Combination of antibody-drug conjugate and parp1 selective inhibitor and use thereof

Abstract

A pharmaceutical product is provided for administration of an anti-HER2 antibody-drug conjugate in combination with a PARP1 selective inhibitor. The anti-HER2 antibody-drug conjugate is an antibody-drug conjugate in which a drug-linker (where A represents the linking position with the antibody) binds to an anti-HER2 antibody via a thioether bond. A therapeutic use and method are also provided, wherein the combination of the antibody-drug conjugate and the PARP1 selective inhibitor is administered to a subject.

Description

technical field

[0001] The disclosure relates to a medicinal product for administering a specific antibody-drug conjugate in combination with a PARP1 selective inhibitor, wherein the antibody-drug conjugate has an anti-tumor drug bound to an anti-HER2 antibody via a linker structure; And to a therapeutic use and method, wherein the specific antibody-drug conjugate is administered to a subject in combination with a PARP1 selective inhibitor. prior art

[0002] The Poly(ADP-ribose) polymerase (PARP) family of enzymes plays important roles in many cellular metabolic processes, such as replication, recombination, chromatin remodeling, and DNA damage Repair (O'Connor MJ, Mol Cell (2015) 60(4): 547-60). Examples of PARP inhibitors and their mechanisms of action are taught eg in WO2004 / 080976.

[0003] PARP1 and PARP2 are the most extensively studied PARPs for their roles in DNA damage modification. PARP1 is activated by DNA damage cleavage and acts to catalyze the addition of poly(ADP-ribose) (PAR) chains to target proteins. This post-translational modification, known as poly(ADP-ribosylation) (PARylation), mediates the recruitment of additional DNA repair factors to the site of DNA damage. After fulfilling this recruiting role, PARP auto-PARylation triggers the release of bound PARP from the DNA, allowing access to other DNA repair proteins to complete the repair. Thus, the binding of PARP to damaged sites, its catalytic activity, and its eventual release from DNA are all important steps for cancer cells to respond to DNA damage induced by chemotherapeutic drugs and radiation therapy (Bai P. Biology of poly(ADP-ribose) polymerases : the factotums of cell maintenance. Mol Cell 2015 ; 58 : 947–58).

[0004] Inhibition of PARP family enzymes has been exploited as a strategy to selectively kill cancer cells by inactivating complementary DNA repair pathways. Many preclinical and clinical studies have confirmed tumor cells with deleterious changes in BRCA1 or BRCA2, which are key tumor suppressor proteins involved in the repair of double-strand DNA breaks (DSBs) through homologous recombination (HR) Small molecule inhibitors of the PARP family of repair enzymes are selectively sensitive. Such tumors have a defective homologous recombination deficiency (HRR) pathway and are dependent on PARP enzyme function for survival. Although PARP inhibitor therapy primarily targets BRCA-mutated cancers, PARP inhibitors have been clinically tested in non-BRCA-mutated tumors that exhibit homologous recombination deficiency (HRD) (Turner N, Tutt A, Ashworth A. Hallmarks of 'BRCAness' in sporadic cancers. Nat Rev Cancer 2004 ; 4 : 814–9).

[0005] It is believed that PARP inhibitors with improved selectivity for PARP1 may have improved efficacy and reduced toxicity compared to non-selective PARP inhibitors. It is also believed that selective strong inhibition of PARP1 leads to the capture of PARP1 on DNA, leading to DNA double-strand breaks (DSBs) through the collapse of replication forks in S-phase. It is also believed that PARP1-DNA trapping is an effective mechanism to selectively kill tumor cells with HRD.

[0006] Antibody-drug conjugates (ADCs), consisting of cytotoxic drugs conjugated to antibodies, can selectively deliver drugs to cancer cells, and thus are expected to cause drug accumulation and kill cancer cells (Ducry, L ., et al., Bioconjugate Chem. (2010) 21, 5-13;Alley, S. C., et al., Current Opinion in Chemical Biology (2010) 14, 529-537;Damle N. K. Expert Opin. Biol. Ther. ( 2004) 4, 1445-1452;Senter P. D., et al., Nature Biotechnology (2012) 30, 631-637;Burris HA., et al., J. Clin. Oncol. (2011) 29(4):398- 405).

[0007] One such antibody-drug conjugate is trastuzumab deruxtecan, which consists of a HER2-targeting antibody and a derivative of exatecan (Ogitani Y. et al. , Clinical Cancer Research (2016) 22(20), 5097-5108; Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). Trastuzumab delutecan (Enhertu®, DS-8201) has shown significant clinical efficacy in HER2-expressing solid tumors, including breast, gastric, colorectal, and non-small cell lung cancers . Notably, DS-8201 has shown promising activity in HER2-low tumors for the above indications. Combination partners for DS-8201 need to be identified to improve efficacy, increase durability of therapeutic response, improve tolerability in patients, and / or reduce dose-dependent toxicity.

[0008] Despite the therapeutic potential of antibody-drug conjugates (such as trastuzumab delutecan) and PARP1 inhibitors, there have been no published reports of test results showing superior efficacy of combining antibody-drug conjugates with PARP1-selective inhibitors. any literature.

[0009] Accordingly, there remains a need for improved therapeutic compositions and methods that enhance the efficacy of existing cancer therapeutics, increase the durability of therapeutic response, improve tolerability to patients, and / or reduce dose-dependent toxicity. Contents of the invention

[0010] [Summary of Reveal]

[0011] The antibody-drug conjugate used in this disclosure, an anti-HER2 antibody-drug conjugate that includes as a component a derivative of the topoisomerase I inhibitor exixitecan, has been shown to be effective when administered alone. It shows excellent anti-tumor effect in the treatment of certain cancers, such as breast cancer and gastritis. In addition, PARP1 inhibitors have been shown to exhibit anti-tumor effects in the treatment of certain cancers. However, it is desirable to provide a medicament and treatment that can achieve superior anti-tumor effects in the treatment of cancer, such as enhanced efficacy, increased durability of therapeutic response and / or reduced dose-dependent toxicity.

[0012] The present disclosure provides a pharmaceutical product that can exhibit excellent anti-tumor effects in the treatment of cancer by administering an anti-HER2 antibody-drug conjugate combined with a PARP1 selective inhibitor. The disclosure also provides a therapeutic use and method wherein an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor are administered to a subject in combination.

[0013] Specifically, the present disclosure relates to the following [1] to

[54] : [1] A pharmaceutical product comprising an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor for combined administration, wherein the anti-HER2 antibody-drug conjugate is a drug-linker represented by the following formula via Antibody-drug conjugates that combine thioether bonds with anti-HER2 antibodies, Wherein A represents the connection position with the antibody; [2] The pharmaceutical product according to [1], wherein the PARP1 selective inhibitor is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof, in: X 1 and X 2 are each independently selected from N and C(H), X 3 is independently selected from N and C (R 4 ), wherein R 4 is H or fluoro, R 1 is C 1-4 alkyl or C 1-4 fluoroalkyl, R is independently selected from H, halo, C 1-4 alkyl, and C 1-4 fluoroalkyl, and R 3 is H or C 1-4 alkyl, The conditions are: When X 1 is N, then X 2 is C(H), and X 3 is C(R 4), When X 2 is N, then X 1=C(H), and X 3 is C(R 4), and When X 3 is N, then both X 1 and X 2 are C(H); [3] The pharmaceutical product as in [2], wherein in formula (I), R 3 is C 1-4 alkyl; [4] The pharmaceutical product as in [3], wherein in formula (I), R 3 is methyl; [5] The pharmaceutical product according to any one of [2] to [4], wherein in formula (I), R 1 is ethyl; [6] The pharmaceutical product according to [1], wherein the PARP1 selective inhibitor is a compound represented by the following formula (Ia) or a pharmaceutically acceptable salt thereof, in R 1 is C 1-4 alkyl, R 2 is selected from H, halo, C 1-4 alkyl, and C 1-4 fluoroalkyl, R 3 is H or C 1-4 alkyl, and R4 is H; [7] The pharmaceutical product according to [6], wherein in formula (Ia), R 2 is H or a halogen group; [8] The pharmaceutical product according to [6], wherein in formula (Ia), R 1 is ethyl, R 2 is selected from H, chloro and fluoro, and R 3 is methyl; [9] The pharmaceutical product according to [1], wherein the PARP1 selective inhibitor is AZD5305 (also known as AZ14170049) represented by the following formula or a pharmaceutically acceptable salt thereof, ;

[10] The pharmaceutical product according to any one of [1] to [9], wherein the anti-HER2 antibody is an antibody comprising a heavy chain comprising an amino acid represented by SEQ ID NO:3 and a light chain CDRH1 [= amino acid residues 26 to 33 of SEQ ID NO: 1] composed of sequence, CDRH2 [= amino acid of SEQ ID NO: 1 composed of amino acid sequence represented by SEQ ID NO: 4 Residues 51 to 58] and CDRH3 [=amino acid residues 97 to 109 of SEQ ID NO: 1] consisting of the amino acid sequence represented by SEQ ID NO: 5, the light chain comprising SEQ ID NO: CDRL1 consisting of the amino acid sequence represented by 6 [=amino acid residues 27 to 32 of SEQ ID NO:2], an amino group consisting of amino acid residues 1 to 3 of SEQ ID NO:7 CDRL2[=amino acid residues 50 to 52 of SEQ ID NO:2] composed of the acid sequence and CDRL3[=amino acid of SEQ ID NO:2 composed of the amino acid sequence represented by SEQ ID NO:8 Acid residues 89 to 97];

[11] The medicinal product according to any one of [1] to [9], wherein the anti-HER2 antibody is an antibody comprising a heavy chain comprising an amino acid represented by SEQ ID NO:9 and a light chain The heavy chain variable region consisting of the sequence [=amino acid residues 1 to 120 of SEQ ID NO:1], the light chain comprising the light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:10 Region [=amino acid residues 1 to 107 of SEQ ID NO:2];

[12] The pharmaceutical product according to any one of [1] to [9], wherein the anti-HER2 antibody is a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2 Indicates the amino acid sequence consisting of the light chain of the antibody;

[13] The pharmaceutical product according to any one of [1] to [9], wherein the anti-HER2 antibody is a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 11 [=SEQ ID NO: 1 Amino acid residues 1 to 449] and the light chain antibody composed of the amino acid sequence represented by SEQ ID NO:2;

[14] The medicinal product according to any one of [1] to

[13] , wherein the anti-HER2 antibody-drug conjugate is represented by the following formula, Wherein "antibody" represents an anti-HER2 antibody bound to a drug-linker via a thioether bond, and n represents the average number of units of drug-linker bound to each antibody molecule in the antibody-drug conjugate, wherein n is in the range of 7 to 8;

[15] The medical product according to any one of [1] to

[14] , wherein the anti-HER2 antibody-drug conjugate is trastuzumab delutecan (DS-8201);

[16] The pharmaceutical product according to any one of [1] to

[15] , wherein the product is a composition comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for simultaneous administration give;

[17] The pharmaceutical product according to any one of [1] to

[15] , wherein the product is a combination preparation comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for sequential use or concurrently;

[18] The pharmaceutical product according to any one of [1] to

[17] , wherein the product is used for treating cancer;

[19] The medical product according to

[18] , wherein the cancer is at least one selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophagus cancer, head and neck cancer, esophagogastric junction gland Cancer, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, Cervical cancer, squamous cell tumor, peritoneal cancer, liver cancer, hepatocellular carcinoma, uterine body cancer, kidney cancer, vulva cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, multiple Glioblastoma, osteosarcoma, sarcoma, and melanoma;

[20] The medicinal product of

[19] , wherein the cancer is breast cancer;

[21] The medicinal product of

[20] , wherein the breast cancer has a HER2 status score of IHC 3+;

[22] The medical product as in

[20] , wherein the breast cancer is breast cancer with low expression of HER2;

[23] The medicinal product of

[20] , wherein the breast cancer has a HER2 status score of IHC 2+;

[24] The medicinal product of

[20] , wherein the breast cancer has a HER2 status score of IHC 1+;

[25] The medicinal product of

[20] , wherein the breast cancer has a HER2 status score of IHC >0 and <1+;

[26] The pharmaceutical product as in

[20] , wherein the breast cancer is triple-negative breast cancer;

[27] The medicinal product of

[18] , wherein the cancer is gastric cancer;

[28] The medicinal product of

[18] , wherein the cancer is colorectal cancer;

[29] The pharmaceutical product as in

[18] , wherein the cancer is lung cancer;

[30] The pharmaceutical product according to

[29] , wherein the lung cancer is non-small cell lung cancer;

[31] The medicinal product of

[18] , wherein the cancer is pancreatic cancer;

[32] The medicinal product of

[18] , wherein the cancer is ovarian cancer;

[33] The medicinal product of

[18] , wherein the cancer is prostate cancer;

[34] The medicinal product of

[18] , wherein the cancer is kidney cancer;

[35] A medicinal product as defined in any one of [1] to

[17] for use in the treatment of cancer;

[36] The medicinal product for use according to

[25] , wherein the cancer is as defined in any one of

[19] to

[34] ;

[37] A use of an anti-HER2 antibody-drug conjugate or a PARP1 selective inhibitor in the manufacture of a drug, the drug is used for combined administration of an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor to treat cancer, wherein the The anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] ;

[38] The use of

[37] , wherein the cancer is as defined in any one of

[19] to

[34] ;

[39] The use according to

[37] or

[38] , wherein the drug is a composition comprising an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor for simultaneous administration;

[40] The use according to

[37] or

[38] , wherein the drug is a combination preparation comprising an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor for sequential or simultaneous administration;

[41] An anti-HER2 antibody-drug conjugate used in combination with a PARP1 selective inhibitor in the treatment of cancer, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as described in [1] to

[15] as defined in either;

[42] The anti-HER2 antibody-drug conjugate as used in

[41] , wherein the cancer is as defined in any one of

[19] to

[34] ;

[43] The anti-HER2 antibody-drug conjugate for use according to

[41] or

[42] , wherein the use comprises sequentially administering the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor;

[44] The anti-HER2 antibody-drug conjugate for use according to

[41] or

[42] , wherein the use comprises simultaneous administration of the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor;

[45] An anti-HER2 antibody-drug conjugate for use in treating cancer in a subject, wherein the treatment comprises administering to the subject separately, sequentially or simultaneously: i) the anti-HER2 antibody-drug conjugate , and ii) a PARP1 selective inhibitor, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] ;

[46] A PARP1 selective inhibitor used in combination with an anti-HER2 antibody-drug conjugate in the treatment of cancer, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as described in [1] to

[15] as defined in any of ];

[47] The PARP1 selective inhibitor for use as in

[46] , wherein the cancer is as defined in any one of

[19] to

[34] ;

[48] ​​The PARP1 selective inhibitor for use according to

[46] or

[47] , wherein the use comprises sequentially administering an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor;

[49] The PARP1 selective inhibitor for use according to

[46] or

[47] , wherein the use comprises simultaneous administration of an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor;

[50] A PARP1 selective inhibitor for use in treating cancer in a subject, wherein the treatment comprises administering to the subject separately, sequentially or simultaneously: i) the PARP1 selective inhibitor, and ii) Anti-HER2 antibody-drug conjugates, wherein the PARP1 selective inhibitor and the anti-HER2 antibody-drug conjugates are as defined in any one of [1] to

[15] ;

[51] A method of treating cancer, comprising administering in combination an anti-HER2 antibody-drug conjugate as defined in any one of [1] to

[15] and a PARP1 selective inhibitor to a subject in need thereof ;

[52] The method of

[51] , wherein the cancer is as defined in any one of

[19] to

[34] ;

[53] The method according to

[51] or

[52] , wherein the method comprises sequentially administering the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor; and

[54] The method according to

[51] or

[52] , wherein the method comprises administering the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor simultaneously. [Beneficial effects of revealing]

[0014] The present disclosure provides a pharmaceutical product, wherein an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor are administered in combination, and the anti-HER2 antibody-drug conjugate has an anti-tumor drug bound to the anti-HER2 antibody through a linker structure; And a therapeutic use and method, wherein the combination of the specific antibody-drug conjugate and PARP1 selective inhibitor is administered to a subject. In this way, the present disclosure can provide a drug and treatment, which can obtain excellent anti-tumor effect in the treatment of cancer. Brief description of the diagram

[0015] Figure 1 is a diagram showing the amino acid sequence (SEQ ID NO: 1) of the heavy chain of an anti-HER2 antibody. Figure 2 is a diagram showing the amino acid sequence (SEQ ID NO: 2) of the light chain of an anti-HER2 antibody. Figure 3 is a diagram showing the amino acid sequence of the heavy chain CDRH1 (SEQ ID NO:3 [=amino acid residues 26 to 33 of SEQ ID NO:1]). Figure 4 is a diagram showing the amino acid sequence of heavy chain CDRH2 (SEQ ID NO: 4 [= amino acid residues 51 to 58 of SEQ ID NO: 1]). Figure 5 is a diagram showing the amino acid sequence of heavy chain CDRH3 (SEQ ID NO:5 [=amino acid residues 97 to 109 of SEQ ID NO:1]). Fig. 6 is a diagram showing the amino acid sequence of the light chain CDRL1 (SEQ ID NO: 6 [= amino acid residues 27 to 32 of SEQ ID NO: 2]). Fig. 7 is a diagram showing the amino acid sequence (SEQ ID NO: 7 [= amino acid residues 50 to 56 of SEQ ID NO: 2]) including the amino acid sequence of the light chain CDRL2 (SAS). Figure 8 is a diagram showing the amino acid sequence of the light chain CDRL3 (SEQ ID NO:8 [=amino acid residues 89 to 97 of SEQ ID NO:2]). Fig. 9 is a diagram showing the amino acid sequence of the heavy chain variable region (SEQ ID NO: 9 [=amino acid residues 1 to 120 of SEQ ID NO: 1]). Figure 10 is a diagram showing the amino acid sequence of the light chain variable region (SEQ ID NO: 10 [= amino acid residues 1 to 107 of SEQ ID NO: 2]). Figure 11 is a diagram showing the amino acid sequence of the heavy chain ((SEQ ID NO: 11 [=amino acid residues 1 to 449 of SEQ ID NO: 1]). 12A and 12B are graphs showing combination matrices obtained from high-throughput screening combining DS-8201 with AZD5305 (AZ14170049; PARP1 selective inhibitor) in cell lines with high HER2 expression. 13A and 13B are graphs showing combination matrices obtained from high-throughput screening combining DS-8201 and AZD5305 in cell lines with low HER2 expression. Figure 14 is a graph showing combined Emax and Loewe synergy score in cell lines treated with DS-8201 combined with AZD5305. Figures 15A and 15B are graphs showing combination matrices for combining DS-8201 and AZD5305 in cell lines with low or high HER2 expression. 16A and 16B show the X-ray diffraction pattern and representative DSC record curve of Synthesis Example 4 Form A, respectively. Figure 17 is a graph showing tumor volumes treated with DS-8201 or AZD5305 alone or in combination with DS-8201 and AZD5305 in vivo. The dashed line represents the end of the AZD5305 dosing period. 18A , 18B and 18C are diagrams showing the combination matrix obtained by high-throughput screening of the combination of DS-8201 and AZD5305 in NSCLC cell lines with low or high HER2 expression. 19A , 19B and 19C are graphs showing the combination matrix obtained by high-throughput screening of the combination of DS-8201 and AZD5305 in urethral cancer cell lines with HER2-mutant expression. Implementation

[0016] In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0017] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to specific compositions or method steps as such may vary. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural forms unless the context clearly requires otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein.

