Biphenylpyridine rigidized compounds with polycyclic heterocycles as dual immune checkpoint inhibitors

Abstract

The present invention relates to small molecules useful therapeutically as immunomodulators that are biphenylpyridine rigidized compounds with polycyclic heterocycles. These compounds disrupt the PD-1 / PD-L1 checkpoint pathway and can also disrupt one or more related checkpoint pathways. These compounds are useful in treating, preventing, or ameliorating a disorder such as a cancer or an infection.

Description

Technical Field

[0001] This invention relates to small molecules of formula (I), which are rigid biphenylpyridine compounds with polycyclic heterocycles, and can be used as immunomodulators in treatment. These compounds disrupt the PD-1 / PD-L1 checkpoint pathway, and preferably also disrupt one or more related pathways. The one or more related checkpoint pathways can be, for example, the VISTA pathway or any other pathway. These compounds can be used to treat, prevent, or improve diseases such as cancer or infections.

[0002]

[0003] (I) Background Technology

[0004] Immune checkpoints (ICs) play a crucial role in regulating immune responses. Inhibitory ICs in the tumor microenvironment (TME) have been implicated in the immune escape of tumor cells. Therefore, recognizing and targeting these inhibitory ICs may be essential for eliminating tumor cells. ICs can be expressed on both immune cells and tumor cells and can modulate immune responses. These molecules are essential for maintaining homeostasis; however, the expression of inhibitory ICs (such as programmed cell death ligand 1 (PD-L1)) can protect tumor cells from antitumor immune responses. Therefore, inhibiting these inhibitory ICs has received particular attention in the treatment of various cancers.

[0005] PD-L1 is a 40 kDa immune checkpoint protein encoded by the CD274 gene in humans. Upon binding to its receptor PD-1, expressed on activated B cells, T cells, and myeloid cells, PD-L1 initiates signaling pathways that lead to downregulation of T cell proliferation and activation, promoting tumor cell escape from T cell-mediated immune surveillance, thereby contributing to cancer severity and progression. PD-L1 has been shown to be effective in various solid tumors (e.g., breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, salivary gland cancer, gastric cancer, glioma, thyroid cancer, thymic carcinoma, head and neck cancer). (References: Brown JA et al., 2003. J. Immunol. 170:1257-66; Dong H et al. 2002. Nat. Med. 8:793-800; Hamanishi J, et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003. Cancer Res. 63:6501-5; Inman BA et al. 2007. Cancer 109:1499-505; Konishi J et al. 2004. Clin. Cancer Res.) PD-L1 is expressed on the liver and has become an attractive target for the development of anticancer therapeutics. PD-L1 expression is also further involved in the escape of immune responses in infectious diseases, such as chronic viral infections including HBV and HIV. (References: 10:5094-100; Nakanishi J et al. 2007. Cancer Immunol. Immunother. 56:1173-82, which are incorporated herein by reference).

[0006] Therefore, PD-L1 also serves as a therapeutic target for various infectious diseases. The therapeutic efficacy of PD-L1 antagonists (and PD-1 antagonists) has been validated in clinical trials. Immune checkpoint inhibitors, such as monoclonal antibodies targeting programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1), have achieved great success in the treatment of various cancers. However, the high production cost of monoclonal antibodies, poor tumor penetration, and the potential to induce autoimmune side effects all limit their application. Nevertheless, there remains a need for more effective, better, and / or selective PD-1 pathway immunomodulators.

[0007] To date, six FDA-approved therapeutic antibodies targeting PD-1 / PD-L1, including nivolumab, pembrolizumab, avelumab, atezolizumab, cimiprimab, and durvalumab, have achieved significant clinical outcomes and durable remissions in their respective indications.

[0008] Despite the unprecedented success of monoclonal antibodies, their inherent drawbacks include high manufacturing costs, low tissue penetration, insufficient oral bioavailability, and immunogenicity. In addition to lower manufacturing costs, higher stability, and better tissue and tumor penetration, small molecule inhibitors offer a better therapeutic index compared to monoclonal drugs, based not only on optimal drug efficacy. Clinical and oral administration parameters are more flexible, and a reasonable half-life can be maintained to avoid systemic immunogenicity. Therefore, small molecule inhibitors can be administered alone or in combination with therapeutic antibodies to provide promising alternative treatment strategies to address drug resistance and low clinical response.

[0009] To date, although numerous patents and publications disclose a range of small molecule inhibitors targeting the PD-1 / PD-L1 pathway, there are currently no FDA-approved small molecule inhibitors for PD-L1. To meet the needs of the domestic market, there is an urgent need to obtain more alternative compounds with novel scaffolds for future clinical applications.

[0010] VISTA (T-cell activated V-domain immunoglobulin inhibitor) is a recognized immunomodulatory receptor. Also known as PD-1 homolog (PD-1H), Dies1 (Dies1), DD1α, Gi24, SISP1, B7-H5, and C10orf54, VISTA is a novel inhibitory IC. VISTA is significantly overexpressed in tumor-infiltrating immune cells in various cancers, including melanoma, gastric cancer, prostate cancer, colorectal cancer, and acute myeloid leukemia.Furthermore, recent findings indicate that VISTA can also be expressed on tumor cells, such as hepatocellular carcinoma, endometrial cancer, ovarian cancer, gastric cancer, and non-small cell lung cancer (Kakavand H, Jackett LA, Menzies AM, Gide TN, Carlino MS, Saw RPM, et al. Modern Pathol and Off JU States Can Acad Pathol Inc (2017) 30(12):1666–76; Kuklinski LF, Yan S, Li Z, Fisher JL, Cheng C, Noelle RJ, et al. Cancer Immunol Immunother CII (2018) 67(7):1113–21; Gao J, Ward JF, Pettaway CA, ShiLZ, Subudhi SK, Vence LM, et al. Nat Med (2017) 23(5):551–5; Xie S, Huang J, Qiao Q, Zang W, Hong S, Tan H, et al. Cancer Immunol Immunother CII (2018) 67(11):1685–94; Wang L, Jia B, Claxton DF, Ehmann WC, Rybka WB, Mineishi S, etal.. Oncoimmunology (2018) 7(9):e1469594; Zhang M, Pang HJ, Zhao W, Li YF, Yan LX, Dong ZY, et al.. BMC Cancer (2018) 18(1):511; Liao H, Zhu H, Liu S,Wang H. Oncol Lett (2018) 16(3):3465–72; Böger C, Behrens HM, Krüger S, Röcken C. Oncoimmunology (2017) 6(4): e1293215. 51. Villarroel-Espindola F, YuX, Datar I, Mani N, Sanmamed M, Velcheti V, et al. Res an Off J Am AssocCancer Res (2018) 24(7):1562–73 (all cited in this paper).

[0011] As a novel inhibitory IC, VISTA can be overexpressed on myeloid lineage cells, lymphoid lineage cells, tumor-infiltrating immune cells, and tumor cells; therefore, it can significantly suppress anti-tumor immune responses. Since VISTA expression on tumor cells can lead to tumor development, and its signaling pathway differs from that of other inhibitory ICs (such as PD-L1), dual blockade of tumor VISTA / PD-L1 can have a synergistic inhibitory effect on tumor development. Combined blockade of inhibitory ICs (such as VISTA / PD-1 blockade) can improve the response rate of affected patients to cancer treatment.

[0012] To date, there are no FDA-approved small molecule inhibitors targeting the PD-1 / PD-L1 and VISTA pathways. CA-170 is the only known drug in clinical trials that can directly target PD-L1 and VISTA.

[0013] In summary, it is crucial to develop novel dual-target PD-1 / PD-L1 and VISTA inhibitors for clinical immunotherapy (such as cancer drugs). Summary of the Invention

[0014] This invention provides rigid biphenylpyridine compounds with terminal polycyclic heterocycles, such as seven- or eight-membered bicyclic spirocyclic heterocycles. These compounds are capable of inhibiting the programmed cell death ligand 1 (PD-1 / PD-L1) signaling pathway and / or the VISTA pathway. These small molecule antagonists of the PD-1 / PD-L1 / VISTA interaction have shown potent antitumor activity both in vitro and in vivo. These compounds have been shown to have efficacy comparable to monoclonal antibodies. Not wishing to be bound by theory, it is hypothesized that these compounds act at least in part by alleviating PD-1 / PD-L1-induced T cell exhaustion.

[0015] This invention provides a compound represented by Formula I or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof:

[0016]

[0017] (I)

[0018] in:

[0019] R1, R2, and R4 are each independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, cycloalkyl, alkenyl, ynyl and -NR'R5;

[0020] R3 is selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C3-6 Cycloalkyl, alkenyl, alkynyl, NR'R5, and -OR6;

[0021] R' is selected from H and C. 1-6 alkyl;

[0022] R5 is selected from hydrogen, C 1-6 Alkyl, cycloalkyl, alkenyl, and ynyl groups containing up to 6 carbon atoms;

[0023] R6 is selected from C 1-6 Alkyl groups and -L2-A;

[0024] L2 does not exist or C 1-6 Alkylene;

[0025] A is selected from aryl and 5- to 14-membered heteroaryl groups, which may optionally be substituted by one or two substituents, each independently selected from cyano, halogen, hydroxyl, and amino groups; and

[0026] L1 is a optionally substituted polycyclic heterocyclic group.

[0027] Compounds of formula (I) include stereoisomers, such as the geometric isomers of compounds of formula (I) as illustrated above.

[0028] The present invention also provides a method for treating, preventing, or improving a disease, the method comprising administering to a subject a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt, or pharmaceutical composition, as defined herein. Similarly, the present invention provides a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt, or pharmaceutical composition, as defined herein, for use in a method for treating, preventing, or improving a disease, as defined herein. Furthermore, the present invention provides the use of a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt, or pharmaceutical composition, as defined herein, in the preparation of a medicament for use in a method for treating, preventing, or improving a disease, as defined herein.

[0029] The present invention also provides an in vitro method, such as an assay method, comprising using a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein:

[0030] (i) Inhibit the PD-1 / PD-L1 checkpoint pathway;

[0031] (ii) Inhibit the activity of VISTA;

[0032] (iii) Reverse T cell exhaustion;

[0033] (iv) Enhance T cell function;

[0034] (v) Increase the CD8+ T cell population;

[0035] (vi) Reduce PD-1 expression;

[0036] (vii) Reduce TIM3 expression;

[0037] (viii) Increase CD107a expression;

[0038] (ix) Increase granzyme B expression; and / or

[0039] (x) Restore IFNg;

[0040] (xi) Enhances T cell-mediated cytotoxicity against tumor cells. Attached Figure Description

[0041] Some embodiments of the invention are described herein by way of example only and with reference to the accompanying drawings. Specific details are now referred to in the drawings, and it should be emphasized that the details shown are by way of example and for the purpose of illustrative discussion of embodiments of the invention. In this regard, the description taken in conjunction with the drawings will make it clear to those skilled in the art how embodiments of the invention can be implemented as illustrated.

[0042] Figure 1 (a): Binding interactions and conformations of NAT-24 at the interface of PD-L1 dimer (PDB 5N2F). Figure 1 (b): Binding interactions and conformations of NAT-25 in VISTA ECD (PDB 6MVL).

[0043] Figure 2 This is a flowchart illustrating the virtual screening of putative PD-1 / PD-L1 inhibitors and substances capable of binding to VISTA as described in the following computational examples. It is a schematic diagram of the stages of the workflow for structure-based virtual screening and identification of small molecule inhibitors of PD-1 / PD-L1 and VISTA using Schrödinger software.

[0044] Figure 3 (a) shows the micro-thermophoretic (MST) dose-response curves of the four compounds described herein binding to His-tagged human PD-L1. (b) shows the melting curves and melting temperature (Tm) shifts of hPD-L1 in the presence of these four compounds.

[0045] Figure 4 (a) IC50 values ​​of Cpd-1, Cpd-2, Cpd-3 and Cpd-4 in HTRF assays, presented as bar graphs. (b) IC50 values ​​of Cpd-1, Cpd-2, Cpd-3 and Cpd-4 in HTRF dimerization assays, presented as bar graphs.

[0046] Figure 5 (a) EC50 values ​​of Cpd-1, Cpd-2, Cpd-3 and Cpd-4 in NFAT reporting bioassays, presented as bar graphs. (b) IC50 values ​​of Cpd-1, Cpd-2, Cpd-3 and Cpd-4 in VISTA / VSIG3 assays, presented as bar graphs.

[0047] Figure 6 This includes the results of the experiments shown in Example 54 using compounds 1 through 4 (referred to as Cp 1, Cp 2, Cp 3, and Cp 4, respectively). Figure 6 (a)-(d) involve the CHO-PDL1 cell line, which was untreated and treated with the compound, followed by 72 hours ( Figure 6 (a)); 48 hours ( Figure 6 (b)); 17 hours ( Figure 6 (c) and 2 hours ( Figure 6 (d) Detection of cell surface PD-L1 by flow cytometry. Figure 6 (e) Involving the MDAMB231 cell line, which was untreated and treated with compounds 1 and 3, and then the cell surface PD-L1 was detected by flow cytometry after 72 hours. Figure 6 (f) The U251 cell line was involved, which was untreated and treated with compounds 1 and 3, and then the cell surface PD-L1 was detected by flow cytometry after 17 hours. Figure 6 (g) represents the HCC827 cell line, which was untreated and treated with compounds 1 and 3, and then the cell surface PD-L1 was detected by flow cytometry after 72 hours.

[0048] Figure 7 The results of the experiments described in Example 55 below are shown, in which apoptosis was measured in an Annexin V assay after co-culturing CD8 T cells with various cell lines and compounds 1 and 3 of the present invention. Figure 7 (a) shows the results using MDAMB231 cells. Figure 7 (b) shows the results using U251 cells. Figure 7 (c) shows the results using HCC827 cells.

[0049] Figure 8 The results of the experiments described in Example 55 below are shown, in which the effects of compounds 1, 2 and 3 according to the invention on exhausted T cells were measured. Figure 8 (a) shows the levels of CD8+ and exhaustion markers PD1 and TIM3 in exhausted T cells. Figure 8(b) through (d) show the levels of these markers after treatment with compounds 1, 2, and 3, respectively. CD8 cell counts increased, while the exhaustion markers PD1 and TIM3 decreased. Figure 8 (e) shows the levels of exhaustion markers PD1 and IFNG in exhausted T cells co-cultured with MDAMB231. Figure 8 (f) and (g) show the levels of these markers when these cultures were treated with compounds 1 and 3 of the present invention. The expression of the depletion marker PD1 was decreased, while the expression of IFNG was restored (increased). Detailed Implementation

[0050] Development of this invention

[0051] As mentioned above, small molecule inhibitors represent an ideal alternative to monoclonal antibody (mAb) drugs in therapies targeting immune checkpoint receptors. Theoretically, small molecules can offer advantages over mAb drugs. Small molecules can provide increased oral bioavailability, bioefficiency, and short half-life activity, which are particularly important for autoimmunity or adverse immune events. Small molecules can also provide a greater diffusion rate within the tumor microenvironment (TME) and the potential to avoid macrophage-mediated resistance observed in anti-PD-1 therapies.

[0052] Small molecules are technically difficult to identify and evaluate. Besides the challenging design, the limited structural resolution of targets has consistently been a challenge in the development of PD-1 / PD-L1 small molecule inhibitors. Prior to 2015, the X-ray structures of human PD-1 / PD-L1 had not been resolved, and the degree of plasticity or the interaction established with PD-L1 in mouse forms could not be assessed. In recent years, several human PD-1 and PD-L1 X-ray structures have been resolved, revealing structural differences in the binding patterns between the mouse and human forms and the plasticity in complex formation.

[0053] The design of inhibitors that directly target the PD-1 / PD-L1 interaction interface is limited by the large, hydrophobic, and flat interface between proteins, which lacks a deep binding pocket. Recently, the distinct X-ray structures of PD-L1 and a class of small molecule inhibitors have been resolved. Bristol Myers Squibb (BMS) compounds were among the first non-peptide compounds capable of inhibiting the PD-1 / PD-L1 interaction; however, they have been reported to have poor drug-like properties. In general, these inhibitors bind to PD-L1, resulting in a deep cylindrical hydrophobic pocket formed at the interface between the two monomers [Zak, KM et al. 2016, as described above].

[0054] The inventors sought to develop novel inhibitors capable of disrupting the PD-1 / PD-L1 binding. Using the computer analysis described in the following computational examples, the inventors identified a novel scaffold structure capable of targeting the PD-L1 dimer interface. This novel scaffold structure is also capable of targeting VISTA.

[0055] As described in the Examples section below, the inventors subsequently employed an interdisciplinary approach to discover novel small molecules capable of modulating PD-1 / PD-L1 interactions. Computer analysis was combined with in vitro, ex vivo, and experimental studies to evaluate the ability of these novel compounds to modulate PD-1 / PD-L1 interactions and enhance T cell function.

[0056] In particular, the inventors recognized that the types of assays previously developed for verifying the efficacy of PD-1 / PD-L1 small molecule inhibitors were very limited. These were biochemical assays used for hit verification and / or engineered cell assays. The inventors decided to use innovative cell-based assays to evaluate compound activity. Initially, the research focused on PD-1 / PD-L1 inhibition, for which different cell lines were selected. Therefore, two types of cancer cells, breast cancer and brain cell lines, were selected for in vitro studies to observe the effect of hit compounds on PD-L1 / PD-1 interactions, and flow cytometry-based internalization assays were established using different concentrations of compounds at different time intervals.

[0057] The inventors also used an in vitro cancer model using different cancer cells and peripheral blood mononuclear cells (PBMCs) / T cells isolated from healthy volunteers.

[0058] These studies led to the identification of novel small molecules capable of promoting T cell activation by targeting the PD-1 / PD-L1 co-inhibitory interaction. These novel small molecules have been shown to have efficacy comparable to approved monoclonal antibodies.

[0059] Experiments have demonstrated that these newly identified small molecules possess a variety of beneficial effects, indicating significant medical potential (e.g., in cancer immunotherapy). These effects include:

[0060] - Reverse T cell exhaustion

[0061] - Restore the upregulation of IFNg, CD107a and granzyme B

[0062] - Increase CD8+ T cell levels, for example, in co-culture with cancer cells.

[0063] - High apoptotic activity, for example in the co-culture of CD8+ T cells with MDAMB231 / U251 / HCC827 cells.

[0064] - Inhibits the binding of PD-1 to PD-L1, for example, in MDAMB231, HCC827, and U251 cell lines, thereby enhancing T cell activation.

[0065] - Suppress VISTA

[0066] - Downregulate the expression of PD-1, a marker of exhaustion on T cells.

[0067] - Reduce the expression of the exhaustion marker TIM3

[0068] - Enhance T cell-mediated cytotoxicity against tumor cells

[0069] In some cases, these effects have been observed to be even greater than those shown by approved monoclonal antibodies.

[0070] definition

[0071] As used in this article, "C" 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group, where "C" is the chain length. 1-6 "" indicates that the part has 1-6 carbon atoms, meaning the group contains 1, 2, 3, 4, 5, or 6 carbon atoms. Typically, C 1-6 alkyl groups or partially C 1-3 Alkyl group or part thereof. C 1-3 The alkyl group or portion is a straight-chain or branched alkyl group or portion containing 1 to 3 carbon atoms. C 1-6 Examples of alkyl groups and moieties include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and 3-methylbutyl. 1-3 Examples of alkyl groups and moieties include methyl, ethyl, n-propyl, and isopropyl. Methyl is preferred. For the avoidance of doubt, when two alkyl moieties are present in a group, the alkyl moieties may be the same or different.

[0072] As used herein, "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing at least one carbon-carbon double bond. Typically, an alkenyl group is C0. 2-6 Alkenyl groups have 2-6 carbon atoms. Examples of alkenyl groups include ethenyl (also known as vinyl) and propenyl.

