders, inflammatory skin disorders, and inflammatory eye disorders.

[0124] In certain embodiments, the compounds of the present invention may also be used for the treatment and / or prevention of autoimmune diseases. In certain embodiments, the compounds of the invention are also suitable for the treatment and / or prevention of fibrotic disorders of the internal organs, such as the lungs, heart, kidneys, bone marrow and especially the liver, and also fibrosis of the skin and fibrotic eye disorders.

[0125] As used herein, the term "fibrotic disorder" includes, inter alia, hepatic fibrosis, cirrhosis of the liver, pulmonary fibrosis, endomyocardial fibrosis, nephropathy, glomerulonephritis, renal interstitial fibrosis, fibrotic lesions resulting from diabetes, myelofibrosis and similar fibrotic disorders, scleroderma, morphea, keloids, hypertrophic scars (also following surgical procedures), nevi, diabetic retinopathy, proliferative vitreoretinopathy and disorders of connective tissue (e.g., sarcoidosis).

[0126] In certain embodiments, the compounds of the present invention are also suitable for controlling post-operative scarring, for example as a result of glaucoma surgery. In certain embodiments, the compounds of the present invention can also be used cosmetically on aging and keratinized skin.

[0127] In certain embodiments, the compounds of the present invention are suitable for the treatment and / or prevention of hepatitis, neoplasms, osteoporosis, glaucoma and gastroparesis. In certain embodiments, the compounds of the present invention are suitable for the treatment and / or prevention of viral infections (e.g., HIV and Kaposi's sarcoma), inflammatory autoimmune diseases (e.g., colitis, arthritis, Alzheimer's disease, glomerulonephritis and wound healing), bacterial, fungal and / or parasitic infections; skin diseases (e.g., psoriasis), hyperplasia-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).

[0128] In another embodiment, the compounds of the present invention can also be used to treat or prevent uterine fibroids (uterine leiomyoma or uterine fibroids) in women. Uterine fibroids are benign tumors of the myometrium, the smooth muscle layer of the uterus. Uterine fibroids grow slowly throughout a woman's life, and their growth depends on the female hormones estradiol and progesterone. Therefore, the highest prevalence of uterine fibroids is approximately 70% and >80% in Caucasian and African-American women, respectively, from age 35 until menopause, when fibroids regress due to declining hormone levels. Approximately 30% and 45% of Caucasian and African-American women, respectively, exhibit heavy menstrual bleeding and pain associated with the menstrual cycle as clinically relevant symptoms attributable to fibrosis (David et al., Eur J Obstet Gynecol Reprod Biol. 199:137-140, 2016). In this regard, heavy menstrual bleeding is defined as blood loss of more than 80 mL during menstrual bleeding. The submucosal location of uterine fibroids, e.g., those located just beneath the endometrium, appears to have an even more severe effect on uterine bleeding, which can lead to anemia in affected women. Furthermore, uterine fibroids, with respect to their symptoms, have a serious impact on the quality of life of affected women.

[0129] In certain embodiments, the compounds of the present invention are useful for the treatment and / or prevention of chronic kidney disease, acute and chronic renal insufficiency, diabetes, inflammatory or hypertensive nephropaties, fibrotic disorders, cardiac dysfunction, angina pectoris, hypertension, pulmonary hypertension, ischemia, vascular disorders, thromboembolic disorders, arteriosclerosis, sickle cell anemia, erectile dysfunction, benign prostatic hyperplasia, urinary disorders associated with benign prostatic hyperplasia, Huntington's, dementia, Alzheimer's and Creutzfeldt-Jakob disease.

[0130] The present invention provides methods for the treatment and / or prevention of chronic kidney disease, acute and chronic renal insufficiency, diabetes, inflammatory or hypertensive nephropaties, fibrotic disorders, cardiac dysfunction, angina pectoris, hypertension, pulmonary hypertension, ischemia, vascular disorders, thromboembolic disorders, arteriosclerosis, sickle cell anemia, erectile dysfunction, benign prostatic hyperplasia, voiding disorders associated with benign prostatic hyperplasia, Huntington's, dementia, Alzheimer's and Creutzfeldt-Jakob disease.

[0131] The present invention further provides the use of the compounds according to the invention for the treatment and / or prevention of disorders, in particular the disorders mentioned above. The present invention further provides a method for the treatment and / or prevention of disorders, in particular the disorders mentioned above, using an effective amount of at least one of the compounds according to the invention.

[0132] Thus, the compounds of the present invention can be utilized to inhibit, block, reduce, or decrease MAP4K1 activity by activating exogenous and / or endogenous ligands for reducing tumor growth and modulating dysregulated immune responses, for example, by blocking immunosuppression and increasing immune cell activation and infiltration in the context of cancer and cancer immunotherapy. The 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, effective to treat the disorder.

[0133] The present invention also provides methods for treating a variety of other disorders in which MAP4K1 is involved, including, but not limited to, disorders caused by dysregulated immune responses, inflammation, infectious disease & cancer vaccination, viral infection, obesity and diet-induced obesity, metabolic disorders, fatty liver, and uterine fibroids. These disorders are well characterized in humans, but also exist in other mammals with similar etiologies and can be treated by administering the pharmaceutical compositions of the present invention. 5. Combination therapy The compounds of the present invention may be used in combination therapy with one or more additional / secondary therapeutic agents suitable to treat the diseases or indications treatable by the subject compounds.

[0134] Thus, in certain embodiments, for example, methods of the invention using compounds of the invention may include administering to a subject in need thereof an additional therapeutic agent. The additional therapeutic agent may be (i) an immunomodulatory agent 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 an antibody specific for a protein expressed by tumor cells; and / or (iii) a composition comprising a tumor antigen or an immunogenic fragment thereof; and / or (iv) a chemotherapeutic agent.

[0135] In certain embodiments, the second therapeutic agent comprises 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 beta-catenin pathway, an inhibitor of the Notch pathway, an inhibitor of the Hedgehog pathway, an inhibitor of Pim kinase, and / or an inhibitor of protein chaperones and cell cycle progression. In certain embodiments, the combination therapy of the present invention reduces the likelihood of drug resistance developing in a cell population and / or reduces the toxicity of treatment.

[0136] In certain embodiments, the HPK1 inhibitor compounds of the present invention are useful in treating cancer by inhibiting the following kinases: 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-II, KDR / F The present invention may be used in combination with one or more inhibitors of LK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR / Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK, and B-Raf.

[0137] In certain embodiments, the HPK1 inhibitor compounds of the present invention are FGFR inhibitors (FGFR1, FGFR2, FGFR3 or FGFR4, e.g., AZD4547, BAY1187982, ARQ087, BGJ398, BIBF1120, TKI258, lucitanib, dovitinib, TAS-120, J J-42756493, Debiol347, INCB54828, INCB62079 and INCB63904), JAK inhibitors (JAK1 and / or JAK2, e.g., ruxolitinib, baricitinib or itacitinib (INCB39110)), IDO inhibitors (e.g., epacadostat and NLG919), LSD1 inhibitors (e.g., GSK2979552, INCB59872 and INCB60003), TDO inhibitors, PI3K-delta inhibitors (e.g., , INCB50797 and INCB50465), PI3K-gamma inhibitors, e.g., PI3K-gamma selective inhibitors, CSF1R inhibitors (e.g., PLX3397 and LY3022855), TAM receptor tyrosine kinase (Tyro-3, Axl and Mer), aryl hydrocarbon receptor (AhR) modulators (e.g., laquinimod, aminoflavone, 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), aminoflavone, NKI150460, indole-3-carbinol, β-naphthoflavone and its dimer, diindolylmethane (DIM), 4-hydroxytamoxifen, leflunomide, raloxifene, tranilast, flutamide, mexiletine, nimodiphine, omeprazole, sulindac, tranilast and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)), angiogenesis inhibitors, interleukin receptor inhibitors, bromo and specific terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors, e.g., OTX015, CPI-0610, INCB54329 and INCB57643), and adenosine receptor antagonists or combinations thereof, for the treatment of cancer.

[0138] In certain embodiments, HPK1 inhibitor compounds of the present invention may be combined with inhibitors of HDAC, such as panobinostat and vorinostat. In certain embodiments, HPK1 inhibitor compounds of the present invention may be combined with inhibitors of c-Met, such as onartumzumab, tivantnib, and capmatinib (INC-280).

[0139] In certain embodiments, HPK1 inhibitor compounds of the present invention may be combined with inhibitors of BTK, such as ibrutinib. In certain embodiments, HPK1 inhibitor compounds of the present invention may be combined with inhibitors of mTOR, such as rapamycin, sirolimus, temsirolimus, and everolimus.

[0140] In certain embodiments, HPK1 inhibitor compounds of the present invention may be combined with inhibitors of MEK, such as trametinib, selumetinib, and GDC-0973. In certain embodiments, HPK1 inhibitor compounds of the present invention may be used in combination with inhibitors of Hsp90 (e.g., tanespimycin), cyclin-dependent kinases (e.g., palbociclib), PARP (e.g., olaparib), and Pim kinases (LGH447, INCB053914, and SGI-1776).

[0141] 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.

[0142] The cGAS (cyclic GMP-AMP synthase)-STING (Stimulator of Interferon Genes) pathway is a component of the innate immune system that detects the presence of cytosolic DNA and, in response, triggers the expression of inflammatory genes, which can lead to the activation of senescence or defense mechanisms. The localization of DNA from its normal nuclear localization to the cytosol is associated with tumorigenesis or viral infection. cGAS is found in the cytosol, and upon direct binding to cytosolic DNA, cGAS dimerizes and catalyzes the production of 2'3'-cGAMP from ATP and GTP. The resulting cGAMP then binds to STING, acting as a second messenger that activates the transcription factor IRF3. IRF3 activation results in the transcription of type 1 IFN-β, which in turn activates numerous downstream target genes, initiating diverse biological responses, such as viral response, tumor surveillance, autoimmunity, and cellular senescence. In many tumor cells, a constitutively active DNA damage response leads to the accumulation of extranuclear DNA and activation of the cGAS / STING pathway. In lymphoma cells, the NKG2D ligand Rae1 has been shown to be upregulated in a STING / IRF3-dependent manner and aid in 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 boost antitumor immunity.

[0143] In certain embodiments, the HPK1 inhibitor compounds of the present invention may be used in combination with one or more immune checkpoint inhibitors. The activation of effector T cells is usually triggered by TCR-recognized antigen peptides presented by MHC complexes. The type and level of activation achieved is then determined by the balance between signals that stimulate effector T cell responses and signals that inhibit them. "Immune system checkpoint" is used herein to refer to any molecular interaction that changes the balance to favor the inhibition of effector T cell responses. That is, it is a molecular interaction that, when it occurs, negatively regulates the activation of effector T cells. Such an interaction can be direct, such as the interaction between a ligand that transmits an inhibitory signal to effector T cells and a cell surface receptor. Alternatively, the interaction can be indirect, such as blocking or inhibiting the interaction between a ligand that otherwise transmits an activation signal to effector T cells and a cell surface receptor, or promoting the upregulation of inhibitory molecules or cells, or enzymatic depletion of metabolic products required by effector T cells, or any combination thereof.

[0144] Examples of immune system checkpoints include a) the interaction between indoleamine 2,3-dioxygenase (IDO1) and its substrate; b) the interaction between PD1 and PD-L1 and / or PD1 and PD-L2; c) the interaction between CTLA-4 and CD86 and / or CTLA-4 and CD80; d) the interaction between B7-H3 and / or B7-H4 and their respective ligands; e) the interaction between HVEM and BTLA; f) the interaction between GAL9 and TIM3; g) the interaction between MHC class I or II and LAG3; and h) the interaction between MHC class I or II and KIR; i) the interaction between OX40 (CD134) and OX40L (CD252); j) the interaction between CD40 and CD40L (CD154); k) the interaction between 4-1BB (CD137) and ligands including 4-1BBL; and l) the interaction between GITR and ligands including GITRL.

[0145] Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules, such as CD20, CD27, CD28, CD39, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, 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.

[0146] A representative checkpoint for purposes of the present invention is checkpoint (b), i.e., the interaction between PD1 and either of its ligands, PD-L1 and PD-L2. PD1 is expressed on effector T cells. Engagement with either ligand results in a signal that downregulates activation. The ligands are expressed by some tumors. PD-L1, in particular, is expressed by many solid tumors, including melanoma. These tumors may therefore downregulate immune-mediated antitumor effects through activation of the inhibitory PD-1 receptor on T cells. Blocking the interaction between PD1 and one or both of its ligands can ablate a checkpoint in the immune response, resulting in an increased antitumor T cell response. Thus, PD1 and its ligands are examples of components of immune system checkpoints that can be targeted in the methods of the present invention.

[0147] Another checkpoint for purposes of the present invention is checkpoint (c), i.e., the interaction between the T cell receptor CTLA-4 and its ligand, the B7 protein (B7-1 and B7-2). CTLA-4 is normally upregulated on the T cell surface after initial activation, and ligand binding results in a signal that inhibits further / continuous activation. CTLA-4 competes with the receptor CD28, also expressed on the T cell surface but which upregulates activation, for binding to the B7 protein. Thus, blocking CTLA-4 interaction with B7 protein, but not CD28 interaction with B7 protein, eliminates one of the checkpoints of the normal immune response and may result in an increased antitumor T cell response. CTLA-4 and its ligand are therefore examples of components of immune system checkpoints that can be targeted in the methods of the present invention.

[0148] In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137.

[0149] 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, TIM3, SIGLEC7, SIGLEC9, and VISTA, and their binding partners (e.g., PD-L1 and PD-L2).

[0150] In some embodiments, the compounds provided herein may be used in combination with one or more agents selected from a KIR inhibitor, a TIGIT inhibitor, a LAIR1 inhibitor, a CD160 inhibitor, a 2B4 inhibitor, and a TGFR beta inhibitor.

[0151] In some embodiments, the compounds provided herein can be used in combination with immune checkpoint inhibitors, typically small organic molecules, small molecule inhibitors (SMIs). For example, in certain embodiments, IDO1 inhibitors include epacadostat (INCB24360), indoximod, GDC-0919 (NLG919) and F001287. Other IDO1 inhibitors include 1-methyltryptophan (1MT).

[0152] In some embodiments, inhibitors of immune checkpoint molecules, also known as "immunomodulatory agents," include any agent that, when administered to a subject, blocks or inhibits the action of an immune system checkpoint and upregulates an immune effector response in the subject, typically a T cell effector response, which may include an anti-tumor T cell effector response.

[0153] The immunomodulatory agent used in the methods of the present invention may block or inhibit any of the above immune system checkpoints. The agent may be an antibody or any other suitable agent that effects such blockage or inhibition. Thus, the agent is generally referred to as an inhibitor of the above checkpoint.

[0154] As used herein, "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") 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(ab')2 ... d Antigen-binding portions of antibodies include fragments, Fv fragments, dAb fragments, and isolated complementarity-determining regions (CDRs). Single-chain antibodies such as scFvs, and heavy-chain antibodies such as VHHs and camelid antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody.