[0018] In addition, "and / or" as used herein will be considered as specific disclosures of each of two specific features or components, including or excluding the other. Thus, the term "and / or" as used herein in the phrase "A and / or B" is intended to include "A and B", "A or B", "A" alone, and "B" alone. Likewise, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provides the skilled artisan with a general dictionary of many of the terms used in this disclosure.

[0020] Units, prefixes, and symbols are indicated in their form accepted by the International System of Units (Système International de Unites (SI)). Numerical ranges include numbers defining the range.

[0021] It should be understood that where any aspect of an aspect is described herein with the word "comprising", other similar aspects described with the word "consisting of" and / or "consisting essentially of" are also provided.

[0022] The terms "inhibit", "block", and "suppress" are used interchangeably herein and refer to any statistically significant reduction in biological activity, including complete blocking of activity. For example, "inhibiting" can mean reducing biological activity by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Cell proliferation can be assayed using art-recognized techniques that measure the rate of cell division, and / or the proportion of cells within a population of cells undergoing cell division, and / or loss from a population of cells due to terminal differentiation or cell death The rate of the cell (for example, thymidine incorporation (thymidine incorporation)).

[0023] The term "subject" refers to any animal (eg, mammal), including but not limited to humans, non-human primates, rodents, etc., that is the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein in reference to human subjects.

[0024] The term "medicinal product" means a preparation in a form that permits the biological activity of the active ingredients, either as a composition containing all active ingredients (for simultaneous administration) or as a combination of separate compositions (combination preparation) each Contains at least one but not all active ingredients (for sequential or simultaneous administration) and does not contain additional ingredients that are unacceptably toxic to subjects to whom the product will be administered. Such products may be sterile. "Simultaneous administration" means that the active ingredients are administered simultaneously. "Sequential administration" means that the active ingredients are administered one after the other in either order with time intervals between single administrations. The time interval may be, for example, less than 24 hours, preferably less than 6 hours, more preferably less than 2 hours.

[0025] Terms such as "treat" or "treat" or "treat" or "alleviate" or "alleviate" refer to both of the following: therapeutic measures; and (2) prophylactic or preventive measures to prevent and / or slow the development of the targeted pathological condition or disorder. Thus, those in need of treatment include those who already have the disorder; those who are prone to have the disorder; and those who need to prevent the disorder. In certain aspects, a subject's cancer is successfully "treated" according to the methods of the present disclosure if the patient exhibits, for example, a complete, partial, or temporary remission of a certain type of cancer.

[0026] The terms "cancer", "tumor", "cancerous", and "malignant" refer to or describe the physiological condition in mammals that is often characterized by unregulated cell growth. Examples of cancers include, but are not limited to, breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, head and neck cancer, esophagogastric junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine Carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, cervical cancer, squamous cell tumor, peritoneal cancer, liver cancer, hepatocellular carcinoma, uterine body cancer, Kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma. Cancers include hematologic malignancies such as acute myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, and solid tumors Such as breast cancer, lung cancer, neuroblastoma and colorectal cancer.

[0027] As used herein, the term "cytotoxic agent" is broadly defined and refers to a substance that inhibits or prevents cell function and / or causes cell destruction (cell death) and / or exerts anti-tumor / anti-proliferative effects. For example, cytotoxic agents prevent, directly or indirectly, the development, maturation or spread of neoplastic tumor cells. The term also includes such agents that cause only cytostatic effects rather than mere cytotoxic effects. The term includes chemotherapeutic agents as specified below, as well as other HER2 antagonists, antiangiogenic agents, tyrosine kinase inhibitors, protein kinase A inhibitors, cytokine family members, radioisotopes, and toxins (such as bacteria, Enzyme-active toxins of fungal, vegetable or animal origin).

[0028] The term "chemotherapeutic agent" is a subset of the term "cytotoxic agent" and includes natural or synthetic compounds.

[0029] According to the methods or uses of the present disclosure, compounds of the present disclosure can be administered to a patient to promote a positive therapeutic response for cancer. The term "positive treatment response" in reference to cancer treatment refers to an improvement in symptoms associated with the disease. For example, improvement in disease can be characterized as a complete response. The term "complete response" refers to the absence of clinically detectable disease and the normalization of any previous test results. Alternatively, improvement in disease may be classified as a partial response. "Positive therapeutic response" encompasses reduction or inhibition of progression and / or duration of cancer, reduction or improvement in severity of cancer, and / or amelioration of one or more symptoms thereof resulting from administration of a compound of the disclosure. In certain aspects, such terms refer to one, two, or three or more outcomes of administration of a compound of the disclosure: (1) Stabilization, reduction or elimination of cancer cell populations; (2) Stabilization or reduction of cancer growth; (3) Impairment of cancer formation; (4) eradication, removal or control of primary, regional and / or metastatic cancer; (5) reduction in mortality; (6) Freedom from disease, recurrence, progression, and / or increase in overall survival, duration, or rate; (7) Increase in response rate, persistence of response, or number of responding or remission patients; (8) Reduced hospitalization rate; (9) Reduced hospital stay; (10) The size of the cancer is maintained and does not increase or increases by less than 10%, preferably less than 5%, preferably less than 4%, preferably less than 2%; and (11) An increase in the number of patients in remission; (12) The amount of adjuvant therapy (for example, chemotherapy or hormonal therapy) that is otherwise needed to treat cancer is reduced.

[0030] Clinical response can be assessed using screening techniques such as PET, magnetic resonance imaging (MRI) scans, radiography, computed tomography (CT) scans, flow cytometry or fluorescence-activated cell sorter (FACS) analysis, histology , gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, RIA, chromatography, and the like. In addition to such positive treatment responses, treated subjects may experience the beneficial effect of ameliorating symptoms associated with the disease.

[0031] Alkyl groups and groups are linear or branched, for example, C 1-8 alkyl, C 1-6 alkyl, C 1-4 alkyl or C 5-6 alkyl. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl, such as methyl or n-hexyl.

[0032] Fluoroalkyl means that one or more H atoms in the alkyl group are replaced by one or more fluorine atoms, for example, C 1-8 fluoroalkyl, C 1-6 fluoroalkyl, C 1-4 fluoroalkyl or C 5 -6 fluoroalkyl. Examples include fluoromethyl (CH 2F-), difluoromethyl (CHF 2-), trifluoromethyl (CF 3-), 2,2,2-trifluoroethyl (CF 3CH 2-), 1, 1-difluoroethyl (CH 3CHF 2-), 2,2-difluoroethyl (CHF 2CH 2-), and 2-fluoroethyl (CH 2FCH 2-).

[0033] Halo means fluoro, chloro, bromo, and iodo. In a specific embodiment, halo is fluoro or chloro.

[0034] As used herein, the phrase "effective amount" means an amount of a compound or composition sufficient to significantly and positively alter the symptoms and / or conditions being treated (eg, provide a positive clinical response). The effective amount of an active ingredient used in a medicinal product will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the nature of the concomitant therapy, the particular active ingredient used, the particular Pharmaceutically acceptable excipients / carriers, similar factors within the knowledge and expertise of the attending physician. In particular, an effective amount of a compound for treating cancer in combination with an antibody-drug conjugate is such that the combination is sufficient to symptomatically alleviate cancer symptoms, slow cancer progression, or reduce disease progression in a patient with cancer symptoms in a warm-blooded animal such as a human. amount of risk.

[0035] In this specification, unless otherwise stated, as used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for use in Contact with human and animal tissues without undue toxicity, irritation, allergic reactions or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0036] It will be appreciated that compounds of formula (I) may form stable pharmaceutically acceptable acid or base salts, in which case administration of the compound as a salt may be appropriate. Examples of acid addition salts include acetate, adipate, ascorbate, benzoate, benzenesulfonate, bicarbonate, bisulfate, butyrate, camphorate, camphorsulfonate, Choline, citrate, cyclamate, diethylenediamine, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxy Ethyl-sulfonate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxymaleate, lactate, malate, maleate, methanesulfonate salt, meglumine, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate, diphosphate, bitter Salicylate, pivalate, propionate, quinate, salicylate, stearate, succinate, sulfamate, sulfanilate, sulfate , tartrate, tosylate (p-toluenesulfonate), trifluoroacetate and undecanoate. Non-toxic, physiologically acceptable salts are preferred, although other salts may be useful, eg, for isolating or purifying a product.

[0037] Salts may be formed by conventional means, such as by reacting the free base form of the product with one or more equivalents of the appropriate acid, in a solvent or medium in which the salt is insoluble or in a solvent such as water, in vacuo or by Removal by lyophilization, or by exchanging the anion of the existing salt for another anion on a suitable ion exchange resin.

[0038] Compounds of formula (I) may possess more than one chiral center, it being understood that this application encompasses all individual stereoisomers, enantiomers and diastereomers and mixtures thereof. Thus, it is to be understood that to the extent that compounds of formula (I) may exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, this application includes within its definition any such optically active or racemic forms. spin form, which has the above-mentioned activity. This application covers all such stereoisomers which have activity as defined herein.

[0039] Thus, throughout the specification, when referring to compounds of formula (I), it is understood that the term compound includes diastereomers, mixtures of diastereomers, and enantiomers that are PARP1 inhibitors.

[0040] It should also be understood that certain compounds of formula (I) and pharmaceutical salts thereof can exist in solvated as well as unsolvated forms, eg, hydrated and anhydrated forms. It is to be understood that the compounds herein encompass all such solvated forms. For clarity, this includes solvated (eg, hydrate) forms of the free form of the compound, as well as solvated (eg, hydrate) forms of the salt of the compound.

[0041] Some of the compounds of formula (I) may be crystalline and may have more than one crystalline form. It is to be understood that the present disclosure encompasses any crystalline or amorphous form or mixture thereof that has PARP1 selective inhibitory activity. It is well known that crystalline materials can be analyzed using conventional techniques such as X-ray powder diffraction (hereinafter referred to as XRPD) analysis and differential scanning calorimetry (DSC).

[0042] Formula (I) as described herein is intended to encompass all isotopes of its constituent atoms. For example, H (or hydrogen) includes any isotopic form of hydrogen, including 1H, 2H (D), and 3H (T); C includes any isotopic form of carbon, including 12C, 13C, and 14C; O includes any isotopic form of oxygen. N includes any isotopic form of nitrogen, including 13N, 14N, and 15N; F includes any isotopic form of fluorine, including 19F and 18F. In one aspect, the compounds of formula (I) include isotopes of the atoms capped therein in amounts corresponding to their naturally occurring abundance. However, in some cases it may be desirable to enrich one or more atoms in a particular isotope that is normally present in lower abundance. For example, 1H is typically present in greater than 99.98% abundance; however, in one aspect, compounds of any of the formulas presented herein may be enriched in 2H or 3H at one or more positions where H is present. In another aspect, when a compound of any of the formulas provided herein is enriched with radioactive isotopes, such as 3H and 14C, the compound may be useful in drug and / or substrate tissue distribution assays. It is understood that this application encompasses all such isotopic forms. [Description of Specific Embodiments]

[0043] Hereinafter, preferred modes for carrying out the present disclosure are described. The specific embodiments described below are only used to illustrate one example of typical specific embodiments of the present disclosure, and are not intended to limit the scope of the present disclosure.

[0044] [1.] [Antibody] [-] [drug conjugates] The antibody-drug conjugate used in this disclosure is an antibody-drug conjugate in which a drug-linker represented by the following formula is bound to an anti-HER2 antibody via a thioether bond, Wherein A represents the linking position with the antibody.

[0045] In this disclosure, the partial structure composed of the linker and the drug in the antibody-drug conjugate is called "drug-linker". The drug-linker is attached to a thiol group (in other words, cysteine) generated at the interchain disulfide bond site of the antibody (two heavy-heavy chains and two heavy-light chains). Sulfur atoms of residues).

[0046] The drug-linker of the present disclosure includes exxitecan (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12, 15-Hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]ind And[1,2-b]quinoline-10,13-dione (also can be expressed as chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-di Hydrogen-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]ind and[1,2-b]quinoline-10,13(9H,15H)-dione)), which is a topoisomerase I inhibitor. Exitecan is a camptothecin derivative with anti-tumor effect, represented by the following formula: .

[0047] Anti-HER2 antibody-drug conjugates used in the present disclosure can also be represented by the following formula: .

[0048] Here, the drug-linker is bound to an anti-HER2 antibody ("Antibody-") via a thioether bond. The meaning of n is synonymous with the so-called average number of drug molecules bound (DAR; drug-to-antibody ratio), indicating the average number of units of drug-linker bound per antibody molecule.

[0049] After migration into cancer cells, the anti-HER2 antibody-drug conjugates used in the present disclosure are cleaved at the linker position to release the compound represented by the following formula: .

[0050] This compound was presumed to be the original source of the antitumor activity of the antibody-drug conjugates used in this disclosure, and topoisomerase I inhibition has been confirmed (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15; 22 (20):5097-5108, Epub 2016 Mar 29).

[0051] The anti-HER2 antibody-drug conjugates used in this disclosure have a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). The bystander effect is exerted by the process by which the antibody-drug conjugates used in the present disclosure are internalized in target-expressing cancer cells, and the released compound then also exerts an anti-tumor effect on cancer cells present in their surroundings but not expressing the target. Even when the anti-HER2 antibody-drug conjugate is used in combination with the PARP1 selective inhibitor according to the present disclosure, it exerts such bystander effect and exhibits excellent antitumor effect.

[0052] [2.] [Antibody] [-] [Antibodies in Drug Conjugates] [] The anti-HER2 antibody in the antibody-drug conjugate used in the present disclosure can be derived from any species, preferably an anti-HER2 antibody derived from human, rat, mouse, or rabbit. Where the antibody is derived from a species other than human, it is preferably chimerized or humanized using known techniques. The anti-HER2 antibody can be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody.

[0053] The antibody in the antibody-drug conjugate used in the present disclosure is preferably an anti-HER2 antibody that has the characteristics of being able to target cancer cells, and is preferably an antibody that has the following characteristics, for example, the characteristics of recognizing cancer cells, and the ability to interact with cancer cells Properties of binding, properties of internalization into cancer cells and / or cytocidal activity against cancer cells.

[0054] The binding activity of anti-HER2 antibodies to cancer cells can be confirmed using flow cytometric analysis techniques. Antibody internalization into cancer cells can be confirmed using the following: (1) Using a secondary antibody (fluorescently labeled) conjugated to the therapeutic antibody, under a fluorescent microscope, the antibody incorporated into the cell is visualized ( Cell Death and Differentiation (2008) 15, 751-761), (2) using secondary antibodies (fluorescently labeled) combined with therapeutic antibodies to measure the intensity of fluorescence incorporated into cells (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay using an immunotoxin conjugated to a therapeutic antibody which, when incorporated into the cell, releases the toxin to inhibit cell growth (Bio Techniques 28:162-165, January 2000). As an immunotoxin, a recombinant complex protein of the catalytic region of diphtheria toxin and protein G can also be used.

[0055] Anti-tumor activity of anti-HER2 antibodies can be confirmed by measuring anti-cytostatic activity. For example, a cancer cell line cultured to overexpress HER2 overexpressing an antibody-targeted protein was added to the culture system at various concentrations to determine the effects on focus formation, colony formation, and spheroid proliferation. inhibitory activity. This antitumor activity can be confirmed in vivo, for example, by administering the antibody to nude mice transplanted with a cancer cell line highly expressing the target protein, and measuring changes in the cancer cells.

[0056] Since the compound in the anti-HER2 antibody-drug conjugate exerts an anti-tumor effect, it is preferable, but not necessary, that the anti-HER2 antibody itself should have an anti-tumor effect. In order to specifically and selectively exert the cytotoxic activity of anti-tumor compounds on cancer cells, it is important and preferable that anti-HER2 antibodies should have the property of internalization to migrate to cancer cells.

[0057] Anti-HER2 antibodies in the antibody-drug conjugates used in the present disclosure can be obtained by known procedures. For example, antibodies of the present disclosure can be obtained using methods commonly practiced in the art, which involve immunizing animals with antigenic polypeptides and collecting and purifying the antibodies produced in vivo. The source of antibodies is not limited to humans, and animals can be immunized with antigens derived from non-human animals such as mice and rats. In this case, antibodies that bind the obtained heterologous antigen can be tested for cross-reactivity with human antigens to screen for antibodies applicable to human diseases.