[0073] As used herein, "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing at least one carbon-carbon triple bond. Typically, the alkynyl group is C0. 2-6 The alkynyl group has 2-6 carbon atoms. Examples of alkynyl groups include ethynyl and propynyl.

[0074] As used in this article, "C" 1-6 "alkylene" refers to a straight-chain or branched alkylene group or portion. Examples include methylene, ethylene, and n-propylene groups and portions.

[0075] As used in this article, "C" 1-6 "Alkoxy" refers to an alkyl ether group. C 1-6 The alkoxy group is of formula C 1-6 alkyl-O- moiety, wherein C 1-6 Alkyl groups are as defined above. Example C 1-6 Alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. Typically, C 1-6 The alkoxy group is of formula C 1-3 C of alkyl-O- 1-3 alkoxy group, wherein C 1-3 Alkyl groups are as defined above. Methoxy groups are preferred.

[0076] As used herein, "cycloalkyl" refers to a non-aromatic saturated hydrocarbon ring. The cycloalkyl moiety typically contains 3 to 8 carbon atoms. Furthermore, the cycloalkyl moiety is typically a monocyclic ring. Preferably, the cycloalkyl group is a C14 ring containing 3 to 6 carbon atoms. 3-6 Cycloalkyl groups. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0077] As used herein, "cycloalkenyl" refers to a non-aromatic unsaturated hydrocarbon ring containing at least one carbon-carbon double bond. The cycloalkenyl moiety typically contains 3 to 8 carbon atoms. Furthermore, the cycloalkenyl moiety is typically a monocyclic ring. Preferably, the cycloalkenyl group is a C14 ring containing 3 to 6 carbon atoms. 3-6 Cycloalkenyl. Examples include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0078] As used herein, the heterocyclic group or portion is a non-aromatic saturated or unsaturated carbocyclic ring in which one or more (e.g., 1, 2, or 3) carbon atoms are replaced by heteroatoms selected from N, O, and S. Preferably, each such heteroatom is selected from N and O. When the heteroatom is S, the sulfur atom may be replaced by O, and may be in the form of, for example, -S(O)- or -S(O)2-. The heterocyclic group portion is typically 3- to 12-membered (i.e., 3 to 12 atoms forming one or more rings). Preferably, the heterocyclic group portion is 7- to 10-membered. The most preferred heterocyclic group portion herein is 7- or 8-membered. If the heterocyclic group or portion is unsaturated, it may contain one or more double bonds. Typically, if the heterocyclic group or portion is unsaturated, it has only one double bond, which is usually a carbon-carbon double bond. Preferably, the heterocyclic group is saturated and does not contain any unsaturated bonds within the ring.

[0079] The heterocyclic group or portion can be monocyclic or polycyclic. When the heterocyclic portion is polycyclic, it typically contains two or three rings; preferably, the polycyclic heterocyclic group contains two rings and can be referred to as a "bicyclic heterocyclic group".

[0080] Polycyclic heterocyclic groups can be spiroheterocyclic groups or portions thereof. As used herein, "spiroheterocyclic group" refers to a polycyclic heterocyclic group that shares a carbon atom (called a spiro atom) between two monocyclic rings. Depending on the number of spiro atoms present (i.e., the atoms shared between the rings), a spiroheterocyclic group can be a monospiroheterocyclic group, a bispiroheterocyclic group, or a multispiroheterocyclic group. Preferably, a bispiroheterocyclic group containing two rings is a preferred spiroheterocyclic group. Exemplary spiroheterocyclic groups include:

[0081] , and .

[0082] Polycyclic heterocyclic groups can be fused heterocyclic groups. As used herein, a “fused heterocyclic group” refers to a polycyclic heterocyclic group in which each ring shares a pair of adjacent carbon atoms with one or more other rings in the polycyclic heterocyclic group system. When the fused heterocyclic group is unsaturated, one or more (preferably 1, 2, 3, or 4) rings may contain one or more (preferably 1, 2, or 3) double bonds. Typically, fused heterocyclic groups are unsaturated. Depending on the number of rings present, the fused heterocyclic group may be, for example, bicyclic, tricyclic, or tetracyclic. Preferably, the fused heterocyclic group is bicyclic. Exemplary fused heterocyclic groups include:

[0083] and .

[0084] As used herein, the "aryl" group or part refers to a monocyclic or polycyclic aromatic carbon ring. Aryl groups are typically C... 6-14 The aryl group contains 6 to 14 carbon atoms. Preferably, the aryl group is C64. 6-10 Aryl group, containing 6 to 10 carbon atoms. Typically, the aryl group is phenyl or naphthyl. Phenyl is preferred.

[0085] As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic ring of 5 to 14 members, typically 5 to 10 members, preferably 5 or 6 members, containing at least one heteroatom selected from N, O, and S. Typically, heteroaryl groups contain 1, 2, or 3 heteroatoms. Preferably, heteroaryl groups are monocyclic. Examples include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thiopheneyl, pyrazolyl, pyrroleyl, oxadiazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolyl, isothiazolyl, thiazolyl, imidazole, pyridazolyl, and pyrazolyl.

[0086] As used herein, halogen refers to fluorine, chlorine, bromine or iodine, preferably chlorine or fluorine.

[0087] As used herein, a pharmaceutically acceptable salt is a salt formed with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, or nitric acid, and organic acids such as citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) hydroxides, and organic bases such as alkylamines, aralkylamines, and heterocyclic amines.

[0088] As used herein, "object" typically refers to a human or animal. A preferred object is a mammal. More preferably, the object is a human.

[0089] Compound of formula (I)

[0090] This article provides a compound, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, represented by Formula I:

[0091]

[0092] (I)

[0093] in:

[0094] R1, R2, and R4 are each independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, cycloalkyl, alkenyl, ynyl and -NR'R5;

[0095] R3 is selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 3-6 Cycloalkyl, alkenyl, alkynyl, NR'R5, and -OR6;

[0096] R' is selected from H and C. 1-6 alkyl;

[0097] R5 is selected from hydrogen, C 1-6 Alkyl, cycloalkyl, alkenyl, and ynyl groups containing up to 6 carbon atoms;

[0098] R6 is selected from C 1-6 Alkyl groups and -L2-A;

[0099] L2 does not exist or C 1-6 Alkylene;

[0100] A is selected from aryl and 5- to 14-membered heteroaryl groups, which may optionally be substituted by one or two substituents, each independently selected from cyano, halogen, hydroxyl, and amino groups; and

[0101] L1 is a optionally substituted polycyclic heterocyclic group.

[0102] Cycloalkyl groups containing up to 6 carbon atoms are typically C10-6 ... 3-6 Cycloalkyl.

[0103] Compounds of formula (I) may be optionally substituted at the indicated positions (e.g., at L1). "Optionally substituted" means that the hydrogen atom may be substituted by a substituent other than hydrogen or not substituted at all. When the substituent is not defined, it is typically selected from halogens, hydroxyl groups, cyano groups, C6 groups, etc. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 cycloalkyl and -NR'2, wherein R' is selected from H and C 1-6 Alkyl groups. When the number of substituents is not defined, they are typically selected from one, two, or three unless the context otherwise specifies.

[0104] Preferably, R' is H (in this document, H and hydrogen are used interchangeably).

[0105] Preferably, R5 is selected from hydrogen and C. 1-6 Alkyl groups, and more preferably selected from hydrogen and C. 1-3 Alkyl group. For example, R5 can be H, methyl, or ethyl.

[0106] In one respect, R1, R2, and R4 are each independently selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, cycloalkyl, alkenyl, ynyl and -NHR5;

[0107] R3 is selected from hydrogen, halogen, cyano, and C. 1-6 Alkyl, cycloalkyl containing up to 6 carbon atoms, alkenyl, ynyl, NHR5 and -OR6;

[0108] -OR6 is C 1-6 alkoxy or -L2-A;

[0109] L2 is absent or methylene; and

[0110] A is an aryl group that is optionally substituted with a cyano group.

[0111] Typically, R1 is selected from halogens, cyano groups, and C. 1-6 Alkyl and C 1-6 Alkyl group. Preferably, R1 is selected from halogen, cyano, C... 1-3 Alkyl and C 1-3 Alkoxy. Most preferably, R1 is a halogen. Therefore, preferably, R1 can be selected from chlorine, fluorine, cyano, methyl, ethyl, isopropyl, n-propyl, methoxy, ethoxy, isopropoxy, and n-propoxy; for example, selected from chlorine, cyano, methyl, and methoxy; especially chlorine.

[0112] Typically, R2 is selected from hydrogen and C. 1-6 Alkyl group. Preferably, R2 is selected from hydrogen and C. 1-3 Alkyl group. Most preferably, R2 is hydrogen. Therefore, R2 is preferably selected from hydrogen, methyl, ethyl, isopropyl, and n-propyl; especially hydrogen.

[0113] Typically, R4 is selected from hydrogen, halogen, cyano, C 1-6 Alkyl and C 1-6 Alkyl group. Preferably, R4 is selected from hydrogen, halogen, C. 1-3 Alkyl and C 1-3 Alkyl group. Most preferably, R4 is selected from hydrogen and C. 1-3 Alkyl group. Therefore, preferably, R4 can be selected from hydrogen, chlorine, fluorine, methyl, ethyl, isopropyl, n-propyl, methoxy, ethoxy, isopropoxy, and n-propoxy; for example, selected from hydrogen, chlorine, fluorine, methyl, and methoxy; especially hydrogen or methyl.

[0114] Typically, R3 is selected from hydrogen, halogens, C 1-6 Alkyl group and -OR6. Preferably, R3 is selected from hydrogen, halogen, C 1-3 Alkyl group and -OR6. Most preferably, R3 is -OR6. Therefore, preferably, R3 can be selected from hydrogen, chlorine, fluorine, methoxy, ethoxy, isopropyl, n-propyl, methoxy, ethoxy, isopropoxy, n-propoxy and -OR6.

[0115] In one respect, R6 can be C 1-6 Alkyl group. Preferably, R6 may be C6. 1-3 Alkyl group. Therefore, preferably, R6 can be methyl, ethyl, isopropyl or n-propyl; especially methyl.

[0116] In another aspect, R6 is L2-A, and A is C, which is optionally substituted with one or two substituents each independently selected from cyano and halogen. 6-10 Aryl group. In this respect, A is preferably a C6 aryl group. It is also preferred that, when one or two substituents are present, they are each cyano groups. It is also preferred that A has 0 or 1 substituent. In this respect, it is most preferred that A is a C6 aryl group substituted with one cyano group.

[0117] In this respect, L2 does not exist or C 1-6 Alkylene moiety. Preferably, L2 is absent or methylene. Most preferably, L2 is methylene.

[0118] More preferably, R6 is C 1-6 Alkyl groups (as discussed above), instead of -L2-A.

[0119] Therefore, R6 is selected from C. 1-6 Alkyl and L2-A, wherein L2 is absent or C 1-6The alkylene moiety, and A is C, which is optionally substituted with one or two substituents, each independently selected from cyano and halogen. 6-10 Aryl. Typically, R6 is selected from C 1-3 The alkyl group and L2-A, wherein L2 is absent or methylene, and A is a C6 aryl group substituted with 0 or 1 cyano group. For example, R6 may be selected from methyl, ethyl, isopropyl, n-propyl, phenyl, benzyl, cyanophenyl, and cyanobenzyl. Preferably, R6 is selected from methyl, ethyl, isopropyl, n-propyl, and cyanobenzyl. Most preferably, R6 is a C6 aryl group. 1-3 Alkyl groups, especially methyl groups.

[0120] Compounds of formula (I) contain an L1 group, which is an optionally substituted polycyclic heterocyclic group. Typically, L1 has the following formula:

[0121]

[0122] in

[0123] n is 0, 1, 2, 3, 4 or 5;

[0124] Wavy lines indicate covalent bonds;

[0125] Two of RA, RB, RC, and RD are linked together to form a group that is optionally formed by one, two, or three independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl and C 1-6 The alkoxy substituent is a 3- to 8-membered cycloalkyl, cycloalkenyl, or heterocyclic ring; and

[0126] (i) The other two of RA, RB, RC and RD are each independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl and C 1-6 alkoxy, or

[0127] (ii) Two of RA, RB, RC and RD are not present, and the carbon atoms between the remaining two of RA, RB, RC and RD are connected by double bonds.

[0128] In aspect (i), the polycyclic heterocyclic group is a spiroheterocyclic group. In aspect (ii), the polycyclic heterocyclic group is a fused heterocyclic group. Typically, L1 is a seven- to ten-membered spiroheterocyclic group or a fused heterocyclic group.

[0129] Preferably, n is 1, 2, or 3. Most preferably, n is 1 or 2.

[0130] Preferably, the two non-connected rings in RA, RB, RC, and RD are each independently selected from hydrogen and C. 1-3 Alkyl group. Most preferably, the two non-linked members of RA, RB, RC, and RD that do not form a ring are hydrogen atoms.

[0131] Preferably, the two cycloalkyl or heterocyclic rings in RA, RB, RC, and RD that are linked together to form a 3- to 8-membered ring are cycloalkyl or heterocyclic rings. When a heterocyclic ring is formed in this way, it is preferred that the heterocyclic ring contains a single heteroatom, most preferably oxygen.

[0132] The ring formed by RA, RB, RC, and RD may or may not be substituted. Typically, it is unsubstituted. However, when substituted, it is preferably substituted with a ring selected from halogens, hydroxyl groups, and C. 1-3 The alkoxy group is substituted. Therefore, preferred substituents include chlorine, fluorine, hydroxyl, methoxy, ethoxy, isopropoxy, and n-propoxy. The most preferred is hydroxyl.

[0133] Examples of L1 include: , , , , , , , , , , , , , , , , , , , , ,and .

[0134] The preferred L1 is selected from , , , ,and .

[0135] The optimal L1 is selected from and .

[0136] Therefore, typically:

[0137] R1 is selected from halogen, cyano, C 1-6 Alkyl and C 1-6 Alkoxy;

[0138] R2 is selected from hydrogen and C. 1-6 alkyl;

[0139] R4 is selected from hydrogen, halogen, cyano, and C. 1-6 Alkyl and C 1-6 Alkoxy;

[0140] R3 is selected from hydrogen, halogens, and C. 1-6 Alkyl groups and -OR6;

[0141] R6 is selected from C 1-3 Alkyl group and L2-A, wherein L2 is absent or methylene, and A is a C6 aryl group substituted with 0 or 1 cyano group; and

[0142] L1 has the following formula:

[0143]

[0144] in

[0145] n is 0, 1, 2, 3, 4 or 5;

[0146] Wavy lines indicate covalent bonds;

[0147] Two of RA, RB, RC, and RD are linked together to form a group that is optionally formed by one, two, or three independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl and C 1-6 The alkoxy substituent is a 3- to 8-membered cycloalkyl, cycloalkenyl, or heterocyclic ring; and

[0148] (i) The other two of RA, RB, RC and RD are each independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl and C 1-6 alkoxy, or

[0149] (ii) Two of RA, RB, RC and RD are not present, and the carbon atoms between the remaining two of RA, RB, RC and RD are connected by double bonds.

[0150] In a preferred aspect:

[0151] R1 is selected from halogen, cyano, C 1-3 Alkyl and C 1-3 Alkoxy;

[0152] R2 is selected from hydrogen and C. 1-3 alkyl;

[0153] R4 is selected from hydrogen, halogens, and C. 1-3 Alkyl and C 1-3 Alkoxy;

[0154] R3 is selected from hydrogen, halogens, and C. 1-3 Alkyl groups and -OR6;

[0155] R6 is selected from C 1-3 Alkyl and cyanobenzyl; and

[0156] L1 has the following formula:

[0157]

[0158] Where n is 1, 2, or 3;

[0159] Two of RA, RB, RC, and RD are linked together to form a compound optionally selected from halogen, hydroxyl, and C. 1-3 Alkoxy substituents of 3- to 8-membered cycloalkyl or heterocyclic rings; and

[0160] The other two of RA, RB, RC, and RD are each independently selected from hydrogen and C. 1-6 alkyl.

[0161] In a highly preferred aspect:

[0162] R1 is a halogen;

[0163] R2 is hydrogen;

[0164] R4 is selected from hydrogen and C. 1-3 alkyl;

[0165] R3 is -OR6;

[0166] R6 is C 1-3 Alkyl groups, especially methyl groups; and

[0167] L1 has the following formula:

[0168]

[0169] Where n is 1 or 2;

[0170] Two of RA, RB, RC, and RD are linked together to form a 3- to 8-membered cycloalkyl ring or heterocyclic ring, wherein the cycloalkyl or heterocyclic ring contains a single heteroatom, i.e., an oxygen atom; and the 3- to 8-membered ring is optionally substituted with a hydroxyl substituent; and

[0171] The other two of RA, RB, RC and RD are each hydrogen.

[0172] Compounds of formula (I) containing one or more chiral centers may be used in enantiomers or diastereomers in pure form, or in mixtures of isomers. For the avoidance of doubt, the compounds of the present invention may be used in any tautomer form. Furthermore, for the avoidance of doubt, compounds of formula (I) may take the form of any stereoisomer (e.g., any geometric isomer) with the structure shown herein.

[0173] The compounds of the present invention can be provided as pharmaceutically acceptable salts. The compounds of the present invention can be provided as hydrates, solvates, or prodrugs. A hydrate is a specific form of a solvate, wherein the solvent molecule associated with the compound is a water molecule. Preferably, the compounds of the present invention are compounds of formula (I) or pharmaceutically acceptable salts thereof.

[0174] Compound of formula (I')

[0175] The compounds of the present invention may be compounds of formula (I'); their geometric isomers or pharmaceutically acceptable compounds; or their salts, hydrates, solvates or prodrugs.

[0176] Compound (I') is a compound of formula (I) in which:

[0177] R1, R2, R3, and R4 are selected from hydrogen, halogen, C1-C6 alkoxy, C1-C6 alkyl, C1-C6 cycloalkyl, alkenyl, alkynyl, NHR4, C1-C3 alkyl, and cyano.

[0178] R3 can also be selected from substituted or unsubstituted aryloxy groups.

[0179] In this context, "alkyl" refers to a straight-chain or branched alkyl group, where C1-C6 groups indicate that the moiety has 1-6 carbon atoms, that is, the group contains 1, 2, 3, 4, 5 or 6 carbon atoms.

[0180] In this context, "alkoxy" refers to an alkyl ether alkyl group, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0181] In this context, "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0182] L1 represents a polycyclic heterocyclic group, including spiroheterocyclic groups and fused heterocyclic groups. A "spiroheterocyclic group" refers to a polycyclic heterocyclic group in which carbon atoms (called spiro atoms) are shared between monocyclic rings, wherein one or more (preferably 1, 2, or 3) ring atoms are heteroatoms selected from nitrogen, oxygen, or sulfur. Depending on the number of spiro atoms shared between the rings, the spiroheterocyclic group can be a monospiroheterocyclic group, a bispiroheterocyclic group, or a polyspiroheterocyclic group. Spiroheterocyclic groups include, but are not limited to:

[0183]

[0184] A "fused heterocyclic group" refers to a polycyclic heterocyclic group in which each ring shares a pair of adjacent carbon atoms with the other rings in the system. One or more (preferably 1, 2, 3, or 4) rings may contain one or more (preferably 1, 2, or 3) double bonds, but none of the rings have a fully conjugated electronic system. One or more (preferably 1, 2, or 3) ring atoms are heteroatoms selected from nitrogen or oxygen, and the remaining ring atoms are carbon atoms. Depending on the number of rings, it can be bicyclic, tricyclic, tetracyclic, or polycyclic. Fused heterocyclic groups include, but are not limited to:

[0185]

[0186] Specific compounds of the present invention

[0187] Compounds of formula (I) can be prepared, for example, according to the reaction schemes outlined in the following “Synthetic Examples”. Eighty-one compounds according to the invention were synthesized. These compounds, along with NMR and mass spectrometry analysis data, are listed in Table 1 below. Each compound is assigned a “Compound ID” number from NAT-1 to NAT-81. In one aspect, compounds of formula (I) can be compounds as listed in Table 1, or their hydrates, prodrugs, hydrates, solvates, or pharmaceutically acceptable salts.