[0155] In certain embodiments, the immunomodulatory agent used in conjunction with the HPK1 inhibitors 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. In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1, such as an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab (MDX-1106), pembrolizumab (Merck3475 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-PD-1 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-L2 F, which binds to PD-1). c fusion protein) or AUNP-12 (anti-PD-1 peptide).

[0156] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, such as 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 antibody includes atezolizumab, avelumab, durvalumab, or MEDI-4736 and MPDL3280A.

[0157] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab, or any of the antibodies disclosed in WO2014 / 207063 (incorporated herein by reference). Other molecules include polypeptides or soluble mutant CD86 polypeptides. In certain embodiments, the antibody is ipilumumab.

[0158] In certain embodiments, the inhibitor of an immune checkpoint molecule is a combination of two or more of the modulators described herein, for example, 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.

[0159] In some embodiments, the inhibitor of an immune checkpoint molecule is an antibody that blocks or inhibits the interaction between 4-1BB and its ligand, including utomilumab. In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CSF1R, such as an anti-CSF1R antibody. In some embodiments, the anti-CSF1R antibody is IMC-CS4 or RG7155.

[0160] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of LAG3, such as an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016, LAG525, IMP321, or GSK2831781.

[0161] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of GITR, such as an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518, MK-4166, MK1248, BMS-986156, MEDI1873, or GWN323.

[0162] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of OX40, such as 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.

[0163] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIM3, e.g., an anti-TIM3 antibody. In some embodiments, the anti-TIM3 antibody is MBG-453.

[0164] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD20, e.g., an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is obinutuzumab or rituximab.

[0165] 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 IDO1, TDO or arginase.Examples of IDO1 inhibitors include epacadostat and NGL919.Examples of arginase inhibitors are CB-1158.

[0166] In some embodiments, the compounds of the invention may be used in combination with bispecific antibodies, in which one domain targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3, CD137, ICOS, CD3, or a TGFβ receptor.

[0167] In some embodiments, the compounds of the present invention may be used in combination with one or more agents for the treatment of diseases 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, ethyleneamine derivatives, alkylsulfonates, nitrosoureas and triazenes, uracil mustard, chlormethine, cyclophosphamide (Cytoxan™), 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).

[0168] The compounds of the present disclosure can also be used in combination with other methods of treating cancer, for example, by chemotherapy, radiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, or surgery. Examples of immunotherapies include cytokine treatment (e.g., interferon, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccines, monoclonal antibodies, adoptive T-cell transfer, oncolytic virotherapy, and immunomodulatory small molecules, including thalidomide or JAK1 / 2 inhibitors.

[0169] The compounds of the invention can be administered in combination with one or more anti-cancer drugs, such as chemotherapeutic agents, including abarelix, abiraterone, afatinib, aflibercept, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, axitinib, azacitidine, bevacizumab, bexarotene, baricitinib, bicalutamide, bleomycin, bortezomib, brivanib, bupallisib, intravenous busulfan, oral busulfan, calsterone, capecitabine, carboplatin, and cyclosporine. Lumustine, cediranib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dacomitinib, dactinomycin, dalteparin sodium, dasatinib, dactinomycin, daunorubicin, decitabine, degarelix, denileukin, denileukin diftitox, deoxycoformycin, dexrazoxane, docetaxel, doxorubicin, droloxafine, dromostanolone propionate, 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, ibritumomab tiuxetan, idarubicin, idelalisib, ifosfamide, imatinib mesylate , interferon alpha 2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mithramycin, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, navelbene, necitumumab, nelarabine, neratinib, nilotinib, nilutamide,Nofetumomab, goserelin, oxaliplatin, paclitaxel, pamidronate, panitumumab, pazopanib, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, piralalisib, pipobroman, plicamycin, ponatinib, prednisone, procarbazine, quinacrine, rasburicase, regorafenib, reloxafine, rituximab, ruxolitinib, sorafenib Any of the following may be used: phenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, tegafur, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, triptorelin, uracil mustard, valrubicin, vandetanib, vinblastine, vincristine, vinorelbine, vorinostat, and zoledronate.

[0170] Other anti-cancer agents include antibody therapeutics such as trastuzumab (Herceptin), antibodies against costimulatory molecules such as CTLA-4 (e.g., ipilimumab or tremelimumab), 4-1BB, antibodies against PD-1 and PD-L1, or antibodies against cytokines (IL-10, TGF-β, etc.). Examples of antibodies against PD-1 and / or PD-L1 that can be combined with compounds of the present disclosure for the treatment of cancer or infections, such as viral, bacterial, fungal, and parasitic infections, include, but are not limited to, nivolumab, pembrolizumab, MPDL3280A, MEDI-4736, and SHR-1210.

[0171] Other anticancer agents include the inhibitor of kinase-related cell proliferation disorders.These kinases include but are not limited to Aurora A, CDK1, CDK2, CDK3, CDK5, CDK7, CDK8, CDK9, ephrin receptor kinase, CHK1, CHK2, SRC, Yes, Fyn, Lck, Fer, Fes, Syk, Itk, Bmx, GSK3, JNK, PAK1, PAK2, PAK3, PAK4, PDK1, PKA, PKC, Rsk and SGK.

[0172] Other anti-cancer 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 also be used in combination with one or more anti-inflammatory agents, steroids, immunosuppressants, or therapeutic antibodies.

[0173] In some embodiments, the compounds of the invention may be used in combination with additional therapeutic agents that directly stimulate immune effector responses, such as cytokines, or tumor-specific adoptively transferred T cell populations, or antibodies specific for proteins expressed by tumor cells.

[0174] 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 either), a tumor-specific adoptively transferred T cell population, or an antibody specific for a protein expressed by tumor cells.

[0175] The cytokine can be an interferon selected from IFNα, ΙΡΝβ, IFNγ, and IFNA, or an interleukin such as IL-2. The chemokine can be an inflammatory mediator selected from CXCL9, 10, and 11, which attracts CXCR3-expressing T cells. Agents that stimulate cytokine or chemokine production can be suitable adjuvants for administration to humans. One example is bacillus Calmette-Guerin (BCG), which is typically administered intravesically (i.e., via a urethral catheter) for the treatment of bladder cancer. The typical dosage regimen for BCG for bladder cancer is once weekly for six weeks, but given its long-standing safety record, it can also be administered indefinitely for maintenance. BCG has been shown to stimulate immune responses to bladder cancer. BCG has also been used as an adjuvant in combination with compositions containing tumor antigens (i.e., cancer vaccines), particularly for colon cancer, when typically administered intradermally. Such use of BCG is also envisioned in the present invention. Tumor-specific adoptive transfer T cell populations directly increase the size of tumor-specific T cell populations in individuals and can be generated by any suitable means. However, typically, the method involves isolating tumor-specific T cells from tumor samples obtained from patients, selectively culturing these cells, and then returning the expanded population of tumor-specific T cells to the patient. Alternatively, tumor-specific T cell populations can be generated by genetic manipulation of T cell receptor loci followed by the proliferation of degenerated cells.

[0176] Antibodies specific for proteins expressed by tumor cells typically stimulate immune activity by binding to tumor cells and promoting their destruction via antibody-dependent cell-mediated cytotoxicity (ADCC). Examples of this type of antibody include anti-CD20 antibodies, such as ofatumumab or rituximab, and anti-CD52 antibodies, such as alemtuzumab.

[0177] Thus, in certain exemplary embodiments, the compounds of the present invention may be used in combination with calcineurin inhibitors, such as cyclosporin A or FK506; mTOR inhibitors, such as rapamycin, 40-0-(2-hydroxyethyl)-rapamycin, biolimus-7, or biolimus-9; ascomycins with immunosuppressive properties, such as ABT-281, ASM981; corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic acid or salts; mycophenolate mofetil; IL-1β inhibitors.

[0178] In another embodiment, a compound of the invention is used in combination with a co-agent that is a PI3 kinase inhibitor. In another embodiment, a compound of the invention is used in combination with a co-agent that affects BTK (Bruton's tyrosine kinase).

[0179] For the treatment of oncological diseases, the compounds of the present invention may be used in combination with B-cell modulators, such as rituximab, BTK or Syk inhibitors, inhibitors of PKC, PI3 kinase, PDK, PIM, JAK and mTOR, and BH3 mimetics.

[0180] In some embodiments, the compounds of the present invention, including salts thereof, may be combined with another immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune-stimulating cytokines. Non-limiting examples of tumor vaccines that may be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MARTI, and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

[0181] In some embodiments, the compounds of the present invention or salts thereof can also be used in combination with vaccination protocols for cancer treatment. In some embodiments, tumor cells are transduced to express GM-CSF. In some embodiments, tumor vaccines include proteins from viruses associated with human cancer, 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 disclosure 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 salts thereof can be used in combination with dendritic cell immunization to activate potent anti-tumor responses.

[0182] In some embodiments, the compounds of the present invention can be used in combination with bispecific macrocyclic peptides that target Feα or Feγ receptor-expressing effector cells to tumor cells. The compounds of the present invention can also be used in combination with macrocyclic peptides that activate host immune responsiveness.

[0183] In some embodiments, the compounds of the present invention may be used in combination with bone marrow transplantation for the treatment of various tumors of hematopoietic origin. Suitable antiviral agents contemplated for use in combination with the compounds of the invention include nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, and other antiviral agents. Examples of suitable NRTIs include zidovudine (AZT); didanosine (ddl); zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir (1592U89); adefovir pivoxil [bis(POM)-PMEA]; lobucavir (BMS-180194); BCH-10652; emtricitabine [(-)-FTC]; beta-L-FD4 (also known as beta-L-D4C, designated beta-L-2',3'-dideoxy-5-fluoro-cytidene); DAPD ((-)-beta-D-2,6-diamino-purine dioxolane); and rhodenosine (FddA). Exemplary suitable NNRTIs include nevirapine (BI-RG-587); delaviradine (BHAP, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442 (1-(ethoxymethyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione); and (+)-calanolide A (NSC-675451) and B. Exemplary suitable protease inhibitors Agents include saquinavir (Ro31-8959); 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.

[0184] It will be appreciated that many of the additional therapeutic agents used in the methods of the invention may be biologics requiring intravenous, intraperitoneal, or depot administration, hi a further embodiment, the compound of the invention is administered orally and the additional therapeutic agent is administered parenterally, e.g., intravenously, intraperitoneally, or as a depot.

[0185] In any of the combination therapies described herein, when two or more pharmaceutical agents are administered to a patient, they may be administered simultaneously, separately, sequentially, or in combination (e.g., in the case of three or more agents).

[0186] In one embodiment, the invention provides a product comprising a compound of the invention, e.g., the subject compound or any subgroup 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 invention or any subgroup thereof and the other therapeutic agent together in the same pharmaceutical composition, or the subject compound or any subgroup thereof and the other therapeutic agent in separate forms, e.g., in the form of a kit.

[0187] In one embodiment, the present invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a subject compound and another of which contains a second therapeutic agent as discussed herein. In one embodiment, the kit includes a means for separately retaining the compositions, such as a container, a divided bottle, or a divided foil packet. An example of such a kit is a blister pack typically used for packaging tablets, capsules, and the like. The kit of the present invention can be used, for example, to administer different oral and parenteral dosage forms, to administer the separate compositions at different dosage intervals, or to adjust the separate compositions to each other. To aid compliance, the kit of the present invention typically includes administration instructions. 6. Compound Screening / Assay Methods The compounds of the present invention inhibit the kinase activity of HPK1, which can be assayed directly using a number of biochemical assays, such as the assay described in Example 1. The IC50 value of any compound can be determined accordingly over a range of inhibitor concentrations. Furthermore, the inhibitory effect of a compound can also be assessed using a biological assay to determine the effect of the compound on cytokine secretion by T cells after TCR and CD28 stimulation.

[0188] For example, Example 5 describes such a functional assay to determine the effect of HPK1 inhibitors on IL-2 and IFN-γ release upon stimulation of pan T cells. Secreted IL-2 and IFN-γ can be measured / quantified by standard ELISA assays. Briefly, pan T cells can be isolated from peripheral blood (PB) mononuclear cells (MNCs) or PBMCs using commercially available kits 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, as well as several gamma / delta T cell subsets. Pan T cells can be isolated using a column-free negative immunomagnetic separation technique.

[0189] The isolated pan T cells were plated at 100,000 cells / well in a 96-well plate and stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies, or PMA / ionomycin as a positive control (or culture medium as a negative control). Different concentrations of test compounds were added to the cells to assess the effect of the compounds on cytokine secretion after TCR stimulation / CD28 costimulation. The stimulated cells were further incubated for two days, after which the supernatant (containing cytokines secreted by the pan T cells) was collected from each well for ELISA assay and quantification of IL-2 and IFN-γ.

[0190] Additional assays can be used to evaluate the ability of any HPK1 inhibitor to inhibit HPK1 or to screen for compounds with HPK1 inhibitory activity. For example, in one assay, inhibition of HPK1 kinase activity can be assayed using a Treg assay (regulatory T cell proliferation assay) as described below. + / CD25 - T cells and CD4 + / CD25 + Regulatory T cells are isolated from peripheral blood mononuclear cells (PBMCs) from humans using a suitable kit, such as that from Thermo Fisher Scientific (Cat. No. 11363D). + / CD25 - T cells are labeled with CFSE (Thermo Fisher Scientific, C34554) according to the protocol provided by the vendor. CFSE-labeled T cells and CD4 + / CD25 + Regulatory T cells, 1 x 10 in RPMI-1640 medium 6 Resuspend 100 μL of CFSE-labeled T cells in 50 μL of CD4 + / CD25 + Regulatory T cells are mixed with or without the compound and treated with 5 μL of anti-CD3 / CD28 beads (Thermo Fisher Scientific, 11132D) and various concentrations of compound diluted in 50 μL of RPMI-1640 medium. The mixed cell populations are cultured for 5 days (37°C, 5% CO2), and proliferation of CFSE-labeled T cells is analyzed on day 5 using a BD LSRFortessa X-20 using the FITC channel. Inhibition of HPK1 by the subject compounds enhances Treg function and inhibits proliferation of CFSE-labeled primary CD4 + / CD25 - It is predicted to inhibit T cell proliferation.

[0191] In another example, inhibition of HPK1 kinase activity can be assayed using the p-SLP-76 S376 HTRF assay (Cisbio), as described below. This HTRF cell-based assay allows for rapid, quantitative detection of SLP-76 phosphorylated at serine 376 by HPK1. Phospho-SLP-76 creates the scaffold on which important signaling complexes are assembled and is a marker of T lymphocyte activation. According to the manufacturer, the phospho-SLP-76 (Ser376) assay uses two antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody is specialized for binding to the phosphorylated S376 motif in SLP-76, while the second antibody is specialized for its ability to recognize SLP-76 independently of its phosphorylation state. Protein phosphorylation allows immune complex formation involving both labeled antibodies, which brings the donor fluorophore into close proximity with the acceptor, thereby generating a FRET signal. The intensity is directly proportional to the concentration of phosphorylated protein present in the sample, providing a means of assessing the phosphorylation state of proteins in a no-wash assay format.