[0058] Alternatively, antibody-producing cells that produce antibodies against the antigen are fused with myeloma cells according to methods known in the art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; and Kennet, R. ed. ., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y. (1980)) to establish fusionomas from which monoclonal antibodies can be obtained.

[0059] Antigens can be obtained by genetically engineering host cells to produce genes encoding antigenic proteins. Specifically, a vector allowing expression of an antigen gene is prepared and transferred to a host cell thereby expressing the gene. Antigens so expressed can be purified. Antibodies can also be obtained by immunizing animals with the above-mentioned genetically engineered antigen-expressing cells or antigen-expressing cell lines.

[0060] The anti-HER2 antibody in the antibody-drug conjugate used in this disclosure is preferably a recombinant antibody artificially modified for the purpose of reducing heteroantigenicity to humans, such as a chimeric antibody or a humanized antibody, or preferably only has An antibody derived from the gene sequence of a human antibody, that is, a human antibody. Such antibodies can be produced using known methods.

[0061] As a chimeric antibody, the variable region and constant region of the antibody in the antibody are derived from different species, for example, a chimeric antibody in which the variable region of an antibody derived from a mouse or rat is joined to the constant region of an antibody derived from a human ( Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0062] As humanized antibodies, antibodies obtained by integrating only the complementarity determining regions (CDRs) of heterologous antibodies into antibodies derived from humans can be exemplified (Nature (1986) 321, pp. 522-525), by CDR Grafting method (CDR-grafting method) An antibody obtained by grafting part of the amino acid residues of the framework of a heterologous antibody and the CDR sequence of a heterologous antibody to a human antibody (WO 90 / 07861), and using a gene Humanized antibody by switch mutagenesis strategy (U.S. Patent No. 5821337).

[0063] As the human antibody, an antibody produced by using a human antibody-producing mouse having a human chromosomal fragment including heavy chain and light chain genes of a human antibody (see, Tomizuka, K. et al., Nature Genetics (1997) 16, p.133-143; Kuroiwa, Y. et. al., Nucl. Acids Res. (1998) 26, p.3447-3448; Yoshida, H. et. al., Animal Cell Technology: Basic and Applied Aspects vol.10, p.69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999;Tomizuka, K. et. al., Proc. Natl . Acad. Sci. USA(2000) 97, p.722-727 etc.). As another alternative, antibodies obtained by phage display, selected from human antibody libraries (see, Wormstone, I. M. et. al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), can be exemplified p.2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1(2), p.189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109 (3), p.427-431 etc.).

[0064] Also included in the present disclosure are modified variants of the anti-HER2 antibodies in the antibody-drug conjugates used in the present disclosure. Modified variants refer to variants obtained by chemically or biologically modifying the antibodies according to the present disclosure. Examples of chemically modified variants include variants comprising attachment of chemical moieties to the amino acid backbone, variants comprising attachment of chemical moieties to N-linked or O-linked carbohydrate chains, and the like. Examples of biologically modified variants include post-translational modifications such as N-linked or O-linked glycosylation, N- or C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine); and variants that add methionine residues to the N-terminus by expression in prokaryotic host cells. Furthermore, the meaning of modified variant also includes labels to enable detection or isolation of antibodies or antigens according to the disclosure, such as enzyme-labeled antibodies, fluorescently-labeled antibodies, affinity-labeled antibodies. The modified variant system of the antibody according to the disclosure is useful for improving the stability and blood retention of the antibody, reducing its antigenicity, detecting or isolating the antibody or antigen, and the like.

[0065] Furthermore, according to the present disclosure, by regulating the modification (glycosylation, defucosylation, etc.) of the glycans linked to the antibody, it is possible to enhance the antibody-dependent cytotoxic activity. Techniques for regulating antibody glycan modifications are known from WO99 / 54342, WO00 / 61739, WO02 / 31140, WO2007 / 133855, WO2013 / 120066 and the like. However, the technology is not limited to these. Anti-HER2 antibodies according to the present disclosure also include antibodies wherein glycan modifications are modulated.

[0066] It is known that the lysine residue at the carboxyl terminus of the heavy chain of antibodies produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705:129-134 (1995)), and it is also known that in cultured mammalian cells The two amino acid residues (glycine and lysine) at the carboxy-terminal of the heavy chain of the antibody produced in the antibody were deleted and the relocated proline residue at the carboxy-terminal was amidated (Analytical Biochemistry, 360:75- 83 (2007)). However, such deletion and modification of the heavy chain sequence does not affect the antigen-binding affinity and effector function (effector function) of the antibody (activation of complement, antibody-dependent cytotoxicity, etc.). Therefore, the anti-HER2 antibody according to the present disclosure also includes such modified antibodies and functional fragments of antibodies, and also includes deletion variants in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and deletion variants variants obtained by amidation of the body (for example, a heavy chain in which the carboxy-terminal proline residue has been amidated), etc. The type of deletion variant having a deletion at the carboxyl terminus of the heavy chain of the anti-HER2 antibody according to the present disclosure is not limited to the above-mentioned variants as long as the antigen-binding affinity and effector function are retained. The two heavy chains constituting the antibody according to the present disclosure may be composed of one type selected from the group consisting of full-length heavy chains and the above-mentioned deletion variants, or may be two types selected from a combination thereof. type constitutes. The quantitative ratio of each deletion variant can be affected by the type and culture conditions of the cultured mammalian cells that produce the anti-HER2 antibody according to the present disclosure; One amino acid residue has been deleted.

[0067] The isotype of the anti-HER2 antibody according to the present disclosure may, for example, be IgG (IgG1, IgG2, IgG3, IgG4), preferably IgG1 or IgG2.

[0068] In this disclosure, the term "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (Human Epidermal Growth Factor Receptor Type 2 (Human Epidermal Growth Factor Receptor Type 2); ErbB-2), preferably by binding to HER2 Binds to have internalization activity in cells expressing HER2.

[0069] Examples of anti-HER2 antibodies include trastuzumab (U.S. Patent No. 5821337) and pertuzumab (pertuzumab) (WO01 / 00245), preferably trastuzumab.

[0070] [3.] [Antibody] [-] [Production of drug conjugates] [] The drug-linker intermediate system used in the production of anti-HER2 antibody-drug conjugates according to the present disclosure is represented by the following formula: .

[0071] The drug-linker intermediate can be expressed as the chemical name N-[6-(2,5-dipentoxy-2,5-dihydro-1H-pyrrol-1-yl)hexyl]glycylaminoglycerol Aminoyl-L-phenylpropanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-two sides Oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]ind [1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide, and can refer to WO2014 / 057687, WO2015 / 098099, WO2015 / 115091 , WO2015 / 155998, WO2019 / 044947, etc. are produced.

[0072] The anti-HER2 antibody-drug conjugates used in this disclosure can be produced by reacting the drug-linker intermediate described above with an anti-HER2 antibody having a thiol group (also known as a sulfhydryl group).

[0073] Anti-HER2 antibodies having a sulfhydryl group can be obtained by methods well known in the art (Hermanson, G. T, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, by using 0.3 to 3 molar equivalents of a reducing agent, such as ginseng (2-carboxyethyl)phosphine hydrochloride (TCEP), in accordance with the interchain disulfide bonds of the antibody, with the antibody in a complex containing a chelating agent such as ethylene disulfide The reaction in a buffer solution of ethylene tetraacetic acid (EDTA) can obtain an anti-HER2 antibody with a sulfhydryl group in which the interchain disulfide bond in the antibody is partially or completely reduced.

[0074] Furthermore, by using 2 to 20 molar equivalents of the drug-linker intermediate according to the anti-HER2 antibody having a sulfhydryl group, 2 to 8 drug molecule conjugates in the anti-HER2 antibody-drug conjugate can be produced.

[0075] The average number of drug molecules bound per anti-HER2 antibody molecule of the resulting antibody-drug conjugate can be determined, for example, by based on the UV absorbance of the antibody-drug conjugate and its conjugate precursor at two wavelengths at 280 nm and 370 nm A method based on the calculation of the measurement of the antibody-drug conjugate (UV method), or a method based on the measurement of the fragments obtained by treating the antibody-drug conjugate with a reducing agent (HPLC method).

[0076] The calculation of the average number of drug molecules bound to each antibody molecule of the anti-HER2 antibody and the drug-linker intermediate and the antibody-drug conjugate can be referred to in WO2014 / 057687, WO2015 / 098099, WO2015 / 115091, According to the description of WO2015 / 155998, WO2017 / 002776, WO2018 / 212136, etc.

[0077] In this disclosure, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate, such that the antibody in the antibody-drug conjugate according to the present disclosure is an anti-HER2 antibody.

[0078] The anti-HER2 antibody is preferably an antibody comprising a heavy chain and a light chain, the heavy chain comprising CDRH1 consisting of amino acid residues 26 to 33 of SEQ ID NO: 1, consisting of CDRH2 consisting of the amino acid sequence consisting of amino acid residues 51 to 58 of NO:1, and the amino acid sequence consisting of amino acid residues 97 to 109 of SEQ ID NO:1 CDRH3; the light chain comprises CDRL1 consisting of an amino acid sequence consisting of amino acid residues 27 to 32 of SEQ ID NO:2, consisting of amino acid residues 50 to 32 of SEQ ID NO:2 CDRL2 consisting of an amino acid sequence consisting of 52, and CDRL3 consisting of an amino acid sequence consisting of amino acid residues 89 to 97 of SEQ ID NO:2; preferably comprising a heavy chain and An antibody with a light chain, the heavy chain comprising a heavy chain variable region consisting of amino acid residues 1 to 120 of SEQ ID NO: 1, the light chain comprising a sequence consisting of amino acid residues 1 to 120 of SEQ ID NO: 1 : a light chain variable region consisting of an amino acid sequence consisting of amino acid residues 1 to 107 of 2; even more preferably a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 1 And an antibody comprising a light chain consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and an antibody consisting of an amino acid sequence represented by SEQ ID NO: 2 and consisting of SEQ ID NO : Antibody of the light chain composed of the amino acid sequence consisting of all amino acid residues 1 to 214 of 2.

[0079] The average number of drug-linker units bound to each antibody molecule in the anti-HER2 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, still more preferably 7 to 8, and still more preferably 7.5 to 8, and more preferably about 8.

[0080] The anti-HER2 antibody-drug conjugate used in the present disclosure can be produced with reference to those described in WO2015 / 115091 and the like.

[0081] In a preferred embodiment, the anti-HER2 antibody-drug conjugate is trastuzumab delutecan (DS-8201).

[0082] [4.PARP1] [Selective Inhibitor] In this disclosure, the term "PARP1 selective inhibitor" refers to a PARP inhibitor that exhibits a superior selectivity for PARP1 over other PARP family members (such as PARP2, PARP3, PARP5a, and PARP6), advantageously being more selective for PARP1 than for PARP1. The selectivity for PARP2 is preferably at least 10 times more selective for PARP1 than for PARP2, more preferably at least 100 times more selective for PARP1 than for PARP2. Preferred examples of PARP1 selective inhibitors may include those disclosed herein.

[0083] Examples of PARP1 selective inhibitors that may be used in accordance with the present disclosure include the azaquinolones of the compounds of formula (I). The azaquinolinones of the compounds of formula (I) described herein have surprisingly high selectivity for PARP1 over other PARP family members such as PARP2, PARP3, PARP5a, and PARP6. Advantageously, the compounds of formula (I) described herein have low hERG activity. It is well known that blockade of cardiac ion channels encoded by the human ether-à-gogo-related gene (hERG) is a risk factor for drug discovery and development, while blockade of hERG can lead to safety issues such as cardiac arrhythmia.

[0084] Therefore, in a preferred embodiment of the PARP1 selective inhibitor used in this disclosure, the PARP1 selective inhibitor is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: in: X 1 and X 2 are each independently selected from N and C(H), X 3 is independently selected from N and C (R 4 ), wherein R 4 is H or fluoro, R 1 is C 1-4 alkyl or C 1-4 fluoroalkyl (preferably ethyl), R is independently selected from H, halo, C 1-4 alkyl, and C 1-4 fluoroalkyl, and R 3 is H or C 1-4 alkyl (preferably C 1-4 alkyl, more preferably methyl), The conditions are: When X 1 is N, then X 2 is C(H), and X 3 is C(R 4), When X 2 is N, then X 1=C(H), and X 3 is C(R 4), and When X 3 is N, both X 1 and X 2 are C(H).

[0085] In a specific embodiment, the PARP1 selective inhibitor used in the disclosure is a compound of formula (Ia): in R 1 is C 1-4 alkyl, R 2 is selected from H, halogen, C 1-4 alkyl, and C 1-4 fluoroalkyl (preferably selected from difluoromethyl, trifluoromethyl , and methyl, or H or halo), R 3 is H or C 1-4 alkyl, and R 4 is H. In the compound of formula (Ia), preferably R 1 is ethyl, R 2 is selected from H, chloro and fluoro, R 3 is methyl, and R 4 is H.

[0086] In another specific embodiment, the PARP1 selective inhibitor used in the disclosure is a compound of formula (Ib): in R 1 is C 1-4 alkyl, R 2 is H or halo, and R 3 is H or C 1-4 alkyl. In the compound of formula (Ib), preferably R 1 is ethyl, R 2 is selected from H, chloro and fluoro, and R 3 is methyl.

[0087] In another specific embodiment, the PARP1 selective inhibitor used in the disclosure is a compound of formula (Ic): in R 1 is C 1-4 alkyl or C 1-4 fluoroalkyl, R 2 is independently selected from H, halogen, C 1-4 alkyl, and C 1-4 fluoroalkyl, R 3 is H or C 1-4 alkyl, and R 4 is H or fluoro.

[0088] In another specific embodiment, the PARP1 selective inhibitor is a compound of formula (Ic), wherein: R is independently selected from ethyl, n-propyl, trifluoromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2-fluoroethyl, and 2,2,2- Trifluoroethyl; R is independently selected from H, methyl, ethyl, trifluoromethyl, difluoromethyl, fluoromethyl, fluoro, and chloro; R is H or methyl, and R4 is H.

[0089] In another specific embodiment, the PARP1 selective inhibitor is a compound of formula (I), or a compound of formula (Ia), (Ib) or (Ic), and has a selectivity to PARP1 better than that to PARP2, preferably The selectivity for PARP1 is at least 10 times greater than the selectivity for PARP2, more preferably the selectivity for PARP1 is at least 100 times greater than the selectivity for PARP2.

[0090] In other specific embodiments, the PARP1 selective inhibitor used in the disclosure is a compound selected from the following or a pharmaceutically acceptable salt thereof: 5-[4-[(3-Ethyl-2-oxo-1H-1,6- Pyridin-7-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(3-Ethyl-2-oxo-1H-1,6- Pyridin-7-yl)methyl]piper-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide, 6-Chloro-5-[4-[(3-ethyl-2-oxo-1H-1,6- Pyridin-7-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piper-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide, 6-chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]pyridine-2-carboxamide 6-Ethyl-5-[4-[(2-Ethyl-3-oxo-4H-quinone Lin-6-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(2-Ethyl-3-oxo-4H-quinone Lin-6-yl)methyl]piper-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide, 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo-4H-quinone Lin-6-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(2-Ethyl-3-oxo-4H-quinone Lin-6-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(2-Ethyl-3-oxo-4H-quinone Lin-6-yl)methyl]piper-1-yl]-6-fluoro-N-methyl-pyridine-2-carboxamide, 5-[4-[(2-Ethyl-3-oxo-4H-quinone Lin-6-yl)methyl]piper-1-yl]-N,6-dimethyl-pyridine-2-formamide, [] 6-chloro-5-[4-[(2-ethyl-3-oxo-4H-quinone Lin-6-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, [] N-Methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinone Lin-6-yl]methyl]piperone-1-yl]pyridine-2-formamide, [] 6-Chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinone Lin-6-yl]methyl]piperone-1-yl]pyridine-2-formamide, [] 6-Fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinone Lin-6-yl]methyl]piperone-1-yl]pyridine-2-formamide, [] N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinone Lin-6-yl)methyl]piperone-1-yl]pyridine-2-carboxamide, 6-Chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinone Lin-6-yl)methyl]piperone-1-yl]pyridine-2-carboxamide, 6-fluoro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinone Lin-6-yl)methyl]piperone-1-yl]pyridine-2-formamide, 5-[4-[(2-Ethyl-7-fluoro-3-oxo-4H-quinone Lin-6-yl)methyl]piper-1-yl]-6-fluoro-N-methyl-pyridine-2-carboxamide, 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinone Lin-6-yl]methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinone Lin-6-yl]methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, [] 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinone Lin-6-yl]methyl]piper-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide, [] 5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinone Lin-6-yl]methyl]piper-1-yl]-N-methyl-pyridine-2-carboxamide, [] 6-fluoro-5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinone Lin-6-yl]methyl]piper-1-yl]-N-methyl-pyridine-2-formamide, [] N-Methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinone Lin-6-yl] methyl] piper-1-yl] pyridine-2-carboxamide, and [] 6-fluoro-N-methyl-5-(4-((3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinone (olin-6-yl)methyl)piperol-1-yl)pyridinamide.

[0091] In another specific embodiment, the PARP1 selective inhibitor used in the disclosure is a compound selected from the following or a pharmaceutically acceptable salt thereof: 6-(Difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piper-1-yl]-N-methyl-6(trifluoromethyl)pyridine-2-carboxamide, 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridine-3-yl)methyl]piper-1-yl]-N,6-dimethyl-pyridine-2-carboxamide, and N-ethyl-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]pyridine-2-carboxamide.