[0188] Table 1: Representative embodiments of this disclosure

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] Specifically, the compound may be selected from:

[0204] , , , , , , , , , , , , , , , , , , , , , , , , ,or ,

[0205] Or its prodrug, hydrate, solvate or pharmaceutically acceptable salt.

[0206] Treatment

[0207] The compounds of the present invention are therapeutically useful. Therefore, the present invention provides a treatment method comprising administering to a subject a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof. Similarly, the present invention provides a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, for the treatment of humans or animals.

[0208] In particular, the compounds of the present invention can interfere with PD-1 / PD-L1 interaction, and therefore can be used to treat conditions related to PD-1 and / or PD-L1 function. Similarly, the compounds of the present invention can enhance T cell function, and therefore can be used to treat conditions requiring enhanced T cell function. Exemplary conditions of this kind include cancer, neurodegenerative diseases, and infectious diseases.

[0209] The compounds can also interfere with additional checkpoint pathways, such as the VISTA checkpoint pathway. Therefore, the compounds of the present invention can be used to interfere with (i) the interaction between PD-1 and PD-L1 and / or (ii) the interaction between VISTA and VSIG-3. Preferably, the compounds of the present invention can be used to interfere with the PD-1 / PD-L1 pathway and the VISTA / VSIG-3 pathway. "Interference" or "disruption" generally refers to the binding of the compounds of the present invention to one of the components of the pathway. For example, the compounds of the present invention can bind to PD-1, PD-L1, VISTA, or VSIG-3.

[0210] For example, the compounds of the present invention can be used to treat conditions associated with PD-L1 overexpression, such as cancers associated with PD-L1 overexpression. Such cancers are known to those skilled in the art and include, in particular, brain cancer, non-small cell lung cancer, triple-negative breast cancer, bladder cancer, gallbladder cancer, liver cancer, and thyroid cancer.

[0211] Similarly, the compounds of the present invention can be used to treat conditions associated with VISTA overexpression, such as cancers associated with VISTA overexpression.

[0212] Therefore, the present invention provides a method for treating, preventing and / or improving a disease, the method comprising administering to a subject a compound of formula (I) as defined herein, or a prodrug thereof, hydrate, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition.

[0213] The method typically involves inhibiting the PD-1 / PD-L1 checkpoint pathway.

[0214] Alternatively or additionally, the method may include inhibiting the activity of VISTA. For example, the method may include interfering with the interaction between VISTA and VSIG-3.

[0215] The compounds of this invention have been shown to reverse T cell exhaustion. For example, they have been shown to be effective in one or more of the following: enhancing T cell function, increasing the CD8+ T cell population, reducing the expression of exhaustion markers PD-L1 and TIM3, increasing CD107a expression, increasing granzyme B expression, and increasing IFNG expression. Therefore, the above methods may include one or more of the following:

[0216] (i) Inhibit the PD-1 / PD-L1 checkpoint pathway;

[0217] (ii) Inhibit the activity of VISTA;

[0218] (iii) Reverse T cell exhaustion;

[0219] (iv) Enhance T cell function;

[0220] (v) Reduces PD-1 expression in exhausted T cells;

[0221] (vi) Reduce the expression of TIM3 in exhausted T cells;

[0222] (vii) Increase the CD8+ T cell population;

[0223] (viii) Increase CD107a expression;

[0224] (ix) Increase granzyme B expression; and / or

[0225] (x) Restore IFNg;

[0226] (xi) Enhances T cell-mediated cytotoxicity against tumor cells.

[0227] In practice, the above-mentioned treatment methods are typically used to treat, improve, or prevent conditions selected from proliferative disorders, cancer, neurodegenerative disorders, autoimmune disorders, or infectious diseases.

[0228] The compounds of this invention have activity that interferes with the PD-1 / PD-L1 pathway. Therefore, the method is preferably a method for treating, improving, or preventing conditions associated with PD-L1 overexpression. Particularly preferably, the condition is cancer associated with PD-L1 overexpression. Exemplary cancers include brain cancer, breast cancer, lung cancer, kidney cancer, bladder cancer, thyroid cancer, liver cancer, and gallbladder cancer. The cancers are most preferably selected from breast cancer, brain cancer, thyroid cancer, and lung cancer.

[0229] Alternatively, the condition may be an infectious disease, such as HIV, septic shock, and any one of hepatitis A, B, C, or D.

[0230] The subject may have the conditions described above or be at risk of having the conditions described above. For example, the subject may have proliferative disorders, cancer, neurodegenerative disorders, or infectious diseases or be at risk of having proliferative disorders, cancer, neurodegenerative disorders, or infectious diseases.

[0231] As described herein, the compounds of the present invention are effective when taken alone. However, these compounds can also be used in methods of co-treatment with another therapeutic agent. Therefore, in one aspect, the methods described herein further include administering an additional therapeutic agent to the subject.

[0232] The other therapeutic agent may be, for example, another small molecule or monoclonal antibody. Preferably, the other therapeutic agent may be an inhibitor of the PD-1 / PD-L1 pathway and / or an inhibitor of the VISTA pathway (e.g., an inhibitor of the interaction between VISTA and VSIG-3).

[0233] In a preferred embodiment, the additional therapeutic agent is selected from nivolumab, pembrolizumab, avelumab, atezolizumab, cimiprimab, and durvalumab. These are currently approved monoclonal antibodies for treating the condition by interfering with the PD-1 / PD-L1 pathway. For example, the method may be a method of treating, improving, and / or preventing the condition by administering to a subject (i) a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, and (ii) at least one of nivolumab, pembrolizumab, avelumab, atezolizumab, cimiprimab, and durvalumab.

[0234] The above two or more therapeutic agents may be administered simultaneously or sequentially.

[0235] This invention provides compounds of formula (I) as defined herein, or their prodrugs, hydrates, solvates, or pharmaceutically acceptable salts, or pharmaceutical compositions thereof, for use in any of the methods described above. For example, this invention provides compounds of formula (I) as defined herein, or their prodrugs, hydrates, solvates, or pharmaceutically acceptable salts, or pharmaceutical compositions thereof, for use in methods of treating, improving, or preventing conditions associated with PD-L1 overexpression. Particularly preferred are cancers associated with PD-L1 overexpression, selected from brain cancer, breast cancer, lung cancer, kidney cancer, bladder cancer, thyroid cancer, liver cancer, and gallbladder cancer.

[0236] The present invention also provides another therapeutic agent for use in a method of co-treatment with a compound of formula (I) as defined herein. For example, the present invention provides:

[0237] Nivolumab, used in treatments comprising administering to a subject (i) a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, and (ii) nivolumab. Typically, the method is a means of treating, improving, or preventing a condition associated with PD-L1 overexpression. Particularly preferred are cancers associated with PD-L1 overexpression, selected from brain cancer, breast cancer, lung cancer, kidney cancer, bladder cancer, thyroid cancer, liver cancer, and gallbladder cancer.

[0238] Similarly, the present invention provides pembrolizumab for use in a treatment method comprising administering to a subject (i) a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, and (ii) pembrolizumab.

[0239] Similarly, the present invention provides avirumab for use in a treatment method comprising administering to a subject (i) a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, and (ii) avirumab.

[0240] Similarly, the present invention provides atezolizumab for use in a treatment method comprising administering to a subject (i) a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, and (ii) atezolizumab.

[0241] Similarly, the present invention provides cimiprimab for use in treatment methods comprising administering to a subject (i) a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, and (ii) cimiprimab.

[0242] Similarly, the present invention provides durvalumab for use in treatment methods comprising administering to a subject (i) a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, and (ii) durvalumab.

[0243] The present invention also provides the use of compounds of formula (I) as defined herein, or their prodrugs, hydrates, solvates, or pharmaceutically acceptable salts, or pharmaceutical compositions thereof, in the preparation of medicaments for use in the methods described above.

[0244] In vitro methods

[0245] The therapeutic applications described above typically involve in vivo methods using the compounds described herein. However, compounds of formula (I) and their prodrugs, hydrates, solvates, and pharmaceutically acceptable salts may also be used in in vitro methods.

[0246] For example, these compounds are particularly useful in assays involving PD-1 / PD-L1 binding. Flow cytometry assays are especially preferred.

[0247] Therefore, the present invention also provides an in vitro method comprising using a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein to:

[0248] (i) Inhibit the PD-1 / PD-L1 checkpoint pathway;

[0249] (ii) Inhibit the activity of VISTA;

[0250] (iii) Reverse T cell exhaustion;

[0251] (iv) Enhance T cell function;

[0252] (v) Reduces PD-1 expression in exhausted T cells;

[0253] (vi) Reduce the expression of TIM3 in exhausted T cells;

[0254] (vii) Increase the CD8+ T cell population;

[0255] (viii) Increase CD107a expression;

[0256] (ix) Increase granzyme B expression; and / or

[0257] (x) Restore IFNg;

[0258] (xi) Enhances T cell-mediated cytotoxicity against tumor cells.

[0259] Example

[0260] The invention will now be described with reference to synthetic, computational, and active embodiments. It should be understood that the invention is not necessarily limited to the details set forth in the following embodiments. Those skilled in the art will understand that the invention can be practiced or carried out in other ways. Therefore, modifications to this embodiment that fall within the meaning and equivalent scope of the claims are intended to be included.

[0261] Materials and methods

[0262] Cell lines, growth conditions, and reagents:

[0263] MDA-MB-231, HCC827, and U251 were all originally purchased from ATCC and maintained according to the instructions.

[0264] CHO-PDL1, CHO-VISTA, and CHO-PD1 cell lines were purchased from Abeomics INC-USA and maintained according to the manufacturer's instructions.

[0265] All cell lines were routinely screened for mycoplasma contamination.

[0266] The PD-1 / PD-L1 binding assay kit was purchased from Cisbio Assays.

[0267] Human IFN-γ GenLISA TM The ELISA 96-well kit was purchased from Krishgen Biosystems, India.

[0268] 4% (v / v) paraformaldehyde, PD-L1 recombinant human protein and Invitrogen SYPRO® orange dye (5000× solution) were purchased from ThermoFisher Scientific.

[0269] MTT® reagent, bovine serum albumin (BSA), and Ficoll® Paque Plus were purchased from Sigma-Aldrich.

[0270] BMS202 was purchased from Selleckchem (Houston, USA).

[0271] The anti-CD3 and anti-CD28 monoclonal antibodies were obtained from Invitrogen.

[0272] Recombinant human IFN-γ and recombinant human IL-2 were purchased from Peprotech. Fluorescent dye-labeled antibodies used for flow cytometry were purchased from Invitrogen and Thermofischer.

[0273] Calculation Example:

[0274] Computer-aided identification of dual small molecule inhibitors of PD-1 / PD-L1 and VISTA

[0275] The inventors used computer simulation analysis, starting with the hPD-L1 IgV domain (5N2F), and based on a virtual screening strategy, combined with free energy (molecular mechanics generalized Born and surface area solvation (MM / GBSA)), and employed molecular dynamics simulations in Schrödinger software to discover active small molecules targeting PD-L1, such as... Figure 1 As shown in Figure A.

[0276] Since the coordinates of the cocrystallization of VISTA and the small molecule inhibitor could not be obtained, the inventors began their investigation by focusing on the binding site at the extracellular domain (ECD) (6MVL) of VISTA, based on mutation data and literature definitions. Subsequently, virtual screening, binding free energy (molecular mechanical generalized Born surface area solvation (MM / GBSA)), and molecular dynamics (MD) simulations using Schrödinger software were performed, leading to the discovery of another active small molecule, such as... Figure 1 As shown in B.

[0277] A collection of approximately 5000 novel structures was proposed and screened using molecular docking at the PD-L1 dimer interface. Structure-based virtual screening for identifying PD-1 / PD-L1 and VISTA small molecule inhibitors was also performed. Figure 2 The process was as follows. First, compounds were pre-filtered based on Lipinski Rule 5. Then, computer screening was performed using the XP scoring function of the GLIDE module in the Schrödinger software. Next, free energy binding was performed using PRIME, and molecular dynamics was analyzed using DESMOND. After visual inspection and ADME filtering, the top-ranking compounds in the binding pocket were further screened.

[0278] Only compounds with favorable binding conformations and that interact with key binding site amino acid residues were retained for further study.

[0279] Synthesis Examples

[0280] All compounds of this invention can be synthesized according to Route 1, wherein the final step is a reductive amination of the corresponding starting material. Route 1 uses substituted benzaldehyde and a suitable amine.

[0281] The following is the synthesis scheme of the compound of this invention:

[0282] NAT-1 (an instance of Route 1)

[0283]

[0284] Example 1: Preparation of 6-chloro-2-methoxy-pyridine-3-carboxaldehyde (intermediate-3)

[0285]

[0286] Stage-1: Preparation of 6-chloro-2-methoxy-pyridine-3-carboxylic acid (intermediate-1)

[0287]

[0288] 40.0 g (0.208 mol) of 2,6-dichloropyridin-3-carboxylic acid was dissolved in 160 mL of tetrahydrofuran and 80 mL of methanol at room temperature. Lithium hydroxide monohydrate (26.18 g, 0.624 mol) was added to the reaction mixture at room temperature, and the reaction was allowed to proceed for 20 hours. The reaction was confirmed to be complete by TLC. The solvent was removed by vacuum distillation. The crude product was diluted with water and acidified with 2N hydrochloric acid (pH 4–5). The solid was filtered and dried in a disc desiccator at 55 °C to give 39.0 g of compound INT-1. 1 H-NMR (DMSO-d6, 400 MHz): δ 13.24 (brs, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 3.93 (s, 3H).

[0289] Stage-2: Preparation of (6-chloro-2-methoxy-3-pyridyl)methanol (Intermediate-2)

[0290]

[0291] Compound INT-1 (38.0 g, 0.2025 mol) was dissolved in tetrahydrofuran (380.0 mL) at room temperature under a nitrogen atmosphere. A 2.4 M solution of lithium aluminum hydride in tetrahydrofuran (101.2 mL, 0.243 mol) was added to the reaction mixture at 0–5 °C. The reaction mixture was stirred at room temperature for 7.30 h. The reaction was confirmed to be complete by TLC. The reaction mixture was cooled to 0 °C and quenched with a saturated aqueous ammonium chloride solution. The reaction mixture was brought to room temperature and filtered. Ethyl acetate was added to the filtrate, the organic layer was separated, and the mixture was distilled under reduced pressure to give 29.4 g of compound INT-2. 1 H-NMR (CDCl3, 400 MHz): δ 7.55 (d, J = 7.2 Hz,1H), 6.91 (d, J = 7.6 Hz, 1H), 4.63 (s, 2H), 3.99 (s, 3H), 2.13 (brs, 1H).

[0292] Stage-3: Preparation of 6-chloro-2-methoxy-pyridine-3-carboxaldehyde (Intermediate-3)

[0293]

[0294] Compound INT-2 (15.0 g, 0.0866 mol) was added to dichloromethane (300.0 mL) at room temperature. Dys-Martin oxidant (43.9 g, 0.1036 mol) was added at room temperature, and the mixture was stirred for 4 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was quenched with 1 N sodium hydroxide and extracted with dichloromethane. The organic layer was distilled under reduced pressure to give the crude compound. This crude compound was purified by silica gel column chromatography (2–5% ethyl acetate / hexane) to give 13.3 g of compound INT-3. 1 H-NMR (CDCl3, 400 MHz): δ10.30 (d, J = 0.8 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.03 (d, J1 = 0.4 Hz J2 =7.6 Hz, 1H), 4.09 (s, 3H).

[0295] Example 2: Preparation of 6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methoxy-pyridine-3-carboxaldehyde (intermediate-7)

[0296]

[0297] Stage 1: Preparation of 2-(2,3-dihydro-1,4-benzodioxane-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentane (Intermediate-4)

[0298]

[0299] 6-Bromo-2,3-dihydro-1,4-benzodioxane (100.0 g, 0.4650 mol) and bis(pinacol)diborone (141.7 g, 0.5580 mol) were dissolved in 1,4-dioxane (1000.0 mL) at room temperature. The reaction mixture was degassed at room temperature for 10 min. Potassium acetate (136.9 g, 1.395 mol) was added, followed by the [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (18.97 g, 0.023 mol). The reaction mixture was degassed at room temperature for 10 min. The reaction mixture was stirred at 100 °C for 6 h. The reaction was confirmed to be complete by TLC. The reaction mixture was cooled to room temperature and filtered through a diatomaceous earth mat. The filtrate was diluted with water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (0–5% ethyl acetate / hexane) to give 124 g of compound INT-4. 1 H-NMR (CDCl3, 400 MHz): δ 7.32 – 7.28 (m,2H), 6.85 (d, J = 8.0 Hz, 1H), 4.27 – 4.23 (m, 4H), 1.32 (s, 9H).

[0300] Stage-2: Preparation of 6-(3-bromo-2-methyl-phenyl)-2,3-dihydro-1,4-benzodioxane (Intermediate-5)

[0301]

[0302] Compound INT-4 (18.0 g, 0.0686 mol) and 1,3-dibromo-2-methylbenzene (18.87 g, 0.0755 mol) were added to tetrahydrofuran (360 mL) at room temperature. The reaction mixture was degassed at room temperature for 10 min. Potassium carbonate (28.40 g, 0.2058 mol), DM water (50.0 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (2.8 g, 0.00343 mol) were added. The reaction mixture was degassed at room temperature for 10 min. The reaction mixture was stirred at 62 °C for 10 h. The reaction was confirmed to be complete by TLC. The reaction mixture was allowed to reach room temperature and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure, and the crude product was purified by silica gel column chromatography (10% ethyl acetate / hexane) to give 12.8 g of compound INT-5. 1 H-NMR (DMSO-d6, 400 MHz): δ 7.59 – 7.57 (dd, J1 =2.4 Hz J2 = 7.2 Hz, 1H), 7.19 – 7.13 (m, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.81(d, J = 2.0 Hz, 1H), 6.77 – 6.75 (dd, J1 = 2.0 Hz J2 = 8.0 Hz, 1H), 4.28 (s,4H), 2.26 (s, 3H).

[0303] Stage-3: Preparation of 2-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentane (intermediate-6)

[0304]

[0305] Compound INT-5 (12.8 g, 0.0419 mol) and bis(pinacol)diboron (12.78 g, 0.0503 mol) were added to 1,4-dioxane (200 mL) at room temperature. The reaction mixture was degassed at room temperature for 10 min. Potassium acetate (12.33 g, 0.1257 mol) was added, followed by the [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (1.71 g, 0.00209 mol). The reaction mixture was degassed at room temperature for 10 min. The reaction mixture was stirred at 100 °C for 3 h. The reaction was confirmed to be complete by TLC. The reaction mixture was allowed to reach room temperature and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure, and the crude product was purified by silica gel column chromatography (0–5% ethyl acetate / hexane) to give 14.0 g of compound INT-6. 1 H-NMR (CDCl3, 400 MHz): δ 7.74 – 7.71 (dd, J1 = 2.0 Hz J2 =7.6 Hz, 1H), 7.26 – 7.24 (m, 1H), 7.19(t, J = 7.2 Hz, 1H), 6.88 (d, J = 8.0Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.76 – 6.73 (dd, J1 = 2.0 Hz J2 = 8.0 Hz, 1H), 4.29 (s, 4H), 2.43 (s, 3H), 1.35 (s, 9H).