[0192] Briefly, Jurkat cells (cultured in RPMI1640 medium containing 10% FBS) were harvested and centrifuged, followed by 3 × 10 6The cells are resuspended in the appropriate medium at 100x cells / mL. Jurkat cells (35 μL) are then added to each well of a 384-well plate. Test compounds are diluted 40-fold with cell culture medium (1 μL of compound is added to 39 μL of cell culture medium). Jurkat cells in the well plate are treated with various concentrations of test compounds (5 μL of diluted compound is added to 35 μL of Jurkat cells, starting at 3 μM for a 1:3 dilution) for 1 hour at 37°C, 5% CO2, followed by treatment with anti-CD3 (5 μg / mL, OKT3 clone) for 30 minutes to activate TCR and HPK1. A 1:25 dilution of 100x blocking reagent (from the p-SLP76 ser376HTRF kit) with 4x lysis buffer (LB) is prepared, and 15 μL of 4x LB buffer containing the blocking reagent is added to each well. The cells are incubated at room temperature for 45 minutes with gentle shaking. Cell lysates (16 μL) are added to a Greiner white plate, treated with p-SLP76 Ser376 HTRF reagent (2 μL donor, 2 μL acceptor), and incubated overnight at 4°C. The next day, homogeneous time-resolved fluorescence (HTRF) is measured using a PHERAstar plate reader. IC50 determinations are performed by fitting a logarithmic curve of percent inhibition versus inhibitor concentration using GraphPad Prism 5.0 software.

[0193] Any of the above assays can be expanded to larger scale or high throughput screening (HTS). Any of the above assays can be used to determine IC50 values ​​for the subject compounds. 7. Pharmaceutical Compositions 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.

[0194] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that aid in the formulation and / or administration to and / or absorption by a subject of an active agent and can be included in the compositions of the present disclosure without causing significant adverse toxicological effects to the subject. Non-limiting examples of pharmaceutically acceptable carriers and excipients include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohol, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations may be sterilized and, if desired, may be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for regulating osmotic pressure, buffers, coloring and / or aromatic substances, which do not adversely react with or interfere with the activity of the compounds provided herein. Those skilled in the art will recognize that other pharmaceutical carriers and excipients are suitable for use with the disclosed compounds.

[0195] These compositions optionally further comprise one or more additional therapeutic agents. Alternatively, the compounds of the present invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic regimens (e.g., Gleevec or other kinase inhibitors, interferon, bone marrow transplant, farnesyltransferase inhibitors, bisphosphonates, thalidomide, cancer vaccines, hormone therapy, antibodies, radiation, etc.). For example, the additional therapeutic agent for co-administration or inclusion in a pharmaceutical composition comprising a compound of the present invention may be another anti-cancer agent or agents.

[0196] As described herein, the compositions of the present invention, as used herein, comprise compounds of the present invention together with pharmaceutically acceptable carriers, including any and all solvents, diluents or other vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, 15th Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1975), discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the present invention, such as producing any undesirable biological effects or otherwise interacting adversely with any other components of the pharmaceutical composition, its use is contemplated 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; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and 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; ethyl alcohol and phosphate buffer; and other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition. 8. Preparation The present invention also encompasses a class of compositions comprising the active compounds of the present invention in combination 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.

[0197] In certain embodiments, the present invention provides a pharmaceutical formulation for treating cancer, particularly the cancers described herein, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.

[0198] 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, together with a pharmaceutically acceptable carrier.

[0199] The compounds of the present invention can be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the intended treatment.The compounds and compositions of the present invention can be administered, for example, orally, transmucosally, topically, rectally, intrapulmonary, for example by inhalation spray, or parenterally, including intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular, transdermal, intraorbital, intrathecal, intraventricular, intratumor, intranasal, intrasternal, implant, inhalation and infusion techniques, in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles.

[0200] Typically, pharmaceutical compositions are tablets or gelatin capsules containing the active ingredient together with a) diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine; b) lubricants such as silica, talc, stearic acid, its magnesium or calcium salts, and / or polyethylene glycol; c) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone; if desired, d) disintegrants such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and / or e) absorbents, colorants, flavorants, and sweeteners. Tablets can be film-coated or enteric-coated according to methods known in the art.

[0201] Compositions suitable for oral administration contain an effective amount of the compounds 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 can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions can contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically elegant and palatable preparation. Tablets may contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a long period of time.For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used.Formulations for oral use can be in the form of 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 the form of 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.

[0202] Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fat emulsions or suspensions. These compositions may be sterilized and / or contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solubility enhancers, salts for regulating osmotic pressure, and / or buffers. In addition, they may contain other therapeutically valuable substances. These compositions are prepared by conventional mixing, granulating, or coating methods, respectively, and contain about 0.1-75%, or about 1-50%, of the active ingredient. Compositions suitable for transdermal application contain an effective amount of a compound of the present invention together with a suitable carrier. Carriers suitable for transdermal delivery include pharmaceutically acceptable absorbable solvents to aid passage through the host's skin. For example, a transdermal device may be in the form of a bandage comprising a backing, a reservoir containing the compound, optionally with a carrier, an optional rate-controlling barrier for delivering the compound to the host's skin at a controlled, predetermined rate over an extended period of time, and a means for securing the device to the skin.

[0203] Compositions suitable for topical application, for example, to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels, or spray formulations, for example, for delivery by aerosol. Such topical delivery systems are particularly suitable for dermal application, for example, for the treatment of skin cancer, or for preventative use, for example, by sun cream, lotion, spray, etc. Thus, they are particularly suitable for use in topical formulations well known in the art, including cosmetics. Such formulations may contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0204] As used herein, topical application can also refer to inhalation or intranasal application, which can be conveniently delivered in the form of a dry powder (either alone, in a mixture, e.g., a dry blend with lactose, or in the form of mixed component particles, e.g., with phospholipids) from a dry powder inhaler, with or without the use of a suitable propellant, or in the form of an aerosol spray presentation from a pressurized container, pump, spray, atomizer, or nebulizer.

[0205] The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients, since water can facilitate the degradation of certain compounds. The anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low-moisture-containing ingredients and low-moisture or low-humidity conditions.Anhydrous pharmaceutical compositions can be prepared and stored so that their anhydrous nature is maintained.Therefore, anhydrous compositions are packaged using materials known to prevent exposure to water, so that they can be included in suitable formulation kits.Suitable packaging examples include, but are not limited to, sealed foils, plastics, unit-dose containers (e.g., vials), blister packs and strip packs.

[0206] The present invention further provides pharmaceutical compositions and dosage forms that comprise one or more agents that reduce the decomposition rate of a compound of the present invention as an active ingredient. Such agents, referred to herein as "stabilizers," include, but are not limited to, antioxidants, such as ascorbic acid, pH buffers, or salt buffers.

[0207] The pharmaceutically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals. The amount of compound to be administered and the dosage regimen for treating disease state with the compound and / or composition of the present invention depend on various factors, including age, weight, sex and medical condition of the patient, disease type, disease severity, route and frequency of administration and the specific compound used.Therefore, dosage regimen can vary widely, but can be routinely determined using standard method.As mentioned above, daily dose can be given in one administration, or can be divided into 2, 3, 4 or more administrations.

[0208] For 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 designated administration route.For oral administration, the compound can be mixed with lactose, sucrose, starch powder, cellulose ester of alkanoic acid, cellulose alkyl ester, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone and / or polyvinyl alcohol, and then tableted or encapsulated for convenient administration.Such capsules or tablets can also contain controlled-release formulations, which can be provided by the dispersion of active compound in hydroxypropylmethylcellulose.

[0209] In the case of skin conditions, it may be preferable to apply a topical preparation of the compounds of the invention to the affected area two to four times daily. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration into the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for application to the eyes, ears, or nose. For topical administration, the active ingredient may constitute 0.001% to 10% w / w of the formulation, e.g., 1% to 2% by weight, and may constitute up to 10% w / w of the formulation, but preferably not more than 5% w / w, more preferably 0.1% to 1%.

[0210] The compounds of the present invention can also be administered by transdermal devices. Preferably, transdermal administration is achieved using a patch of either the reservoir and porous membrane type or the solid matrix type. In either case, the active agent is continuously delivered from the reservoir or microcapsules through a membrane to an active agent permeable adhesive that contacts the recipient's skin or mucosa. When the active agent is absorbed through the skin, a controlled and predetermined flow of the active agent is administered to the recipient. In the case of microcapsules, the encapsulating agent can also function as the membrane. The oil phase of the emulsions of the present invention can be constituted from known ingredients by known methods.

[0211] The phase may contain only an emulsifier, but may also contain a mixture of at least one emulsifier with a fat or oil, or both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both a fat and an oil. Taken together, the emulsifier, with or without a stabilizer, constitutes the so-called emulsifying wax, and the wax together with the oil and fat constitutes the so-called emulsifying ointment base, which forms the oily dispersed phase of the cream formulation. Emulsifiers and emulsion stabilizers suitable for use in the formulations of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate, alone or in combination with waxes or other materials known in the art.

[0212] The selection of an oil or fat suitable for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active ingredient in most oils likely to be used in pharmaceutical emulsion formulations is very low. Therefore, creams should preferably be non-greasy, non-staining, washable products with a viscosity suitable to avoid leakage from tubes or other containers. Linear or branched acid monobasic or dibasic alkyl esters, such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or blends of branched esters, can be used. These can be used alone or in combination depending on the properties required.

[0213] Alternatively, high melting point lipids such as white liquid paraffin and / or liquid paraffin or other mineral oils can be used. Formulations suitable for topical application 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.

[0214] The active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% and especially about 1.5% w / w. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions may be prepared from sterile powders or granules using one or more of the carriers or diluents described for use in oral formulations, or other suitable dispersing 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 arts. The active ingredient may also be administered by injection as a composition with a suitable carrier, including saline, dextrose, or water, or with cyclodextrins (i.e., Captisol), cosolvent solubilization (i.e., propylene glycol), or micelle solubilization (i.e., Tween 80).

[0215] Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0216] For pulmonary administration, the pharmaceutical composition can be administered in the form of an aerosol or by an inhaler including a dry powder aerosol. Suppositories for rectal administration of drugs can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and propylene glycol, which is solid at ordinary temperatures but becomes liquid at the rectal temperature and therefore melts in the rectum to release the drug.

[0217] The pharmaceutical compositions may be subjected to conventional pharmaceutical procedures, such as sterilization, and / or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. In addition, tablets and pills may be prepared with enteric coatings. Such compositions may also contain adjuvants, such as wetting agents, sweeteners, flavoring agents, and flavoring agents. The pharmaceutical compositions of the present invention comprise a compound of a formula described herein or a pharmaceutically acceptable salt thereof; an additional agent selected from a kinase inhibitor (such as a small molecule, polypeptide, antibody), an immunosuppressant, an anti-cancer 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.

[0218] Alternative compositions of the present invention include a compound of the formula described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. Such compositions can optionally include one or more additional therapeutic agents, including, for example, kinase inhibitors (small molecules, polypeptides, antibodies, etc.), immunosuppressants, anti-cancer agents, antiviral agents, anti-inflammatory agents, antifungal agents, antibiotics, or anti-vascular hyperproliferation compounds.

[0219] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with a compound of the invention, which does not destroy the pharmacological activity of the compound of the invention, and which is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of a compound of the invention. Pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of the invention include ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS), such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms, such as Tween or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, phosphate), sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat. Cyclodextrins, such as u-, p-, and y-cyclodextrin, or chemically modified derivatives, such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-cyclodextrin, or other solubilizing derivatives, may also be advantageously used to enhance delivery of the compounds of the formulae described herein.

[0220] Pharmaceutical compositions can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, emulsions, and aqueous suspensions, dispersions and solutions.For oral use of tablets, commonly used carriers include lactose and corn starch.Lubricants such as magnesium stearate are also typically added.For oral administration in capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions and / or emulsions are orally administered, the active ingredient, which may be suspended or dissolved in an oil phase, is combined with an emulsifier and / or suspending agent.

[0221] If desired, certain sweetening, flavoring and / or coloring agents can be added.The pharmaceutical compositions can include formulations utilizing liposome or microencapsulation technology, various examples of which are known in the art.

[0222] Pharmaceutical compositions can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents, examples of which are well known in the art. 9. Treatment kit One aspect of the present invention relates to a kit for conveniently and effectively carrying out or using the method according to the present invention. Generally, a pharmaceutical pack or kit includes one or more containers filled with one or more of the components of the pharmaceutical composition of the present invention. Such kits are particularly suitable for delivering solid oral forms such as tablets or capsules. Such kits preferably include several unit doses and may also include a card with the dosages for intended use. If desired, a memory aid, for example, in the form of numbers, letters, or other markings or a calendar insert, indicating the days of the treatment schedule on which the dosages can be administered may be provided. Such containers may optionally be accompanied by notices in the form required by government agencies regulating the manufacture, use, or sale of pharmaceutical products, which notices reflect the agency's approval for manufacture, use, or sale for human administration.

[0223] The following representative examples contain important additional information, examples, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof. These examples are intended to help illustrate the invention and are not intended to, and should not be construed as, limiting its scope. Indeed, various modifications of the invention, and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art upon review of this document, including the examples that follow and the references to the scientific and patent literature cited therein.

[0224] The contents of the cited references are incorporated herein by reference to help illustrate the state of the art. Furthermore, for purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS Edition, Handbook of Chemistry and Physics, 75th Edition, inside pages. Furthermore, general principles of organic chemistry, as well as specific functional 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 entireties. 10.Synthesis Scheme The compounds of the present invention can be prepared by those skilled in the art according to art-recognized techniques and procedures. More specifically, the compounds of the present invention can be prepared as shown in the schemes, methods and examples shown below. Those skilled in the art will recognize that the individual steps in the following schemes may be modified to provide compounds of the present invention. Reagents and starting materials are readily available to those skilled in the art. All substituents are as previously defined unless otherwise specified. [Example]

[0225] The following are abbreviations and their meanings used herein: Ac: Acetyl Boc: tert-butoxycarbonyl EtOAc: ethyl acetate DCM: dichloromethane ACN: acetonitrile THF: tetrahydrofuran DMSO: dimethyl sulfoxide MeOH: Methanol EtOH: ethanol DMAP: 4-(dimethylamino)pyridine DIPEA: N,N-diisopropylethylamine NMR: nuclear magnetic resonance LC-MS: Liquid chromatography mass spectrometry TLC: Thin Layer Chromatography TCR: T-cell receptor BCR: B cell receptor mM: millimolar concentration μM: micromolar mL: milliliter ng: nanogram nM: nanomolar concentration nm: nanometer I C 50 :50% inhibitory concentration (Half maximal inhibitory concentration) OD: optical density A. Biological Examples Biological Example 1 HPK1 Biochemical Assay This example uses the ADP-GLO™ Kinase Assay to measure the effect of potential HPK1 inhibitor compounds on HPK1 kinase activity.