[0092] In a preferred embodiment, the PARP1 selective inhibitor used in the present disclosure is the compound AZD5305 represented by the following formula (5-[4-[(7-ethyl-6-oxo-5H-1, 5- Pyridine-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide) or a pharmaceutically acceptable salt thereof: .

[0093] [5.] [Antibody] [-] [drug conjugates with] [PARP1] [Combination of Selective Inhibitors] In the first combination embodiment of the present disclosure, the anti-HER2 antibody-drug conjugate combined with the PARP1 selective inhibitor is an antibody-drug combination in which the drug-linker represented by the following formula is bound to the anti-HER2 antibody through a thioether bond things, Wherein A represents the linking position with the antibody.

[0094] In another combination embodiment, the anti-HER2 antibody-drug conjugate as defined in the above first combination embodiment and the compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof selectively inhibit PARP1 Agents are combined: in: X 1 and X 2 are each independently selected from N and C(H), X 3 is independently selected from N and C (R 4 ), wherein R 4 is H or fluoro, R 1 is C 1-4 alkyl or C 1-4 fluoroalkyl, R is independently selected from H, halo, C 1-4 alkyl, and C 1-4 fluoroalkyl, and R 3 is H or C 1-4 alkyl, The conditions are: When X 1 is N, then X 2 is C(H), and X 3 is C(R 4), When X 2 is N, then X 1=C(H), and X 3 is C(R 4), and When X 3 is N, both X 1 and X 2 are C(H).

[0095] In another combination embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with the PARP1 selective inhibitor as defined above, wherein in formula (I), R 3 is C 1-4 alkyl.

[0096] In another combination embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with the PARP1 selective inhibitor as defined above, wherein in formula (I), R 3 is methyl.

[0097] In another combination embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with the PARP1 selective inhibitor as defined above, wherein in formula (I), R 1 is ethyl.

[0098] In another combination specific embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor, and the PARP1 selective inhibitor is a compound represented by the following formula (Ia) or a pharmaceutically acceptable salt thereof : in R 1 is C 1-4 alkyl, R 2 is selected from H, halo, C 1-4 alkyl, and C 1-4 fluoroalkyl, R 3 is H or C 1-4 alkyl, and R4 is H.

[0099] In another combination embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with the PARP1 selective inhibitor as defined above, wherein in formula (Ia), R 2 is H or a halogen group.

[0100] In another combination embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with the PARP1 selective inhibitor as defined above, wherein in formula (Ia), R 1 is ethyl, and R 2 is selected from H, Chloro and fluoro, and R3 is methyl.

[0101] In another combination embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor, wherein the PARP1 selective inhibitor is AZD5305 represented by the following formula or a pharmaceutically acceptable salt thereof: .

[0102] In the specific embodiment of each of the above-mentioned combination specific embodiments, the anti-HER2 antibody comprises a heavy chain and a light chain, and the heavy chain comprises CDRH1 composed of the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: CDRH2 composed of the amino acid sequence represented by SEQ ID NO: 4 and CDRH3 composed of the amino acid sequence represented by SEQ ID NO: 5, the light chain comprising CDRL1 composed of the amino acid sequence represented by SEQ ID NO: 6 , CDRL2 consisting of the amino acid sequence consisting of amino acid residues 1 to 3 of SEQ ID NO:7, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:8. In another embodiment of each of the above combination embodiments, the anti-HER2 antibody comprises a heavy chain and a light chain, and the heavy chain comprises a variable heavy chain consisting of the amino acid sequence represented by SEQ ID NO:9. region, the light chain comprises a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:10. In another embodiment of each of the above combination embodiments, the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and the amino acid represented by SEQ ID NO: 2 The light chain composed of the sequence. In another embodiment of each of the above combination embodiments, the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 11 and the amino acid represented by SEQ ID NO: 2 The light chain composed of the sequence.

[0103] In a particularly preferred embodiment of the combination disclosed in the present disclosure, the anti-HER2 antibody-drug conjugate is trastuzumab delutecan (DS-8201) and the PARP1 selective inhibitor is a compound represented by the following formula: , which is also identified as AZD5305.

[0104] [6.] [Combined uses and methods of treatment] Described below are pharmaceutical products and therapeutic uses and methods wherein an anti-HER2 antibody-drug conjugate according to the present disclosure and a PARP1 selective inhibitor are administered in combination.

[0105] The pharmaceutical products and therapeutic uses and methods of the present disclosure may be characterized in that the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are included as active ingredients in different formulations and administered at the same time or at different times; or The antibody-drug conjugate and PARP1 selective inhibitor may be contained and administered in a single formulation as active ingredients.

[0106] In the disclosed pharmaceutical products and methods of treatment, a single PARP1 selective inhibitor used in the present disclosure may be administered in combination with an anti-HER2 antibody-drug conjugate, or two or more different PARP1 selective inhibitors may be combined with Antibody-drug conjugates are administered in combination.

[0107] The pharmaceutical products and methods of treatment of the present disclosure can be used to treat cancer, and preferably can be used to treat at least one cancer selected from the group consisting of: breast cancer (including triple negative breast cancer, and luminal breast cancer) ), gastric cancer (also known as gastric adenocarcinoma), colorectal cancer (also known as colorectal cancer, including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, head and neck cancer (including salivary gland cancer and pharyngeal cancer), gastroesophageal junction adenocarcinoma, biliary tract cancer (including bile duct cancer), Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer Carcinoma, gastrointestinal stromal tumor, cervical cancer, squamous cell tumor, peritoneal cancer, liver cancer, hepatocellular carcinoma, uterine body cancer, kidney cancer, vulva cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma , plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma, and may preferably be used for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer , colorectal cancer, lung cancer (preferably non-small cell lung cancer), pancreatic cancer, ovarian cancer, prostate cancer, and kidney cancer.

[0108] The presence or absence of the HER2 tumor marker can be determined, for example, by collecting tumor tissue from cancer patients to prepare formalin-fixed paraffin-embedded (FFPE) specimens and subjecting the specimens to detection of gene products (proteins). Assays, for example, immunohistochemistry (IHC), flow cytometry, or Western blot (Western blot), or assays for gene transcription, for example, in situ hybridization (ISH), quantitative PCR (q- PCR), or microarray analysis, or assays by collecting cell-free circulating tumor DNA (ctDNA) from cancer patients and subjecting the ctDNA to methods such as next-generation sequencing (NGS).

[0109] The pharmaceutical products and methods of treatment of the present disclosure may be used in HER2 expressing cancers, which may be HER2 overexpressing cancers (high or intermediate) or may be HER2 low expressing cancers.

[0110] In this disclosure, the term "cancer overexpressing HER2" is not particularly limited, as long as it is recognized as a cancer overexpressing HER2 by those skilled in the art to which the present invention belongs. Preferred examples of cancers that overexpress HER2 may include cancers with a HER2 expression score of 3+ in the IHC method, and cancers with a HER2 expression score of 2+ in the IHC method and HER2 expression in in situ hybridization (ISH) Cancers that were judged positive. The in situ hybridization methods disclosed herein include fluorescence in situ hybridization (FISH) and dual-color in situ hybridization (DISH).

[0111] In this disclosure, the term "cancer with low expression of HER2" is not particularly limited, as long as it is recognized as a cancer with low expression of HER2 by those skilled in the art to which the present invention belongs. Preferable examples of cancers with low HER2 expression may include cancers with a HER2 expression score of 2+ in the IHC method and judged to be HER2 negative in the in situ hybridization method, and a HER2 expression score of 1 in the IHC method + cancer.

[0112] The method of assessing the degree of HER2 expression by IHC method, or the method of determining whether HER2 expression is positive or negative by in situ hybridization method is not particularly limited, as long as it is recognized by those with ordinary knowledge in the technical field of the present invention. Examples of this method may include those described in the 4th edition of the HER2 Detection Guidelines, Breast Cancer (developed by the Japanese Pathology Board for Optimal Use of HER2 for Breast Cancer).

[0113] Cancer, particularly with regard to the treatment of breast cancer, may be HER2 overexpressing (high or intermediate) or low expressing breast cancer, or triple negative breast cancer, and / or may have a HER2 status score of IHC 3+, IHC 2+, IHC 1+ or IHC >0 and <1+.

[0114] The pharmaceutical products and methods of treatment of the present disclosure are preferably used in mammals, but more preferably in humans.

[0115] The anti-tumor effect of the pharmaceutical products and treatment methods disclosed herein can be established by transplanting cancer cells into test animals to prepare models and measuring the effect of reducing tumor volume or prolonging lifespan by applying the pharmaceutical products and treatment methods disclosed herein confirmed. Moreover, the effect of the combined use of the antibody-drug conjugate used in the present disclosure and the PARP1 selective inhibitor can be compared by administering the antibody-drug conjugate used in the present disclosure or the PARP1 selective inhibitor alone. Antitumor effect was confirmed.

[0116] The anti-tumor effect of the pharmaceutical products and treatment methods disclosed herein can be confirmed in clinical trials using any of the following evaluation methods: by Response Evaluation Criteria in Solid Tumors (RECIST) evaluation method, WHO evaluation method , Macdonald evaluation method, weight measurement, and other methods; and can be judged based on the following indicators: complete response (Complete response; CR), partial response (Partial response; PR), progressive disease (Progressive disease; PD), objective response Rate (Objective Response Rate; ORR), duration of response (Duration of response; DoR), progression-free survival (Progression-Free Survival; PFS), overall survival (Overall Survival; OS), etc.

[0117] By using the above method, it is possible to confirm the superiority of the anti-tumor effect of the pharmaceutical product and treatment method of the present disclosure over existing pharmaceutical products and treatment methods for cancer treatment.

[0118] The pharmaceutical products and treatment methods disclosed herein can delay the development of cancer cells, inhibit their growth, and further kill cancer cells. These effects can save cancer patients from symptoms caused by cancer or achieve improvement in the quality of life (QOL) of cancer patients and achieve therapeutic effects by maintaining the life of cancer patients. Even if the pharmaceutical products and treatment methods disclosed herein fail to kill cancer cells, they can still achieve higher QOL and longer-term survival of cancer patients by inhibiting or controlling the growth of cancer cells.

[0119] The medical product of the present disclosure can be expected to exert a therapeutic effect by performing systemic treatment on a patient and by locally applying it to cancer tissue.

[0120] In another aspect, the pharmaceutical products and methods of treatment of the present disclosure provide for use as adjuvants in cancer treatment using ionizing radiation or other chemotherapeutic agents. For example, in the treatment of cancer, the treatment may comprise simultaneous or sequential administration of ionizing radiation or other chemotherapeutic agents to a subject in need of treatment with a therapeutically effective amount of a medicinal product.

[0121] The pharmaceutical products and methods of treatment of the present disclosure can be used as adjuvant chemotherapy in combination with surgery. The pharmaceutical products of the present disclosure can be administered before surgery for the purpose of reducing tumor size (known as preoperative adjuvant chemotherapy or neoadjuvant therapy), or can be used after surgery to prevent tumor recurrence Administered for purpose (called postoperative adjuvant chemotherapy, or adjuvant therapy).

[0122] In another aspect, the pharmaceutical products of the present disclosure can be used to treat cancer that lacks Homologous Recombination (HR)-dependent DNA DSB repair activity. The HR-dependent DNA DSB repair pathway repairs double-strand breaks (DSBs) in DNA via a homology mechanism to reform a continuous DNA helix (K.K. Khanna and S.P. Jackson, Nat. Genet. 27(3): 247-254 (2001)). Components of the HR-dependent DNA DSB repair pathway include, but are not limited to, ATM (NM_000051), RAD51 (NM_002875), RAD51L1 (NM_002877), RAD51C (NM_002876), RAD51L3 (NM_002878), DMC1 (NM_007068), XRCC2 (NM _005431), XRCC3 (NM_005432), RAD52 (NM_002879), RAD54L (NM_003579), RAD54B (NM_012415), BRCA1 (NM_007295), BRCA2 (NM_000059), RAD50 (NM_005732), MRE11A (NM_00559 0) and NBS1 (NM_002485). Other proteins involved in the HR-dependent DNA DSB repair pathway include regulators such as EMSY (Hughes-Davies, et al., Cell, [115,] pp523-535). The HR component is also described in Wood, et al., Science, [291,] 1284-1289 (2001). Cancers deficient in HR-dependent DNA DSB repair may comprise or consist of one or more cancer cells that, relative to normal cells, have a reduced or eliminated ability to repair DNA DSBs by this pathway , that is, the activity of the HR-dependent DNA DSB repair pathway may be reduced or eliminated in one or more cancer cells. The activity of one or more components of the HR-dependent DNA DSB repair pathway can be abolished in one or more cancer cells in an individual with a HR-dependent DNA DSB repair-deficient cancer. Components of the HR-dependent DNA DSB repair pathway are well characterized in the art (see, e.g., Wood, et al., Science, [291,] 1284-1289 (2001)) and includes the components listed above.

[0123] In some embodiments, the cancer cells may have BRCA1 and / or BRCA2 deficient phenotype (phenotype), that is, the activity of BRCA1 and / or BRCA2 in the cancer cells is reduced or disappeared. Cancer cells with this phenotype may lack BRCA1 and / or BRCA2, i.e. the expression and / or activity of BRCA1 and / or BRCA2 in cancer cells may be reduced or eliminated, for example by means of mutations or pleomorphisms in encoding nucleic acids , or by means of amplification, mutation, or polymorphism of genes encoding regulators, such as the EMSY gene encoding BRCA2 regulators (Hughes-Davies, et al., Cell, [115,] 523-535). BRCA1 and BRCA2 are known tumor suppressors, and their wild-type alleles are often lost in tumors of heterozygous carriers (Jasin M., Oncogene, [21(58),] 8981-93 (2002);Tutt, et al., Trends Mol Med., [8 (12),] 571-6, (2002)). The association of BRCA1 and / or BRCA2 mutations with breast cancer is well characterized in the art (Radice, P.J., Exp Clin Cancer Res., [21(3 Suppl),] 9-12 (2002)). Amplification of the EMSY gene, which encodes a BRCA2-binding factor, has also been linked to breast and ovarian cancer. Carriers of BRCA1 and / or BRCA2 mutations are also at increased risk of certain cancers, including breast, ovarian, pancreatic, prostate, blood, gastrointestinal, and lung cancers. In some embodiments, the individual is heterozygous for one or more variations, such as mutations and polymorphisms, in BRCA1 and / or BRCA2 or regulators thereof. Detection of BRCA1 and BRCA2 variants is well known in the art and described, for example, in EP 699 754, EP 705 903, Neuhausen, S.L. and Ostrander, E.A., Genet. Test, [1,]75-83 (1992); Chappnis, P.O. and Foulkes, W.O., Cancer Treat Res, [107,] 29-59 (2002); Janatova M., et al., Neoplasma, 50(4), 246-505 (2003); Jancarkova, N., Ceska Gynekol., [68{1),]11-6 (2003)). The assay for the amplification of the BRCA2-binding factor EMSY is described in Hughes-Davies, et al., Cell, [115,] 523-535).

[0124] Cancer-associated mutations and polymorphisms can be detected at the nucleic acid level by detecting the presence of variant nucleic acid sequences, or at the protein level by detecting the presence of variant (i.e. mutant or allelic variant) polypeptides on detection.

[0125] The pharmaceutical products of the present disclosure can be administered in the presence of at least one pharmaceutically suitable ingredient. Depending on the dosage, administration concentration, etc. of the antibody-drug conjugate and PARP1 selective inhibitor used in the present disclosure, pharmaceutically suitable components can be appropriately selected and applied from formulation additives generally used in the art. Anti-HER2 antibody-drug conjugates used in the present disclosure can be used, for example, as pharmaceuticals containing buffers such as histidine buffers, vehicles such as sucrose and trehalose, and surfactants such as polysorbate 80 and 20. Product delivery. Pharmaceutical preparations containing the antibody-drug conjugates used in this disclosure can be preferably used as injections, more preferably as aqueous injections or lyophilized injections, and even more preferably as lyophilized injections. In the case that the medicinal product containing the anti-HER2 antibody-drug conjugate used in the present disclosure is an aqueous injection, the aqueous injection can preferably be diluted with a suitable diluent, and then administered as an intravenous infusion. Examples of diluents include glucose solution, physiological saline, etc., preferably glucose solution, more preferably 5% glucose solution. In the case where the pharmaceutical product disclosed herein is a freeze-dried injection, the required amount of the freeze-dried injection pre-dissolved in water for injection can be preferably diluted with a suitable diluent and administered as an intravenous infusion. Examples of diluents include glucose solution and physiological saline, preferably glucose solution, more preferably 5% glucose solution.

[0126] Examples of administration routes applicable to administering the pharmaceutical products of the present disclosure may include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes, preferably intravenous routes.

[0127] The anti-HER2 antibody-drug conjugates used in this disclosure can be administered to humans at intervals of 1 to 180 days, preferably at intervals of one week, two weeks, three weeks or four weeks, more preferably at intervals of three weeks give. Anti-HER2 antibody-drug conjugates used in the present disclosure may be administered at a dose of about 0.001 to 100 mg / kg per administration, and may preferably be administered at a dose of 0.8 to 12.4 mg / kg per administration . For example, the anti-HER2 antibody-drug conjugate can be dosed at 0.8 mg / kg, 1.6 mg / kg, 3.2 mg / kg, 5.4 mg / kg, 6.4 mg / kg, 7.4 mg / kg, or 8 mg / kg every three It is administered once a week, and can preferably be administered once every three weeks at a dose of 5.4 mg / kg or 6.4 mg / kg.