[0306] Stage-4: Preparation of 6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methoxy-pyridine-3-carboxaldehyde (intermediate-7)

[0307]

[0308] Compounds INT-6 (1.0 g, 0.0028 mol) and INT-3 (0.53 g, 0.0031 mol) were added to 1,4-dioxane (20 mL) at room temperature. The reaction mixture was degassed at room temperature for 10 min. Potassium carbonate (1.17 g, 0.0085 mol), DM water (6.0 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (0.11 g, 0.00014 mol) were added. The reaction mixture was degassed at room temperature for 10 min. The reaction mixture was stirred at 95 °C for 8 h. The reaction was confirmed to be complete by TLC. The reaction mixture was allowed to reach room temperature and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure, and the crude product was purified by silica gel column chromatography (15% ethyl acetate / hexane) to give 0.71 g of compound INT-7. 1 H-NMR (CDCl3, 400 MHz): δ 10.41 (d, J = 0.8 Hz, 1H), 8.18(d, J = 7.6 Hz, 1H), 7.42 – 7.40 (m, 1H), 7.32 – 7.30 (m, 2H), 7.18 – 7.16(dd, J1 = 0.8 Hz J2 = 7.6 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 2.0Hz, 1H), 6.84 – 6.82 (dd, J1 = 2.0 Hz J2 = 8.4 Hz, 1H), 4.31 (s, 4H), 4.10 (s,3H), 2.28 (s, (3H). m / z: 362.31 (M+H) +

[0309] Example 3: Preparation of 3-[[6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl]methyl]-3-azabicyclo[3.2.0]hepta-6-ol (NAT-1)

[0310]

[0311] Compound INT-7 (0.15 g, 0.00041 mol) and compound 3-azabicyclo[3.2.0]heptan-6-ol·HBr salt (0.161 g, 0.00083 mol) were dissolved in dichloromethane (10.0 mL) at room temperature. Acetic acid (0.1 mL) was added to the reaction mixture, and the mixture was stirred at 80–85 °C for 4 hours. The reaction mixture was brought to room temperature, and sodium triacetoxyborohydride (0.263 g, 0.0012 mol) was added. The mixture was stirred at 80–85 °C for 3–4 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was diluted with DM water (15 mL) and extracted with dichloromethane (20 mL). The organic layer was washed with 10% sodium bicarbonate solution (15 mL) and then with DM water (15 mL). The organic layer was separated and distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (5-10% methanol / MDC) to obtain 30 mg of solid NAT-1. 1 H NMR (DMSO-d6, 400 MHz): δ 7.97 (d, J = 7.6 Hz,1H), 7.38-7.36 (m, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.21-7.19 (m, 1H), 7.13 (d,J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.84-6.80 (m, 2H), 4.65 (brs, 1H), 4.28 (s, 4H), 4.13 (brs, 1H), 3.90 (s, 3H), 3.74-3.60 (m, 2H), 2.86 (d, J =5.6 Hz, 1H), 2.75 (d, J = 8.0 Hz, 1H), 2.43-2.47 (m, 2H), 2.19 (s, 3H), 2.06-2.03 (m, 1H), 1.81-1.78 (m, 1H). m / z: 459.37 (M+H) +

[0312] NAT-2

[0313]

[0314] intermediate preparation

[0315] Example 4: Preparation of 3-[(6-chloro-3-formyl-2-pyridyl)oxymethyl]benzylnitrile (Intermediate-11)

[0316]

[0317] Stage 1: Preparation of 6-chloro-2-[(3-cyanophenyl)methoxy]pyridine-3-carboxylic acid (intermediate-8)

[0318]

[0319] Add tetrahydrofuran (96.0 mL) and sodium hydride (3.3 g, 0.1375 mol) to a round-bottom flask at room temperature. Cool the reaction mixture to 15–20 °C. Dissolve 3-(hydroxymethyl)benzyl nitrile (9.15 g, 0.0687 mol) in tetrahydrofuran (96.0 mL) and add it to the reaction mixture. Stir at 15–20 °C for 15–20 minutes. Dissolve 2,6-dichloro-4-methylpyridin-3-carboxylic acid (12.0 g, 0.2426 mol) in tetrahydrofuran (96 mL) and add it to the reaction mixture. Stir at 15–20 °C for 15–20 minutes. Raise the reaction mixture to room temperature and stir for 3 hours. Confirm the reaction is complete by TLC. Quench the reaction mixture with 1 N hydrochloric acid (120.0 mL) and extract twice with ethyl acetate (120 mL × 2). Wash the organic layer with 5% sodium bicarbonate and acidify the organic layer with 50% acetic acid solution. A white solid was observed to form, and the mixture was stirred for 30 minutes. The solid was filtered and dried in a hot air oven at 45°C for 4 hours to obtain 12.2 g of compound INT-8. 1 H NMR (DMSO-d6, 400 MHz): δ 13.24 (s, 1H), 8.21 (d, J =8.0 Hz, 1H), 7.99 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz,1H), 7.62 (t, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 5.48 (s, 2H).

[0320] Stage-2: Preparation of methyl 6-chloro-2-[(3-cyanophenyl)methoxy]pyridine-3-carboxylate (intermediate-9)

[0321]

[0322] Add a solution of 12.0 g (0.0415 mol) of dimethylformamide (120.0 mL) to a round-bottom flask at room temperature. Add potassium carbonate (17.2 g, 0.1247 mol) to the reaction mixture at 25-30 °C. Stir for 5-10 minutes. Slowly add iodomethane (12.9 mL, 0.2078 mol) to the reactants at 25-30 °C and stir for 3-4 hours. Filter the reactants and slowly add them to DM water (240.0 mL). Observe the formation of a solid. Filter the solid and wash it with DM water (200.0 mL). Dry the compound in a hot air oven at 60 °C for 4 hours to obtain 12.2 g of compound INT-9. 1 H NMR (DMSO-d6, 400MHz): δ 8.26 (d, J = 8.0 Hz, 1H), 7.98 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 5.50 (s, 2H), 3.84 (s, 3H).

[0323] Stage-3: Preparation of 3-[[6-chloro-3-(hydroxymethyl)-2-pyridyl]oxymethyl]benzylnitrile (Intermediate-10)

[0324]

[0325] A solution of INT-9 (12.2 g, 0.0403 mol) in tetrahydrofuran (1220.0 mL) and methanol (122.0 mL) was added to a round-bottom flask at 25-30 °C. Sodium borohydride (6.1 g, 0.1612 mol) was slowly added to the reactants. The reaction mixture was stirred for 16-17 hours. The solvent in the reactants was removed by distillation, and DM water (120.0 mL) was added, followed by stirring for 10 minutes. The mixture was extracted with ethyl acetate (120 mL), and the organic solvent was removed by distillation to give 11.1 g of compound INT-10. 1 H NMR (DMSO-d6, 400 MHz): δ 7.93(s, 1H), 7.83-7.78 (m, 3H), 7.61 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz,1H), 5.40 (s, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.50 (d, J = 8.0 Hz, 2H).

[0326] Stage-4: Preparation of 3-[(6-chloro-3-formyl-2-pyridyl)oxymethyl]benzylnitrile (Intermediate-11)

[0327]

[0328] Compound INT-10 (11 g, 0.40 mol) was added to dichloromethane (165.0 mL) at 25–30 °C. Dys-Martin oxidant (20.3 g, 0.048 mol) was added at 0–5 °C, and the mixture was stirred for 4 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was quenched with 1 N sodium hydroxide and extracted with dichloromethane (165 mL). The organic layer was distilled under reduced pressure to give 10.2 g of compound INT-11. 1 H NMR (DMSO-d6, 400 MHz): δ 10.26 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.05(s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 8.0Hz, 1H), 7.32 (d, J = 8.0Hz, 1H), 5.55 (s, 2H).

[0329] Example 5: Preparation of 3-[[6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-3-formyl-2-pyridyl]oxymethyl]benzylnitrile (intermediate-12)

[0330]

[0331] Compounds INT-11 (0.4 g, 0.0145 mol) and INT-6 (0.51 g, 0.0145 mol) were dissolved in 1,4-dioxane (80.0 mL) at 25–30 °C. The reaction mixture was degassed for 10 min at 25–30 °C. Potassium carbonate (0.604 g, 0.044 mol), DM water (4.0 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (0.3 g, 0.00036 mol) were added. The reaction mixture was degassed for 10 min at 25–30 °C. The reaction mixture was stirred at 95 °C for 8 h. The reaction was confirmed to be complete by TLC. The reaction mixture was allowed to reach room temperature and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (100–200 mesh) to give 0.32 g of compound INT-12. 1H NMR (DMSO-d6, 400 MHz): δ 10.40 (s, 1H), 8.23 ​​(d, J = 7.6 Hz, 1H), 8.04 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.81-7.79(m, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.40-7.32 (m, 3H), 7.28-7.26 (m, 1H), 6.93(d, J = 8.0 Hz, 1H), 6.94-6.80 (m, 2H), 5.61 (s, 2H), 4.29 (s, 4H), 2.1 (s,3H). m / z: 463.35 (M+H) +

[0332] Example 6: Preparation of 3-[[6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-3-formyl-2-pyridyl]oxymethyl]benzylnitrile (NAT-2)

[0333]

[0334] Compound INT-12 (0.15 g, 0.00032 mol) and compound 3-azabicyclo[3.2.0]heptan-6-ol·HBr salt (0.125 g, 0.00064 mol) were dissolved in dichloromethane (10 mL) at 25–30 °C. Acetic acid (0.1 mL) was added to the reaction mixture, and the mixture was stirred at 80–85 °C for 4 hours. The reaction mixture was brought to 25–30 °C, and sodium triacetoxyborohydride (0.206 g, 0.00097 mol) was added. The mixture was stirred at 80–85 °C for 3–4 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was diluted with DM water (150.0 mL) and extracted with dichloromethane (150.0 mL). The organic layer was washed with 10% sodium bicarbonate (150 mL) and then with DM water. The organic layer was separated and distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (5-10% methanol / MDC) to obtain 30 mg of solid NAT-2. 1H NMR (400 MHz, DMSO-d6): 8.01(d, J = 7.6 Hz, 1H), 7.92 (s, 1H), 7.85-7.76 (m, 2H), 7.62-7.58 (m, 1H),7.30-7.27 (m, 2H), 7.21-7.15 (m, 2H), 6.92 (d, J = 8.4 Hz, 1H), 6.83-6.78 (m,2H), 5.46 (s, 2H), 4.65 (m, 1H), 4.28 (s, 4H), 4.13-4.11 (m, 1H), 3.84-3.67(m, 2H), 2.87-2.75 (m, 2H), 2.43-2.47 (m, 2H), 2.21-2.17 (m, 1H), 2.06 (s,3H), 1.81-1.78 (m, 1H). m / z: 560.44 (M+H) +

[0335] NAT-3

[0336]

[0337] intermediate preparation

[0338] Example 7: Preparation of 6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methoxy-pyridine-3-carboxaldehyde (intermediate-7)

[0339]

[0340] For the preparation of intermediate-7, see NAT-1.

[0341] Example 8: Preparation of 6-[[6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane (NAT-3)

[0342]

[0343] Compound INT-7 (2.75 g, 0.0076 mol) and compound 2-oxa-6-azaspiro[3.3]heptane (0.83 g, 0.00837 mol) were dissolved in dimethylformamide (30.0 mL) at 25–30 °C. Acetic acid (2.7 mL) was added to the reaction mixture, and the mixture was stirred at 25–30 °C for 16 hours. Sodium triacetoxyborohydride (4.83 g, 0.0228 mol) was added at 25–30 °C, and the mixture was stirred for 3–4 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was diluted with DM water (275 mL) and extracted with dichloromethane (275 mL). The organic layer was washed with 10% sodium bicarbonate (275 mL) and then with DM water. The organic layer was separated and distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (5-10% methanol / MDC) to obtain 1.6 g of solid NAT-3. 1 H NMR (CDCl 3, 400 MHz): δ 7.55 (d, J = 7.6 Hz, 1H), 7.38-7.36 (m, 1H), 7.29-7.22 (m, 2H), 7.0 (d, J = 7.6 Hz, 1H), 6.92-6.88 (m, 2H), 6.84-6.82 (m, 1H), 4.77 (s, 4H), 4.30 (s, 4H), 3.96 (s, 3H), 3.57 (s, 2H), 3.49 (s, 4H), 2.25 (s, 3H). m / z: 445.3 (M+H) +

[0344] NAT-4

[0345]

[0346] intermediate preparation

[0347] Example 9: Preparation of 6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methoxy-pyridine-3-carboxaldehyde (intermediate-7)

[0348]

[0349] For the preparation of intermediate-7, see NAT-1.

[0350] Example 10: Preparation of 2-[[6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl]methyl]-6-oxa-2-azaspiro[3,4]octane (NAT-4)

[0351]

[0352] Compound INT-7 (2.0 g, 0.00553 mol) and compound 6-oxa-2-azaspiro[3.4]octane ditrifluoroacetate (2.45 g, 0.0071 mol) were dissolved in dimethylformamide (20 mL) at 25–30 °C. N,N-diisopropylethylamine (3.3 mL, 0.0193 mol) was added to the reaction mixture, and the mixture was stirred at 25–30 °C for 16 hours. Sodium triacetoxyborohydride (2.34 g, 0.011 mol) was added at 25–30 °C, and the mixture was stirred for 3–4 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was diluted with DM water (200 mL) and extracted with dichloromethane (200 mL). The organic layer was washed with 10% sodium bicarbonate, followed by washing with DM water. The organic layer was separated and distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (5-10% methanol / MDC) to obtain 1.0 g of solid NAT-4. 1 H NMR (CDCl 3, 400 MHz): δ 7.71 (d, J = 7.6Hz, 1H), 7.38-7.36 (m, 1H), 7.30-7.24 (m, 2H), 7.05 (d, J = 7.2 Hz, 1H), 6.92-6.88 (m, 2H), 6.84-6.82 (m, 1H), 4.30 (s, 4H), 4.0-3.97 (m, 5H), 3.89(s, 2H), 3.83-3.73 (m, 6H), 2.25 (s, 3H), 2.21 (t, J = 7.2 Hz, 2H). m / z:459.3 (M+H) +

[0353] NAT-5

[0354]

[0355] intermediate preparation

[0356] Example 11: Preparation of 3-[(6-chloro-3-formyl-2-pyridyl)oxymethyl]benzylnitrile (Intermediate-11)

[0357]

[0358] For the preparation of intermediate-11, see NAT-2.

[0359] Example 12: Preparation of 2-(2,3-dihydro-1,4-benzodioxane-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentane (intermediate-4)

[0360]

[0361] For the preparation of intermediate-4, see NAT-1.

[0362] Example 13: Preparation of 2-chloro-6-(2,3-dihydro-1,4-benzodioxane-6-yl)benzylnitrile (Intermediate-13)

[0363]

[0364] INT-4 (60.0 g, 0.2289 mol) and 2,6-dichlorobenzyl nitrile (59.0 g, 0.3433 mol) were dissolved in 1,4-dioxane (900.0 mL) at 25–30 °C. The reaction mixture was degassed for 10 minutes at 25–30 °C. Potassium carbonate (94.9 g, 0.686 mol), DM water (180 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (9.34 g, 0.114 mol) were added. The reaction mixture was degassed for 10 minutes at 25–30 °C. The reaction mixture was stirred at 90–95 °C for 10 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was brought to 25–30 °C and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water (600 mL) and extracted with ethyl acetate (600 mL × 2). The organic layer was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (10% ethyl acetate / hexane) to give 51.2 g of compound INT-13. I HNMR: (400MHz, CDCl3): δ 7.54-7.50 (t, J=8.0 Hz, 1H), 7.47-7.44 (m, 1H), 7.35 (d, J=7.6Hz, 1H), 7.06-7.02 (m, 2H), 6.96 (d, J=8.4 Hz, 1H), 4.31 (s, 4H).

[0365] Example 14: Preparation of 2-(2,3-dihydro-1,4-benzodioxane-6-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentan-2-yl)benzylnitrile (Intermediate-14)

[0366]

[0367] INT-13 (12.8 g, 0.0736 mol) and bis(pinacol)diboron (22.4 g, 0.0883 mol) were dissolved in 1,4-dioxane (200 mL) at 25–30 °C. The reaction mixture was degassed for 10 min at 25–30 °C. Potassium acetate (21.6 g, 0.2208 mol) was added, followed by the [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (4.2 g, 0.0051 mol). The reaction mixture was degassed for 10 min at 25–30 °C. The reaction mixture was stirred at 90–95 °C for 16 h. The reaction was confirmed to be complete by TLC. The reactants were brought to 25–30 °C and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water (200 mL) and extracted with ethyl acetate (200 mL × 2). The organic layer was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (0-5% ethyl acetate / hexane) to give 15.1 g of compound INT-14. 1 HNMR: (DMSO-d6, 400MHz): δ 7.76 (d, J=8.0 Hz, 1H), 7.71 (t, J=7.6 Hz, 1H), 7.62 (d, J=7.6 Hz,1H), 7.04 (s, 1H), 6.99 (m, 2H), 4.3 (s, 4H), 1.3 (s, 12H).

[0368] Example 15: Preparation of 2-[6-[(3-cyanophenyl)methoxy]-5-formyl-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxane-6-yl)benzylnitrile (Intermediate-15)

[0369]

[0370] INT-11 (4.0 g, 0.0146 mol) and compound INT-14 (5.6 g, 0.0154 mol) were dissolved in 1,4-dioxane (80 mL) at 25–30 °C. The reaction mixture was degassed for 10 min at 25–30 °C. Potassium carbonate (6.07 g, 0.044 mol), DM water (40.0 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (0.23 g, 0.00029 mol) were added. The reaction mixture was degassed for 10 min at room temperature. The reaction mixture was stirred at 95 °C for 8 h. The reaction was confirmed to be complete by TLC. The reaction mixture was allowed to reach room temperature and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (15% ethyl acetate / hexane) to give 4.7 g of compound INT-15. 1 H NMR (DMSO-d6, 400 MHz): δ 10.38 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.04 (s, 1H), 7.90-7.80 (m, 4H), 7.72 (d, J = 8.0 Hz, 1H), 7.68-7.66 (m, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.14 (s, 1H), 7.09 (d, J =8.0 Hz, 1H) 7.02 (d, J = 8.0 Hz, 1H), 5.72 (s, 2H), 4.31 (s, 4H).

[0371] Example 16: Preparation of 2-[6-[(3-cyanophenyl)methoxy]-5-(6-oxa-2-azaspiro[3.4]oct-2-ylmethyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxane-6-yl)benzyl nitrile (NAT-5)

[0372]

[0373] INT-15 (0.2 g, 0.00042 mol) and compound 6-oxa-2-azaspiro[3.4]octane ditrifluoroacetate (0.214 g, 0.00063 mol) were dissolved in ethyl acetate (4 mL) at 25–30 °C. N,N-diisopropylethylamine (0.26 mL, 0.00152 mol) was added to the reaction mixture, and the mixture was stirred at 25–30 °C for 16 hours. Sodium triacetoxyborohydride (0.134 g, 0.00063 mol) was added at 25–30 °C, and the mixture was stirred for 3–4 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was diluted with DM water (10 mL). The organic layer was separated, washed with 10% sodium bicarbonate, and then washed with DM water. The organic layer was separated and distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (5-10% methanol / MDC) to obtain 20 mg of solid NAT-5. 1 H NMR (DMSO-d6, 400 MHz): δ 7.94 (s, 1H), 7.85-7.74 (m, 4H), 7.71-7.67 (m, 1H), 7.63-7.57 (m, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.11 (s, 1H),7.07 (d, J = 8.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 5.57 (s, 2H), 4.31 (s,4H), 3.73 (s, 2H), 3.66-3.63 (m, 4H), 3.26 (s, 4H), 2.04 (t, J = 8.0 Hz, 2H).m / z: 571.6 (M+H) +

[0374] NAT-6

[0375]

[0376] intermediate preparation

[0377] Example 17: Preparation of 2-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentane (intermediate-6)

[0378]

[0379] For the preparation of intermediate-6, see NAT-1.