[0226] The ADP-GLO™ Kinase Assay (Promega Corp., Madison, WI) measures ADP formed from a 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 a luciferase reaction. The luminescence produced correlates with kinase activity. A representative experimental setup is described below, although some adjustments can be made for individual assays. Materials and Equipment 1. Reagents

[0227] [Table 1]

[0228] 2. Equipment and supplies

[0229] [Table 2]

[0230] 3. Plate Setting Serial 3-fold dilutions of compound 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. 4. Procedure 1. Buffer Preparation 40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT Buffer Stock

[0231] [Table 3]

[0232] Add 20 mL of 1 M Tris and 10 mL of 1 M MgCl to 470 mL of ddH2O to obtain a buffer stock and store at RT. 2. Fresh 1 * Prepare assay buffer

[0233] [Table 4]

[0234] 3. Compound preparation 1) Compounds were diluted to 1 mM by mixing 10 μL of the 10 mM respective compound stock with 90 μL of DMSO.

[0235] 2) The compound was then diluted 3-fold (5 μL to 10 μL dilutions) with BRAVO for 10 doses. The highest compound concentration (conc.) was 1 mM (100x), and the DMSO concentration was 100%.

[0236] 3) 100 nL of each diluted compound sample is transferred to a 384-well plate (Corning-4512) by ECHO. 4) Centrifuge the plate at 1,500 rpm for 1 minute.

[0237] 4. Preparation of 2x ATP-MBP mixture 20μM ATP, 0.2μg / μL MBP in kinase buffer (final concentration: 10μM ATP, 0.1μg / μL MBP)

[0238] [Table 5]

[0239] 5. Preparation of 2x HPK1 Working Solution with Assay Buffer The final HPK1 concentration was 0.6 ng / μL. For more potent compounds, lower concentrations of HPK1 were used (0.26 ng / μL to 0.065 ng / μL).

[0240] Add 6.5 μL / well of 2×HPK1 working solution and centrifuge at 1,500 rpm for 1 minute. Add 7.5 μL / well of 2× ATP substrate and centrifuge at 1,500 rpm for 1 minute.

[0241] 8. Incubate at 25°C for 1 hour (or 6 hours for highly potent compounds). Add 9.5 μL / well of ADP-GLO™ Reagent to stop the kinase reaction and deplete unconsumed ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0242] 10. Add 10 μL of Kinase Detection Reagent to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0243] 11. Record the luminescence signal on an Envision plate reader (384-CTG). 5. Data Analysis The percent (%) inhibition at each compound concentration was calculated based on and relative to the signals of high and low control wells contained within each assay plate. High control wells served as 0% inhibition, and low control wells containing no compound but DMSO (final concentration = 0.5%) served as 100% inhibition. Concentration and % inhibition values ​​for the test compounds 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) for the reference peptide / compound was assessed in each experiment as a means of quality control. If the endpoint value was within three-fold of the expected value, the experiment was considered acceptable. Biological Example 2 PKC-Theta Biochemical Assay This example measured the effect of potential HPK1 inhibitor compounds on PKC-theta kinase activity using the ADP-GLO™ Kinase Assay. A representative experimental setup is described below, although some adjustments can be made for individual assays. Materials and Equipment 1. Reagents

[0244] [Table 6]

[0245] 2. Equipment and supplies

[0246] [Table 7]

[0247] 3. Plate Setting Serial 3-fold dilutions of compound 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. 4. Procedure 1. Buffer Preparation 40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT Buffer Stock

[0248] [Table 8]

[0249] Add 20 mL of 1 M Tris and 10 mL of 1 M MgCl to 470 mL of ddH2O to obtain a buffer stock and store at RT. 2. Fresh 1 * Prepare assay buffer

[0250] [Table 9]

[0251] 3. Compound preparation 1) Compounds were diluted to 1 mM by mixing 10 μL of the 10 mM respective compound stock with 90 μL of DMSO.

[0252] 2) The compound was then diluted 3-fold (5 μL to 10 μL dilutions) in BRAVO for 10 doses. The highest compound concentration was 1 mM (100x), and the DMSO concentration was 100%.

[0253] 3) 50 nL of each diluted compound sample is transferred to a 384-well plate (Corning-4512) by ECHO. 4) Centrifuge the plate at 1,500 rpm for 1 minute.

[0254] 4. Preparation of 2x Enzyme Working Solution in Kinase Buffer

[0255] [Table 10]

[0256] 5. Preparation of 2x ATP-sub mixture

[0257] [Table 11]

[0258] 6. Add 2.5 μL / well of 2x enzyme working solution and centrifuge at 1,500 rpm for 1 minute. 7. Add 2.5 μL / well of 2× ATP substrate and centrifuge at 1,500 rpm for 1 minute.

[0259] 8. Incubate at 25°C for 60 minutes. Add 9.5 μL / well of ADP-GLO™ Reagent to stop the kinase reaction and deplete unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0260] 10. Add 10 μL of Kinase Detection Reagent to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0261] 11. Record the luminescence signal on an Envision plate reader (384-USL). 5. Data Analysis The percent (%) inhibition at each compound concentration was calculated based on and relative to the signals of high and low control wells contained within each assay plate. High control wells served as 0% inhibition, and low control wells containing no compound but DMSO (final concentration = 0.5%) served as 100% inhibition. Concentration and % inhibition values ​​for the test compounds 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) for the reference peptide / compound was assessed in each experiment as a means of quality control. If the endpoint value was within three-fold of the expected value, the experiment was considered acceptable. Biological Example 3 TBK1 Biochemical Assay This example measured the effect of potential HPK1 inhibitor compounds on TBK1 kinase activity using the ADP-GLO™ Kinase Assay. A representative experimental setup is described below, although some adjustments can be made for individual assays. Materials and Equipment 1. Reagents

[0262] [Table 12]

[0263] 2. Equipment and supplies

[0264] [Table 13]

[0265] 3. Plate Setting See above. 4. Procedure 1. Buffer Preparation 40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT Buffer Stock

[0266] [Table 14]

[0267] Add 20 mL of 1 M Tris and 10 mL of 1 M MgCl to 470 mL of ddH2O to obtain a buffer stock and store at RT (room temperature). 2. Fresh 1 * Prepare assay buffer

[0268] [Table 15]

[0269] 3. Compound preparation 1) Compounds were diluted to 1 mM by mixing 10 μL of 10 mM respective compound stock with 90 μL of DMSO.

[0270] 2) The compound was then diluted 3-fold (5 μL to 10 μL dilutions) in BRAVO for 10 doses. The highest compound concentration was 1 mM (100x), and the DMSO concentration was 100%.

[0271] 3) 50 nL of each diluted compound sample is transferred to a 384-well plate (Corning-4512) by ECHO. 4) Centrifuge the plate at 1,500 rpm for 1 minute.

[0272] 4. Preparation of 2x Enzyme Working Solution in Kinase Buffer

[0273] [Table 16]

[0274] 5. Preparation of 2x ATP-sub mixture

[0275] [Table 17]

[0276] 6. Add 2.5 μL / well of 2x enzyme working solution and centrifuge at 1,500 rpm for 1 minute. 7. Add 2.5 μL / well of 2× ATP substrate and centrifuge at 1,500 rpm for 1 minute.

[0277] 8. Incubate at 25°C for 60 minutes. Add 9.5 μL / well of ADP-GLO™ Reagent to stop the kinase reaction and deplete unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0278] 10. Add 10 μL of Kinase Detection Reagent to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0279] 11. Record the luminescence signal on an Envision plate reader (384-USL). 5. Data Analysis The percent (%) inhibition at each compound concentration was calculated based on and relative to the signals of high and low control wells contained within each assay plate. High control wells served as 0% inhibition, and low control wells containing no compound but DMSO (final concentration = 0.5%) served as 100% inhibition. Concentration and % inhibition values ​​for the test compounds 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) for the reference peptide / compound was assessed in each experiment as a means of quality control. If the endpoint value was within three-fold of the expected value, the experiment was considered acceptable. Biological Example 4 JAK3 Biochemical Assay This example measured the effect of potential HPK1 inhibitor compounds on JAK3 kinase activity using the ADP-GLO™ Kinase Assay. A representative experimental setup is described below, although some adjustments can be made for individual assays. Materials and Equipment 1. Reagents

[0280] [Table 18]

[0281] 2. Equipment and supplies

[0282] [Table 19]

[0283] 3. Procedure 1. Prepare JAK3 Kinase Buffer DTT and BSA are freshly added to the buffer (final concentrations: 40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT).

[0284] [Table 20]

[0285] 2. Compound preparation For test compounds, compounds were diluted to 1 mM by mixing 5 μL of 10 mM compound stock with 45 μL of DMSO. The compound solution was then serially diluted 3-fold for 10 doses. The highest compound concentration was 1 mM (100×) and the DMSO concentration was 100%.

[0286] 50 nL of each of the 10 doses of diluted compound was added to a 384-well assay plate (Corning #4512) by ECHO. For positive controls, 50 nL of 1 mM reference compound was added to a 384-well assay plate (Corning #4512) by ECHO.

[0287] For negative controls, 50 nL of DMSO was transferred to the 384-well assay plate as a negative control. The assay plate was centrifuged at 1,500 rpm for 1 minute.

[0288] 3. Compounds were transferred by ECHO according to the following layout: Reference and test compounds 1–15: serial 3-fold dilutions (10 doses) from 10 μM to 0.508 nM; negative control: 0.78 ng JAK3, 4 μM ATP, and 0.2 μg / μL Poly(E4Y1), 1% DMSO; positive control: highest dose of reference compound, 0.78 ng JAK3, 0.2 μg / μL Poly(E4Y1), 4 μM ATP, 1% DMSO.

[0289] 4. Add 2.5 μL / well of 2x JAK3 working solution using a pipette (Thermo, 30 μL multichannel) and centrifuge at 1,500 rpm for 1 minute. 5. Add 2.5 μL / well of 2×ATP-Poly(E4Y1) working mixture using a pipette (Thermo, 30 μL multichannel) and centrifuge at 1,500 rpm for 1 minute.

[0290] 6. Incubate the assay plate at 25°C for 60 minutes. Add 7.5 μL / well of ADP-GLO™ Reagent by Bravo to stop the kinase reaction and deplete unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0291] 8. Add 10 μL of Kinase Detection Reagent by Bravo to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0292] 9. Luminescent signals are recorded on an Envision plate reader (384-USL). 10. Process data using XL-fit. % Inhibition = [1 - (Test wells - Negative control) / (Positive control - Negative control)] x 100%. 5. Data Analysis The percent (%) inhibition at each compound concentration is calculated based on and relative to the signal of negative and positive control wells contained within each assay plate. Negative control wells served as 0% inhibition, and positive control wells served as 100% inhibition. Concentration and % inhibition values ​​for the test compound are plotted, and the concentration of compound required for 50% inhibition (IC50) is determined using a three-parameter logistic dose-response equation. The endpoint value (IC50) for the reference peptide / compound is assessed in each experiment as a quality control measure. An experiment was considered acceptable if the endpoint value was within three-fold of the expected value. Biological Example 5 ZAP70 Biochemical Assay Protocol This example measured the effect of potential HPK1 inhibitor compounds on ZAP70 kinase activity using the ADP-GLO™ Kinase Assay. A representative experimental setup is described below, although some adjustments can be made for individual assays. Materials and Equipment 1. Reagents

[0293] [Table 21]

[0294] 2. Equipment and supplies

[0295] [Table 22]

[0296] 3. Procedure 1. ZAP70 Kinase Buffer Preparation Add DTT and BSA fresh to the buffer (final concentrations: 40 mM Tris pH 7.5; 20 mM MgCl, 0.1 mg / ml BSA, 50 μM DTT, 2 mM MnCl).

[0297] [Table 23]

[0298] 2. Compound preparation For test compounds, compounds were first diluted to 1 mM by mixing 5 μL of 10 mM compound stock with 45 μL of DMSO. These compounds were then diluted 3-fold for 10 doses. The highest compound concentration was 1 mM (100×) and the DMSO concentration was 100%.

[0299] 50 nL of compound solution was transferred by ECHO into a 384-well assay plate (Corning #4512). For the positive control, 50 nL of 1 mM staurosporine was transferred to the 384-well assay plate as a positive control.

[0300] For the negative control, 50 nL of DMSO was transferred to the 384-well assay plate as a negative control. The assay plate was centrifuged at 1,500 rpm for 1 minute. 3. Compounds were transferred by ECHO according to the following layout:

[0301] Staurosporine and test compounds 1-15: serial 3-fold dilutions (10 doses) from 10 µM to 0.508 nM; negative control: 6.25 ng of ZAP70, 10 µM of ATP, and 0.4 µg / µL of poly(vinyl alcohol), 1% DMSO; positive control: 10 µM of staurosporine, 6.25 ng of ZAP70, 10 µM of ATP, and 0.4 µg / µL of poly(vinyl alcohol), 1% DMSO.

[0302] 4. Prepare a 2x ATP-Poly(E4Y1) mixture in kinase buffer: 20 µM ATP, 0.8 µg / µL Poly(E4Y1) (final concentrations: 10 µM ATP, 0.4 µg / µL Poly(E4Y1).

[0303] 5. Prepare a 2x ZAP70 working solution: (2.5 ng / µL) in kinase buffer (final concentration was 1.25 ng / µL). 6. Add 2.5 μL / well of 2x ZAP70 working solution using a pipette (Thermo, 30 μL multichannel) and centrifuge at 1,500 rpm for 1 minute.

[0304] 7. Add 2.5 μL / well of 2x ATP-Poly(E4Y1) working mixture using a pipette (Thermo, 30 μL multichannel) and centrifuge at 1,500 rpm for 1 minute.

[0305] 8. Incubate the assay plate at 25°C for 60 minutes. Add 9.5 μL / well of ADP-GLO™ Reagent by Bravo to stop the kinase reaction and deplete unconsumed ATP after 1 hour. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0306] 10. Add 10 μL of Kinase Detection Reagent by Bravo to convert ADP to ATP. Centrifuge at 1,500 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0307] 11. Record the luminescence signal on an Envision plate reader (384-USL). 12. Process data using XL-fit. % Inhibition = [1 - (Test wells - Negative control) / (Positive control - Negative control)] x 100%. 5. Data Analysis The percent (%) inhibition at each compound concentration is calculated based on and relative to the signal of negative and positive control wells contained within each assay plate. Negative control wells served as 0% inhibition, and positive control wells served as 100% inhibition. Concentration and % inhibition values ​​for the test compound are plotted, and the concentration of compound required for 50% inhibition (IC50) is determined using a three-parameter logistic dose-response equation. The endpoint value (IC50) for the reference peptide / compound is assessed in each experiment as a quality control measure. An experiment was considered acceptable if the endpoint value was within three-fold of the expected value. Biological Example 6 LCK Biochemical Assay Protocol This example measured the effect of potential HPK1 inhibitor compounds on Lck kinase activity using the ADP-GLO™ Kinase Assay. A representative experimental setup is described below, although some adjustments can be made for individual assays. Materials and Equipment 1. Reagents

[0308] [Table 24]

[0309] 2. Equipment and supplies

[0310] [Table 25]

[0311] 3. Procedure 1. LCK Kinase Buffer Preparation Add DTT and BSA fresh to the buffer (final concentrations: 40 mM Tris pH 7.5; 20 mM MgCl, 0.1 mg / ml BSA, 50 μM DTT, 2 mM MnCl).