[0128] PARP1 selective inhibitors can be administered in suitable dosages by any suitable route of administration. The size of the dose required for the therapeutic treatment of a particular disease state will necessarily vary depending upon the subject being treated, the route of administration and the severity of the disease being treated. For more information on routes of administration and dosage regimens, reference can be made to Comprehensive Medicinal Chemistry, Volume 5, Chapter 25.3 (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0129] The compound of formula (I) or a pharmaceutically acceptable salt thereof is usually administered orally in the form of a pharmaceutical preparation comprising the active ingredient or a pharmaceutically acceptable salt or solvate thereof in a pharmaceutically acceptable dosage form , or solvates of such salts. The compositions may be administered in varying dosages depending on the disorder and patient to be treated.

[0130] Pharmaceutical formulations of the compounds of formula (I) above may be prepared for oral administration, especially in the form of tablets or capsules, and are particularly concerned with technologies aimed at providing colon-targeted drug release (Patel, M. M. Expert Opin. Drug Deliv. . 2011, 8 (10), 1247-1258).

[0131] Pharmaceutical formulations of compounds of formula (I) above may be conveniently administered in unit dosage form and may be prepared by any methods well known in the art of pharmacy, for example, as in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985).

[0132] Pharmaceutical formulations of compounds of formula (I) suitable for oral administration may comprise one or more physiologically compatible carriers and / or excipients excipients and may be in solid or liquid form. Tablets and capsules can be prepared with binders, fillers, lubricants and / or surfactants such as sodium lauryl sulfate. Liquid compositions may contain conventional additives such as suspending agents, emulsifying agents and / or preservatives. Liquid compositions can be enclosed in, for example, gelatin to provide unit dosage forms. Solid oral dosage forms include lozenges, two-piece hard shell capsules and soft elastic gelatin (SEG) capsules. Such two-piece hard shell capsules can be prepared, for example, by filling gelatin or hydroxypropylmethylcellulose (HPMC) shells with a compound of formula (I).

[0133] Dry shell formulations of compounds of formula (I) generally comprise gelatin at a concentration of about 40% to 60% w / w, plasticizers (such as glycerin, sorbitol or propylene glycol) at a concentration of about 20% to 30%, and about 30% to 30% 40% concentration of water. Other materials such as preservatives, dyes, opacifiers and flavorings may also be present. Liquid fill materials include: solid drug that has been dissolved, dissolved, or dispersed (using a suspending agent such as beeswax, hydrogenated castor oil, or polyethylene glycol 4000) or liquid drug in a vehicle or combination of vehicles such as Mineral oil, vegetable oil, triglycerides, glycols, polyols and surfactants.

[0134] A suitable daily dosage of a compound of formula (I) or a pharmaceutically acceptable salt thereof in human therapy is about 0.0001-100 mg / kg body weight. Oral formulations are preferred, especially tablets or capsules, which may be formulated by methods known to those skilled in the art to provide dosages of active compound in the range of 0.1 mg to 1000 mg. [Example]

[0135] The present disclosure is specifically described in view of the examples shown below. However, the present disclosure is not limited to these examples. Furthermore, it must not be interpreted in a restrictive manner.

[0136] [PARP1] [Synthesis Example of Selective Inhibitor] The synthesis examples 1 to 32 described below of the PARP1 selective inhibitor are as examples 1 to 32 of WO2021 / 013735.

[0137] General Experimental Conditions [] Unless otherwise stated, 1H NMR spectra were obtained at 27°C using a Bruker 300 MHz, 400 MHz, or 500 MHz spectrometer; chemical shifts are expressed in parts per million (ppm, δ units) and are referred to the residual mono-1H of the solvent Isotopologue (CHCl 3: 7.24 ppm; CHDCl 2: 5.32 ppm; CD 3S(=O)CD 2H: 2.49 ppm). Coupling constants are given in units of Hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br s (broad singlet) . LC-MS was performed using a Waters UPLC equipped with a Waters SQD mass spectrometer or a Shimadzu LC-20AD LC-20XR LC-30AD equipped with a Shimadzu 2020 mass spectrometer. Unless otherwise stated, reported molecular ions correspond to [M+H]+; for molecules with multiple isotopic patterns (Br, Cl, etc.), reported values ​​are at the lowest isotopic mass obtained unless otherwise stated value.

[0138] Flash chromatography was performed using the following methods: straight phase flash chromatography (straight phase flash chromatography) on SP1 TMPurification system from Biotage™, CombiFlash®Rf from ISCO or on Gilson system from Thermo Fisher using normal phase two Silica FLASH+ TM (40M, 25M or 12 M) or SNAP TMKP-Sil Cartridges (340, 100, 50 or 10), Flash Column silica-CS columns from Agela, C18-flash columns or standard flash chromatography . In general, all solvents used were commercially available and of analytical grade. Anhydrous solvents are routinely used for the reactions. The phase separator (Phase Separator) used in these examples is an ISOLUTE® phase separator column. The following named intermediates and examples were named using ACD / Name 12.01 from Advanced Chemistry Development, Inc. (ACD / Labs). Starting materials were obtained from commercial sources or prepared via literature methods.

[0139] X-ray Powder Diffraction (XRPD) Analysis XRPD analysis was performed using a Bruker D8 diffractometer, commercially available from Bruker AXS Inc™ (Madison, Wisconsin). XRPD spectra were obtained by loading a sample of the material for analysis (approximately 10 mg) on ​​a single silicon crystal wafer holder (e.g., Bruker Silicon Zero Background X-ray Diffraction Sample Holder) and loading it with the aid of a microscope. The slide spreads the sample into a thin layer. The sample was spun at 30 revolutions per minute (to improve counting statistics) and measured at X with a wavelength of 1.5406 Angstroms (i.e., about 1.54 Angstroms) generated by a copper elongated focus tube operating at 40 kV and 40 mA. radiation exposure. Expose the sample for 1 s per 0.02 degree 2-theta increment (continuous scan mode) over the range from 5 degrees to 40 degrees 2-theta in theta-theta mode. D8 has a run time of ~15 min. XRPD 2Θ values ​​may vary within reasonable limits, such as within ±0.2°, and XRPD intensities may vary when measuring substantially identical crystalline forms for a variety of reasons including, for example, preferred orientation. The principles of XRPD are described in publications, for example, Giacovazzo, C. et al. (1995), Fundamentals of Crystallography, Oxford University Press; Jenkins, R. and Snyder, R. L. (1996), Introduction to X-Ray Powder Diffractometry , John Wiley & Sons, New York; and Klug, H. P. & Alexander, L. E. (1974), X-ray Diffraction Procedures, John Wiley and Sons, New York.

[0140] DSC analysis For samples prepared according to standard methods, DSC analysis was performed using a Q SERIES™ Q1000 DSC calorimeter available from TA INSTRUMENTS® (New Castle, Delaware). Samples (approximately 2 mg) were weighed into aluminum sample pans and transferred to the DSC. The instrument was purged with nitrogen at 50 mL / min and data was collected between 22°C and 300°C using a dynamic heating rate of 10°C / min. Thermal data were analyzed using standard software (eg, Universal v.4.5A from TA INSTRUMENTS®).

[0141] The following abbreviations are used: AcOH=acetic acid; aq=aqueous; BAST=bis(2-methoxyethyl)aminosulfur trifluoride; Boc2O=di-tert-butyl dicarbonate; Boc=tertiary butoxy Carbonyl; CDCl 3=deuterochloroform; CD 3OD=deuteromethanol; CH 3NO 2=nitromethane; DCE=1,2-dichloroethane; DCM=dichloromethane; DEA=diethylamine; DEAD=even Diethyl azodicarboxylate; Dess–Martin periodinane [=]1,1,1-Phen(acetyloxy)-1,1-dihydro-1,2-phenyliodo-3-(1 H)-one; DIPEA= N,N-diiso Propylethylamine; DMAP=2,6-dimethylaminopyridine; DMF= N,N-dimethylformamide; DMSO=dimethylsulfoxide; DMSO-d 6=deuterated dimethyl Pyridine; DPPA=diphenylphosphoryl azide; dppf=1,1'-bis(diphenylphosphino)ferrocene; DIAD=di-isopropyl(E)-diazene-1,2- Dicarboxylate; DSC=differential scanning calorimetry; DTAD=di-tertiary butyl(E)-diazene-1,2-dicarboxylate; ee=excess of enantiomer; eq.= Equivalent; ESI = electrospray ionization; Et2O = diethyl ether; EtOAc or EA = ethyl acetate; EtOH = ethanol; FA = formic acid; Grubbs (Grubbs) catalyst (1,3-two 2,4,6-tri Tolyl imidazolin-2-ylidene)(tricyclohexylphosphine)ruthenium dichloride; h=hour; HATU=(dimethylamino)-N,N-dimethyl(3-oxide-1 H- [1,2,3]triazolo[4,5-b]pyridyl)methaneimine cation hexafluorophosphate; HCl=hydrochloric acid; H2O2=hydrogen peroxide; HP=high pressure; IPA=iso Propanol; LC = liquid chromatography; LiClO 4 = lithium perchlorate; mmol = millimolar; mCPBA = m-chloroperbenzoic acid; MeOH = methanol; min = minutes; MeCN or CH3CN = acetonitrile; MeNO 2 = Nitromethane; MS=mass spectrometry; NMP=N-methyl-2-pyrrolidone; NMR=nuclear magnetic resonance; Pd / C=palladium on carbon; ); PdCl 2(dppf)=1,1'-bis(di-tertiary butylphosphino)ferrocene palladium dichloride; PE=petroleum ether; PPh 3=triphenylphosphine; rt=room temperature; Rt or RT=retention time; Ruphos Pd G3=methanesulfonic acid (2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2′- Amino-1,1′biphenyl)]palladium(II); sat=saturated; SFC=supercritical fluid chromatography; T3P=2,4,6-tripropyl-1,3,5,2 , 4,6-trioxatriphosphinane 2,4,6-trioxide (2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide ); TBTU=2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate; TFA = trifluoroacetic acid; THF=tetrahydrofuran; TLC=thin layer chromatography; TMS=trimethylsilyl; Xantphos=4,5-bis(diphenylphosphino)-9,9-dimethyl; CBr4= Carbon tetrabromide; HCl=hydrochloric acid; HBr=hydrobromic acid; Cs2CO3=cesium carbonate; MgSO4=magnesium sulfate; NaHCO3=sodium bicarbonate; DDQ=2,3-dichloro-5,6-dicyano- 1,4-Benzoquinone; SOCl2=thionyl chloride; DIBAL-H=diisobutylaluminum hydride; NH4HCO3=ammonium bicarbonate; BINAP=2,2′-bis(diphenylphosphino)-1,1 '-binaphthyl.

[0142] [Synthesis of starting materials and intermediates] []

[0143] [ , intermediate , ] [ , 2 , ] [ , : , ] 7-Bromo-3-ethyl-1H-1,6- pyridin-2-one Add butyryl chloride (0.143 mL, 1.37 mmol) dropwise to 4-amino-6-bromo-pyridine-3-carbaldehyde ( [intermediate] [1], 250 mg, 1.24 mmol), DIPEA (1.086 mL, 6.22 mmol) and DMAP (30.4 mg, 0.25 mmol) in a stirred solution of CH 2 Cl 2 (5 mL). The resulting solution was stirred at rt for 4 h. Over 2 eq of butyryl chloride was added and the reaction was continued for another 24 h. The reaction was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to provide crude product. 1.5 mL of MeOH was added and the solid (product) was filtered off, washed with 1 mL of MeOH to afford 7-bromo-3-ethyl-1H-1,6- Pyridine-2-one ( [intermediate] [2], 167 mg, 53.1%). 1H NMR (DMSO-d6) 1.17 (3H, t), 2.45 - 2.50 (2H, m, overlapping with the solvent DMSO peak), 7.35 (1H, s), 7.82 (1H, s), 8.63 (1H, s), 12.09 (1H, br s); m / z (ES +) [M+H] +=252.

[0144] [ , intermediate , ] [ , 3 , ] [ , : , ] 3-ethyl-7-vinyl-1H-1,6- pyridin-2-one Add PdCl2(dppf) (37.6 mg, 0.05 mmol) to 7-bromo-3-ethyl-1H-1,6- Pyridine-2-one ( [intermediate] [2], 130 mg, 0.51 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (0.105 mL, 0.62 mmol) and K 2CO 3 (213 mg, 1.54 mmol) in 1,4-di alkane (4 mL) / water (1.333 mL), and the resulting mixture was stirred at 90°C for 1 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate and concentrated to provide crude product. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 20% MeOH in DCM. The product fractions were concentrated to dryness under reduced pressure to give 3-ethyl-7-vinyl-1H-1,6- Pyridine-2-one ( [intermediate] [3], 93 mg, 90 %). 1H NMR (DMSO-d6) 1.18 (3H, t), 2.53 (2H, m, overlapping with solvent DMSO peak), 5.49 (1H, dd), 6.27 (1H, dd), 6.84 (1H, dd), 7.15 ( 1H, s), 7.81 (1H, s), 8.78 (1H, s), 12.00 (1H, br s) ; m / z (ES +) [M+H] +=201.

[0145] [ , intermediate , ] [ , 4 , ] : 3-ethyl-2-oxo-1H-1,6- pyridine-7-carbaldehyde Osmium tetroxide (0.024 mL, 3.00 µmol) in HO was added to 3-ethyl-7-vinyl-1H-1,6- Pyridine-2-one ( [intermediate] [3], 30 mg, 0.15 mmol), 2,6-lutidine (0.035 mL, 0.30 mmol) and sodium periodate (128 mg, 0.60 mmol) in THF (1 mL) / water (0.200 mL) solution and stirred overnight at rt. The reaction was diluted with water and extracted with ethyl acetate, and the filtrate was concentrated to dryness. The resulting residue was purified by flash silica chromatography with a gradient of 0 to 15% MeOH in DCM. The product fractions were concentrated under reduced pressure to give 3-ethyl-2-oxo-1H-1,6- Pyridine-7-carbaldehyde ( [intermediate] [4], 24.00 mg, 79 %). 1H NMR (DMSO-d6) 1.20 (3H, t), 2.55 - 2.62 (2H, m, overlapping with the solvent DMSO peak), 7.73 (1H, s), 7.95 (1H, s), 9.03 (1H, s), 10.00 (1H, s), 12.32 (1H, br s); m / z (ES +) [M+H] +=203.

[0146] [ , intermediate , ] [ , 5 , ] [ , : , ] 3-ethyl-7-(hydroxymethyl)-1H-1,6- pyridin-2-one [ , , ] Slowly add sodium borohydride (61.4 mg, 1.62 mmol) to 3-ethyl-2-oxo-1H-1,6- Pyridine-7-carbaldehyde ( [intermediate] [4], 82 mg, 0.41 mmol) in methanol (2 mL), and the resulting mixture was stirred at room temperature for 1 h. Methanol was removed under vacuum and the resulting residue was purified by flash silica chromatography with a gradient of 0 to 35% MeOH in DCM. The product fractions were concentrated under reduced pressure to afford 3-ethyl-7-(hydroxymethyl)-1H-1,6- Pyridine-2-one ( [intermediate] [5], 68.0 mg, 82%). [] 1H NMR (500MHz, DMSO-d6) 1.18 (3H, t), 2.52 - 2.55 (2H, m, overlapping with the solvent DMSO peak), 4.59 (2H, br s), 5.52 (1H, br s), 7.33 (1H , s), 7.80 (1H, s), 8.71 (1H, s), 12.01 (1H, br s) ; m / z (ES +) [M+H] +=205.

[0147] [ , intermediate , ] [ , 6 , ] : 7-(bromomethyl)-3-ethyl-1H-1,6- pyridin-2-one Add CBr4 (928 mg, 2.80 mmol) to 3-ethyl-7-(hydroxymethyl)-1H-1,6- Pyridine-2-one ( [intermediate] [5], 381 mg, 1.87 mmol) and triphenylphosphine (734 mg, 2.80 mmol) in CH2Cl2 (18.656 ml) were stirred and the resulting solution was stirred at 0°C for 2 hours. The reaction was concentrated and the resulting residue was purified by flash silica chromatography with a gradient of 0 to 15% MeOH in DCM. The product fractions were concentrated under reduced pressure to give 7-(bromomethyl)-3-ethyl-1H-1,6- as a white solid. Pyridine-2-one ( [intermediate] [6], 386 mg, 77%) (contains triphenylphosphine oxide, which is difficult to separate). This compound was carried on to the next step without further purification. m / z (ES +) [M] +=267.

[0148] [ , Synthesis example , ] [ , 1 , ] : 5-[4-[(3-Ethyl-2-oxo-1H-1,6- Pyridin-7-yl)methyl]piperidine-1-yl]-N-methyl-pyridine-2-formamide [ , , ]

[0149] Add DIPEA (0.059 mL, 0.34 mmol) to 7-(bromomethyl)-3-ethyl-1H-1,6- Pyridine-2-one ( [intermediate] [6], 30 mg, 0.11 mmol) and N-methyl-5-piperone-1-yl-pyridine-2-formamide 2HCl ( [intermediate]

[13] , 42.8 mg, 0.15 mmol) in a stirred solution of acetonitrile (1 mL). The resulting solution was stirred at 70°C for 2 hours. The solvent was removed under vacuum and the resulting crude material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 0 to 90% acetonitrile in water, 0.1% NH4OH as additive). The product fractions were concentrated to dryness under reduced pressure to give 5-[4-[(3-ethyl-2-oxo-1H-1,6- Pyridine-7-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example] [1], 23.60 mg, 51.7%). 1H NMR (500MHz, DMSO-d6) 1.18 (3H, br t), 2.54 (2H, m, overlapping with solvent DMSO peak), 2.67 (4H, br s), 2.79 (3H, br d), 3.38 (4H, br s), 3.75 (2H, br s), 7.34 (1H, s), 7.42 (1H, br dd), 7.77 - 7.88 (2H, m), 8.29 (1H, br d), 8.40 (1H, br d ), 8.75 (1H, s), 11.60 - 12.11 (1H, m); m / z (ES +) [M+H] +=407.