[0380] Example 18: Preparation of 2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]pyridine-3-carboxylic acid (intermediate-16)

[0381]

[0382] 2,6-Dichloropyridin-3-carboxylic acid (15.0 g, 0.0781 mol) and INT-6 (27.5 g, 0.0781 mol) were dissolved in a mixture of 1,4-dioxane (150 mL) and ethanol (150 mL) at 25–30 °C. The reaction mixture was degassed for 10 min at 25–30 °C. Potassium carbonate (37.7 g, 0.2733 mol), DM water (150.0 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (2.74 g, 0.0039 mol) were added. The reaction mixture was degassed for 10 min at 25–30 °C. The reaction mixture was stirred at 95 °C for 7 h. The reaction was confirmed to be complete by TLC. The reactants were distilled under reduced pressure to give a crude product. The crude product was diluted with DM water (150.0 mL) and extracted with ethyl acetate (150 mL). The aqueous layer was acidified with 4N hydrochloric acid and extracted with ethyl acetate (150 mL × 2). The organic layer was distilled under reduced pressure to give 32.0 g of compound INT-16. 1 H NMR(DMSO-d6, 400 MHz): δ 13.79 (brs, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.68 (d, J =8.0 Hz, 1H), 7.39 – 7.33 (m, 2H), 7.29 – 7.27 (dd, J = 2.4 Hz 7.2 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.85 – 6.80 (m, 2H), 4.28 (s, 4H), 2.15 (s, 3H).

[0383] Example 19: Preparation of [2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-3-pyridyl]methanol (intermediate-17)

[0384]

[0385] INT-16 (32.0 g, 0.0838 mol) was dissolved in tetrahydrofuran (320.0 mL) at 25–30 °C and cooled to 0–10 °C. A BH3·DMS complex solution (2 M in THF) was slowly added to the reactants. After completion, the temperature of the reactants was raised to 65–70 °C and maintained for 8 hours. The reaction was confirmed to be complete by TLC. The reactants were cooled to 0–10 °C, quenched with saturated ammonium chloride solution (320.0 mL), and extracted with ethyl acetate (320 mL). The organic solvent was separated and distilled under reduced pressure. The crude compound was purified by silica gel column chromatography (ethyl acetate / hexane) using 100–200 mesh to give 27.9 g of compound INT-17. 1 H NMR (DMSO-d6,400 MHz): δ 8.02 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.31 (d, J =4.4 Hz, 2H), 7.25 (m, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.84 – 6.79 (m, 2H), 5.59 (t, J = 5.6 Hz, 1H), 4.60 (d, J = 5.6 Hz, 2H), 4.28 (s, 4H), 2.12 (s, 3H).

[0386] Example 20: Preparation of 2-chloro-3-(chloromethyl)-6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]pyridine (intermediate-18)

[0387]

[0388] INT-17 (2.0 g, 0.00543 mol) was dissolved in dichloromethane (30.0 mL) at 25–30 °C. Thionyl chloride (0.75 mL, 0.0108 mol) was added dropwise to the reactants at 25–30 °C. The reaction mixture was maintained at 25–30 °C for 6 hours. The reaction was confirmed to be complete by TLC. The reaction was quenched with ice water (20 mL) and extracted with ethyl acetate (30 mL). The organic solvent was separated and distilled under reduced pressure to give 1.8 g of compound INT-18. 1 H NMR (CDCl 3,400 MHz): δ 7.91 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.34-7.31 (m, 1H), 7.29-7.27 (m, 2H), 6.91 (d, J =8.4 Hz, 1H), 6.86 – 6.85 (m, 1H), 6.82-6.79 (m, 1H), 4.75 (s, 2H), 4.30 (s, 4H), 2.20 (s, 3H).

[0389] Example 21: Preparation of 6-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane (NAT-6)

[0390]

[0391] INT-18 (0.9 g, 0.00232 mol) was dissolved in acetonitrile (10.0 mL) at 25–30 °C. 2-oxa-6-azaspiro[3.3]heptane (0.34 g, 0.00349 mol) was added at 25–30 °C, followed by N,N-diisopropylamine (0.8 mL, 0.00464 mol). The reaction was maintained at 85–90 °C for 5 hours. The reaction was confirmed to be complete by TLC. The solvent was distilled under reduced pressure to give the crude compound. The crude compound was diluted with DM water (10 mL) and extracted with ethyl acetate (10.0 mL × 2). The organic solvent was distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / hexane) at 100–200 mesh to give 0.7 g of compound NAT-6. 1 H NMR (CDCl 3, 400 MHz): δ 7.77 (d, J = 8.0 Hz, 1H), 7.35 (d, J =7.6 Hz, 1H), 7.32 – 7.29 (m, 1H), 7.27 – 7.25 (m, 2H), 6.90 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.82 – 6.79 (dd, J = 2.0 Hz 8.0 Hz, 1H), 4.75(s, 4H), 4.30 (s, 4H), 3.70 (s, 2H), 3.52 (s, 4H), 2.18 (s, 3H). m / z: 449.4(M+H) +

[0392] NAT-7

[0393]

[0394] Example 22: Preparation of 2-chloro-3-(chloromethyl)-6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]pyridine (intermediate-18)

[0395]

[0396] For the preparation of intermediate-18, see NAT-6.

[0397] Example 23: Preparation of 2-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-3-pyridyl]methyl]-6-oxa-2-azaspiro[3,4]octane (NAT-7)

[0398]

[0399] INT-18 (0.9 g, 0.00232 mol) was dissolved in acetonitrile (10.0 mL) at 25–30 °C. 6-oxa-2-azaspiro[3,4]octane difluoroacetate (1.19 g, 0.00349 mol) was added at 25–30 °C, followed by N,N-diisopropylamine (1.21 mL, 0.00696 mol). The reaction was maintained at 85–90 °C for 5 hours. The reaction was confirmed to be complete by TLC. The solvent was distilled under reduced pressure to give the crude product. The crude compound was diluted with DM water (10.0 mL) and extracted with ethyl acetate (10.0 mL × 2). The organic solvent was distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / hexane) at 100–200 mesh to give 0.7 g of compound NAT-7. 1 H NMR (CDCl 3,400 MHz,): δ 7.82 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.32 – 7.24 (m, 3H), 6.90 (d, J = 8.4 Hz, 1H), 6.85(d, J = 2.0 Hz, 1H), 6.80 (dd, J = 2.0 Hz 8.4 Hz, 1H), 4.30 (s, 4H), 3.89 (s,2H), 3.81 (t, J = 6.8 Hz, 2H), 3.77 (s, 2H), 3.38 (s, 4H), 2.19 (s, 3H), 2.15(t, J = 6.8 Hz, 2H). m / z: 463.1(M+H) +

[0400] NAT-8

[0401]

[0402] intermediate preparation

[0403] Example 24: Preparation of 2-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentane (intermediate-6)

[0404]

[0405] For the preparation of intermediate-6, see NAT-1.

[0406] Example 25: Preparation of (6-chloro-2-methyl-3-pyridyl)methanol (intermediate-19)

[0407]

[0408] 6-Chloro-2-methylpyridin-3-carboxylic acid (5.0 g, 0.02914 mol) was dissolved in tetrahydrofuran (100 mL) at 25–30 °C under a nitrogen atmosphere. A 2.0 M solution of the BH3.DMS complex in tetrahydrofuran (17.4 mL, 0.0349 mol) was added to the reaction mixture at 0–5 °C. The reaction mixture was stirred at 60–65 °C for 10 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was cooled to 0–5 °C and quenched with 2N hydrochloric acid aqueous solution. The reaction mixture was heated to 25–30 °C and extracted with ethyl acetate (100 mL). The organic layer was washed with DM water (100 mL). Ethyl acetate was distilled under reduced pressure to give 3.7 g of compound INT-19. 1HNMR (CDCl 3, 400 MHz): δ 7.75 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 4.5 (s, 4H), 2.38 (s, 3H).

[0409] Example 26: Preparation of [6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methyl-3-pyridyl]methanol (intermediate-20)

[0410]

[0411] INT-19 (0.5 g, 0.00317 mol) and compound INT-6 (1.45 g, 0.0412 mol) were dissolved in 1,4-dioxane (40 mL) at 25–30 °C. The reaction mixture was degassed for 10 min at 25–30 °C. Potassium carbonate (1.31 g, 0.00951 mol), DM water (2.0 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (0.051 g, 0.000063 mol) were added. The reaction mixture was degassed for 10 min at 25–30 °C. The reaction mixture was stirred at 90–95 °C for 7 h. The reaction was confirmed to be complete by TLC. The reaction mixture was brought to 20–25 °C and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (15% ethyl acetate / hexane) to give 0.74 g of compound INT-20. 1 H NMR (CDCl 3, 400 MHz): δ 7.78(d, J = 7.6 Hz, 1H), 7.33 – 7.28 (m, 3H), 7.20 – 7.18 (m, 1H), 6.92 (d, J =8.0 Hz, 1H), 6.83-6.78 (m, 2H), 5.27 (t, J = 5.2 Hz, 1H), 4.57 (d, J = 5.6Hz, 2H), 4.28 (s, 4H), 2.47 (s, 3H), 2.10 (s, 3H). m / z: 348.1 (M+H) +

[0412] Example 27: Preparation of 3-(chloromethyl)-6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methylpyridine (intermediate-21)

[0413]

[0414] INT-20 (0.74 g, 0.00213 mol) was dissolved in dichloromethane (15 mL) at 25–30 °C. Thionyl chloride (0.29 mL, 0.00426 mol) was added dropwise to the reactants at 25–30 °C. The reaction mixture was maintained at 25–30 °C for 4 hours. The reaction was confirmed to be complete by TLC. The reaction was quenched with 10% sodium carbonate solution (10.0 mL). The organic solvent was distilled under reduced pressure to give 0.64 g of compound INT-21. ¹H NMR (CDCl₂) 3, 400 MHz): δ 7.73 (d, J = 8.0 Hz, 1H), 7.33 – 7.28 (m, 4H), 6.94-6.90 (m, 2H), 6.85-6.83 (m, 1H), 6.83-6.78 (m, 1H), 4.70 (s, 2H), 4.32 (s, 4H), 2.74 (s, 3H), 2.20 (s, 3H). m / z: 366.3 (M+H) +

[0415] Example 28: Preparation of 6-[[6-[3-(2,3-dihydro-1,4-benzodioxane-6-yl)-2-methyl-phenyl]-2-methyl-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane (NAT-8)

[0416]

[0417] INT-21 (0.9 g, 0.00090 mol) was dissolved in acetonitrile (10 mL) at 25–30 °C. 2-oxa-6-azaspiro[3.3]heptane (0.071 g, 0.00099 mol) was added at 75–80 °C, followed by triethylamine (0.25 mL, 0.00180 mol), and the reaction was allowed to proceed for 4 hours. The reaction was confirmed to be complete by TLC. The solvent was distilled under reduced pressure to obtain the crude product. The crude compound was diluted with DM water (10 mL) and extracted with ethyl acetate (10 mL × 2). The organic solvent was distilled under reduced pressure to obtain the crude compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / hexane) using 100–200 mesh to obtain 0.24 g of compound NAT-8. 1 H NMR (CDCl) 3,400 MHz): δ 7.60 (d, J = 7.6 Hz, 1H), 7.33 – 7.29 (m, 2H), 7.26 – 7.21(m, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 1.6 Hz, 1H), 6.83 (dd, J =2.0 Hz, 8.4 Hz, 1H), 4.79 (s, 4H), 4.32 (s, 4H), 3.59 (s, 2H), 3.47 (s, 4H), 2.57 (s, 3H), 2.17 (s, 3H). m / z: 429.6 (M+H) +

[0418] NAT-22 and NAT-23

[0419]

[0420] Example 29: Preparation of 6-chloro-2-methoxy-pyridine-3-carboxylic acid (intermediate-1)

[0421] For the preparation of intermediate-1, see NAT-1.

[0422] Example 30: Preparation of (6-chloro-2-methoxy-3-pyridyl)methanol (intermediate-2)

[0423] For the preparation of intermediate-2, see NAT-1.

[0424] Example 31: Preparation of 2-(2,3-dihydro-1,4-benzodioxane-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentane (intermediate-4)

[0425] For the preparation of intermediate-4, see NAT-1.

[0426] Preparation of intermediate 2-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentane (intermediate-23)

[0427]

[0428] Example 32: Preparation of 6-(3-bromo-2-chlorophenyl)-2,3-dihydro-1,4-benzodioxane (intermediate-22)

[0429]

[0430] Compound INT-4 (128.9 g, 0.4650 mol) and 1,3-dibromo-2-chlorobenzene (125.7 g, 0.4650 mol) were added to 1,4-dioxane (1290 mL) at room temperature. The reaction mixture was degassed at room temperature for 10 min. Potassium carbonate (192.6 g, 1.395 mol), DM water (200.0 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (9.49 g, 0.011625 mol) were added. The reaction mixture was degassed at room temperature for 10 min. The reaction mixture was stirred at 80–85 °C for 10 h. The reaction was confirmed to be complete by TLC. The reaction mixture was allowed to reach room temperature and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure, and the crude product was purified by silica gel column chromatography (10% ethyl acetate / hexane) to give 100 g of compound INT-22. 1 H NMR (400 MHz, CDCl3): δ 7.77 – 7.74(dd, J = 1.6 Hz, 7.6 Hz, 1H), 7.37 – 7.35 (dd, J = 1.6 Hz, 7.6 Hz, 1H), 7.31(t, J = 7.6 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.86– 6.84 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.28 (s, 4H).

[0431] Example 33: Preparation of 2-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentane (intermediate-23)

[0432] Compound INT-22 (100 g, 0.191 mol) and bis(pinacol)diboron (94.0 g, 0.229 mol) were added to 1,4-dioxane (1000 mL) at room temperature. The reaction mixture was degassed at room temperature for 10 min. Potassium acetate (90.8 g, 0.5741 mol) was added, followed by the [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (6.3 g, 0.0077 mol). The reaction mixture was degassed at room temperature for 10 min. The reaction mixture was stirred at 100 °C for 7 h. The reaction was confirmed to be complete by TLC. The reaction mixture was allowed to reach room temperature and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure, and the crude product was purified by silica gel column chromatography (5-15% ethyl acetate / hexane) to give 65 g of compound INT-23. 1 H NMR (400 MHz, CDCl3): δ 7.59 – 7.57 (dd, J = 2.0 Hz, 8.4Hz, 1H), 7.34 – 7.32 (dd, J = 2.0 Hz, 7.6 Hz, 1H), 7.27 – 7.23 (m, 1H), 6.92(d, J = 1.6 Hz, 2H), 6.89 – 6.88 (m, 1H), 4.29 (s, 4H), 1.38 (s, 12H).

[0433] Example 34: Preparation of [6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-2-methoxy-3-pyridyl]methanol (intermediate-24)

[0434]

[0435] INT-2 (23 g, 0.1324 mol) and compound INT-23 (59.2 g, 0.1589 mol) were dissolved in 1,4-dioxane (276 mL) at 25–30 °C. The reaction mixture was degassed for 10 min at 25–30 °C. Potassium carbonate (54.8 g, 0.3972 mol), DM water (69 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (2.7 g, 0.0033 mol) were added. The reaction mixture was degassed for 10 min at 25–30 °C. The reaction mixture was stirred at 90–95 °C for 7 h. The reaction was confirmed to be complete by TLC. The reaction mixture was brought to 20–25 °C and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (15% ethyl acetate / hexane) to give 45.0 g of compound INT-24. 1 H NMR (400 MHz, DMSO-d6): δ 7.82 -7.80 (d, J = 7.6Hz, 1H), 7.53 – 7.51 (dd, J = 2.8 Hz, 7.6 Hz, 1H), 7.48 –7.44 (t, J = 7.6 Hz, 1H), 7.40 – 7.38 (dd, J = 2.0 Hz, 7.6 Hz, 1H), 7.24 (d,J = 7.6 Hz, 1H), 6.95 – 6.93 (m, 2H), 6.91 – 6.88 (dd, J = 1.6 Hz, 8.4 Hz,1H), 5.23 (t, J = 5.6 Hz, 1H), 4.51 (d, J = 5.6 Hz, 2H), 4.29 (s, 4H), 3.89(s, 3H). m / z: 384.2 (M+), 386.2 (M+2H) +.

[0436] Example 35: Preparation of 6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-3-(chloromethyl)-2-methoxy-pyridine (intermediate-25)

[0437]

[0438] INT-24 (24.0 g, 0.0703 mol) was dissolved in dichloromethane (540 mL) at 25–30 °C. Thionyl chloride (16.72 g, 0.1406 mol) was added dropwise to the reactants at 10–15 °C. The reaction mixture was maintained at 20–25 °C for 4 hours. The reaction was confirmed to be complete by TLC. The reaction was quenched with 10% sodium carbonate solution (10.0 mL). The organic solvent was distilled under reduced pressure to give 21.0 g of compound INT-25. 1 H NMR (400 MHz, CDCl3): δ 7.74 – 7.72 (d, J = 7.6 Hz, 1H), 7.53 – 7.50 (dd, J = 2.8 Hz, 7.2 Hz, 1H), 7.38 – 7.33 (m, 2H), 7.26 – 7.22 (m, 1H), 7.00– 6.99 (m, 1H), 6.94 – 6.93 (m, 2H), 4.66 (s, 2H), 4.31 (s, 4H), 4.04 (s,3H). m / z: 402.1 (M+), 404.1 (M+2H)+, 406.1 (M+4H)+.