[0312] [Table 26]

[0313] 2. Compound preparation For test compounds, compounds were first diluted to 1 mM by mixing 5 μL of 10 mM compound stock with 45 μL of DMSO. The compound solution was then serially diluted 3-fold for 10 doses. The highest compound concentration was 1 mM (100×), and the DMSO concentration was 100%.

[0314] For the positive control, staurosporine was diluted to 300 μM by mixing 3 μL of 10 mM stock into 97 μL of DMSO. 50 nL of compound solution and 300 μM staurosporine were transferred to a 384-well assay plate (Corning #4512) by BRAVO.

[0315] For the negative control, 50 nL of DMSO was transferred to the 384-well assay plate as a negative control. The assay plate was centrifuged at 1,500 rpm for 1 minute. 3. Compounds were transferred by ECHO according to the following layout:

[0316] Staurosporine and test compounds 1-15: serial 3-fold dilutions (10 doses) from 10 µM to 0.508 nM; negative control: 7 ng LCK, 20 µM ATP, and 0.4 µg / µL poly(Asp-1), 1% DMSO; positive control: 3 µM staurosporine, 7 ng LCK, 20 µM ATP, and 0.4 µg / µL poly(Asp-1), 1% DMSO.

[0317] 4. Prepare a 2x ATP-Poly(E4Y1) mixture: 40 µM ATP, 0.8 µg / µL poly(E4Y1) in kinase buffer (final concentrations: 20 µM ATP, 0.4 µg / µL poly(E4Y1).

[0318] 5. Prepare a 2x LCK working solution (2.8 ng / µL) in kinase buffer (final concentration was 1.4 ng / µL). 6. Add 2.5 μL / well of 2x LCK working solution using a pipette (Thermo, 30 μL multichannel) and centrifuge at 1,000 rpm for 1 minute.

[0319] 7. Add 2.5 μL / well of 2×ATP-Poly(E4Y1) working mixture using a pipette (Thermo, 30 μL multichannel) and centrifuge at 1,000 rpm for 1 minute.

[0320] 8. Incubate the assay plate at 25°C for 60 minutes. Add 9.5 μL / well of ADP-GLO™ Reagent by Bravo to stop the kinase reaction and deplete unconsumed ATP after 1 hour. Centrifuge at 1,000 rpm for 1 minute. Incubate at 25°C for 40 minutes.

[0321] 10. Add 10 μL of Kinase Detection Reagent by Bravo to convert ADP to ATP. Centrifuge at 1,000 rpm for 1 minute. Incubate at 25°C for 30 minutes.

[0322] 11. Record the luminescence signal on an Envision plate reader (384-USL). 12. Process data using XL-fit. % Inhibition = (negative control - test wells) / (negative control - positive control) x 100%. 4. Data Analysis The percent (%) inhibition at each compound concentration is calculated based on and relative to the signal of negative and positive control wells contained within each assay plate. Negative control wells served as 0% inhibition, and positive control wells served as 100% inhibition. Concentration and % inhibition values ​​for the test compound are plotted, and the concentration of compound required for 50% inhibition (IC50) is determined using a three-parameter logistic dose-response equation. The endpoint value (IC50) for the reference peptide / compound is assessed in each experiment as a quality control measure. An experiment was considered acceptable if the endpoint value was within three-fold of the expected value. Biology Example 7 MAP4K3 Protein Kinase Assay This example provides an assay protocol for measuring the phosphorylation of a peptide substrate by the protein kinase MAP4K3.

[0323] Briefly, MAP4K3, its substrates, and cofactors (ATP and Mg 2+ ) are mixed in the wells of a microtiter plate and incubated for 5 hours at 25°C. At the end of the incubation, the reaction is quenched by the addition of a buffer containing EDTA. The substrate and product are separated and electrophoretically quantified using Caliper Life Sciences' microfluidic-based LabChip 3000 Drug Discovery System.

[0324] For this assay, the MAP4K3 substrate is FAM-GAGRLGRDKYKTLRQIRQ-NH2 (FAM is carboxyfluorescein). The peptide substrate is preferably >98% pure by capillary electrophoresis.

[0325] A typical assay setup and conditions are provided below. 1. Add 5 μL of 2× Enzyme Buffer (or control) to wells of a 384-well plate.

[0326] 2. Add 100 nL of 100x compound. Enzyme and compound may be pre-incubated at this point if desired. Add 3.5 μL of 2× substrate buffer.

[0327] 4. Incubate the plate at 25°C for 5 hours. The reaction is terminated by adding 5.40 μL of 1.25× stop buffer. 6. Create a job in the Caliper LabChip® 3000 Drug Discovery System using the values ​​in the table below.

[0328] 12 Separation conditions for Sipper Chip

[0329] [Table 27]

[0330] 7. Load the plate and begin electrophoresis using a blue laser (480 nm) for excitation and a green CCD (520 nm) (CCD2) for detection. The assay is carried out under the following reaction conditions: 5 total hours; 25°C, in the presence of 20 mM of the 100% inhibitor EDTA.

[0331] 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 beta-glycerophosphate, 20 μM ATP, 1% DMSO (compound-derived), 0.5 μM FAM-GAGRLGRDKYKTLRQIRQ-NH2, and 0.5 nM MAP4K3 enzyme.

[0332] It should be noted that the specific activity of MAP4K3 varies between lots and the enzyme concentration may need to be adjusted to obtain approximately 10-20% conversion of substrate to product. The substrate and product peptides present in each sample are electrophoretically separated using a LabChip 3000 capillary electrophoresis instrument. Two fluorescent peaks are observed as the substrate and product peptides separate. The change in relative fluorescence intensity of the substrate and product peaks is the measured parameter, reflecting enzyme activity. Capillary electropherograms (RDA acquisition files) are analyzed using HTS Well Analyzer software (Caliper Life Sciences). The kinase activity of each sample is determined as the product-to-sum ratio (PSR), i.e., P / (S+P), where P is the peak height of the product peptide and S is the peak height of the substrate peptide.

[0333] For each compound, enzyme activity is measured at various concentrations (12 compound concentrations spaced at 3x dilution intervals). Negative control samples (0% - inhibition in the absence of inhibitor) and positive control samples (100% - inhibition in the presence of 20 mM EDTA) are collected in quadruplicate and used to calculate the % inhibition value for each compound at each concentration.

[0334] Percent inhibition (P inh ) is determined using the following formula: P inh =(PSR 0% -PSR inh ) / (PSR 0% -PSR 100 %)×100 Here, PSR inh is the sum of the products in the presence of the inhibitor, and PSR 0% is the average product sum ratio in the absence of inhibitor, and PSR 100% is the average sum product ratio of the 100% inhibition control samples.

[0335] The IC50 values ​​of inhibitors are determined by fitting the inhibition curves (Pinh vs. inhibitor concentration) with a four-parameter sigmoidal dose-response model using XL-fit4 software (IBDS).

[0336] Some materials and buffers used in the assay are listed below for reference.

[0337] [Table 28]

[0338] Biology Example 8 Compound Efficacy Study on IL2 and IFN-γ Released by Human Pan T Cells This example illustrates assay methods that can be used to determine the effect of compounds on IL2 and IFN-γ release using pan-T cell and ELISA assay formats. Materials and Equipment 1. Reagents

[0339] [Table 29]

[0340] 2. Equipment and supplies

[0341] [Table 30]

[0342] 3. Procedure Procedure for pan-T isolation and reagent preparation (Day 0) 1. Cell Growth Medium RPMI1640:ATCC, Cat#30-2001 10% FBS: Gibco, Cat# 10099141 1%Pen-Strep:Gibco, Cat#15140122 1% Non-Essential Aminos: Gibco, Cat# 11140050 Beta-mercaptoethanol: Gibco, Cat# 21985023 2. Pan-T Isolation Buffer Preparation Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA stock solution (#130-091-376) 20-fold with automated MACS® rinse solution. Keep the buffer at 4°C. Degas the buffer before use, as air bubbles may clog the column.

[0343] [Table 31]

[0344] 3. Thawing Frozen PBMCs 1) Preheat the medium in a 37°C water bath. 2) The cells are rapidly thawed in a 37°C water bath.

[0345] 3) Add pre-warmed medium to the 15 mL tube and transfer the cells to the tube. 4) Centrifuge at 300 x g for 8 minutes (0 increase, 0 decrease in centrifugation). 5) Wash the PBMCs with rinse buffer.

[0346] 6) Centrifuge the PBMCs at 300 x g for 8 min and wash twice (centrifugation increase 9, decrease 1). 7) Resuspend the cells in pan T cell isolation buffer and count the number of cells.

[0347] 4. Pan T Cell Isolation Staining cells with microbead cocktail 1) Prepare cells and determine cell number. Filter cells through a 70 μm cell strainer.

[0348] 2) Cell pellets were collected in 10 7 Resuspend in 40 μL of buffer per 100 μL of total cells. 3)10 7Add 10 μL of pan T cell biotin-antibody cocktail per total cells. 4) Mix well and incubate in a freezer (on ice) for 10 minutes.

[0349] 5)10 7 Add 30 μL of buffer per total cells. 6)10 7 Add 20 μL of pan-T cell microbead cocktail per 100 μL of total cells. 7) Mix well and incubate in a freezer (on ice) for 15 minutes.

[0350] 8) Proceed to subsequent magnetic cell separation. NOTE: a. Work quickly, keep cells cold, and use pre-chilled solutions (2-8°C). b. The volume of a given magnetic label should be a maximum of 10 7 of total cells. If working with fewer cells, use the same volumes as indicated. c. If working with higher cell numbers, scale up all reagent volumes and therefore total volume. d. For optimal performance, it is important to obtain a single-cell suspension before magnetic labeling.

[0351] Subsequent manual cell separation 1) Place the LS column in the magnetic field of the appropriate MACS Separator. For details, refer to the respective MACS Column data sheet.

[0352] 2) Prepare the column by rinsing with 3 mL of buffer. 3) Apply the cell suspension to the column and collect the flow-through, which represents enriched T cells.

[0353] 4) Wash the column with 5 mL of buffer. Collect the unlabeled cells that pass through, representing enriched T cells. Note: Always wait until the column reservoir is empty before proceeding to the next step.

[0354] 5. Pan T Cell FACS 1) Take 50 μL of PBMCs and pan-T cells into FACS tubes. 2) Incubate cells with anti-human CD3 / CD4 / CD8 antibody (1 μL / 1 μL / 1 μL / tube) for 20 min at 4° C. For unstained controls, incubate cells with FACS staining buffer.

[0355] 3) Wash twice with cold staining buffer (PBS containing 0.2% BSA and 1 mM EDTA). 4) Perform FACS. CD3 + , CD3 + CD4 + , and CD3 + CD8 + Gate for population % analysis.

[0356] 5) If the purity of pan T cells is higher than 90%, dilute the cell suspension to one million cells / mL with an appropriate volume of cell culture medium. 6) Aliquot the cell suspension into sterile disposable reservoirs for later use. Compound and anti-human CD3 / CD28 preparation procedure (Day 0) 1. Compound preparation Compound serial dilution (source plate 1000x) Compounds were solubilized in 100% DMSO to a concentration of 10 mM, then serially diluted 3-fold to 8 dose points.

[0357] 4x Compound Dose Preparation (Interplate: Corning-3599) 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).

[0358] 2. Anti-human CD3 (stock concentration 6.76 mg / ml): Store at 4°C. 1) Dilute anti-human CD3 in PBS to a final concentration of 0.5 μg / mL. 2) Add 50 μL / well of CD3 to each well except for the positive and negative control wells, which have no CD3 / CD28 stimulation.

[0359] 3) Incubate for 2 hours at 37°C in a 5% CO2 incubator. 4) Remove 50 μL of antibody solution from the cell culture plate. Rinse each well twice with 200 μL of sterile PBS each time.

[0360] 3. Anti-human CD28 (4x) preparation The antibody was diluted to 2 μg / mL (4×) from a stock concentration of 11.07 mg / mL in culture medium.

[0361] 4. PMA / Inomycin Preparation (4x) 1) Dilute PMA to 400 ng / mL (8x) in medium. 2) Dilute ionomycin to 8 μM (8×) in medium.

[0362] 3) Mix an equal volume of PMA with ionomycin to obtain a 4x mixture. Cell stimulation procedure (day 0) 1.1×10 5 Transfer the cell suspension at 100 cells / well (100 μL / well) into a 96-well plate (cell plate: Greiner-655180).

[0363] 2. Add 50 μL / well of anti-human CD28 for test compounds and ZPE / HPE controls. Add 50 μL / well of 4×PMA / ionomycin solution or culture medium for positive or negative controls, respectively.

[0364] 3. Add 50 μL / well of compound to the cell plate according to the plate map shown below. For ZPE / HPE controls, add 50 μL / well of 0.4% DMSO solution or 0.4 μM RGT003-026 (4x), respectively. For positive or negative controls, add 50 μL / well of culture medium.

[0365] 4. Incubate the plates for 48 hours. IL-2 and IFN-γ ELISA procedure Day 1: Coating plates 1) Coat microwells with 100 μL per well of IL-2 and IFN-γ capture antibodies diluted in coating buffer. See lot-specific Instructions / Analysis Certificate for recommended antibody coating dilutions. Seal the plate and incubate overnight at 4°C. Day 2: Sample collection 1) After 48 hours of incubation in a 37°C, 5% CO2 incubator, the cell plate is centrifuged at 1,000 rpm for 10 minutes. 100 μL of supernatant per well is collected and then used for IL-2 and IFN-γ ELISA assays. The supernatant can be stored at -80°C, and the IL-2 and IFN-γ ELISA assays can be performed the next day. The supernatant may need to be diluted 30-40 times to ensure that the assays do not exceed the linear range of the IL-2 and IFN-γ standard curves. 2) Add 100 μL / well of fresh medium (containing anti-CD28, P / I and compounds) to the plate. 3) Plate map: Day 3-4: IL-2 and IFN-γ ELISA: 1) Aspirate wells and wash three times with ≥ 300 μL / well of wash buffer. After the final wash, invert the plate and blot with absorbent paper to remove residual buffer. 2) Block plates with ≧200 μL / well of Assay Diluent. Incubate at RT for 1 hour. 3) Aspirate / wash as in step 2. 4) Prepare standards in assay diluent. IL-2 standard stock preparation Add 1 mL of deionized water to the vial (235 ng / vial) for a stock concentration of 235 ng / mL. Take 10 μL of the standard stock per vial and freeze at -80°C. IFN-γ standard stock preparation Add 1 mL of deionized water to the vial (145 ng / vial) for a stock concentration of 145 ng / mL. Take 10 μL of the standard stock per vial and freeze at -80°C. Preparation of IL-2 / IFNγ standard curve Dilute the standard sample to a maximum concentration of 500 pg / mL. Then perform two-fold serial dilutions to 10 doses (including a blank control). Transfer various concentrations of the standard to an ELISA plate, 100 μL / well. 1) Pipette 100 μL of each standard, sample, and control into the appropriate wells. Seal the plate and incubate for 2 hours at RT. 2) Aspirate / wash as in step 2, but wash a total of 5 times. 3) Add 100 μL of Working Detector (Detection Antibody + SAv-HRP Reagent) to each well. Seal the plate and incubate at RT for 1 hour. 4) Aspirate / wash as in step 2, but for a total of 7 washes. Note: For this final wash step, soak the wells in wash buffer for 30 seconds to 1 minute for each wash. 5) Add 100 μL of substrate solution to each well. Incubate the plate (without plate sealer) for 30 minutes at room temperature in the dark. 6) Add 50 μL of stop solution to each well. 7) Read the absorbance at 450 nm within 30 minutes after stopping the reaction. The OD at 450 nm was normalized to the OD at 570 nm. 4. Data Analysis For biochemical assays, the percent (%) inhibition at each concentration of compound was calculated based on and relative to the signal of the HPE and ZPE wells contained within 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.