[0150] [ , Synthesis example , ] [ , 2 , ] [ , : , ] 5-[4-[(3-ethyl-2-oxo-1H-1,6- Pyridin-7-yl)methyl]piperidine-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide

[0151] Add DIPEA (0.082 mL, 0.47 mmol) to 7-(bromomethyl)-3-ethyl-1H-1,6- Pyridine-2-one ( [intermediate] [6], 25 mg, 0.09 mmol) and 6-fluoro-N-methyl-5-piperone-1-yl-pyridine-2-carboxamide, HCl ( [intermediate]

[23] , 28.3 mg, 0.10 mmol) in a stirred solution of acetonitrile (2 mL). The resulting solution was stirred at 70°C for 2 hours. Solvent was removed under vacuum. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 20% MeOH in DCM. The product fractions were concentrated under reduced pressure to afford 5-[4-[(3-ethyl-2-oxo-1H-1,6- Pyridine-7-yl)methyl]piperone-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide ( [synthesis example] [2], 17.00 mg, 42.8%). 1H NMR (500MHz, DMSO-d6) 1.18 (3H, t), 2.52 - 2.55 (2H, m, overlapping with the solvent DMSO peak), 2.64 (4H, br s), 2.77 (3H, d), 3.20 (4H, br s), 3.70 (2H, s), 7.32 (1H, s), 7.59 (1H, dd), 7.80 (1H, s), 7.86 (1H, d), 8.31 - 8.49 (1H, m), 8.73 ( 1H, s), 11.93 (1H, br s); m / z (ES +) [M+H] +=425.

[0152] [ , Synthesis example , ] [ , 3 , ] : 6-chloro-5-[4-[(3-ethyl-2-oxo-1H-1,6- Pyridin-7-yl)methyl]piperidine-1-yl]-N-methyl-pyridine-2-formamide

[0153] Add DIPEA (0.082 mL, 0.47 mmol) to 7-(bromomethyl)-3-ethyl-1H-1,6- Pyridine-2-one ( [intermediate] [6], 25 mg, 0.09 mmol) and 6-chloro-N-methyl-5-piperone-1-yl-pyridine-2-carboxamide, 2HCl( [intermediate]

[47] , 33.7 mg, 0.10 mmol) in acetonitrile (2 mL) and the resulting solution was stirred at 70°C for 2 hours. Solvent was removed under vacuum. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 20% MeOH in DCM. The product fractions were concentrated under reduced pressure to afford 6-chloro-5-[4-[(3-ethyl-2-oxo-1H-1,6- Pyridine-7-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example] [3], 19.20 mg, 46.5%). 1H NMR (500MHz, DMSO-d6) 1.18 (3H, t), 2.53 (2H, m, overlapping with the solvent DMSO peak), 2.66 (4H, br s), 2.80 (3H, d), 3.15 (4H, br s ), 3.72 (2H, s), 7.33 (1H, s), 7.68 (1H, d), 7.81 (1H, s), 7.95 (1H, d), 8.43 (1H, br d), 8.74 (1H, s ), 11.93 (1H, s); m / z (ES +) [M+H] +=441.

[0154]

[0155] [ , intermediate , ] [ , 8 , ] : ethyl 6-formyl-5-nitro-pyridine-3-carboxylate Stir 6-methyl-5-nitro-pyridine-3-carboxylic acid ethyl ester at 110°C ( [intermediate] [7], 10 g, 47.58 mmol) and selenium dioxide (7.92 g, 71.36 mmol) in 1,4-bis A mixture of alkanes (50 mL) for 20 h. The reaction mixture was cooled to room temperature, filtered through a pad of celite and the celite was washed with ethyl acetate. The combined filtrates were concentrated and the resulting residue was purified by flash silica chromatography with a gradient of 0% to 70% ethyl acetate in hexanes. The product fractions were concentrated under reduced pressure to afford ethyl 6-formyl-5-nitro-pyridine-3-carboxylate as a brown oil ( [intermediate] [8], 9.70 g, 91 %). 1H NMR (500 MHz, chloroform-d) 1.48 (3H, t), 4.54 (2H, q), 8.81 (1H, d), 9.51 (1H, d), 10.32 (1H, s); m / z (ES +) [M] +=224.

[0156] [ , intermediate , ] [ , 9 , ] : ethyl 6-[(E)-2-ethoxycarbonylbut-1-enyl]-5-nitro-pyridine-3-carboxylate (mixture of E / Z isomers) To a stirred solution of sodium hydride (9.63 g, 240.89 mmol) (60% in mineral oil) in anhydrous THF (100 mL) was added 2-(diethoxyphosphate) dropwise at 0 °C with an addition funnel Ethyl butyrate (60.8 g, 240.89 mmol) gave a gray mixture. The resulting mixture was stirred at 0°C for 10 minutes and allowed to warm to room temperature over 10 minutes and stirred at 40°C for 5 minutes. The reaction mixture was cooled to -78°C, and ethyl 6-formyl-5-nitro-pyridine-3-carboxylate ( [intermediate] [8], 22.5 g, 100.37 mmol) in 100 ml THF solution. The mixture was quenched with saturated NH 4 Cl solution, extracted with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated to provide crude product. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 50% ethyl acetate in hexane. The product fractions were concentrated under reduced pressure to give ethyl 6-[(E)-2-ethoxycarbonylbut-1-enyl]-5-nitro-pyridine-3-carboxylate as a yellow oil ( [intermediate] [9], 24.30 g, 75 %) (1:1 mixture of E / Z isomers). 1H NMR (500 MHz, chloroform-d) 1.13 (3H, t), 1.18 (3H, t), 1.23 (3H, t), 1.37 (3H, t), 1.45 (6H, q), 2.57 (2H, qd ), 2.66 (2H, q), 4.11 - 4.24 (2H, m), 4.32 (2H, q), 4.45 - 4.56 (4H, m), 7.08 (1H, s), 7.85 (1H, s), 8.86 ( 2H, dd), 9.26 (1H, d), 9.43 (1H, d); m / z (ES +) [M] +=322.

[0157] [ , intermediate , ] [ , 10 , ] : 7-ethyl-6-oxo-7,8-dihydro-5H-1,5- Ethyl pyridine-3-carboxylate 6-[(E)-2-Ethoxycarbonylbut-1-enyl]-5-nitro-pyridine-3-carboxylic acid ethyl ester (1:1 mixture of E / Z isomers) ( [intermediate] [9], 3.75 g, 11.63 mmol), Pd / C (1.857 g, 1.75 mmol) (10%) in ethanol (30 mL) mixture degassed, filled with H2 (balloon), and at room temperature under H2 gas environment The reaction was stirred overnight. The mixture was filtered through a bed of celite and the bed was washed with ethanol. After concentration, add in two 4M HCl in alkanes (15 ml) was added to the resulting residue and the mixture was stirred at room temperature for 30 minutes. The mixture was diluted with ether and the solid was filtered off, washed with diethyl ether and dried under vacuum to give 7-ethyl-6-oxo-7,8-dihydro-5H-1,5- as a white solid. Ethyl pyridine-3-carboxylate ( [intermediate]

[10] , 2.260 g, 78%). 1H NMR (500 MHz, DMSO-d6) 0.94 (3H, t), 1.33 (3H, t), 1.41 - 1.51 (1H, m), 1.69 - 1.81 (1H, m), 2.41 - 2.48 (1H, m) , 2.94 (1H, dd), 3.20 (1H, dd), 4.35 (2H, t), 7.67 (1H, d), 8.61 (1H, d), 10.32 (1H, s); m / z (ES +) [M+H] +=249.

[0158] [ , intermediate , ] [ , 11 , ] : 7-ethyl-6-oxo-5H-1,5- Ethyl pyridine-3-carboxylate 7-Ethyl-6-oxo-7,8-dihydro-5H-1,5- Ethyl pyridine-3-carboxylate ( [intermediate]

[10] , 2.26 g, 9.10 mmol) dissolved in 1,4-bis In alkane (40 mL), DDQ (2.273 g, 10.01 mmol) was added and the mixture was stirred at reflux for 3 h. The solvent was removed under reduced pressure, saturated NaHCO 3 solution was added and the residue was stirred at room temperature for 1 hr. The solid was filtered off, washed with water and then with 10 ml of diethyl ether. The resulting solid was dried under vacuum to give 7-ethyl-6-oxo-5H-1,5- Ethyl pyridine-3-carboxylate ( [intermediate]

[11] , 1.738 g, 78 %). 1H NMR (500 MHz, DMSO-d6) 1.14 - 1.28 (3H, m), 1.35 (3H, t), 2.58 (2H, q), 4.38 (2H, q), 7.83 (1H, s), 8.17 (1H , s), 8.90 (1H, s), 12.05 (1H, s); m / z (ES +) [M+H] +=247.

[0159] [ , intermediate , ] [ , 12 , ] : 3-ethyl-7-(hydroxymethyl)-1H-1,5- pyridin-2-one Lithium aluminum hydride (2 M in THF (29.2 mL, 58.47 mmol)) was added dropwise to 7-ethyl-6-oxo-5H in THF (150 mL) at 0 °C during 45 min under nitrogen -1,5- Ethyl pyridine-3-carboxylate ( [intermediate]

[11] , 7.2 g, 29.24 mmol). The resulting mixture was stirred at 0°C for 1.5 hours. The reaction mixture was quenched by the dropwise addition of 1 M aq HCl (29 mL). The reaction mixture was concentrated and the solid was diluted with water (-150 ml) and 29 ml of 1M HCl solution to give a yellow suspension. The solid was collected by filtration, washed with water, diethyl ether, and dried to give the crude product as a yellow solid (contaminated with some inorganic salts). This solid was suspended in a mixture of methanol and DCM (2:1 ) (400 ml) and heated to reflux. The solid was filtered off. This solid was resuspended in methanol / DCM mixture and this procedure was repeated 5 times to remove most of the product from this mixture. The combined filtrates were then concentrated until about 100 ml and the solid was collected by filtration, washed with diethyl ether, and dried under vacuum to yield 3-ethyl-7-(hydroxymethyl)-1H-1,5- Pyridine-2-one ( [intermediate]

[12] , 4.35 g, 72.8 %). 1H NMR (500 MHz, DMSO-d6) 1.18 (3H, t), 2.52 - 2.56 (2H, m), 4.61 (2H, d), 5.44 (1H, t), 7.61 (1H, s), 7.74 (1H , s), 8.37 (1H, s), 11.87 (1H, br s); m / z (ES+) [M+H]+=205.3.

[0160] [ , Synthesis example , ] [ , 4 , ] [ , : , ] 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperidine-1-yl]-N-methyl-pyridine-2-formamide

[0161] Add thionyl chloride (6.41 mL, 88.14 mmol) dropwise to 3-ethyl-7-(hydroxymethyl)-1,5- Pyridine-2(1H)-one ( [intermediate]

[12] , 3 g, 14.69 mmol) and N,N-dimethylformamide (0.114 mL, 1.47 mmol) were suspended in CH 2 Cl 2 (60 mL) and the resulting solution was stirred at room temperature for 6 hours. The mixture was concentrated to dryness to give crude 7-(chloromethyl)-3-ethyl-1H-1,5- Pyridine-2-one ( [intermediate]

[17] ). Add DIPEA (12.83 mL, 73.45 mmol) to 7-(chloromethyl)-3-ethyl-1H-1,5- Pyridine-2-one ( [intermediate]

[17] , crude product from above), potassium iodide (0.488 g, 2.94 mmol) and N-methyl-5-piper-1-yl-pyridine-2-formamide, 2HCl ( [intermediate]

[13] [,] 4.31 g, 14.69 mmol) in a stirred solution of acetonitrile (50.00 mL). The resulting solution was stirred at 80°C for 2 hours. Solvent was removed under vacuum. The crude material was diluted with water, basified with aqueous NaHCO3 and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to provide crude product. The resulting residue was purified by flash silica chromatography with a gradient of 0 to 15% MeOH in DCM. The product fractions were concentrated under reduced pressure to afford 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridine-3-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example] [4], 3.93 g, 65.8 %). 1H NMR (500MHz, DMSO-d6) 1.19 (3H, t), 2.53 - 2.59 (6H, m), 2.79 (3H, d), 3.33 - 3.39 (4H, m), 3.66 (2H, s), 7.39 ( 1H, dd), 7.64 (1H, s), 7.76 (1H, s), 7.83 (1H, d), 8.27 (1H, d), 8.36 - 8.40 (1H, m), 8.41 (1H, d), 11.85 (1H, s); m / z (ES +) [M] +=406.

[0162]

[0163] [ , intermediate , ] [ , 14 , ] : 7-(bromomethyl)-3-ethyl-1H-1,5- pyridin-2-one Add CBr4 (219 mg, 0.66 mmol) to 3-ethyl-7-(hydroxymethyl)-1H-1,5- Pyridine-2-one ( [intermediate]

[12] , 90 mg, 0.44 mmol) and triphenylphosphine (173 mg, 0.66 mmol) in a stirred solution of CH2Cl2 (4 mL). The resulting solution was stirred at 0°C for 2 hours. The reaction was concentrated in vacuo and the resulting residue was purified by flash silica chromatography with a gradient of 0 to 15% MeOH in DCM. The product fractions were concentrated under reduced pressure to give 7-(bromomethyl)-3-ethyl-1H-1,5- Pyridine-2-one ( [intermediate]

[14] , 84 mg, 71.4%) (contains triphenylphosphine oxide, which is difficult to separate). This compound was carried on to the next step without further purification. m / z (ES +) [M] +=267.

[0164] [ , Synthesis example , ] [ , 5 , ] : 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperidine-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide

[0165] Add DIPEA (0.082 mL, 0.47 mmol) to 7-(bromomethyl)-3-ethyl-1H-1,5- Pyridine-2-one ( [intermediate]

[14] , 25 mg, 0.09 mmol) and 6-fluoro-N-methyl-5-piperone-1-yl-pyridine-2-carboxamide, 2HCl( [intermediate]

[23] , 32.0 mg, 0.10 mmol) in a stirred solution of acetonitrile (2 mL). The resulting solution was stirred at 70°C for 2 hours. Solvent was removed under vacuum. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 20% MeOH in DCM. The product fractions were concentrated under reduced pressure to give 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridine-3-yl)methyl]piperone-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide ( [synthesis example] [5], 13.00 mg, 33%). 1H NMR (500MHz, DMSO-d6) 1.19 (3H, t), 2.55 (2H, m, overlapping with solvent DMSO peak), 2.58 (4H, br d), 2.77 (3H, d), 3.19 (4H, br s ), 3.67 (2H, s), 7.57 (1H, dd), 7.63 (1H, s), 7.76 (1H, s), 7.85 (1H, d), 8.32 - 8.49 (2H, m), 11.85 (1H, s);m / z (ES +) [M+H] +=425.

[0166] [ , Synthesis example , ] [ , 6 , ] : 6-chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperidin-1-yl]-N-methyl-pyridine-2-formamide

[0167] Add DIPEA (0.082 mL, 0.47 mmol) to 7-(bromomethyl)-3-ethyl-1H-1,5- Pyridine-2-one ( [intermediate]

[14] , 25 mg, 0.09 mmol) and 6-chloro-N-methyl-5-piperone-1-yl-pyridine-2-carboxamide ( [intermediate]

[48] , 26.2 mg, 0.10 mmol) in a stirred solution of acetonitrile (2 mL). The resulting solution was stirred at 70°C for 2 hours. Solvent was removed under vacuum. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 20% MeOH in DCM. The product fractions were concentrated under reduced pressure to afford 6-chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridine-3-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example] [6], 19.80 mg, 48.0%). 1H NMR (500MHz, DMSO-d6) 1.19 (3H, t), 2.55 (2H, m, overlapping with solvent DMSO peak), 2.58 - 2.65 (4H, m), 2.79 (3H, d), 3.13 (4H, br s), 3.68 (2H, s), 7.63 (1H, d), 7.67 (1H, d), 7.76 (1H, s), 7.94 (1H, d), 8.34 - 8.50 (2H, m), 11.85 (1H , s); m / z (ES +) [M+H] +=441.

[0168]

[0169] [ , intermediate , ] [ , 16 , ] : methyl 5-piperone-1-ylpyridine-2-carboxylate will be in two HCl (4.67 mL, 18.67 mmol) in alkanes was added to tertiary butyl 4-(6-methoxycarbonyl-3-pyridyl)piperoxo-1-carboxylate ( [intermediate]

[15] , 600 mg, 1.87 mmol) in MeOH (1 mL) was stirred and the resulting solution was stirred at rt for 18 hours. The solvent was removed under vacuum to give methyl 5-piperazol-1-ylpyridine-2-carboxylate, 2HCl ( [intermediate]

[16] , 543 mg, 99 %). 1H NMR (500 MHz, DMSO-d6) 3.20 (4H, br s), 3.71 (4H, br s), 3.85 (3H, s), 7.58 (1H, br d), 7.99 (1H, br d), 8.43 (1H, br s), 9.73 (2H, br), 11.29 - 11.75 (1H, br) ; m / z (ES +) [M+H] +=222.