[0439] Example 36: Preparation of 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-6-oxa-2-azaspiro[3,4]octane (NAT-22)

[0440]

[0441] INT-25 (16.8 g, 0.04176 mol) was dissolved in acetonitrile (168.0 mL) at 25–30 °C. 6-oxa-2-azaspiro[3.4]octane ditrifluoroacetate (21.3 g, 0.0626 mol) or INT-32 (7.07 g, 0.0625 mol) was added at 25–30 °C, followed by N,N-diisopropylamine (26.9 g, 0.2088 mol). The reaction was maintained at 65–70 °C for 5 hours. The reaction was confirmed to be complete by TLC. The solvent was distilled under reduced pressure to give the crude product. The crude compound was diluted with DM water (168.0 mL) and extracted with ethyl acetate (168.0 mL). The organic solvent was distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / hexane) at 100–200 mesh to give 11.0 g of compound NAT-22. 1H NMR (400 MHz, DMSO-d6): δ 7.71 (d, J = 7.2 Hz, 1H), 7.53 – 7.51 (m, 1H), 7.45 (t, J = 8.8 Hz, 1H), 7.40 – 7.37 (m, 1H), 7.21 (d, J = 7.6 Hz, 1H), 6.95 – 6.88 (m, 3H), 4.30 (s,4H), 3.88 (s, 3H), 3.73 (s, 2H), 3.64 (t, J = 6.8 Hz, 2H), 3.57 (brs, 2H), 3.24 (brs, 4H), 2.03 (t, J = 6.8 Hz, 2H). m / z: 479.1 (M+H)+ and 481.1(M+2H)+

[0442] Example 37: Preparation of 6-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane (NAT-23)

[0443]

[0444] INT-25 (15.0 g, 0.04176 mol) was dissolved in acetonitrile (150.0 mL) at 25–30 °C. 2-oxa-6-azaspiro[3.3]heptane (7.39 g, 0.0745 mol) was added at 25–30 °C, followed by N,N-diisopropylamine (9.7 g, 0.0745 mol). The reaction was maintained at 65–70 °C for 5 hours. The reaction was confirmed to be complete by TLC. The solvent was distilled under reduced pressure to give the crude product. The crude compound was diluted with DM water (168.0 mL) and extracted with ethyl acetate (150.0 mL). The organic solvent was distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / hexane) at 100–200 mesh to give 11.5 g of compound NAT-23. 1H NMR (400 MHz, CDCl3): δ 7.57 (d, J = 7.6 Hz, 1H), 7.52-7.49 (m,1H), 7.37 – 7.30 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.0 (s, 1H), 6.93 (t, J =10.0 Hz, 2H), 4.77 (s, 4H), 4.31 (s, 4H), 3.98 (s, 3H), 3.58 (s, 2H), 3.49(s, 4H). m / z: 465.0 (M+H) + and 467.0 (M+2H) +

[0445] NAT-24 and NAT-25

[0446]

[0447] Example 38: Preparation of intermediate (6-chloro-2-methoxy-4-methyl-3-pyridyl)methanol (INT-27)

[0448]

[0449] Step 1: Preparation of 6-chloro-2-methoxy-4-methylpyridine-3-carboxylic acid (INT-26)

[0450]

[0451] 60% NaH (52.38 g, 1.3104 mol) was dissolved in tetrahydrofuran (1080 mL) at 25–30 °C under a nitrogen atmosphere. The reaction mixture was cooled to 5–10 °C. 2,6-Dichloro-4-methylpyridin-3-carboxylic acid (108 g, 0.5241 mol) was dissolved in tetrahydrofuran (540 mL) and slowly added to the reaction mixture at 5–10 °C. The reaction mixture was maintained at 60–65 °C for 4 hours. The reaction was confirmed to be complete by TLC. DM water was slowly added to the reaction mixture. The crude product was diluted with water and acidified with 2N hydrochloric acid (pH 1–2) at 5–10 °C. The solid was filtered and dried in a disc desiccator at 75 °C to give 95.0 g of compound INT-26. 1 H-NMR(DMSO-d6, 400 MHz): δ 13.44 (s, 1H), 7.09 (s, 1H), 3.87 (s, 3H), 2.27 (s,3H). m / z: 202.1 [M+H] + and 204.1 [M+2H] +

[0452] Step 2: Preparation of intermediate (6-chloro-2-methoxy-4-methyl-3-pyridyl)methanol (INT-27)

[0453]

[0454] INT-26 (94.0 g, 0.4662 mol) was dissolved in tetrahydrofuran (940 mL) at 25–30 °C under a nitrogen atmosphere. A 2.0 M solution of the BH3.DMS complex in tetrahydrofuran (466.25 mL, 0.9325 mol) was added to the reaction mixture at 0–5 °C. The reaction mixture was stirred at 60–65 °C for 4 hours. The reaction was confirmed to be complete by TLC. The reaction mixture was cooled to 0–5 °C and quenched with 10% ammonium chloride solution. The reaction mixture was heated to 25–30 °C and extracted with ethyl acetate (100 mL). The organic layer was washed with DM water (100 mL). The solid was filtered and dried in a disc desiccator at 70 °C to give 86 g of compound INT-27. 1 H NMR (DMSO-d6, 400 MHz): δ 6.96 (s, 1H), 4.44 (s, 2H), 3.84 (s, 3H), 2.34(s, 3H). m / z: 188.1 [M+H] + and 190.1 [M+2H] +

[0455] Example 39: Preparation of 2-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaboranecyclopentane (intermediate-23)

[0456]

[0457] For the preparation of intermediate-23, see NAT-22.

[0458] Example 40: Preparation of [6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-2-methoxy-5-methyl-3-pyridyl]methanol (intermediate-28)

[0459]

[0460] INT-23 (86 g, 0.2307 mol) and compound INT-27 (34.63 g, 0.1846 mol) were dissolved in 1,4-dioxane (860 mL) at 25–30 °C. The reaction mixture was degassed for 10 min at 25–30 °C. Potassium carbonate (95.33 g, 0.6923 mol), DM water (172 mL), and then [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride DCM complex (4.71 g, 0.00576 mol) were added. The reaction mixture was degassed for 10 min at 25–30 °C. The reaction mixture was stirred at 90–95 °C for 6 h. The reaction was confirmed to be complete by TLC. The reaction mixture was brought to 20–25 °C and filtered through a diatomaceous earth mat. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (15% ethyl acetate / hexane) to give 58.8 g of compound INT-28. 1 H NMR (DMSO-d6, 400 MHz): δ 7.49-7.43 (m, 2H), 7.38 (dd, J = 2.0 Hz, 1.6 Hz, 1H), 7.06 (s, 1H), 6.95-6.92 (m, 2H), 6.89 (dd, J = 2.0 Hz, 1.6 Hz, 1H), 4.80 (t, J = 5.2 Hz, 1H), 4.54 (d, J = 5.2 Hz, 2H), 4.28 (s, 4H), 3.92 (s, 3H), 2.40 (s, 3H). m / z:398.2 (M+H) + and 400.2 (M+2H) +

[0461] Example 41: Preparation of 2-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-5-(chloromethyl)-6-methoxy-3-methylpyridine (intermediate-29)

[0462]

[0463] INT-28 (15.5 g, 0.0389 mol) was dissolved in dichloromethane (155 mL) at 25–30 °C. Thionyl chloride (11.30 mL, 0.1558 mol) was added dropwise to the reactants at 10–15 °C. The reaction mixture was maintained at 20–25 °C for 4 hours. The reaction was confirmed to be complete by TLC. The reaction was quenched with 10% sodium carbonate solution (10.0 mL). The organic solvent was distilled under reduced pressure to give 14.8 g of compound INT-29. 1H NMR (DMSO-d6, 400 MHz): δ 7.49 (dd, J = 2.4 Hz, 2.4 Hz, 1H), 7.37-7.31 (m, 2H), 7.06 (s, 1H), 6.99-6.98 (m, 2H), 6.93 (dd, J = 2.4 Hz, 2.4Hz, 1H), 4.74 (s, 2H), 4.31 (s, 4H), 4.02 (s, 3H), 2.45 (s, 3H). m / z: 416 (M+), 418.2 (M+2H)+, 420.2 (M+4H)+.

[0464] Example 42: Preparation of 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-2-methoxy-5-methyl-3-pyridyl]methyl]-6-oxa-2-azaspiro[3,4]octane (NAT-24)

[0465]

[0466] INT-29 (14.8 g, 0.0355 mol) was dissolved in acetonitrile (148.0 mL) at 25–30 °C. 6-oxa-2-azaspiro[3.4]octane ditrifluoroacetate (7.04 g, 0.0711 mol) or INT-32 (6.02 g, 0.0532 mol) was added at 25–30 °C, followed by N,N-diisopropylamine (24.72 mL, 0.1422 mol). The reaction was maintained at 70–75 °C for 5 hours. The reaction was confirmed to be complete by TLC. The solvent was distilled under reduced pressure to give the crude product. The crude compound was diluted with DM water (148.0 mL) and extracted with ethyl acetate (148.0 mL). The organic solvent was distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / hexane) at 100–200 mesh to give 10.7 g of compound NAT-24. 1H NMR (400 MHz, CDCl3): δ 7.49 (dd, J = 2.0 Hz, 5.2 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.04 (s,1H), 7.0 – 6.98 (m, 1H), 6.94 – 6.93 (m, 2H), 4.31 (s, 4H), 3.96 (s, 3H), 3.82 (s, 2H), 3.76 (t, J = 7.2 Hz, 2H), 3.69 (brs, 2H), 3.31 (brs, 4H), 2.41(s, 3H), 2.08 (t, J = 7.2 Hz, 2H). m / z: 493.1 (M+H) + and 495.1 (M+2H) +

[0467] Example 43: Preparation of 6-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxane-6-yl)phenyl]-2-methoxy-5-methyl-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane (NAT-25)

[0468]

[0469] INT-29 (10.6 g, 0.02546 mol) was dissolved in acetonitrile (106.0 mL) at 25–30 °C. 2-oxa-6-azaspiro[3.3]heptane (13.03 g, 0.03819 mol) was added at 25–30 °C, followed by N,N-diisopropylamine (22.19 g, 0.1273 mol). The reaction was maintained at 65–70 °C for 5 hours. The reaction was confirmed to be complete by TLC. The solvent was distilled under reduced pressure to give the crude product. The crude compound was diluted with DM water (106.0 mL) and extracted with ethyl acetate (106.0 mL). The organic solvent was distilled under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography (ethyl acetate / hexane) at 100–200 mesh to give 7.2 g of compound NAT-25. 1H NMR (400 MHz, CDCl3): δ 7.49 (dd, J = 2.0 Hz, 5.2 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.03 (s, 1H), 7.01 – 6.98 (m, 1H), 6.93 – 6.92 (m, 2H),4.74 (s, 4H), 4.31 (s, 4H), 3.96 (s, 3H), 3.60 (s, 2H), 3.46 (s, 4H), 2.37(s, 3H). m / z: 479.1 (M+H) + and 481.1 (M+2H) +

[0470] The salt formation of NAT-24 is described below.

[0471] Example 44: Preparation of intermediate 6-oxa-2-azaspiro[3.4]octane INT-32

[0472]

[0473] Preparation of intermediate 6-oxa-2-azaspiro[3.4]octane INT-32

[0474]

[0475] Example 45: Preparation of intermediate 2-benzyl-6-oxa-2-azaspiro[3.4]oct-3-one INT-30

[0476] Step-I: Dissolve 100 g (0.861 mol) of tetrahydrofuran-3-carboxylic acid in 1000 mL of dichloromethane at 25-30 °C. Add pyridine (1.38 mL, 0.017 mol) at 25-30 °C. Add thionyl chloride (65.0 mL, 0.895 mol) dropwise to the reactants over approximately 20-30 minutes at 25-30 °C. Maintain the reaction mixture at 25-30 °C for 4 hours.

[0477] Step 2: Dissolve 1,3,5-tribenzyl-1,3,5-triazine (101.59 g, 0.284 mol) in dichloromethane (500 mL) at 25-30 °C. Add BF3 diethyl ether compound (271 g, 0.861 mol) over approximately 10-15 minutes at 25-30 °C. Maintain the reaction mixture at 25-30 °C for 2 hours. Add triethylamine (503.8 mL, 3.615 mol) to the reaction mixture from Step 1 over approximately 30-40 minutes at 25-30 °C, and maintain for 30 minutes. Add the reaction mixture from Step 2 to the reaction mixture from Step 1 over approximately 15-20 minutes at -67.5 ± 2.5 °C, and maintain for 20 minutes. Raise the temperature of the reaction mixture to 2.5 ± 2.5 °C and maintain for 20 minutes. Add 500 mL of DM water at 2.5 ± 2.5 °C and raise the temperature to 25–30 °C. Stir the reaction mixture at 25–30 °C for 15–16 hours. Separate the dichloromethane layer, wash with 500 mL of 2 M KHSO4 solution, then wash with 500 mL of saturated NaHCO3 aqueous solution and 500 mL of DM water. Distill the dichloromethane layer at 45–50 °C to give 122.3 g of INT-30. 1 H NMR (400 MHz, CDCl3): δ 7.38-7.21 (m, 5H), 4.45-4.34 (m, 2H), 4.03-3.95 (m, 2H), 3.92-3.83(m, 2H), 3.25-3.18 (m, 2H), 2.46-2.38 (m, 1H), 2.14-2.07 (m, 1H). m / z: 218.3(M+H) +

[0478] Example 46: Preparation of intermediate 2-benzyl-6-oxa-2-azaspiro[3.4]octane hydrochloride INT-31

[0479] Tetrahydrofuran (900.0 mL) and aluminum chloride (88.3 g, 0.662 mol) were added to the reactants over approximately 20–25 minutes at -22.5 ± 2.5 °C under a nitrogen atmosphere. LiAlH4 (2.0 M in THF) solution (331 mL, 0.662 mol) was added to the reactants over approximately 30–40 minutes at -22.5 ± 2.5 °C under a nitrogen atmosphere. The reactants were maintained at 25–30 °C for 1 hour. INT-30 (120 g, 0.552 mol) dissolved in tetrahydrofuran (900 mL) was added to the reactants over approximately 30–40 minutes at -10 ± 2.5 °C. The reactants were maintained at 0 ± 5 °C for 2 hours. The reaction was confirmed to be complete by TLC. Ethyl acetate (180 mL) was added to the reactants over approximately 15–20 minutes at -5 ± 5 °C. The reactants were maintained at -5 ± 5 °C for 12 hours. Ethanolamine (240 mL, 1.104 mol) was added to the reactants over approximately 25–30 minutes at -5 ± 5 °C. The reactants were maintained at 27.5 ± 2.5 °C for 14 hours. The reactants were filtered, and tetrahydrofuran was distilled at 47.5 ± 2.5 °C. DM water (444 mL) and dichloromethane (1200 mL) were added at 27.5 ± 2.5 °C, and the mixture was stirred for 15 minutes. The dichloromethane layer was separated and distilled at 47.5 ± 2.5 °C to give the crude compound (GC purity 87.0%).

[0480] The crude compound was dissolved in ethyl acetate (1370 mL) at 27.5 ± 2.5 °C. IPA·HCl (274 mL) was added at 12.5 ± 2.5 °C. The reaction mixture was maintained at 27.5 ± 2.5 °C for 40 min. 2-Benzyl-6-oxa-2-azaspiro[3.4]octane hydrochloride was filtered, and the salt was washed with n-heptane (685 mL) to obtain hydrochloride INT-31 (HPLC purity 99.0%).

[0481] 2-Benzyl-6-oxa-2-azaspiro[3.4]octane hydrochloride was added to ethyl acetate (1370 mL) and alkalized to pH 11-12 with 2N NaOH solution at 12.5 ± 2.5 °C. The organic layer was separated and distilled at 47.5 ± 2.5 °C to give 44.4 g of INT-31 base (HPLC purity 99.0%). 1 H NMR (400 MHz, DMSO-d6): δ 7.31-7.20 (m, 5H),3.68 (s, 2H), 3.62 (t, J = 8.0 Hz, 2H), 3.52 (s, 2H), 3.13-3.09 (m, 4H), 1.98(t, J = 8.0 Hz, 2H). m / z: 204.1 (M+H) +

[0482] Example 47: Preparation of intermediate 6-oxa-2-azaspiro[3.4]octane INT-32

[0483] INT-31 (144 g, 0.708 mol) and ethanol (2880 mL) were dissolved in an autoclave at 27.5 ± 2.5 °C. 10% Pd / C (72 g) was added to the reactants under a nitrogen atmosphere at 27.5 ± 2.5 °C. The reaction mixture was maintained at 27.5 ± 2.5 °C under a hydrogen atmosphere (70 psi) for 24 hours. The reaction was confirmed to be complete by TLC. The reactants were filtered through a diatomaceous earth pad under a nitrogen atmosphere and washed with ethanol (720 mL). The filtrate was distilled at 42.5 ± 2.5 °C to give 68 g of INT-32. 1 H NMR (400 MHz, DMSO-d6): δ 3.69 (s, 2H), 3.62 (t, J = 8.0 Hz, 2H), 3.40 (s, 4H), 2.71 (s,1H), 1.99 (t,J = 8.0 Hz, 2H).

[0484] Active Examples

[0485] As a preliminary summary, the method used to test the competitive binding ability of the compounds of the present invention to PD-1 / PD-L1 is as follows:

[0486] The affinity of these compounds for the target protein was determined using biophysical methods such as microthermophoresis (MST) and differential scanning fluorometry (DSF).

[0487] A series of in vitro assays were performed to determine the immunomodulatory effects of the compounds—these included cell-free assays, homogeneous time-resolved fluorescence (HTRF) for PD1 / PDL1 axis disruption and identification of dimerization.

[0488] Cell-based assays involving PBMCs / T cells / exhausted T cells / CD8+ T cells were also performed. These assays were performed using flow cytometry to measure CD8+ T cell populations, IFNg secretion, internalization, and apoptosis. Thus, the immunomodulatory properties of these compounds were determined.

[0489] In addition, in some cases, HTRF dimerization assays were performed to determine PDL1 dimerization.

[0490] Example 48:

[0491] Binding affinity of compounds was determined by microthermophoresis (MST).

[0492] Experimental materials:

[0493] Dye kit: MonolithTM The series of protein labeling kits used were RED-tris-NTA second generation and RED-NHS second generation; capillary tubes: MO-K022 Monolith NT.115 capillary tubes; buffer: pH 7.4, PBST; proteins: human hPD-L1 / B7-H1 (19-134) protein, His Tag (MALS verified), human PD-L1 (19-134), His Tag was expressed by human 293 cells (HEK293). It contains AA Phe 19 - Tyr 134, predicted N-terminus: Phe 19; VISTA / B7-H5 protein, human, recombinant (His Tag), encoding the DNA sequence of human Gi24 (AAH20568.1) (Met1-Ala194) with a C-terminal multi-histidine tag; human B7-1 / CD80 protein, His Tag (MALS verified), human B7-1, His Tag is expressed by human 293 cells (HEK293). It contains AA Val 35 - Asn 242. Predicted N-terminus: Val 35 B71-H5228; MO.Affinity Analysis v2.2.4 software.

[0494] Experimental model: Monolith NT.115

[0495] Experimental procedures (binding affinity of compounds with human PD-L1 / human VISTA / human B7-1)

[0496] The binding affinity between human PD-L1 / human VISTA / human B7-1 and the compounds synthesized in this invention was determined by micro-thermophoresis (MST). Proteins were labeled according to the instructions of the Monolith RED-Tris-NTA second-generation protein labeling kit / RED-NHS second-generation protein labeling kit (Nano Temper Technologies). The compounds were diluted to solutions ranging from 10.0 µM to 305.0 pM (10 µM, diluted 16 times, each time by a factor of two, to a final concentration of 305.0 pM). The compound solutions and labeled proteins were homogenized at a 1:1 ratio at room temperature, incubated in the dark for 10 minutes, and the mixture was analyzed using a glass capillary tube. Data were processed using MO.Affinity Analysis v2.2.4 software to obtain the binding affinity values ​​between the compounds and proteins.

[0497] The obtained hPD-L1 and compound Kd values ​​are shown in Table 2. The ranges are as follows: A: <50 nM; B: 50 nM - 100 nM; C: 100 nM - 200 nM; D: 200 nM - 600 nM; E: >600 nM.

[0498] Table 2: Binding affinity of compounds to hPD-L1 protein

[0499]

[0500] Exemplary data for the four compounds of this invention (all belonging to Class A in the table above) are shown in Figure 3 These compounds are referred to below as compound 1, compound 2, compound 3, and compound 4.

[0501] The Kd values ​​of the obtained hVISTA protein and compounds are shown in Table 3. The ranges are as follows: A: <50 nM; B: 50 nM - 200 nM; C: 200 nM - 1000 nM.

[0502] Table 3: Binding affinity of compounds to hVISTA protein

[0503]

[0504] The Kd values ​​of the obtained human B7-1 / CD80 protein are shown in Table 4. The ranges are as follows: A: <50 nM; B: 50 nM - 200 nM; C: 200 nM - 1000 nM; D: 1.0 µM - 2.0 µM.