[0366] In the cell assay, the response of each compound concentration to IL-2 or IFN-γ (data not shown) was calculated relative to the DMSO control. EC2x represents the concentration of compound that gave a 200% response (2-fold), and EC50 was calculated using GraphPad Prism. Biological Example 9 Kinetic Solubility Assay This protocol is designed to measure the kinetic solubility of test articles in assay buffer. This study will be conducted in accordance with international bioethical standards, the World Medical Association Declaration of Helsinki. 1. Reagents Control: 10mM propranolol in DMSO 10mM ketoconazole in DMSO 10mM tamoxifen in DMSO Deionized 18.2MΩ-cm MQ ultrapure water Phosphoric acid, 85% (H3PO4) Dimethyl sulfoxide Assay Buffer 100 Phosphate buffer, pH 7.4 2. Procedure Calibration curve preparation: 300 μM compound solution: add 6 μL of compound stock solution to 192 μL of MeOH / HO (1:1).

[0367] Prepare a working solution in MeOH / H2O (1:1).

[0368] [Table 32]

[0369] 1) Prepare a stock solution for the test compound in DMSO at a concentration of 10 mM. Dilute the stock solution in triplicate into 100 mM phosphate buffer in 1.5 ml Eppendorf tubes to a target concentration of 100 μM with a final DMSO concentration of 1%.

[0370] 2) 4 μL of 10 mM DMSO stock solution to 396 μL of assay buffer. 3) The sample tube is left shaking (1000 rpm) at room temperature for 1 hour. 4) The sample tube is centrifuged at 12,000 rpm (Eppendrof-5424, approximately 13,500 g) for 10 minutes to precipitate undissolved particles.

[0371] 5) Transfer the supernatant to new tubes at various dilutions (undiluted, 10x diluted, 100x diluted or other dilution factors). 6) Add 5 μL of working solution and diluted supernatant to 95 μL ACN (containing IS).

[0372] Samples are analyzed by LC-MS-MS against a calibration curve. 3. Data Analysis A standard calibration curve is prepared in methanol / HO (1:1). The test compound concentration in the supernatant after 1 h of incubation is determined from the calibration curve constructed by plotting the peak area against the nominal concentration with a working range of 0.02 µM to 60 µM and used to evaluate aqueous solutions of the test compound. Biological Example 10 MDCK Permeability Assay This assay is intended to generate screening data only and to estimate the intestinal absorption potential of drug candidates. This study will be conducted in accordance with the international bioethical standards of the World Medical Association Declaration of Helsinki. device ◆24-well cell culture plate (PET membrane): Millipore ◆24-well feeder tray: Millipore ◆ Pipettors and tubes (Eppendorf) ◆Millicell ERS System ◆96-well U-shaped plate (Greiner) ◆96-well microplate (Greiner) ◆37℃ CO2 incubator ◆FlexStation 3 (Molecular Devices) reagent Cell line: MDCK (ATCC) or MDCK MDR1 (NKI).

[0373] Cell culture growth medium (MEM + 10% FBS + 1% NEAA): Prepare growth medium by adding 50 mL FBS and 5 mL NEAA to 445 mL of MEM, or adjust the final volume according to actual needs.

[0374] Trypsin-EDTA (Invitrogen, Cat# 25200-072) Assay and Dosing Solution Buffers: Hanks' balanced salt solution (HBSS, Invitrogen) containing 25 mM HEPES, pH 7.4 Hanks' balanced salt solution (HBSS, Invitrogen)

[0375] [Table 33]

[0376] Preparation of donor buffer: From A to B: 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).

[0377] 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).

[0378] HBSS buffer containing 5 µM Lucifer Yellow (LY): Add 125 µL LY (2 mM) to 50 mL HBSS buffer (pH 7.4). From B to A: HBSS containing 0.3% DMSO: Add 150 µL DMSO to 50 mL HBSS buffer (pH 7.4).

[0379] HBSS buffer containing 0.1% DMSO: Add 50 µL DMSO to 50 mL HBSS buffer (pH 7.4). HBSS buffer without DMSO. Preparation of receiver buffer: For direction A to B: Prepare HBSS buffer containing 0.4% DMSO: add 200 µL DMSO to 50 mL HBSS buffer (pH 7.4).

[0380] For the B to A direction: Prepare HBSS buffer containing 0.4% DMSO and 5 µM LY: add 200 uL DMSO and 125 µL LY (2 mM) to 50 mL HBSS buffer (pH 7.4). Control: The following formula: (actual weight / molecular weight) / mL of solvent = 10 mM Prepare 10 mM stock concentrations of compounds in DMSO and Lucifer Yellow in assay buffer using the following:

[0381] Reference compounds: erythromycin, metoprolol, atenolol Preparation of donor solution (10 μM for compounds and 5 μM for Lucifer Yellow):

[0382] [Table 34]

[0383] The diluted solution is centrifuged at 4000 rpm for 5 minutes. The supernatant is collected for compound dosing. Prepare compound solutions with standard curve (3 µM / 1 µM / 0.2 µM / 0.04 µM / 0.01 µM / 0.005 µM): 20x solution: ≫15 μL(10mM)+485μL(MeOH:H2O=1:1)---500μL(300μM) ≫200μL(300μM)+800μL(MeOH:H2O=1:1)---1000μL(60μM) ≫200μL(60μM)+400μL(MeOH:H2O=1:1)---600μL(20μM) ≫200μL(20μM)+800μL(MeOH:H2O=1:1)---1000μL(4μM) ≫200μL(4μM)+800μL(MeOH:H2O=1:1)---1000μL(0.8μM) ≫200μL(0.8μM)+600μL(MeOH:H2O=1:1)---800μL(0.2μM) ≫200μL(0.2μM)+200μL(MeOH:H2O=1:1)---400μL(0.1μM) 1x solution: 3 µL of 20x solution (0.1–60 µM) + 57 µL 0.4% DMSO HBSS + 60 µL ACN containing IS (200 ng / mL osalmid). research design ◆ Test concentration: Determined by the sponsor Incubation temperature and time points: 0 and 60 minutes at 37°C ◆Sample size: 1 to 3 procedure Routine culture and maintenance Stock cultures were maintained in MEM + 10% FBS + 1% NEAA and grown at 75 cm 2 They are grown in tissue culture treated flasks and split (passaged) twice a week to maintain the desired confluence.

[0384] For maintenance passages: Routinely distribute trypsinized cells into new flasks at a standard passage ratio of 1:20. Subculture Protocol: Remove and discard the culture medium.

[0385] Rinse the cell layer twice with 0.25% (w / v) trypsin-0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitors. Add 2.0–3.0 mL of trypsin-EDTA solution to the flask and observe the cells under an inverted microscope until the cell layer is dispersed (usually within 5–15 min).

[0386] (Note: To avoid clumping, do not agitate the cells by tapping or rocking the flask; wait for the cells to detach. Cells that are difficult to detach can be incubated at 37°C to facilitate dispersion.) Add 6.0-8.0 ml of complete growth medium and aspirate the cells by gently pipetting.

[0387] Add an appropriate aliquot of the cell suspension to a new culture vessel. Incubate the culture at 37 °C. Seeding assay plates MDCK assay plates are seeded 2–4 days before the assay. 24-well plates are seeded with 400 μL of apical chamber volume (2.2 × 10 5 / mL) in 0.88×10 5 Cells are seeded into the 24-well base chamber at a density of 1000 cells / well in 25 mL of growth medium. Assay plates are generally refilled with growth medium 24 hours prior to the assay. Preparation of assay plates and transepithelial electrical resistance (TEER) measurements MDCK assay plates are rinsed with HBSS+ buffer before performing the assay. After rinsing, fresh HBSS+ is added to the assay plate in a 400 μL apical chamber volume and 0.8 mL HBSS+ basal chamber volume. A Millicell ERS system ohmmeter is used to measure the electrical resistance across the monolayer (TEER of 100 ohms*cm). 2 If supercells are used). Preparation of standard curve Preparation of cell plates: ◆ 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 apical wells based on the plate map.

[0388] ♦ Prepare a fresh base plate by adding 800 μL of receiver solution (for A to B) or 900 μL of donor solution (for B to A) to the wells of a new 24-well plate.

[0389] Place the top and bottom plates in a 37°C incubator. Preparation of assay plates: After 5 minutes, transfer 100 μL of sample from all donors (both A to B and B to A) to the appropriate wells of the DO sample plate, and transfer 100 μL of sample from all top chambers (A to B donors and B to A receivers) into the appropriate wells of the Lucifer Yellow DO (DO LY) microplate.

[0390] ♦ The top plate is loaded onto the base plate to begin the transport process. At 90 minutes, separate the apical and basal plates and transfer 100 μL of sample from all donors (for both A to B and B to A) into the appropriate wells of a new sample plate in D90, and transfer 200 μL of sample from all receivers into the appropriate wells of a sample plate in R90. Transfer 100 μL of sample from all basal chambers (receivers from A to B and donors from B to A) into the appropriate wells of a new microplate in Lucifer Yellow R90 (R90 LY).

[0391] ♦ LY permeability is determined by reading D0 LY and R90 LY using a fluorescent plate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. ◆Sample preparation: ≫For receiver solution: 60 μL of sample + 60 μL of ACN containing IS (200 ng / mL osalmid) Donor solution: 6 μL sample + 54 μL 0.4% DMSO / HBSS + 60 μL ACN containing IS (200 ng / mL osalmid) calculation Transepithelial electrical resistance (TEER) = (resistance sample -resistance blank ) × effective membrane area Lucifer Yellow Transparency: P app =(V A / (area×time))×([RFU]accepter -[RFU] blank ) / (([RFU] initial,dоnоr -[RFU] blank ) × dilution factor) × 100 7.1 Plate drug transport assay using the following formula: Transepithelial electrical resistance (TEER) = (resistance sample -resistance blank ) × effective membrane area Lucifer Yellow Transparency: P app =(V R / (area×time))×([RFU] accepter -[RFU] blank ) / (([RFU] initial,dоnоr -[RFU] blank ) × dilution factor) Drug Permeability: P app =(V R / (area × time) × ([drug] receiver / (([drugs] initial,dоnоr ) × dilution factor) In the formula, V R is the volume of 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 transport time in seconds.

[0392] Percentage recovery = 100 x (total compounds in donor at 90 min x dilution factor + total compounds in receiver at 90 min) / (total compounds at 0 min x dilution factor) Data analysis The apparent permeability coefficient (P app ) is calculated using the following formula: P app =(V R / (area × time) × ([drug] receiver / (([drugs] initial,dоnоr ) × dilution factor) In the formula, V Ris the volume of 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 transport time in seconds. Test compound concentrations in the receiver and donor sides are determined from standard calibration curves constructed by plotting peak areas against nominal concentrations.

[0393] The outflow rate is the mean apical to basal (AB) P app Data and Basal to Apical (BA)P app Calculate from the data: Flow rate = P app(B-A) / P app(A-B) .

[0394] Transepithelial electrical resistance (TEER) measurements were used to determine tight junction formation between cells, with TEER values ​​of 100 Ω cm 2 In cases where the cell monolayer is too large, a cell monolayer is used. To assess the integrity of the cell monolayer, 5 μM of Lucifer Yellow is co-treated with the test compound in the donor compartment. The P of the co-treated Lucifer Yellow app is 5 x 10 -6 If it is greater than cm / sec, the data point is excluded from the calculation. Biological Example 11 Metabolic Stability Assay in Liver Microsomes This assay is to determine the stability of test compounds in liver microsomes. This study is conducted in accordance with international bioethical standards, the World Medical Association Declaration of Conformity. Liver microsome sample Human, Sprague Dawley rat, CD-1 mouse, beagle dog, and cynomolgus monkey liver microsome samples purchased from BD Gentest or RILD. device Incubator (37℃) Centrifuge (Eppendorf 5810) Eppendorf pipettes and tubes 96-well plate (Greiner) reagent Compound stock solutions: 10 mM test compound in DMSO, stored at -80°C Assay Buffer 0.1M potassium phosphate buffer, pH 7.4: Buffer A: 1.0 L of 0.1 M monobasic potassium phosphate buffer containing 1.0 mM EDTA - Buffer B: 1.0 L of 0.1 M dibasic potassium phosphate buffer containing 1.0 mM EDTA - Buffer C (K phosphate buffer): 0.1 M potassium phosphate buffer, 1.0 mM EDTA, pH 7.4 obtained by titrating 700 mL of buffer B with buffer A while monitoring with a pH meter. Spiking Solution - Prepare 3x test compound or positive control solutions: 500 μM spike solution: add 10 μL of 10 mM DMSO stock solution to 190 μL ACN.

[0395] - 1.5 μM spiking solution in microsomes (0.75 mg / mL): Add 1.5 μL of 500 μM spiking solution and 18.75 μL of 20 mg / mL liver microsomes to 479.75 μL of K-phosphate buffer. Other solutions - 6mM NADPH in 0.1M K phosphate buffer: - Dissolve 25.1 mg of NADPH tetrasodium salt in 5 mL of K phosphate buffer. procedure 30 μL of 1.5 μM spike solution containing 0.75 mg / ml microsome solution is added to wells labeled 45 minutes, 30 minutes, 15 minutes, 5 minutes and 0 minutes. The plate is pre-incubated at 37° C. for 5 minutes.

[0396] Add 15 μL of NADPH stock solution (6 mM) to all wells to begin timing. Immediately add 135 μL of ACN containing IS to the wells labeled time 0.

[0397] At 5, 15, 30 and 45 minutes, 135 μL of ACN containing IS is added to each well. At the end of the incubation, 15 μL of NADPH stock solution (6 mM) is added to the wells labeled time 0.

[0398] After quenching, the reaction mixture is centrifuged at 3220 x g for 10 minutes. 50 μL of supernatant is transferred from each well into a 96-well sample plate containing 50 μL of ultrapure water (Millipore) for LC / MS analysis.

[0399] [Table 35]

[0400] calculation Results are expressed as % test compound remaining at each time point: % remaining = peak area ratio t x / peak area ratio t0 (wherein t0 = 0 min incubation t x = given incubation time) The in vitro half-life is reported in minutes and is calculated as follows: T 1 / 2 =-(0.693 / slope) Slope = ln(% remaining) vs. incubation time where x = 0 min and y intercept = 100%.