[0170] [ , intermediate , ] [ , 18 , ] [ , : , ] 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl) methyl] piperidine-1-yl] pyridine-2-carboxylic acid methyl ester Add DIPEA (944 µl, 5.40 mmol) to 7-(chloromethyl)-3-ethyl-1H-1,5- Pyridine-2-one, HCl ( [intermediate]

[17] , 200 mg, 0.77 mmol), sodium iodide (11.57 mg, 0.08 mmol) and methyl 5-piperone-1-ylpyridine-2-carboxylate, 2HCl ( [intermediate]

[16] , 250 mg, 0.85 mmol) in a stirred solution of acetonitrile (6774 µl). The resulting solution was stirred at 80°C for 3 hours. The solvent was removed under vacuum, 0.4 mL of saturated sodium bicarbonate solution and 1.5 mL of acetonitrile were added and the reaction was stirred for 10 min. The solid was filtered off and washed with 2 mL of water followed by 1 mL of acetonitrile to give 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridine-3-yl)methyl]piperone-1-yl]pyridine-2-carboxylic acid methyl ester ( [intermediate]

[18] , 158 mg, 50.2%). 1H NMR (500MHz, DMSO-d6) 1.19 (3H, br t), 2.54 - 2.61 (6H, m), 3.40 (4H, br s), 3.66 (2H, s), 3.81 (3H, s), 7.35 ( 1H, br dd), 7.62 (1H, s), 7.75 (1H, s), 7.88 (1H, br d), 8.28 - 8.47 (2H, m), 12.03 (1H, br); m / z (ES + ) [M+H] +=408.

[0171] [ , Synthesis example , ] [ , 7 , ] : 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperidin-1-yl]pyridine-2-formamide

[0172] Add ammonia (in methanol) (4 mL, 28.00 mmol) to 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridine-3-yl)methyl]piperone-1-yl]pyridine-2-carboxylic acid methyl ester ( [intermediate]

[18] , 60 mg, 0.15 mmol) and the resulting solution was heated to 50°C for 24 h (sealed tube). The reaction was cooled to room temperature, the solid was filtered off and washed with 2 mL of methanol to obtain 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]pyridine-2-formamide ( [synthesis example] [7], 88 mg, 90 %). 1H NMR (500MHz, DMSO-d6) 1.19 (3H, t), 2.56 (6H, m, overlapping with solvent DMSO peak), 3.35 (4H, br d), 3.66 (2H, s), 7.30 (1H, br s ), 7.40 (1H, dd), 7.64 (1H, s), 7.76 (2H, s), 7.85 (1H, d), 8.28 (1H, d), 8.41 (1H, d), 11.61 - 11.98 (1H, m) ;m / z (ES +) [M+H] +=393.

[0173]

[0174] [ , intermediate , ] [ , 20 , ] [ , : , ] methyl 5-bromo-6-fluoro-pyridine-2-carboxylate An oven-dried flask was charged with methyl 5-bromopyridine-2-carboxylate ( [intermediate]

[19] , 6 g, 27.77 mmol). Silver(II) fluoride (14.18 g, 97.21 mmol) was added and the mixture was stirred at room temperature overnight. The reaction mixture was filtered through filter paper and washed with DCM. The filtrate was concentrated to give a light brown solid. The residue was suspended in a mixture of DCM and sat. NH 4 Cl solution and the white suspension was filtered off. The organic layer was separated, and the aqueous layer was extracted with DCM (100 ml x 2). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated. The resulting residue was purified by flash silica chromatography with a gradient of 0 to 25% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give methyl 5-bromo-6-fluoro-pyridine-2-carboxylate ( [intermediate]

[20] , 5.98g yield 90%). 1H NMR (500 MHz, chloroform-d) 4.01 (3H, s), 7.93 (1H, d), 8.15 (1H, t); m / z (ES +) [M] +=234.

[0175] [ , intermediate , ] [ , 21 , ] [ , : , ] tertiary butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperone-1-carboxylate Stir tertiary butyl piper-1-carboxylate (13.11 g, 70.41 mmol), methyl 5-bromo-6-fluoro-pyridine-2-carboxylate ( [intermediate]

[20] , 10.985 g, 46.94 mmol), RuphosPd-G3 (2.5 g, 2.99 mmol) and Cs 2CO 3 (38 g, 116.63 mmol) in 1,4-di The mixture in alkanes (200 mL) overnight. The mixture was diluted with water and ethyl acetate, and the layers were separated. The aqueous layer was extracted with DCM (100 ml x 2). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 100% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to afford tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperoxa-1-carboxylate as a yellow solid ( [intermediate]

[21] ,14.00 g,88 %);1H NMR (500 MHz, chloroform-d) 1.51 (9H, s), 3.16 - 3.32 (4H, m), 3.58 - 3.72 (4H, m), 3.98 (3H, s), 7.29 - 7.34 (1H, m), 8.00 (1H, d); m / z (ES +) [M+H] +=340.

[0176] [ , intermediate , ] [ , 22 , ] [ , : , ] 4-[2-Fluoro-6-(methylcarbamoyl)-3-pyridyl]piperone-1-carboxylate tertiary butyl 4-(2-Fluoro-6-methoxycarbonyl-3-pyridyl)piperone-1-carboxylic acid in methylamine (120 mL, 36.80 mmol, 33 wt% in ethanol) was stirred at r.t. Butyl ester ( [intermediate]

[21] , 12.49 g, 36.80 mmol) for 24 hours (sealed tube). Solvent was removed under reduced pressure. The residue was dissolved in DCM and filtered through a bed of silica gel, washing with ethyl acetate. The filtrate was concentrated and dried under vacuum to give tert-butyl 4-[2-fluoro-6-(methylaminoformyl)-3-pyridyl]piperoxa-1-carboxylate as a yellow solid ( [intermediate]

[22] , 12.45 g, 100%). 1H NMR (500 MHz, DMSO-d6) 1.42 (9H, s), 2.77 (3H, d), 3.04 - 3.16 (4H, m), 3.43 - 3.56 (4H, m), 7.59 (1H, dd), 7.80 - 7.93 (1H, m), 8.41 (1H, q); m / z (ES +) [M+H] +=340.

[0177] [ , intermediate , ] [ , 23 , ] [ , : , ] 6-Fluoro-N-methyl-5-piperone-1-yl-pyridine-2-carboxamide Add HCl (4M in di alkane, 100 ml, 400.00 mmol) to tertiary butyl 4-[2-fluoro-6-(methylaminoformyl)-3-pyridyl]piperone-1-carboxylate ( [intermediate]

[22] , 12.5 g, 36.94 mmol) in 1,4-di in a solution of alkane (50 mL). The reaction was stirred for 5 h while the temperature was warming to room temperature to give a yellow suspension. The suspension was diluted with ether and the solid was filtered off and washed with ether. The solid was dried under vacuum to give 6-fluoro-N-methyl-5-piperol-1-yl-pyridine-2-carboxamide, 2HCl ( [intermediate]

[23] , 11.42 g, 99 %). 1H NMR (500 MHz, DMSO-d6) δ ppm 2.8 (d, J=4.6 Hz, 3 H) 3.3 (br s, 4 H) 3.4 (br d, J=4.4 Hz, 4 H) 7.6 - 7.7 (m , 1 H) 7.9 (d, J=8.1 Hz, 1 H) 8.4 (br d, J=4.4 Hz, 1 H) 9.0 - 9.3 (m, 2 H); m / z (ES +) [M+H ] +=239

[0178]

[0179] [ , intermediate , ] [ , 15 , ] [ , : , ] 4-(6-Methoxycarbonyl-3-pyridyl)piperone-1-carboxylate tertiary butyl ester Ruphos Pd G3 (4.07 g, 4.86 mmol) was added to methyl 5-bromopyridine-2-carboxylate ( [intermediate]

[19] , 30 g, 138.87 mmol), tertiary butyl piper-1-carboxylate (27.2 g, 145.81 mmol), Cs 2CO 3 (90 g, 277.73 mmol) in 1,4-bis Alkanes (200 mL) and the mixture was stirred at 110 °C for 6 hrs under N 2 gas atmosphere. The mixture was then cooled to room temperature, diluted with water, extracted with ethyl acetate (150 ml x 3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. To this filtrate was added 3-(diethylenetriamino)propyl-functionalized silica gel (12 g, 1.3 mmol / g loading) and the mixture was stirred at r.t. for 1 hr. The mixture was filtered, and the filtrate was concentrated to ~100 ml. The crystalline yellow solid was filtered off, washed with diethyl ether and dried under vacuum to give tertiary-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperoxo-1-carboxylate as a yellow solid ( [intermediate]

[15] , 26.36 g, 82 mmol, 59.1%). 1H NMR (500 MHz, Chloroform-d) 1.50 (9H, s), 3.31 - 3.42 (4H, m), 3.56 - 3.68 (4H, m), 3.98 (3H, s), 8.04 (1H, d), 8.37 (1H, d); m / z (ES +) [M+H] +=322.

[0180] [ , intermediate , ] [ , 24 , ] [ , : , ] 4-[6-(Methylaminoformyl)-3-pyridyl]piperyl-1-carboxylate tertiary butyl ester Add methylamine (100 ml, 1155.26 mmol, 40% in water) to tertiary-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperoxa-1-carboxylate ( [intermediate]

[15] [,] 36 g, 112.02 mmol) in MeOH (100 mL) and the reaction was stirred at room temperature for 4 hs to give a white suspension. The mixture was concentrated, the residue was partitioned between sat. NH 4 Cl solution and DCM, and the layers were separated. The aqueous layer was extracted with DCM, the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated to give 4-[6-(methylaminoformyl)-3-pyridyl]piperone as a yellow solid - tertiary butyl formate ( [intermediate]

[24] , 35.9 g, 100%). 1H NMR (500 MHz, Chloroform-d) 1.49 (9H, s), 3.02 (3H, d), 3.26 - 3.35 (4H, m), 3.58 - 3.67 (4H, m), 7.23 (1H, dd), 7.81 (1H, br d), 8.07 (1H, d), 8.16 (1H, d); m / z (ES +) [M+H] +=321.

[0181] [ , intermediate , ] [ , 13 , ] [ , : , ] N-Methyl-5-piperone-1-yl-pyridine-2-carbamide formate Add HCl (4M in di alkane, 150 ml, 600.00 mmol) to tertiary butyl 4-[6-(methylaminoformyl)-3-pyridyl]piper-1-carboxylate ( [intermediate]

[24] , 35.9 g, 112.05 mmol) in MeOH (50 mL) and stirred the resulting orange suspension at r.t for 4 hr. About 80 ml of solvent was removed under reduced pressure and the mixture was diluted with ether and hexane (200 ml, 1 / 1). The solids were collected by filtration, washed with hexanes, dried and dried under vacuum to give N-methyl-5-piperol-1-yl-pyridine-2-carboxamide, 2HCl salt as a yellow solid ( [intermediate]

[13] , 37.0 g, 100%). 1H NMR (500 MHz, DMSO-d6) 2.79 (3H, d), 3.22 (4H, br s), 3.53 - 3.67 (4H, m), 7.51 (1H, dd), 7.91 (1H, d), 8.33 ( 1H, d), 8.50 (1H, br s), 9.19 - 9.49 (2H, m); m / z (ES +) [M+H] +=221

[0182]

[0183] [ , intermediate , ] [ , 26 , ] [ , : , ] 4-(1-Methoxycarbonylpropylamino)-3-nitro-benzoic acid methyl ester Sodium bicarbonate (27.0 g, 321.39 mmol) was added portionwise to methyl 4-fluoro-3-nitrobenzoate ( [intermediate]

[25] , 16 g, 80.35 mmol), and methyl 2-aminobutyrate, HCl (14.81 g, 96.42 mmol) in a stirred mixture of THF (100 mL). The reaction mixture was stirred overnight at room temperature. The reaction was quenched by adding water, extracted with ethyl acetate. The combined organic layers were washed with saturated aq. NaHCO 3 solution, the organic layer was dried over MgSO 4 and concentrated to dryness to give 4-(1-methoxycarbonylpropylamino)-3-nitro as a bright yellow solid - Methyl benzoate ( [intermediate]

[26] , 22.86 g, 96 %). 1H NMR (500MHz, DMSO-d6) 0.91 (3H, t), 1.75 - 2.12 (2H, m), 3.75 (3H, s), 3.85 (3H, s), 4.63 - 4.82 (1H, m), 7.15 ( 1H, d), 8.00 (1H, dd), 8.52 - 8.76 (2H, m).

[0184] [ , intermediate , ] [ , 27 , ] [ , : , ] 2-ethyl-3-oxo-2,4-dihydro-1H-quinone Methyl phen-6-carboxylate Pd / C (4.15 g, 3.90 mmol) was added portionwise to methyl 4-(1-methoxycarbonylpropylamino)-3-nitro-benzoate ( [intermediate]

[26] , 23.1 g, 77.97 mmol) in MeOH (300 mL) was stirred and the resulting slurry was stirred at room temperature under H 2 gas for 30 h. Methanol was removed under vacuum, 150 mL of DMF was added and the mixture was stirred for 10 min. The palladium catalyst was filtered off on Celite, washed with 50 mL of DMF (material has very low solubility in organic solvents such as MeOH / DCM / EtOAc). The filtrate was concentrated in Genevac to give 2-ethyl-3-oxo-2,4-dihydro-1H-quinone as a gray solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[27] , 15.80 g, 87 %). The material was analyzed by NMR and used in the next step without purification. 1H NMR (500MHz, DMSO-d6) 0.91 (3H, t), 1.63 - 1.73 (2H, m), 3.75 (3H, s), 3.90 (1H, td), 6.71 (1H, d), 6.84 (1H, s), 7.33 (1H, d), 7.41 (1H, dd), 10.39 (1H, s); m / z (ES +) [M] +=235.

[0185] [ , intermediate , ] [ , 28 , ] [ , : , ] 2-Ethyl-3-oxo-4H-quinone Methyl phen-6-carboxylate Add DDQ (15.87 g, 69.92 mmol) to 2-ethyl-3-oxo-2,4-dihydro-1H-quinone Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[27] , 15.6 g, 66.59 mmol) in 1,4-di a suspension in alkanes (150 mL). The reaction mixture was stirred overnight at room temperature. The mixture was added slowly to saturated aq NaHCO 3 solution (~500 ml) and stirred at room temperature for 20 min. The precipitate was filtered, washed with water (100 ml) and dried to yield 2-ethyl-3-oxo-4H-quinone as an off-white solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[28] , 11.40 g, 73.7 %). 1H NMR (500 MHz, DMSO-d6) 1.23 (3H, t), 2.83 (2H, q), 3.89 (3H, s), 7.73 - 7.86 (2H, m), 7.89 (1H, d), 12.45 (1H , s); m / z (ES +) [M+H] +=233.

[0186] [ , intermediate , ] [ , 29 , ] [ , : , ] 3-Ethyl-7-(hydroxymethyl)-1H-quinone Lin-2-one Lithium aluminum hydride (2 M in THF (49.1 mL, 98.17 mmol)) was added dropwise to 2-ethyl-3-oxo-4H-quinone at 0 °C under nitrogen atmosphere over a period of 50 min. Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[28] , 11.4 g, 49.09 mmol) in a slurry in THF (350 mL). The resulting mixture was stirred at 0°C for 1.5 hours. The reaction mixture was poured slowly into 1 M aq HCl (300 mL) at 0 °C. The reaction mixture was extracted with ethyl acetate (-300 ml x 2) followed by DCM / methanol (5:1) (150 ml x 3). The combined organic layers were concentrated to 300 ml and diluted with ether (200 ml) to give a suspension. The solid was collected by filtration, washed with ether, and dried under vacuum to yield 3-ethyl-7-(hydroxymethyl)-1H-quinone Lin-2-one ( [intermediate]

[29] , 8.00 g, 80 %). 1H NMR (500 MHz, DMSO-d6) 1.22 (3H, t), 2.80 (2H, q), 4.59 (2H, s), 5.19 - 5.61 (1H, m), 7.19 (1H, dd), 7.28 (1H , s), 7.66 (1H, d), 12.28 (1H, br s); m / z (ES +) [M+H] +=205.

[0187] [ , intermediate , ] [ , 30 , ] [ , : , ] 7-(bromomethyl)-3-ethyl-1H-quinone Lin-2-one Add hydrogen bromide (60 ml, 48 wt% in water) to 3-ethyl-7-(hydroxymethyl)-1H-quinone Lin-2-one ( [intermediate]

[29] , 7.8 g, 38.19 mmol) (a clear brown solution was produced) and the mixture was stirred at 80 °C for 8 hrs, the reaction mixture was cooled to room temperature, and poured into 150 mL of ice water to obtain an off-white precipitate. The solid was filtered under vacuum and washed with water followed by diethyl ether, and dried to give 7-(bromomethyl)-3-ethyl-1H-quinone in 80% purity as a beige solid. Lin-2-one ( [intermediate]

[30] , 11.10 g, 84 %). 1H NMR (500 MHz, DMSO-d6) 1.20 (3H, t), 2.79 (2H, q), 4.79 (2H, s), 7.27 - 7.38 (2H, m), 7.69 (1H, d), 12.34 (1H , br s); m / z (ES +) [M] +=267.0.

[0188]

[0189] [ , intermediate , ] [ , 32 , ] [ , : , ] tertiary butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperone-1-carboxylate Stir tertiary butyl piperazine-1-carboxylate at 110°C ( [intermediate]

[31] , 2.57 g, 13.80 mmol), 6-bromo-5-fluoro-pyridine-2-carboxylic acid methyl ester (1.9 g, 8.12 mmol) and potassium carbonate (1.459 g, 10.55 mmol) in DMF (20 mL) The mixture was 5 hours, LCMS indicated complete conversion. The mixture was cooled to r.t, diluted with DCM and water, and the layers were separated. The aqueous layer was extracted twice with DCM and the combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 50% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to afford tert-butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperoxa-1-carboxylate ( [intermediate]

[32] , 2.200 g, 67.7%). 1H NMR (500 MHz, Chloroform-d) 1.50 (9H, s), 3.05 - 3.20 (4H, m), 3.58 - 3.72 (4H, m), 3.98 (3H, s), 7.31 (1H, d), 8.06 (1H, d); m / z (ES +) [M+H] +=400.