[0505] Table 4: Binding affinity of compounds to human B7-1 / CD80 protein

[0506]

[0507] Example 49

[0508] Determination of thermal displacement (ΔTm) by differential scanning fluorescence (DSF) – the effect of compounds on thermal stability

[0509] Experimental materials:

[0510] Reagent kit: Protein Thermal Shift TM Dye Kit (Applied Biosystems (ThermoFisher Scientific) #4461146), Protein Thermal Shift Dye, Protein Thermal ShiftBuffer; MicroAmp TMOptical 8-Tube Strip with Attached Optical Caps, 0.2 mL (Applied Biosystems (Thermo Fisher Scientific) Cat# A30588); Protein: Human hPD-L1 / B7-H1 (19-134) protein, His Tag (MALS verified), Human PD-L1 (19-134), His Tag expressed by human 293 cells (HEK293). It contains AA Phe 19 - Tyr 134, predicted N-terminus: Phe 19.

[0511] Experimental Model: QuantStudio TM 3 Real-Time PCR System (Software version 1.4.0) AppliedBiosystems (Applied Biosystems (Thermo Fisher Scientific))

[0512] Experimental steps

[0513] The compound was dissolved in DMSO at a concentration of 10 mM. Lyophilized human hPD-L1 protein was dissolved in distilled water at a concentration of 40 µM. hPD-L1 was used at a final concentration of 10 µM; the compound dissolved in DMSO was added to the protein sample to a final concentration of 50 µM. Protein incubated alone with DMSO served as a control. 1000X protein thermal shift dye was diluted with protein thermal shift buffer to prepare 8X protein thermal shift dye, which was used immediately and protected from light to reduce photobleaching. Assay buffer (PBS), protein, and compound were added to the wells. The PCR plate was sealed and then centrifuged at 1000 rpm for 1 minute. After an incubation period (5 minutes at room temperature) to allow the protein and compound to equilibrate, the protein thermal shift dye was mixed with the protein. The PCR plate was placed in a PCR instrument and run at 25 °C for 2 minutes, then scanned to 95 °C at 0.05 °C / s and heated at 95 °C for 2 minutes. The thermal denaturation assay was performed in a total volume of 20 µL. All samples were run in triplicate.

[0514] Table 5 Summary of Tm and ΔTm for NAT-compounds / human PD-L1 protein

[0515]

[0516] Example 50: Determination of the IC50 value of a compound by in vitro homogeneous time-resolved fluorescence (HTRF) PD1 / PDL-1 binding assay

[0517] Experimental materials

[0518] PD1 / PD-L1 binding kit (CisBio #64PD1PEG); assay plate: 384-well white opaque proxiplate (Perkin Elmer #6008280); centrifuge tubes; serum pipettes; Easy pet.

[0519] Experimental protocol

[0520] Follow the steps given in the PD1 / PD-L1 binding kit.

[0521] Results: The IC50 values ​​obtained by HTRF measurement are shown in Table 6.

[0522] The ranges are as follows: A: <50 nM; B: 50 nM < IC50 < 250 nM; C: 250 nM < IC50 < 600 nM; D: >600 nM.

[0523] Table 6: IC50 values ​​of compounds in HTRF assays

[0524]

[0525] Example 51: Determination of IC50 value of test compound by in vitro HTRF dimerization of PDL1 / PDL-1 binding assay

[0526] Experimental materials

[0527] PDL1 FC Tag Protein; PDL1 HIS Tag Protein; Allophycocyanin; XL665; Assay plate: 384-well white opaque Proxiplate (Perkin Elmer #6008280); Centrifuge tubes; Serum pipettes; Easy pet.

[0528] Experimental plan:

[0529] Add 2 µL of compound (10X) to 2% DMSO in the assay plate. Prepare compound dilution in europium detection buffer (provided in the kit). Add 4 µL of diluted Tag1-PDL-1 HIS Tag to all wells except the buffer wells. Dilute the Tag1-PDL-1 HIS Tag stock solution (40 µM) to 200 nM (40 nM / well) with europium detection buffer. Add 4 µL of diluted Tag-2-PDL-1 FC Tag to all wells except the buffer and negative control wells. Dilute the Tag2-PD-L1 FC Tag stock solution (1923 nM) to 60 nM (12 nM / well) with europium detection buffer. Add 2 µL of europium detection buffer to the positive control wells, 6 µL to the negative control wells, and 20 µL to the buffer control wells. Add 10 µL of premixed Anti-Tag1 XL665 antibody and Anti-Tag2 allophycocyanin reagent to all wells, except for the buffer wells. Dilute the Anti-Tag1 XL665 antibody 625 nM to 12 nM (3 nM / well) with europium detection buffer, and dilute the Anti-Tag2 allophycocyanin reagent stock solution 2960 nM to 230 nM (58 nM / well). Seal the plate and incubate at room temperature for 1 hour. After incubation, remove the plate seal and read the plate using an HTRF® compatible plate reader.

[0530] Table 7: IC50 values ​​of lead compounds in HTRF dimerization assays

[0531]

[0532] Example 52: Determination of EC50 value of compound in intracellular PD1 / PDL-1 blocking bioassay (NFAT)

[0533] Experimental materials

[0534] PD-1 / PD-L1 Blockade Bioassay Kit (Promega #J1250); Assay plate: 96-well white transparent plate (Corning 3610); Centrifuge tubes; Serum pipettes; Easy PET; Microplate reader

[0535] Experimental protocol: Follow the steps given in the screening assay kit.

[0536] Table 8: EC50 values ​​of lead compounds in NFAT assays

[0537]

[0538] Example 53: Determination of the binding activity of the compound in the VISTA / VSIG3 biotinylation inhibition assay

[0539] Experimental materials:

[0540] VSIG-3: VISTA [Biotinylated] Inhibitor Screening Assay Kit; Catalog No. #79782 (BPS Bioscience); PBS (phosphate-buffered saline); chemiluminescence meter or microplate reader capable of reading chemiluminescence; adjustable micropipette and sterile pipette tips.

[0541] Experimental procedure: Follow the steps given in the screening assay kit.

[0542] Table 9: IC50 values ​​of lead compounds in VISTA / VSIG3 assays

[0543]

[0544] Experiment 54: PDL1 internalization—Selected cell lines were incubated with selected compounds to assess compound binding affinity, thereby evaluating internalization. Results are shown in Figure 4 .

[0545] Experimental Scheme-1:

[0546] CHO-PDL1 (high PDL1 expression) cancer cells (0.1×10⁻⁶) 6 Cells were seeded in 6-well plates and incubated for 24 hours. The next day, cells were treated for 17 hours with the test compound (5 µM) for PDL1 inhibition, DMSO (background), and atezolizumab (1 µM, positive control). Non-confluent cell cultures were trypsinized into single-cell suspensions, washed with PBS, and centrifuged. Cells were then stained with a PDL1 fluorescent probe (1 µg / 100 µL) at 4°C for 30 minutes, washed twice, and resuspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analyzed using flow cytometry.

[0547] Internalization efficiency (%) = [(Fluorescence intensity of control group - Fluorescence intensity of experimental group) / Fluorescence intensity of control group] × 100%

[0548] Table 10: Internalization of lead compound in CHO-PDL1 cells (PDL1 expression) at different incubation time points

[0549]

[0550] As shown in this paper, the maximum reduction in PD-L1 accessibility occurred after different compound treatments at the longest incubation time (72 hours) (except for compound 3), so subsequent experiments were conducted by incubating the compounds for 72 hours.

[0551] Experimental Scheme-2:

[0552] CHO-PDL1 cells were digested with trypsin. Cells were resuspended in complete culture medium and placed in 15 mL centrifuge tubes. Cells were centrifuged at 1000 rpm for 5 minutes and resuspended in FACS staining buffer (1x PBS, containing 2% FBS) to a concentration of 2×10⁻⁶ cells / mL. 7 Cells / mL. Dispense the cell suspension into 1.5 mL EP tubes, 50 µL per tube (1 × 10⁻⁶ cells / mL). 6 (cells). Compound 3 (5 µM) and cell suspension diluted with FACS staining buffer were combined to a final volume of 100 µL, ensuring the concentration of Compound 3 in the combined solution was within the appropriate range (5 µM). The mixture was pipetted and incubated on ice for 60 min. Cells were washed with 1 mL of FACS staining buffer and then centrifuged at 300 × g for 5 min at 4 °C to remove unbound compound. After adding 200 µL of FACS staining buffer to each tube, the tubes were incubated at 37 °C for 2 h to observe the internalization of the compound bound to the cell surface. At each time point, another sample tube incubated at 4 °C served as a negative control, as antibody internalization was very weak under these conditions. To terminate internalization, 1 mL of ice-cold FACS staining buffer was added to each tube. The tubes were then immersed in an ice bath and centrifuged at 300 g for 5 min at 4 °C. Then, 100 µL of secondary antibody solution containing 5 µL of anti-PDL1 was added to each sample. The tubes were incubated at 4 °C in the dark for 30 min. Cells were washed with 1 mL of FACS staining buffer and centrifuged at 300 × g for 5 min at 4 °C to remove unbound secondary antibody. Cells were washed twice and then resuspended in 300 µL of ice-cold FACS staining buffer. Fluorescence intensity was detected by flow cytometry. The degree of internalization of NAT-24 bound to the cell surface was determined by the percentage decrease in mean fluorescence intensity (MFI) of the sample incubated at 37 °C compared to the control sample incubated at 4 °C. The internalization efficiency of each antibody in cells was calculated using the following formula: Internalization efficiency (%) = [(Fluorescence intensity of control group - Fluorescence intensity of experimental group) / Fluorescence intensity of control group] × 100%

[0553] Table 11: Internalization observed after 2 hours of incubation of the lead compound with CHO-PDL1 (PDL1-expressing) cells

[0554]

[0555] Experimental Scheme-3:

[0556] MDAMB231 (high PD-L1 expression) cancer cells (0.1 × 10⁻⁶) 6Cells were seeded in 6-well plates and incubated for 24 hours. The next day, cells were treated for 17 hours with the test compound (3 µM) for PDL1 inhibition, DMSO (background), and atezolizumab (1 µM, positive control). Non-confluent cell cultures were trypsinized into single-cell suspensions, washed with PBS, and centrifuged. Cells were then stained with a PDL1 fluorescent probe (1 µg / 100 µL) at 4°C for 30 minutes, washed twice, and resuspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analyzed using flow cytometry.

[0557] Internalization efficiency (%) = [(Fluorescence intensity of control group - Fluorescence intensity of experimental group) / Fluorescence intensity of control group] × 100%

[0558] Table 12: Internalization of the lead compound at different time points after incubation with MDAMB231 cells

[0559]

[0560] Experimental Scheme-3:

[0561] U251 (PD-L1 expression) cancer cells (0.1×10 6 Cells were seeded in 6-well plates and incubated for 24 hours. The next day, cells were treated for 17 hours with the test compound (10 µM) for PD-L1 inhibition, DMSO (background), and atezolizumab (positive control). Non-confluent cell cultures were trypsinized into single-cell suspensions, washed with PBS, and centrifuged. Cells were then stained with a PD-L1 fluorescent probe (1 µg / 100 µL) at 4°C for 30 minutes, washed twice, and resuspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analyzed using flow cytometry.

[0562] PD-L1 internalization in cells was measured by MFI reduction relative to the background.

[0563] Internalization efficiency (%) = [(Fluorescence intensity of control group - Fluorescence intensity of experimental group) / Fluorescence intensity of control group] × 100%

[0564] Table 13: Internalization of the lead compound at different time points after incubation with U251 cells

[0565]

[0566] Experimental Scheme-4:

[0567] 8505C (thyroid cancer, PDL1 expression) cancer cells (0.1×10⁻⁶) 6Cells were seeded in 6-well plates and incubated for 24 hours. The next day, cells were treated for 17 hours with the test compound (10 µM) for PDL1 inhibition, DMSO (background), and atezolizumab (positive control). Non-confluent cell cultures were trypsinized into single-cell suspensions, washed with PBS, and centrifuged. Cells were then stained with a PDL1 fluorescent probe (1 µg / 100 µL) at 4°C for 30 minutes, washed twice, and resuspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analyzed using flow cytometry.

[0568] PD-L1 internalization in cells was measured by MFI reduction relative to the background.

[0569] Internalization efficiency (%) = [(Fluorescence intensity of control group - Fluorescence intensity of experimental group) / Fluorescence intensity of control group] × 100%

[0570] Table 14: Internalization observed at 17 hours after incubation of the lead compound with 8505C cells

[0571]

[0572] Experimental Scheme-4:

[0573] HCC827 (thyroid cancer, PDL1 expression) cancer cells (0.1×10⁻⁶) 6 Cells were seeded in 6-well plates and incubated for 24 hours. The next day, cells were treated for 17 hours with the test compound (5 µM) for PDL1 inhibition, DMSO (background), and atezolizumab (positive control). Non-confluent cell cultures were trypsinized into single-cell suspensions, washed with PBS, and centrifuged. Cells were then stained with a PDL1 fluorescent probe (1 µg / 100 µL) at 4°C for 30 minutes, washed twice, and resuspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analyzed using flow cytometry.

[0574] PD-L1 internalization in cells was measured by MFI reduction relative to the background.

[0575] Internalization efficiency (%) = [(Fluorescence intensity of control group - Fluorescence intensity of experimental group) / Fluorescence intensity of control group] × 100%

[0576] Table 15: Internalization of the lead compound at 72 hours after incubation with HCC827 cells

[0577]

[0578] Experiment 55: Demonstrating that lead compounds reverse T cell exhaustion by restoring IFNg through PD1 downregulation.

[0579] a. T cell exhaustion experimental protocol

[0580] - In a suitable tissue culture plate or flask, in a culture medium at a concentration of 1-1.5 × 10⁻⁶. 6 Start with 1 purified T cell / mL.

[0581] - Add Dyna beads Human T-Activator CD3 / CD28 (Thermo) at a 1:1 bead-to-cell ratio.

[0582] - Add 30 U / mL rIL-2.

[0583] - Incubate in a humid CO2 incubator at 37°C, depending on the specific experimental requirements.

[0584] - Examine the culture daily, noting cell size and shape. Cell shrinkage and reduced proliferation rate are commonly observed in depleted cell cultures.

[0585] - Count the cells every 48 hours and restimulate them with a fresh batch of CD3 / CD28 Dynabeads.

[0586] - After two or three stimulations, Dynabeads are removed by magnetic separation using LS columns.

[0587] - Count the exhausted T cells and freeze them for subsequent experiments.

[0588] a. Experimental Protocol-1 – Treatment of exhausted T cells with compounds in the absence of cancer cells (PD1, TIM3, and CD8 were measured by flow cytometry)

[0589] *Depleted T cells (10 5 Ex T cells were plated (10 µM) and activated with (CD3+CD28) and incubated with the compound for 72 hours. The supernatant was collected for FACS analysis to examine T cell exhaustion reversal. By treating exhausted T cells with the compound (in the absence of cancer cells), the expression of early exhaustion marker PD1 and late exhaustion marker TIM3 in Ex T cells decreased, while the CD8 population increased.

[0590] Table 16

[0591]

[0592] b. Experimental Protocol 2 – Treatment of exhausted T cells co-cultured with MDAMB231 cells with compounds (IFNg recovery and PD1 expression were measured by flow cytometry)

[0593] Exhausted T cells were plated and co-cultured with MDA-MB-231 cancer cells for 72 hours in the presence of the test compound (3µM, 1µM, 0.3µM). Atezolizumab was used as a positive control (0.3µM). The T cell to cancer cell ratio was 3:1. The supernatant containing T cells was collected, and the restored IFNg level and PD1 expression of activated T cells in the supernatant were detected by flow cytometry. The highest IFNg restoration and PD1 expression downregulation were observed at a test compound concentration of 0.3µM.

[0594] Table 17:

[0595]

[0596] Experiment 55: T cell-mediated cytotoxicity against tumor cells

[0597] Apoptosis assay (based on Annexin V flow cytometry)

[0598] Exemplary results are shown in Figure 5 and Figure 6 .

[0599] Experimental protocol for isolating human peripheral blood mononuclear cells (PBMCs) from whole blood of healthy volunteers

[0600] 1. Process whole blood cells from healthy donors on a density gradient (such as Ficoll Hypaque) to enrich mononuclear cells.

[0601] 2. Recover the "white membrane layer" containing mononuclear cells by washing the cells twice with excess DPBS to remove any residual separation medium. This can be done by centrifuging the cells at 200×g for 10 minutes.

[0602] 3. After the second washing step, break up the cell pellet by tapping the tube and resuspend the cells in H-Lyse buffer diluted to a 1X concentration with sterile distilled water from the R&D Systems® Human Erythrocyte Lysing Kit (catalog number WL1000). Use a rapid vortex tube (10 mL of 1X H-Lyse solution is recommended for every 250 million cells).

[0603] 4. Incubate the cells at room temperature for 10 minutes, then fill the tube with 1X wash buffer from the lysis kit. Note: The wash buffer must also be diluted to a 1X concentration with sterile water before use.

[0604] 5. Centrifuge the cells at 200×g for 10 minutes, then resuspend the cells in a small amount of 1X MagCellect buffer.

[0605] 6. Perform cell counting, then adjust the cell concentration to 1×10⁶ cells / year using cold 1X MagCellect buffer. 8 Cells / mL.

[0606] Human CD8 T cell isolation assay protocol (Reagent kit: Cat no: MAGH112)

[0607] 1. Take 1×10 8 Transfer 1 cell (1 mL volume) to a 15 mL centrifuge tube. Add 100 µL of MagCellect human CD8+ T cell biotinylated antibody mixture. Gently mix the cell-antibody suspension, avoiding the formation of air bubbles, and incubate at 2–8 °C for 15 minutes.

[0608] 2. Add 200µL of Streptavidin Ferrofluid to the cell suspension, mix gently, and incubate at 2-8°C for 15 minutes.

[0609] 3. At the end of the incubation period, add 0.7 mL of 1X MagCellect buffer to adjust the reaction volume in the tube to 2 mL. Gently mix to ensure that all reactants in the tube are suspended.

[0610] 4. Perform magnetic separation.

[0611] 5. Place the column in the magnetic field of a suitable MACS separator.

[0612] 6. Rinse the column with 3 mL of buffer solution to prepare.

[0613] 7. Prepare the cell suspension (maximum 10...) 9 Add (number) cells to the column. Collect the effluent containing unlabeled cells, i.e., enriched CD8 T cells.

[0614] 8. Wash the column with 3 mL of buffer. Collect the unlabeled cells that have passed through the column, i.e., the enriched CD8 T cells, and combine them with the effluent from step 7.

[0615] 9. Centrifuge the effluent at 300g for 10 minutes.

[0616] 10. Count the cells and freeze them in 90% FBS and 10% DMSO in LN2.

[0617] Experimental Protocol 1 – Apoptosis assay of MDAMB231 and CD8+ T cells treated with lead compound (Annexin V assay).

[0618] - CD8+ T cells isolated from PBMCs were collected in a 1:1 ratio (3 × 10⁻⁶ cells / mL). 5MDAMB231 cancer cells were plated in a 96-well plate.

[0619] - T cells were stimulated with anti-CD3 and anti-CD28 antibodies. Cells were co-cultured at 37°C for 20 hours in the presence of the compound (3 µM) and mAb (1 µM).

[0620] - Wash cells in FACS buffer, then resuspend in Annexin binding buffer containing FITC-conjugated Annexin V. Mix and incubate at room temperature for 10 minutes. Wash cells in binding buffer, resuspend in binding buffer, and add propidium iodide (PI).

[0621] - Flow cytometry analysis was performed. When CD8+ T cells were co-cultured with MDAMB231 cells, compounds-1 and-3 showed more apoptotic signatures.

[0622] Table 18

[0623]

[0624] Experimental Protocol 2 – Apoptosis assay of U251 and CD8+ T cells treated with lead compound (Annexin V assay).

[0625] - CD8+ T cells isolated from PBMCs were collected in a 1:1 ratio (3 × 10⁻⁶ cells / mL). 5 U251 cancer cells were plated in a 96-well plate.