[0401] In vitro intrinsic clearance, Cl' int is as follows: 1 / 2 It was calculated from. Cl' int =(0.693 / T 1 / 2 ) × (1 / (microsomal protein concentration (0.5 mg / mL)) × magnification The magnifications used for each species are listed in Table 11-2.

[0402] Predicted hepatic clearance for each species was calculated from half-lives using a well-stirred model. CL hep =(Q H ×Cl' int ×f ub ) / (Q H +Cl' int ×f ub )(in the formula, Q H is hepatic blood flow (mL / min / kg) (Table 11-2), f ub is the fraction of unbound drug in plasma, Cl' int is the in vitro intrinsic clearance).

[0403] [Table 36]

[0404] Data analysis The percent remaining at each incubation time point is calculated by comparing the peak area ratio (compound peak area / internal standard peak area) at TO (0 min incubation). The ln peak area ratio is plotted against time and the slope of the line is determined. The compound half-life and metabolism are calculated based on natural log-linear regression of relative peak area versus time. The half-life and intrinsic clearance in microsomes (Cl int ) is calculated using the following formula:

[0405] In Vitro T 1 / 2 (min)=0.693 / disappearance rate constant (k) Cl int (μL / min / mg of microsomal protein) = (0.693 / In Vitro T 1 / 2 ) (incubation volume / mg of microsomes) A well-stirred model is used to determine predicted clearance of the test compound for each species from calculated half-lives.

[0406] A positive control is included in the assay. If the positive control is not within the specified limits, the compound value is excluded. Biological Example 12 Comparison of Compounds and Analogs of the Present Disclosure Based on the protocols provided in this disclosure, the biological properties of the compounds of the present disclosure and several compound analogs were tested. The test results are summarized in Tables 12-1 and 12-2 below. According to Table 1, the compounds of the present disclosure (Examples 1, 2, 4, and 5) exhibited excellent HPK1 inhibitory activity (less than 1 nM). They also showed good efficacy in inducing IL-2 production. Furthermore, Table 12-2 shows that these compounds unexpectedly possessed better overall properties than their close analogs (Compounds 7-10) when considering solubility, permeability, and metabolic stability.

[0407] [Table 37-1]

[0408] [Table 37-2]

[0409] [Table 38-1]

[0410] [Table 38-2]

[0411] B. Synthesis Examples Equipment Description NMR spectra were obtained at 400 MHz ( 1 H), 376MHz( 19 F) or 75MHz ( 13Measurements were made on a Varian Mercury spectrometer operating at 1000 kHz (C). The solvent used for the samples is specified in the experimental procedures for each compound. Chemical shifts are expressed in parts per million (ppm, δ units). Coupling constants are in hertz (Hz). Splitting patterns indicate apparent multiplicity and are designated as s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), or br (broad).

[0412] The following system was used for LCMS: Agilent 6120 (Binary Gradient Module pump), XBridge analytical column C 18 , 5 μm, 4.6 × 50 mm, 25 °C, injection volume 5 μL, 2 mL / min, gradient of acetonitrile in 0.1% aqueous ammonium acetate according to the following timing:

[0413] [Table 39]

[0414] Experimental Procedures: All reactions were carried out under an atmosphere of dry nitrogen unless otherwise specified. TLC plates were visualized with ultraviolet light. Flash chromatography refers to column chromatography on silica gel (40-60 μm) using a glass column. Alternatively, automated chromatography was performed using a Biotage SP1 or Biotage Isolera system with ultraviolet detection at 220 or 254 nm and using Biotage normal-phase or reverse-phase silica cartridges. Further details can be found under the appropriate experimental procedures.

[0415] General Methods and Preparation The compounds and intermediates of the present disclosure are prepared by the synthetic methods described herein. In experimental procedures, variations in reaction conditions, such as temperature, solution concentration, solvent volume, application of microwave conditions, reaction time, or combinations thereof, are recognized as part of the present invention, and alternative suitable acids, bases, reagents, coupling reagents, solvents, etc., in addition to those specifically described, can be used and are included within the scope of the present disclosure. All possible geometric isomers, stereoisomers, and salt forms are recognized as being within the scope of the present disclosure. Intermediate 0

[0416] [ka]

[0417] 1-(tert-butyl) 3-methyl 3-methylindoline-1,3-dicarboxylate

[0418] [ka]

[0419] Step 1: To a solution of 1-(tert-butyl) 3-methylindoline-1,3-dicarboxylate (320 mg, 1.15 mmol) and iodomethane (491.36 mg, 3.46 mmol) in DMF (8 mL) was added sodium hydride (50.77 mg, 1.27 mmol, 60% in mineral oil) at 25 °C. The mixture was stirred at 25 °C for 2 h, 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 give 1-(tert-butyl) 3-methyl 3-methylindoline-1,3-dicarboxylate as a pale yellow oil (244 mg, 70% yield). LC-MS(ESI)m / z:236[M+H-56] + . Intermediate 1

[0420] [ka]

[0421] Methyl 2-(3-methylindolin-3-yl)acetate

[0422] [ka]

[0423] Step 1: To a solution of 1-(tert-butyl) 3-methyl 3-methylindoline-1,3-dicarboxylate (730 mg, 2.51 mmol) in THF (20 mL) was added a solution of sodium hydroxide (3.01 g, 75.17 mmol) in water (1.41 mL). The mixture was stirred at room temperature for 16 hours, acidified with 1 M aqueous HCl, and extracted with EtOAc (100 mL × 3). The organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to give crude 1-tert-butoxycarbonyl-3-methylindoline-3-carboxylic acid as a yellow oil (620 mg, 85% yield). LC-MS (ESI) m / z: 222 [M+H-56] + .

[0424] Step 2: To a mixture of 1-tert-butoxycarbonyl-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 h 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) (2 M solution in diethyl ether) was slowly added to the above solution. The reaction mixture was stirred under N for 2 h, 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 give tert-butyl 3-(2-diazoacetyl)-3-methylindoline-1-carboxylate as a yellow oil (200 mg, 56% yield).

[0425] 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 under N2 for 1.5 h and then 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 give 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]. + .

[0426] Step 4: A mixture of tert-butyl 3-(2-methoxy-2-oxoethyl)-3-methylindoline-1-carboxylate (100 mg, 0.278 mmol) and HCl / EA (4 M, 1.5 mL) in DCM (3 mL) was stirred at room temperature for 2 h and concentrated in vacuo to give crude methyl 2-(3-methylindolin-3-yl)acetate without further purification. LC-MS (ESI) m / z: 206 [M+H] + .

[0427] Alternative synthesis methods:

[0428] [ka]

[0429] Step 1: To a solution of 3-methylindolin-2-one (20 g, 135.89 mmol) in THF (200 mL) was added NaH (6.52 g, 163.09 mmol, 60% suspension in mineral oil) over 30 min at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, followed by the dropwise addition of a solution of di-tert-butyl carbonate (29.07 g, 133.18 mmol) in THF (50 mL). The reaction mixture was stirred for 1 h, diluted with saturated aqueous NH4Cl (50 mL), and extracted with CHCl (100 mL × 2). The combined 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 (PE / EA=9 / 1) to give tert-butyl 3-methyl-2-oxoindoline-1-carboxylate (30 g, 74% yield) as a yellow oil. LC-MS (ESI) m / z: 192 [M+H] + .

[0430] 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) over 30 min at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, followed by the dropwise addition of methyl 2-bromoacetate (18.93 g, 123.74 mmol). The reaction mixture was stirred for 1 h, quenched with saturated aqueous NH Cl (100 mL), and extracted with CHCl (200 mL × 2). The combined organic phase was dried over anhydrous Na SO 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 give 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] + .

[0431] Step 3: A mixture of 4 M 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 CHCl (200 mL) was stirred at room temperature for 2 hours and concentrated in vacuo. The residue was partitioned between CHCl (200 mL) and saturated aqueous NaHCO (50 mL). The separated aqueous layer was extracted with CHCl (50 mL × 3), and the combined organic layer was dried over anhydrous NaSO and filtered. The filtrate was concentrated in vacuo to give methyl 2-(3-methyl-2-oxoindolin-3-yl)acetate (17.6 g, 82% yield). LC-MS (ESI) m / z: 220 [M+H] + .

[0432] Step 4: A mixture of methyl 2-(3-methyl-2-oxoindolin-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 give methyl 2-(3-methyl-2-thioxoindolin-3-yl)acetate (15.5 g, 74% yield) as a white solid. LC-MS (ESI) m / z: 236 [M+H] + .

[0433] Step 5: To a mixture of methyl 2-(3-methyl-2-thioxoindolin-3-yl)acetate (9.5 g, 40.37 mmol) and nickel chloride (10.46 g, 80.75 mmol) in THF (70 mL) and methanol (70 mL) at 0 °C, NaBH (9.16 g, 242.24 mmol) was added portionwise over 1 h. The resulting mixture was stirred for 10 min and filtered through a pad of Celite. 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 give methyl 2-(3-methylindolin-3-yl)acetate (6.5 g, 75% yield) as a yellow oil. LC-MS (ESI) m / z: 206 [M+H] + . Intermediate 2

[0434] [ka]

[0435] 6-Methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[0436] [ka]

[0437] Step 1: To a mixture of 2-(3-methoxyphenyl)ethanamine (20.0 g, 132.27 mmol, 19.23 mL) in aqueous HCl (1 N, 192 mL, 192 mmol), an aqueous solution of formaldehyde (37 wt%, 41.64 g, 529.08 mmol) was added. The mixture was stirred at 60° C. for 1 hour, cooled to 0° C., and basified by the dropwise addition of 50% aqueous NaOH (17.44 g, 218 mmol) at 0° C. The resulting mixture was stirred overnight at room temperature and filtered. The filtrate was concentrated to give bis(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)methane as a white solid (23 g, 100% yield).

[0438] Step 2: To a suspension of bis(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)methane (28 g, 82.73 mmol) in i-PrOH (200 mL) was added concentrated HCl (15.20 g, 182.01 mmol, 15.3 mL) dropwise at 0 °C, and the mixture was stirred at room temperature for 18 h. MTBE (70 mL) was added to the mixture, and the suspension was stirred at room temperature for another 4 h. After filtration, the cake was washed with a mixture of MTBE / i-PrOH (100 mL, 1 / 1 v / v) and dried to give 6-methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride as a white solid, which was suspended in DCM (300 mL). Saturated aqueous NaHCO3 (500 mL) was added to the mixture, and the mixture was stirred at room temperature for 2 h. After separation, the aqueous layer was extracted with DCM (50 mL × 4). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated to give 6-methoxy-1,2,3,4-tetrahydroisoquinoline as a yellow oil (10 g, 75% yield). LC-MS (ESI) m / z: 164 [M+H] + .

[0439] Step 3: To a solution of 6-methoxy-1,2,3,4-tetrahydroisoquinoline (1.63 g, 9.99 mmol) in MeOH (30 mL) was added aqueous formaldehyde (37% w / w, 4.8 g, 59.92 mmol, 1.67 mL) at room temperature. The mixture was stirred at room temperature for 15 min, cooled to 0 °C, and NaBH (1.13 g, 29.96 mmol) was added in portions. The mixture was stirred at room temperature for 3 h, quenched with ice-water (10 mL), and extracted with DCM (20 mL × 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 give 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] + .

[0440] Step 4: To a pre-cooled (0 °C) solution of concentrated sulfuric acid (4 mL) was added 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline (2 g, 11.28 mmol) and guanidine nitrate (1.17 g, 9.59 mmol) sequentially at 0 °C. The resulting mixture was stirred at 0 °C for 30 min, quenched with ice-water (20 mL), basified with aqueous NaOH (4 N) to pH 10-11, and extracted with DCM (50 mL × 4). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (PE 100% v / v, PE / EA = 1 / 1 v / v, EA 100% v / v, then DCM / MeOH = 20 / 1, v / v) to give 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] + .

[0441] Step 5: To a solution of 6-methoxy-2-methyl-7-nitro-1,2,3,4-tetrahydroisoquinoline (4.2 g, 14.40 mmol) in EA (32 mL), HO (16 mL), and EtOH (144 mL), Fe powder (5.5 g, 93.99 mmol) and ammonium chloride (793.96 mg, 13.08 mmol) were added. The mixture was stirred at 60 °C for 48 h, cooled to room temperature, and filtered. The cake was washed with methanol (30 mL × 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 give 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine hydrochloride as a yellow solid (4.5 g, 100% yield). LC-MS(ESI)m / z:193[M+H] + .

[0442] Alternative synthesis methods:

[0443] [ka]

[0444] Step 1: To a solution of triethylamine (13.38 g, 132.27 mmol, 18.44 mL), 2-(3-methoxyphenyl)ethanamine (10 g, 66.13 mmol), and DMAP (807.96 mg, 6.61 mmol) in DCM (100 mL) was added BocO (15.88 g, 72.75 mmol, 16.70 mL) slowly at 0 °C. The mixture was then stirred at room temperature for 16 h, diluted with ice-water (200 mL), and extracted with EtOAc (3 × 200 mL). The combined organic phase was washed with brine (100 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1) to give 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] + .

[0445] 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 CHCl (50 mL) was added a solution of TfO (8.64 g, 30.64 mmol, 5.15 mL) in CHCl (5 mL) at −78 °C. After 20 min, BH·EtO (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 h, and carefully quenched with saturated NaHCO solution. The resulting mixture was extracted with CHCl (3 × 100 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous NaSO, 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 give 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] + .

[0446] Step 3: To a solution of 6-methoxy-3,4-dihydroisoquinolin-1(2H)-one (3.8 g, 21.44 mmol, 9.62 mL) in concentrated HSO (50 mL) was added HNO (2.16 g, 22.29 mmol, 65% purity) dropwise at -20 °C. The mixture was stirred at -20 °C to -25 °C for 3 h and poured into ice-water (300 mL). The aqueous layer was extracted with dichloromethane (3 × 200 mL). The combined organic layers were concentrated in vacuo, and the residue was purified by flash column chromatography (SiO, petroleum ether / ethyl acetate / methanol = 10 / 1 / 0 to 10 / 10 / 1) to give 6-methoxy-7-nitro-3,4-dihydroisoquinolin-1(2H)-one (2.02 g, 40.78% yield). LC-MS(ESI)m / z:223[M+H] + .

[0447] Step 4: To a solution of 6-methoxy-7-nitro-3,4-dihydroisoquinolin-1(2H)-one (2.02 g, 9.09 mmol) in THF (200 mL) was added 1 M BH3 / THF (45.46 mmol, 45.5 mL). The mixture was stirred under reflux for 20 hours and carefully quenched with methanol (30 mL). The resulting solution was concentrated in vacuo. The residue was heated in 2 N HCl (50 mL) at 80 °C for 3 hours, cooled, basified with aqueous ammonium hydroxide, and extracted with dichloromethane (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give 6-methoxy-7-nitro-1,2,3,4-tetrahydroisoquinoline (1.89 g, 100.00% yield). LC-MS (ESI) m / z: 209 [M+H] + .

[0448] Step 5: 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) was added formaldehyde (1.64 g, 54.46 mmol, 1.51 mL) at room temperature. The mixture was stirred at room temperature for 15 minutes and cooled to 0 °C. NaBH (1.03 g, 27.23 mmol) was then added portionwise to the above mixture. The resulting mixture was stirred at room temperature for 3 hours, quenched with ice water (10 mL), and extracted with DCM (20 mL × 5). The combined 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 give 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] + .

[0449] Step 6: 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), HO (1 mL), and EtOH (10 mL) was added Fe (2.19 g, 39.19 mmol) and ammonium chloride (284.74 mg, 5.32 mmol). The mixture was stirred at 60 °C for 16 h, cooled to room temperature, and filtered. The cake was washed with methanol (30 mL × 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 give 6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine hydrochloride as a yellow solid (0.85 g, 75.6% yield). LC-MS (ESI) m / z: 193 [M+H] + .

[0450] Intermediate 3

[0451] [ka]

[0452] 6-Fluoro-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine

[0453] [ka]

[0454] Step 1: A mixture of 6-fluoro-3,4-dihydro-2H-isoquinolin-1-one (20.0 g, 121.09 mmol) in HSO (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. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 7.9 Hz, 1H), 8.33 (d, J = 27.5 Hz, 1H), 7.63 (d, J = 11.8 Hz, 1H), 3.43 (td, J = 6.6, 2.8 Hz, 2H), 3.04 (t, J = 6.5 Hz, 2H).LCMS(ESI + )[(M+H) + ]:211.

[0455] Step 2: 6-Fluoro-7-nitro-3,4-dihydro-2H-isoquinolin-1-one (25.2 g, 119.91 mmol) was added to 1 M borane tetrahydrofuran (599.54 mmol, 600 mL). The mixture was stirred under reflux for 20 hours, cooled to room temperature, and carefully quenched with methanol (150 mL). The resulting solution was concentrated in vacuo. The residue was stirred in 2N aqueous HCl (500 mL) at 80 °C for 3 hours, cooled to room temperature, basified to pH 8-9 with ammonium hydroxide solution, and extracted with dichloromethane (500 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo to give 6-fluoro-7-nitro-1,2,3,4-tetrahydroisoquinoline (22.3 g, 113.67 mmol, 94.8% yield) as a yellow solid. LCMS (ESI) + )[(M+H) + ]:197.

[0456] 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 g, 454.69 mmol) was added in portions to the mixture. 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 layer 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 v / v) to give 6-fluoro-2-methyl-7-nitro-3,4-dihydro-1H-isoquinoline (21.3 g, 101.33 mmol, 89.1% yield) as a yellow oil. + )[(M+H) + ]:211.

[0457] 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 HO (40 mL), ammonium chloride (37.22 g, 709.31 mmol) and iron powder (56.59 g, 1.01 mol, 7.20 mL) were added. The mixture was stirred at 60 °C for 3 h, cooled to room temperature, and filtered. The solid 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 give 6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-amine (17.3 g, 95.99 mmol, 94.7% yield) as a yellow solid. LCMS (ESI) + )[(M+H) + ]:181.

[0458] Example 1

[0459] [ka]

[0460] 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 Step 1: To a mixture of intermediate 1 (67 mg, 0.248 mmol) in n-BuOH (3 mL), 2,4,5-trichloropyrimidine (45.5 mg, 0.248 mmol) and DIPEA (96.19 mg, 0.744 mmol) were slowly added. The mixture was stirred at 100 °C for 16 h, quenched with ice-cold water (20 mL), and extracted with EA (30 mL × 3). The organic phase was washed with brine (20 mL), dried over anhydrous NaSO, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (DCM / PE = 0 to 100%) to give methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate (80 mg, 83.3% yield) as a yellow solid. LC-MS(ESI)m / z:352[M+H] + .

[0461] 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), Intermediate 2 (51.5 mg, 0.227 mmol) and TsOH·HO (43.2 mg, 0.227 mmol) were slowly added. The mixture was stirred at 100 °C for 16 h, quenched with saturated aqueous NaHCO (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 NaSO, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (DCM / MeOH = 0 to 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 (500 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.98 (s, 1H), 7.59 (s, 1H), 7.30 (t, J = 8.8 Hz, 2H), 7.12 (s, 1H), 6.98 (s, 1H), 6.74 (s, 1H), 4.38 (d, J = 10.7 Hz, 1H), 4.05 (d, J = 10.7 Hz, 1H), 3.78 (s, 3H), 3.50 (s, 3H), 3.27 (s, 2H), 2.78 (dd, J = 10.3, 4.8 Hz, 3H), 2.68 (s, 1H), 2.55 (t, J = 5.7 Hz, 2H), 2.30 (s, 3H), 1.36 (s, 3H). 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 (2 N, 1 mL) in THF (5 mL) was stirred at room temperature for 16 h, adjusted to pH ∼5 with formic acid, and extracted with DCM (30 mL × 3). The combined organic phase was 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 (500 MHz, DMSO-d6) δ 8.25 (s, 1H), 7.99 (s, 1H), 7.57 (s, 1H), 7.29 (dd, J = 17.2, 7.6 Hz, 2H), 7.11 (t, J = 7.3 Hz, 1H), 6.98 (d, J = 7.3 Hz, 1H), 6.75 (s, 1H), 4.42 (d, J = 10.7 Hz, 1H), 4.02 (d, J = 10.6 Hz, 1H), 3.78 (s, 3H), 3.30 (s, 2H), 2.78 (s, 2H), 2.69 (d, J = 15.4 Hz, 1H), 2.58 (d, J = 6.1 Hz, 3H), 2.32 (s, 3H), 1.34 (s, 3H). Examples 2 and 3

[0462] [ka]

[0463] (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 Method 1: Step 1: 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) was separated by SFC chiral separation to give (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)-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 (67 mg, 63 mg), respectively. The stereochemistry was not fully determined.

[0464] The component corresponds 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. LC-MS(ESI)m / z:508[M+H] + Chiral-HPLC retention time: 1.829 min; ee value: 98.3%.

[0465] The component corresponds to peak 1: (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. LC-MS(ESI)m / z:508[M+H] + Chiral HPLC retention time: 3.427 min; ee value: >99%.

[0466] SFC separation conditions: Apparatus: SFC-80 (Thar, Waters) Column: AD-H 20 * 250mm, 10μm (Daicel) Column temperature: 35℃ Mobile phase: CO2 / EtOH (0.2% methanol / ammonia) = 50 / 50 Flow rate: 80g / min Back pressure: 100bar Detection wavelength: 214 nm Cycle time: 6.9 minutes Step 2: (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)-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 were reacted with 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 in the same manner as in Step 3 of Example 1. The compounds were each hydrolyzed under the conditions described above 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 and 33.8 mg, respectively).

[0467] Example 2: ((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 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)acetic acid) corresponds to the product from peak 1 of the SFC separation in step 1 of method 1.

[0468] LC-MS(ESI)m / z:494[M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.00 (s, 1H), 7.57 (s, 1H), 7.30 (dd, J = 13.7, 7.7 Hz, 2H), 7.11 (t, J = 7.6 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 6.75 (s, 1H), 4.42 (d, J = 10.7 Hz, 1H), 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%.

[0469] 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) corresponds to the product from peak 2 of the SFC separation in step 1 of method 1.

[0470] LC-MS(ESI)m / z:494[M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.00 (s, 1H), 7.56 (s, 1H), 7.29 (dd, J = 13.6, 7.4 Hz, 2H), 7.10 (t, J = 7.1 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 6.75 (s, 1H), 4.41 (d, J = 10.8 Hz, 1H), 4.02 (d, J = 10.8 Hz, 1H), 3.77 (s, 3H), 3.29 (s, 2H), 2.77 (t, J = 5.6 Hz, 2H), 2.69 (d, J = 15.4 Hz, 1H), 2.57 (dd, J = 13.1, 7.1 Hz, 3H), 2.30 (d, J = 11.1 Hz, 3H), 1.34 (s, 2H). Chiral HPLC retention time: 1.132 min; ee value: >99%.

[0471] Method 2: Step 1: SFC chiral separation of methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate gave (R)-methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate and (S)-methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate.

[0472] The component corresponds to peak 1: LC-MS (ESI) m / z: 352 [M+H] + Chiral HPLC retention time: 1.191 min (peak 1); ee value: >99%. The component corresponds to peak 2: LC-MS (ESI) m / z: 352 [M+H] + Chiral HPLC retention time: 1.519 min (peak 2); ee value: >99%.

[0473] SFC separation conditions: Apparatus: SFC-80 (Thar, Waters) Column: AD-H 20 *250mm, 10μm (Daicel) Column temperature: 35℃ Mobile phase: CO2 / isopropanol (0.2% methanol / ammonia) = 87 / 13 Flow rate: 80g / min Back pressure: 100bar Detection wavelength: 214 nm Cycle time: 2.8 minutes Additional large batches of SFC chiral separation of methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate to give (R)-methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate and (S)-methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate were performed under similar or identical conditions. (One stereoisomer as representative of steps 2 and 3) Step 2: A mixture of Intermediate 2 (1.36 g, 5.96 mmol), (S)-methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate or (R)-methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate (2.1 g, 5.96 mmol, derived from peak 2 of the SFC separation in Step 1 of Method 2), and TsOH HO (1.13 g, 5.96 mmol) in t-butanol (25 mL) was stirred at 100 °C for 12 h, cooled to room temperature, poured into saturated aqueous NaHCO (10 mL), and extracted with EA (30 mL × 3). The organic phase was dried over anhydrous NaSO 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 give (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, 56.3% yield) as a light yellow solid. LC-MS (ESI) m / z: 508 [M+H] + .

[0474] Step 3: A mixture of lithium hydroxide hydrate (264 mg, 6.3 mmol) in water (5 mL) 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 ∼3 with acetic acid. The resulting mixture was purified by preparative HPLC (MeCN / 10 mM NH4HCO3) to afford 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 pale yellow solid, which originated from peak 2 of the SFC separation of step 1 of method 2. LC-MS (ESI) m / z: 494 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.02 (s, 1H), 7.63 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.02 (t, J = 8.0 Hz, 1H), 6.80 (s, 1H), 4.48 (d, J = 10.8 Hz, 1H), 4.08 (d, J = 10.8 Hz, 1H), 3.83 (s, 3H), 2.83 (t, J = 5.6 Hz, 2H), 2.74 (d, J = 15.2 Hz, 1H), 2.66-2.59 (m, 3H), 2.37 (s, 3H), 1.40 (s, 3H). Chiral HPLC retention time: 1.132 min; ee value: >99%.

[0475] Example 4

[0476] [ka]

[0477] 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 Step 1: A solution of methyl 2-(1-(2,5-dichloropyrimidin-4-yl)-3-methylindolin-3-yl)acetate (195.4 mg, 0.555 mmol), Intermediate 3 (100 mg, 0.555 mmol), and TsOH HO (105.5 mg, 0.555 mmol) in i-PrOH (4 mL) was stirred at 120 °C for 16 h, cooled to room temperature, and neutralized to pH 8 with saturated aqueous NaHCO. The mixture was extracted with DCM / MeOH (v / v, 10 / 1, 20 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 (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] + .

[0478] 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) and a mixture of aqueous LiOH (2N, 4 mL) in THF (4 mL) was stirred at room temperature for 5 hours and at 50° C. for 16 hours, adjusted to pH 5 with formic acid, and extracted with DCM / MeOH (10 / 1, 20 mL×3). The combined organic phase was 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] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.22 (s, 1H), 7.35-7.24 (m, 3H), 7.07-6.92 (m, 3H), 4.41 (d, J = 10.7 Hz, 1H), 4.03 (d, J = 10.8 Hz, 1H), 3.36 (s, 2H), 2.78 (d, J = 5.4 Hz, 2H), 2.68 (d, J = 15.5 Hz, 1H), 2.60-2.54 (m, 3H), 2.32 (s, 3H), 1.33 (s, 3H). Examples 5 and 6

[0479] [ka]

[0480] Method 2 of Examples 2 and 3 was followed. Step 1: To a solution of the first eluate (peak 1) of ((R)-methyl 2-[1-(2,5-dichloropyrimidin-4-yl)-3-methyl-indolin-3-yl]acetate or (S)-methyl 2-[1-(2,5-dichloropyrimidin-4-yl)-3-methyl-indolin-3-yl]acetate) (27.8 g, 78.93 mmol) in n-butanol (anhydrous, 99.9%, 600 mL), 6-fluoro-2-methyl-3,4-dihydro-1H-isoquinolin-7-amine (Intermediate 3, 17.07 g, 94.71 mmol) and 4-methylbenzenesulfonic acid (14.95 g, 86.82 mmol) were added as described in Step 1 of Method 2 in Examples 2 and 3. The mixture was stirred at 120 ° C. for 20 hours. After completion of the reaction 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 x 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 give (R)-methyl 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]acetate or (S)-methyl 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]acetate (22.6 g, 45.57 mmol, 57.7% yield, mixed with the butyl ester) as a yellow solid. LCMS (ESI + )[(M+H) + ]:496.

[0481] Step 2: A mixture of (R)-methyl 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]acetate or (S)-methyl 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]acetate (22.6 g, 45.57 mmol) and aqueous lithium hydroxide solution (5.46 g, 227.83 mmol) (1N in HO) 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 (NHHCO) 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 peak 1 of the SFC separation in Step 1 of Method 2 of Examples 2 and 3, which resulted in a more potent product.

Claims

1. Formula I 【Chemical 1】 or a pharmaceutically acceptable salt or stereoisomer thereof.

2. Formula IA 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

3. Formula IB 【Chemistry 3】 or a pharmaceutically acceptable salt thereof.

4. Formula II 【Chemistry 4】 or a pharmaceutically acceptable salt or stereoisomer thereof.

5. Formula IIA 【Chemistry 5】 or a pharmaceutically acceptable salt thereof.

6. Formula IIB 【Chemistry 6】 or a pharmaceutically acceptable salt thereof.

7. A compound described in any one of claims 2, 3, 5, and 6, or a pharmaceutically acceptable salt thereof, wherein the compound has an optical purity of 70% or more.

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

9. The compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the compound has an optical purity of 99% or more.

10. 10. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1 to 9, 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 9, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

12. 10. A combination comprising a therapeutically effective amount of a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, and one or more therapeutically active agents.

13. 10. A pharmaceutical composition for treating cancer, comprising an effective amount of a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof.

14. 14. The pharmaceutical composition of claim 13, wherein the cancer is selected from breast cancer, colorectal cancer, lung cancer, ovarian cancer, and pancreatic cancer.

15. 10. A pharmaceutical composition for inhibiting HPK1 activity in a subject, comprising an effective amount of a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof.

16. 16. The pharmaceutical composition of claim 15, wherein the subject has cancer and the cancer is to be treated.

17. 17. The pharmaceutical composition of claim 16, wherein the cancer is selected from breast cancer, colorectal cancer, lung cancer, ovarian cancer, and pancreatic cancer.

Images