[0626] - T cells were stimulated with anti-CD3 and anti-CD28 antibodies. Cells were co-cultured at 37°C for 20 hours in the presence of the compound (10 µM) and mAb (1 µM).

[0627] - Wash cells in FACS buffer, then resuspend in Annexin binding buffer containing FITC-conjugated Annexin V. Mix and incubate at room temperature for 10 minutes. Wash cells in binding buffer, resuspend in binding buffer, and add propidium iodide (PI).

[0628] - Flow cytometry analysis was performed. When CD8+ T cells were co-cultured with U251 cells, compounds-1 and-3 showed more apoptotic characteristics.

[0629] Table 19

[0630]

[0631] Experimental Protocol 3 – Apoptosis assay of HCC827 and CD8+ T cells treated with lead compound (Annexin V assay).

[0632] - CD8+ T cells isolated from PBMCs were collected in a 1:1 ratio (3 × 10⁻⁶ cells / mL). 5 HCC827 cancer cells were plated in a 96-well plate.

[0633] - T cells were stimulated with anti-CD3 and anti-CD28 antibodies. Cells were co-cultured at 37°C for 20 hours in the presence of the compounds and mAb (1 µM).

[0634] - Wash cells in FACS buffer, then resuspend in Annexin binding buffer containing FITC-conjugated Annexin V. Mix and incubate at room temperature for 10 minutes. Wash cells in binding buffer, resuspend in binding buffer, and add propidium iodide (PI).

[0635] - Flow cytometry analysis was performed. When CD8+ T cells were co-cultured with HCC827 cells, compounds-1 and-3 showed more apoptotic characteristics.

[0636] Table 20

[0637]

[0638] Experiment 56: T cells were activated by co-culturing with MDAMB231 / U251 cells and treating them with the test compound, resulting in the restoration of IFNg and an increase in T cell markers.

[0639] Experimental Scheme-1

[0640] PBMCs isolated from three healthy donors were used in this experiment. The PBMCs were resuspended in T cell culture medium one day prior to co-culture and cultured overnight at 37°C. MDAMB231 cells were pre-cultured with 200 ng / mL of [agent / sample] prior to co-culture. −1 Human recombinant interferon IFN-γ was treated overnight. PBMCs were treated with 0.1 × 10⁻⁶ IFN-γ. 6 PBMCs were seeded at a density of 100 cells / well and stimulated with CD3, CD28, and IL2. PBMCs were co-cultured with MDAMB231 cells at a 2:1 cell ratio in the presence of the test compound (3 µM) and Mab for 72 hours. Cells were then stained with fluorescently labeled antibodies and analyzed by flow cytometry.

[0641] Table 21

[0642]

[0643] Experimental scheme 2:

[0644] U251(1.5×10 5In 6-well plates, cells were treated with 500 IU / mL IFNg for 24 hours, co-cultured with activated T cells and a compound (10 µM) (2:1) for 72 hours, and the supernatant was analyzed by flow cytometry to examine CD8+ activation markers.

[0645] Table 22

[0646]

[0647] The above experiment was demonstrated in a T cell from a specimen donor.

[0648] Experiment 57:

[0649] ADME and pharmacokinetics of the lead compound

[0650] Experimental Scheme 1

[0651] Permeability of the lead compound was determined in the MDR1-MDCK monolayer.

[0652] In vitro transport studies were performed using MDCK monolayers transfected with MDR1 cells cultured for 5 days, obtained from the Netherlands Cancer Institute. Cells were passaged every other day at a ratio of 1:3 to 1:5 and grown in DuPont modified Eagle medium supplemented with 10% FBS and 1% Pen-Strip antibiotic. For transport studies, cells were seeded at a density of 500,000 cells / well onto polycarbonate Transwell membranes (Millipore). The medium was changed 24 hours after seeding, and cells were cultured for another 4 days before transport experiments were performed. Donor solutions were prepared by diluting a stock solution of the test compound in transport medium (HBSS buffer containing 10 mM HEPES, pH 7.4). The receiving solution was the same HBSS buffer containing 10 mM HEPES, pH 7.4. Transport of the test compound (5 µM) was measured in duplicate in two directions [apex-to-basal (A→B) and basal-to-apex (B→A)]. The permeability coefficient of the membrane transport of the test compound was determined using the following equation:

[0653] Papp (cm / s) = (Vr / C0) (1 / S) (dC / dt)

[0654] (Papp = apparent permeability, Vr = volume of medium in the receiving chamber, C0 = PAR of the test drug in the receiving chamber, S = surface area of ​​a monolayer, dC / dt = change in PAR of the drug in the receiving chamber over time); 24-well area = 0.7 cm² 2 Peak area ratio = Analyte peak area / IS peak area

[0655] The external displacement ratio is defined as:

[0656] Outflow ratio = (Papp BA) / (Papp AB)

[0657]

[0658] Table 23: Permeability of lead compounds in the MDR1-MDCK layer

[0659] Conclusion: None of the lead compounds are PgP substrates and all exhibit moderate permeability.

[0660] Experimental scheme 2

[0661] Time-dependent inhibition (TDI) of CYP 3A4 by the lead compound in human liver microsomes.

[0662] Stock solution preparation:

[0663] Preparation of standard working solutions:

[0664] • Preparation of working stock solutions: 10 mM of the test compound and 100 mM of metoprolol were prepared using DMSO.

[0665] • The test compound and the reference standard mebendazole were tested at final concentrations of 1 µM and 10 µM.

[0666] • The final protein concentration was maintained at 0.1 mg / mL.

[0667] Buffer preparation: 66.7 mM potassium phosphate buffer (pH 7.4) was used as the assay buffer and prepared by mixing appropriate volumes of 1 M K2HPO4 and 1 M KH2PO4 stock buffer.

[0668] The experiment was conducted under the following conditions.

[0669] Table 24

[0670]

[0671] Methodology (n=2):

[0672] Preparation of incubation mixture

[0673] 1. Thaw the microsomes while keeping them on ice. 2. Prepare a microsome suspension in potassium phosphate buffer to achieve the desired final protein concentration. 3. Gently mix the contents by inverting the container and equilibrate at 37±1°C for 5 minutes.

[0674] Experimental steps

[0675] Initial incubation steps:

[0676] 1. Add 898.8 µL of the above incubation mixture to three polypropylene tubes labeled “Test,” “Test Control,” and “Positive Control”; 2. Spike 1.12 µL of the 10 mM and 100 mM “Test” and “Positive” compounds for +NADPH and -NADPH incubation, respectively; 3. Similarly, spike 1.12 µL of DMSO to the tube labeled “Test Control”; 4. Add 100 µL of 10 mM NADPH to the +NADPH incubation and 100 µL of buffer to the -NADPH incubation; 5. Mix the incubation mixture and incubate for 1, 5, 10, 20, 30, and 40 minutes.

[0677] Secondary incubation steps:

[0678] 6. At each time point, transfer two equal aliquots (2 × 20 µL) from each of the initial incubation mixtures to a secondary incubation mixture containing midazolam (2.5 µM); 7. Further, add 20 µL of NADPH to all tubes and incubate in a shaking water bath at 37 ± 1 °C for 10 minutes; 8. After the incubation period, add 300 µL of quenching solution to terminate the reaction; 9. Gently vortex the tubes and centrifuge at 4000 rpm for 20 minutes at 4 °C; 10. Collect the supernatant and submit the samples to an LC-MS / MS system; 11. Use an LC-MS / MS system to estimate the metabolite formation of each probe substrate.

[0679] Data Analysis:

[0680] Remaining percentage: [Average peak area ratio at each time point / Average peak area ratio at 0 minutes] × 100

[0681] Bioanalysis:

[0682] Perform bioanalysis on LC-MS / MS under the following conditions.

[0683]

[0684]

[0685] Table 25

[0686]

[0687] Table 26

[0688]

[0689] NI—No inhibition

[0690] Conclusion—CYP3A4 is the major human enzyme responsible for phase I drug metabolism. Inhibition of CYP3A4 may lead to drug toxicity, drug-drug interactions, and other adverse reactions, which are generally undesirable. All compounds have minimal risk of drug-drug interactions.

[0691] Experimental scheme 3

[0692] Plasma protein binding of the lead compound in mouse and human plasma

[0693] step:

[0694] To assess the ability of the compound to bind plasma proteins, plasma protein binding assays were performed using the most common equilibrium dialysis method. The compound was tested in mouse and human plasma at a final concentration of 3 µM. An aliquot of 150 µL of plasma containing the compound was added to the first half (plasma side) well of a 96-well micro equilibrium dialysis apparatus. An aliquot of 150 µL of 100 mM sodium phosphate buffer (pH 7.4) was added to the other half (buffer side) well of the 96-well HT equilibrium dialysis apparatus. The plates containing plasma and buffer were equilibrated at 37 ± 1 °C for 4.5 h with continuous shaking on a track shaker. After the incubation period, samples were taken from each half well. Proteins were precipitated with an organic solvent. The samples were centrifuged, and the supernatant was analyzed by LC-MS / MS.

[0695] in conclusion:

[0696] All compounds exhibited high binding rates in mouse and human plasma, with binding fractions greater than 99.50%. All compounds showed good stability and recovery in plasma. For compounds targeting the brain, in vitro high-protein plasma binding posed the least risk.

[0697] Experimental scheme 4

[0698] In vitro assessment of the lead compound's potential for PXR activation in DPX2 cell plating culture

[0699] Research Design

[0700] 1. Cell Seeding Preparation

[0701] 1) Prepare DPX2 cell culture medium using DPX2 cell culture medium containing 10% FBS.

[0702] 2) Culture DPX2 cells in a T-75 flask at 37°C, 5% CO2, and 95% relative humidity. Once the cells reach 80-90% confluence, isolate and passage them.

[0703] 3) Rinse the cells in the T-75 flask with 5 mL PBS, aspirate dry, add 1.5 mL trypsin, and incubate at 37°C for about 5 minutes or until the cells detach and float. Add excess serum-containing medium to inactivate the trypsin.

[0704] 4) Transfer the cell suspension to a conical tube and centrifuge at 150×g for 5 minutes to pellet the cells. Then centrifuge the cells at 3.2×10⁻⁶. 5 Resuspend cells at a density of 100 cells / mL in seeding medium. Transfer 25 μL to each well of a 384-well cell culture plate. Incubate the plate at 37°C for 24 hours.

[0705] 2. Incubate with the test compound

[0706] 1) Prepare stock solutions of the test compound and the inducer in DMSO. The final concentration of the positive control rifampin is 10 μM. The working concentration of the test compound is set as required. The final concentration of DMSO in the treatment group should be 0.1%.

[0707] 2) Remove the plate from the incubator and add 25 nL of negative control, inducer, or test substance solution to each well, in triplicate. Return the plate to the incubator and incubate for 24 hours.

[0708] 3) Before initiating substrate experiments, check cell morphology and monolayer integrity to ensure that the monolayer has research-acceptable quality.

[0709] 3. Quantitative determination of PXR activation

[0710] 1) After 48 hours of treatment, the culture can be used for quantitative determination of PXR activation.

[0711] 2) CellTiter-Fluor TM The cell viability assay kit and One-Glo luciferase assay system were brought to room temperature. GF-AFC substrate (10 μL) was transferred to an assay buffer container (10 mL) to form the 2X reagent, which was then diluted to the 1X reagent by adding 10 mL of PBS. The ONE-Glo luciferase transfer assay substrate was transferred to the ONE-Glo luciferase assay buffer.

[0712] 3) Remove the plate from the incubator and aspirate the culture medium from each well. Add 1× CellTiter-Fluor... TM Pour the contents of the cell viability assay reagent tube into a sterile culture medium tank. Gently add 25 μL of reagent to each well using a multichannel pipette. Incubate the plate at 37°C for 30 minutes.

[0713] 4) Remove the 384-well plate from the incubator and allow it to cool briefly to ambient temperature. Measure the fluorescence value of each well using a microplate reader in fluorescence mode (excitation wavelength 400 nm, emission wavelength 505 nm).

[0714] 5) Pour the ONE-Glo assay reagent into the culture medium tank, adding 25 μL of reagent to each well. Carefully shake the plate to mix the reagent in the wells. After 5 minutes, read the luminescence value of each well using a luminescence detector.

[0715] 4. Data Analysis

[0716] All calculations were performed using Microsoft Excel.

[0717] Normalized luciferase activity was determined by RLU / RFU, where RLU refers to the relative luminescence units per well in three replicates for each test compound at each dose, and RFU refers to the relative fluorescence units per well in three replicates for each test compound at each dose. The RLU and RFU for the solvent were the average values ​​of the three replicates for the solvent sample.

[0718] The fold activation of mRNA levels is determined by the following formula:

[0719]

[0720] The percentage (%) is determined by the following formula:

[0721] Control percentage (%) = (activation factor) 化合物 / Activation factor 阳性对照 ) * 100

[0722] Vitality (%) is determined by the following formula:

[0723] Vitality (%) = (RFU) 化合物 / RFU DMSOl ) * 100

[0724] Table 27

[0725]

[0726] Conclusion: None of the lead compounds were CYP3A4 inducers.

[0727] Experiment 58:

[0728] Experimental plan:

[0729] Assessing the cardiac risk of lead compounds using hERG measurements

[0730] Table 28

[0731]

[0732] Conclusion—Based on hERG assays, the lead compound is reasonably safe and does not exhibit cardiotoxicity. Safety margin (IC50 > 10 µM); E-4031 is an experimental Class III antiarrhythmic drug that blocks hERG-type potassium channels.

Claims

1. A compound represented by Formula I, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof: (I) in: R1, R2, and R4 are each independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, cycloalkyl, alkenyl, ynyl and -NR'R5; R3 is selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 3-6 Cycloalkyl, alkenyl, alkynyl, NR'R5, and -OR6; R' is selected from H and C. 1-6 alkyl; R5 is selected from hydrogen, C 1-6 Alkyl, cycloalkyl, alkenyl, and ynyl groups containing up to 6 carbon atoms; R6 is selected from C 1-6 Alkyl groups and -L2-A; L2 does not exist or C 1-6 Alkylene; A is selected from aryl and 5- to 14-membered heteroaryl groups, which can optionally be substituted by one or two substituents, each independently selected from cyano, halogen, hydroxyl, and amino groups; and L1 is a optionally substituted polycyclic heterocyclic group.

2. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to claim 1, wherein: R1, R2, and R4 are each independently selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, cycloalkyl, alkenyl, ynyl and -NHR5; R3 is selected from hydrogen, halogen, cyano, and C. 1-6 Alkyl, cycloalkyl containing up to 6 carbon atoms, alkenyl, ynyl, NHR5 and -OR6; -OR6 is C 1-6 alkoxy or -L2-A; L2 is absent or methylene; and A is an aryl group that is optionally substituted with a cyano group.

3. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to claim 1 or 2, wherein R1 is selected from halogens, cyano groups, C... 1-6 Alkyl and C 1-6 Alkyl groups; preferably selected from halogens, cyano groups, and C4 groups. 1-3 Alkyl and C 1-3 Alkyl groups; and more preferably halogens.

4. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any one of claims 1 to 3, wherein R2 is selected from hydrogen and C. 1-6 Alkyl group, preferably hydrogen.

5. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any one of claims 1 to 4, wherein R3 is selected from hydrogen, halogen, C 1-6 Alkyl groups and -OR6; preferably selected from hydrogen, halogens, C 1-3 Alkyl groups and -OR6.

6. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any one of claims 1 to 5, wherein R4 is selected from hydrogen, halogen, cyano, C 1-6 Alkyl and C 1-6 Alkoxy groups; preferably selected from hydrogen, halogens, and C. 1-3 Alkyl and C 1-3 Alkoxy groups; and more preferably selected from hydrogen and C. 1-3 alkyl.

7. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any one of claims 1 to 6, wherein R6 is C 1-6 alkyl.

8. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any one of claims 1 to 6, wherein R6 is L2-A, and A is a C group optionally substituted with one or two substituents each independently selected from cyano and halogen. 6-10 Aryl; preferably, A is a C6 aryl group optionally substituted with a cyano group.

9. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any one of claims 1 to 6 or 8, wherein L2 is a methylene group.

10. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any one of claims 1 to 9, wherein L1 has the formula: in n is 0, 1, 2, 3, 4 or 5; Wavy lines indicate covalent bonds; Two of RA, RB, RC, and RD are linked together to form a group that is optionally formed by one, two, or three independently selected from halogen, hydroxyl, cyano, C 1-6 Alkyl and C 1-6 The alkoxy substituent is a 3- to 8-membered cycloalkyl, cycloalkenyl, or heterocyclic ring; and (i) The other two of RA, RB, RC and RD are each independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 Alkyl and C 1-6 alkoxy, or (ii) Two of RA, RB, RC and RD are not present, and the carbon atoms between the remaining two of RA, RB, RC and RD are connected by double bonds.

11. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any one of claims 1 to 10, wherein L1 is selected from: , , , , , , , , , , , , , , , , , , , , ,and ; Preferred selection , , , ,and ; And more preferably selected from and .

12. The compound according to any one of claims 1 to 11, or its prodrug, hydrate, solvate, or pharmaceutically acceptable salt, wherein the compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , ,or .

13. A method for treating, preventing, or improving a disease, the method comprising administering to a subject a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined in any one of claims 1 to 12.

14. The method of claim 13, wherein the method includes suppressing the PD-1 / PD-L1 checkpoint pathway.

15. The method of claim 13 or 14, wherein the method includes inhibiting the activity of VISTA.

16. The method according to any one of claims 13 to 15, wherein the method comprises one or more of the following: (i) Inhibit the PD-1 / PD-L1 checkpoint pathway; (ii) Inhibit the activity of VISTA; (iii) Reverse T cell exhaustion; (iv) Enhance T cell function; (v) Reduces PD-1 expression in exhausted T cells; (vi) Reduce the expression of TIM3 in exhausted T cells; (vii) Increase the CD8+ T cell population; (viii) Increase CD107a expression; (ix) Increase granzyme B expression; and / or (x) Restore IFNg; (xi) Enhances T cell-mediated cytotoxicity against tumor cells.

17. The method according to any one of claims 13 to 16, wherein the method is a method for treating, improving and / or preventing a condition selected from proliferative diseases, cancer, neurodegenerative diseases or infectious diseases.

18. The method according to any one of claims 13 to 17, wherein the method is a method for treating, improving and / or preventing conditions associated with PD-L1 overexpression, preferably wherein the condition is cancer associated with PD-L1 overexpression.

19. The method according to any one of claims 13 to 18, wherein the method is a method for treating, improving and / or preventing cancers selected from brain cancer, breast cancer, lung cancer, kidney cancer and bladder cancer; preferably breast cancer, brain cancer, thyroid cancer and lung cancer.

20. The method according to any one of claims 13 to 19, wherein the method further comprises administering an additional therapeutic agent to the subject.

21. The method of claim 20, wherein the additional therapeutic agent is selected from nivolumab, pembrolizumab, avelumab, atezolizumab, cimiprimab, and durvalumab.

22. A compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined in any one of claims 1 to 12, for use in the method as defined in any one of claims 13 to 21.

23. Use of a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined in any one of claims 1 to 12 in the preparation of a medicament for use in the method as defined in any one of claims 13 to 21.

24. An in vitro method comprising using a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined in any one of claims 1 to 12 to: (i) Inhibit the PD-1 / PD-L1 checkpoint pathway; (ii) Inhibit the activity of VISTA; (iii) Reverse T cell exhaustion; (iv) Enhance T cell function; (v) Reduces PD-1 expression in exhausted T cells; (vi) Reduce the expression of TIM3 in exhausted T cells; (vii) Increase the CD8+ T cell population; (viii) Increase CD107a expression; (ix) Increase granzyme B expression; and / or (x) Restore IFNg; (xi) Enhances T cell-mediated cytotoxicity against tumor cells.

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