[0190] [ , intermediate , ] [ , 33 , ] [ , : , ] 4-[2-Bromo-6-(methylaminoformyl)-3-pyridyl]piperyl-1-carboxylate tertiary butyl ester Inject the sealed pressure bottle into 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperone-1-formic acid tertiary butyl ester ( [intermediate]

[32] , 2.2 g, 5.50 mmol) and methylamine (22 ml, 176.72 mmol) (33 w.t% in ethanol) and the mixture was heated at 60°C for 2 hours, LCMS indicated complete conversion. The mixture was concentrated and the resulting residue was purified by flash silica chromatography with a gradient of 0% to 80% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to afford tert-butyl 4-[2-bromo-6-(methylaminoformyl)-3-pyridyl]piperoxa-1-carboxylate as a white solid ( [intermediate]

[33] , 2.200 g, 100 %). 1H NMR (500 MHz, Chloroform-d) 1.50 (9H, s), 3.02 (3H, d), 3.05 - 3.14 (4H, m), 3.56 - 3.74 (4H, m), 7.36 (1H, d), 7.68 (1H, br d), 8.11 (1H, d); m / z (ES +) [M+H] +=399.

[0191] [ , intermediate , ] [ , 34 , ] [ , : , ] 4-[6-(Methylaminoformyl)-2-vinyl-3-pyridyl]piperyl-1-carboxylate tertiary butyl Stirring 4-[2-bromo-6-(methylaminoformyl)-3-pyridyl]pipera-1-carboxylic acid tertiary butyl ester at 100°C under N 2 ( [intermediate]

[33] , 200 mg, 0.50 mmol), tributyl (vinyl) stannane (0.161 ml, 0.55 mmol) and the second generation XPhos Pd cycle (19.71 mg, 0.03 mmol) in 1,4-bis Mixture of alkanes (5 ml) for 2.5 hr, LCMS indicated complete conversion. The mixture was diluted with DCM, washed with saturated NH 4 Cl, the organic layer was dried (anhydrous Na 2 SO 4 ), filtered and concentrated. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 80% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to afford tert-butyl 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperoxo-1-carboxylate as a white solid ( [intermediate]

[34] , 174 mg, 100%). m / z (ES +) [M+H] +=347

[0192] [ , intermediate , ] [ , 35 , ] [ , : , ] 4-[2-Ethyl-6-(methylcarbamoyl)-3-pyridyl]piperyl-1-carboxylate tertiary butyl Add Pd / C (53.5 mg, 0.05 mmol) (10 wt% dry basis, wet load) to 4-[6-(methylaminoformyl)-2-vinyl-3-pyridyl]piperone 1-Tertiary butyl carboxylate ( [intermediate]

[34] , 174 mg, 0.50 mmol) in MeOH (6 mL). The flask was degassed and refilled with H2 (balloon). The mixture was stirred overnight at r.t. LCMS indicated that the reaction was not complete. More Pd / C (53.5 mg, 0.05 mmol) was added and the resulting mixture was stirred at r.t under H2 gas atmosphere for 5hrs. The mixture was filtered through a pad of celite, washed with methanol, and the filtrate was concentrated to dryness to give 4-[2-ethyl-6-(methylaminoformyl)-3-pyridyl]piperide as a colorless residue. 𠯤 -Tertiary butyl formate ( [intermediate]

[35] , 172 mg, 98 %). 1H NMR (500 MHz, chloroform-d) 1.37 (3H, t), 1.51 (9H, s), 2.82 - 2.95 (6H, m), 3.05 (3H, d), 3.57 - 3.73 (4H, m), 7.39 (1H, d), 7.93 - 8.13 (2H, m); m / z (ES +) [M] +=348.

[0193] [ , intermediate , ] [ , 36 , ] [ , : , ] 6-Ethyl-N-methyl-5-piperyl-1-yl-pyridine-2-formamide Stirring tertiary butyl 4-[2-ethyl-6-(methylaminoformyl)-3-pyridyl]piperone-1-carboxylate at r.t ( [intermediate]

[35] , 172 mg, 0.49 mmol) in HCl (4M in di alkanes, 8 ml, 32.00 mmol) for 1 hr to give a white suspension. The mixture was diluted with diethyl ether and the solid was filtered off and dried under vacuum to give 6-ethyl-N-methyl-5-piperol-1-yl-pyridine-2-carboxamide, 2HCl ( [intermediate]

[36] , 159 mg, 100%). 1H NMR (500 MHz, DMSO-d6) 1.31 (3H, t), 2.74 - 2.86 (5H, m), 3.00 - 3.14 (4H, m), 3.24 (4H, br s), 7.57 (1H, d), 7.82 (1H, d), 8.43 (1H, br d), 9.20 (2H, br s); m / z (ES +) [M+H] +=249.

[0194] [ , Synthesis example , ] [ , 8 , ] [ , : , ] 6-ethyl-5-[4-[(2-ethyl-3-oxo-4H-quinone phyllin-6-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-formamide

[0195] Add DIPEA (0.203 mL, 1.17 mmol) to 6-ethyl-N-methyl-5-piperazol-1-yl-pyridine-2-carboxamide, 2HCl ( [intermediate]

[36] , []75 mg, 0.23 mmol) and 7-(bromomethyl)-3-ethyl-1H-quinone Lin-2-one ( [intermediate]

[30] , 69.3 mg, 0.23 mmol) in acetonitrile (3 mL). The resulting mixture was stirred at 60 °C for 3 hrs, LCMS indicated complete conversion. The mixture was cooled to r.t., concentrated and the residue was purified on a Gilson reverse phase column (0 to 95% ACN / water / 0.1% TFA, run for 15 minutes, collected from 5 to 9 minutes). Fractions containing product were concentrated and the residue was dissolved in methanol and DCM. 300 mgs of tetraalkylammonium carbonite polymer linkage (40-90 mesh, 2.5-3.5 mmol / g) and the mixture were stirred for 10 min at r.t. The mixture was then filtered and washed with methanol. The filtrate was concentrated, redissolved in a water / CAN mixture and this mixture was lyophilized to dryness to yield 6-ethyl-5-[4-[(2-ethyl-3-oxo -4H-quinone Lin-6-yl) methyl] piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example] [8], 60.0 mg, 59.1%). 1H NMR (500 MHz, DMSO-d6) 1.22 (3H, t), 1.30 (3H, t), 2.54 - 2.69 (2H, m), 2.72 - 2.86 (7H, m), 2.93 (4H, br s), 3.26 (2H, s), 3.64 (2H, s), 7.17 - 7.33 (2H, m), 7.52 (1H, d), 7.69 (1H, br d), 7.80 (1H, d), 8.40 (1H, br d), 12.25 (1H, br s); m / z (ES +) [M+H] +=435.

[0196]

[0197] [ , intermediate , ] [ , 37 , ] [ , : , ] 4-[6-(Methylaminoformyl)-2-(trifluoromethyl)-3-pyridyl]piperyl-1-carboxylate tertiary butyl ester To a well stirred mixture of silver(I) fluoride (176 mg, 1.39 mmol) in DMF (2 mL) was added trimethyl(trifluoromethyl)silane (0.247 mL, 1.67 mmol) at room temperature. This mixture was stirred for 20 min, then copper powder (133 mg, 2.09 mmol) was added thereto. After stirring for 4 h, the reaction mixture turned blue (an indicator of the formation of CuCF 3 ). Add tertiary butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperone-1-carboxylate ( [intermediate]

[33] , 150 mg, 0.38 mmol) to this mixture and stirred the resulting mixture at 90°C for 18 hr to obtain a brown suspension. LCMS indicated complete conversion. The mixture was diluted with ethyl acetate and the solid was filtered off. The filtrate was washed with water followed by brine. The organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 70% EtOAc in hexanes. Concentration of the product fractions to dryness under reduced pressure gave 4-[6-(methylaminoformyl)-2-(trifluoromethyl)-3-pyridyl]piperazine-1- as a yellow residue. Tertiary butyl formate ( [intermediate]

[37] , 146 mg, 100%). 1H NMR (500 MHz, Chloroform-d) 1.50 (9H, s), 2.93 - 3.03 (4H, m), 3.05 (3H, d), 3.55 - 3.69 (4H, m), 7.71 (1H, d), 7.81 (1H, br d), 8.33 (1H, d); m / z (ES +) [M+H] +=389.

[0198] [ , intermediate , ] [ ,38 , ] [ , : , ] N-methyl-5-piperone-1-yl-6-(trifluoromethyl)pyridine-2-formamide Stirring 4-[6-(methylaminoformyl)-2-(trifluoromethyl)-3-pyridyl]piperone-1-carboxylic acid tertiary butyl ester at r.t ( [intermediate]

[37] , 146 mg, 0.38 mmol) in HCl (4M in di alkane, 8 ml, 32.00 mmol) for 2 hr. LCMS indicated complete conversion. The solvent was concentrated to a volume of 2 ml, and the mixture was diluted with ether / hexane (15 ml, 5 / 1). The solid was filtered off and dried under vacuum to afford N-methyl-5-piperol-1-yl-6-(trifluoromethyl)pyridine-2-carboxamide, 2HCl ( [intermediate]

[38] , 127 mg, 94 %). 1H NMR (500 MHz, DMSO-d6) 2.83 (3H, d), 3.21 (8H, br s), 8.09 (1H, d), 8.23 ​​(1H, d), 8.46 (1H, br d), 9.08 (2H , br d);m / z (ES +) [M+H] +=289.

[0199] [ , Synthesis example , ] [ , 9 , ] [ , : , ] 5-[4-[(2-ethyl-3-oxo-4H-quinone phyllin-6-yl)methyl]piperone-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-formamide

[0200] Add DIPEA (0.121 mL, 0.69 mmol) to N-methyl-5-piperol-1-yl-6-(trifluoromethyl)pyridine-2-carboxamide, 2HCl ( [intermediate]

[38] , 50 mg, 0.14 mmol) and 7-(bromomethyl)-3-ethylquine Lin-2(1H)-one ( [intermediate]

[30] , 46.2 mg, 0.14 mmol) in acetonitrile (3 mL) and this mixture was stirred at 60°C for 3 hr. The mixture was cooled to r.t., concentrated and the residue was purified on a Gilson reverse phase column (0 to 95% ACN / water / 0.1% TFA). Fractions containing product were concentrated at room temperature. The residue was then dissolved in methanol and DCM, followed by the addition of 250 mg of tetraalkylammonium carbonate polymer linkage (40-90 mesh, 2.5-3.5 mmol / g) and the mixture was stirred at room temperature for 10 min. The solid was then filtered off, washed with methanol and the filtrate was concentrated to give a solid. This solid was then redissolved in a water / CH CN mixture and lyophilized to dryness to give 5-[4-[(2-ethyl-3-oxo-4H-quinone as a white solid Lin-6-yl) methyl] piper-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide ( [synthesis example] [9], 40.0 mg, 60.9 %). 1H NMR (500 MHz, chloroform-d) 1.40 (3H, t), 2.70 (4H, br s), 2.98 - 3.08 (5H, m), 3.12 (4H, br s), 3.72 (2H, br s), 7.29 - 7.32 (1H, m), 7.37 (1H, dd), 7.74 (1H, d), 7.79 - 7.88 (2H, m), 8.33 (1H, d), 11.06 (1H, br s); m / z (ES +) [M+H] +=475.

[0201]

[0202] [ , intermediate , ] [ , 39 , ] [ , : , ] 4-[2-formyl-6-(methylaminoformyl)-3-pyridyl]piperyl-1-carboxylate tertiary butyl Add osmium tetroxide (0.050 mL, 6.35 µmol) in HO to tert-butyl 4-[6-(methylaminoformyl)-2-vinyl-3-pyridyl]piperoxo-1-carboxylate Esters ( [intermediate]

[34] , 110 mg, 0.32 mmol), 2,6-lutidine (0.074 mL, 0.64 mmol) and sodium periodate (272 mg, 1.27 mmol) in THF (5 mL) / water (1 mL) / tert-butanol (0.304 mL, 3.18 mmol) and the mixture was stirred at rt overnight to give a yellow suspension. LCMS and TLC indicated complete conversion. The reaction was diluted with water and extracted with ethyl acetate. After concentration, the resulting residue was purified by flash silica chromatography with a gradient of 0% to 100% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to afford 4-[2-formyl-6-(methylaminoformyl)-3-pyridyl]piperazol-1-carboxylic acid tert-butyl as a yellow solid Esters ( [intermediate]

[39] , 100 mg, 90 %). 1H NMR (500 MHz, Chloroform-d) 1.50 (9H, s), 3.07 (3H, d), 3.14 - 3.29 (4H, m), 3.66 - 3.79 (4H, m), 7.49 (1H, d), 7.86 (1H, br d), 8.28 (1H, d), 10.10 (1H, s). m / z (ES +) [M+H] +=349.

[0203] [ , intermediate , ] [ , 40 , ] [ , : , ] 4-[2-(Difluoromethyl)-6-(methylaminoformyl)-3-pyridyl]piperyl-1-carboxylate tertiary butyl tertiary butyl 4-[2-formyl-6-(methylaminoformyl)-3-pyridyl]piperone-1-carboxylate ( [intermediate]

[39] , 99 mg, 0.28 mmol) was cooled to 0°C, DAST (0.710 mL, 0.71 mmol) (1M in DCM) was added, and the resulting mixture was stirred at room temperature for 3hr. TLC and LCMS indicated complete conversion. The reaction was quenched by adding sat. NaHCO 3 solution dropwise and extracted with DCM. The combined organics were dried over anhydrous Na2SO4, filtered and concentrated to give crude product. The resulting residue was purified by flash silica chromatography with a gradient of 0% to 100% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to afford 4-[2-(difluoromethyl)-6-(methylaminoformyl)-3-pyridyl]piperazine-1-carboxylic acid as an off-white solid Tertiary butyl ester ( [intermediate]

[40] ,94 mg,89 %). 1H NMR (500 MHz, chloroform-d) 1.51 (9H, s), 2.89 - 3.03 (4H, m), 3.06 (3H, d), 3.54 - 3.73 (4H, m), 6.82 - 7.16 (1H, m), 7.64 (1H, d), 7.94 (1H, br d), 8.29 (1H, d); m / z (ES +) [M+H] +=371.

[0204] [ , intermediate , ] [ , 41 , ] [ , : , ] 6-(Difluoromethyl)-N-methyl-5-piperyl-1-yl-pyridine-2-formamide Stirring 4-[2-(difluoromethyl)-6-(methylaminoformyl)-3-pyridyl]piperone-1-carboxylic acid tertiary butyl ester at r.t ( [intermediate]

[40] , 92 mg, 0.25 mmol) in HCl 4M (in 1,4-di (6 ml, 24.00 mmol)) to give an orange suspension for 1.5 hr, the mixture was diluted with diethyl ether, filtered, and the solid redissolved in methanol, concentrated to dryness to yield 6-(difluoro Methyl)-N-methyl-5-piperone-1-yl-pyridine-2-carboxamide, 2HCl ( [intermediate]

[41] , 56.0 mg, 65.7 %). 1H NMR (500 MHz, DMSO-d6) 2.83 (3H, d), 3.03 - 3.23 (5H, m), 3.30 (4H, br s), 7.06 - 7.49 (1H, m), 7.92 (1H, d), 8.13 (1H, d), 8.43 (1H, br d), 9.00 (2H, br d); m / z (ES +) [M+H] +=271.

[0205] [ , Synthesis example , ] [ , 10 , ] [ , : , ] 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo-4H-quinone phyllin-6-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-formamide

[0206] Add DIPEA (0.127 mL, 0.73 mmol) to 6-(difluoromethyl)-N-methyl-5-piperone-1-yl-pyridine-2-carboxamide, 2HCl ( [intermediate]

[41] , []50 mg, 0.15 mmol) and 7-(bromomethyl)-3-ethylquine Lin-2(1H)-one ( [intermediate]

[30] , 48.6 mg, 0.15 mmol) in a suspension in acetonitrile (3 mL). The resulting mixture was stirred at 60 °C for 3 hr, LCMS indicated complete conversion. The mixture was concentrated and the residue was purified on a Gilson reverse phase column (0 to 95% ACN / water / 0.1% TFA). Fractions containing product were concentrated at room temperature. The residue was then dissolved in methanol and DCM, followed by the addition of 250 mg of tetraalkylammonium carbonate polymer linkage (40-90 mesh, 2.5-3.5 mmol / g) and the mixture was stirred at room temperature for 10 min. The solid was then filtered off, washed with methanol and the filtrate was concentrated to give a solid. This solid was then redissolved in a water / CHCN mixture and lyophilized to dryness to afford 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo- 4H-quinone Lin-6-yl) methyl] piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example]

[10] , 50.0 mg, 75%). 1H NMR (500 MHz, Chloroform-d) 1.40 (3H, t), 2.72 (4H, br s), 2.97 - 3.17 (9H, m), 3.73 (2H, s), 6.84 - 7.15 (1H, m), 7.32 (1H, s), 7.37 (1H, d), 7.64 (1H, d), 7.83 (1H, d), 7.95 (1H, br d), 8.29 (1H, d), 11.32 - 11.62 (1H, m );m / z (ES +) [M+H] +=457.

[0207]

[0208] [ , Synthesis example , ] [ , 11 , ] : 5-[4-[(2-Ethyl-3-oxo-4H-quinone phyllin-6-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-formamide

[0209] Add 7-(bromomethyl)-3-ethylquine to a 20 mL vial Lin-2(1H)-one ( [intermediate]

[30] , 0.147 g, 0.55 mmol) and N-methyl-5-piperone-1-yl-pyridine-2-carboxamide, 2HCl ( [intermediate]

[13] , 0.161 g, 0.55 mmol). The vial was sealed, evacuated, and refilled with N. Acetonitrile (3 mL) and DIPEA (0.481 mL, 2.75 mmol) were added to the vial and placed on a hot plate preheated to 70°C. The reaction mixture was stirred at the same temperature for 2 hours and cooled to room temperature. The reaction volume was reduced to 1 / 3 of its initial volume under vacuum and aqueous NaHCO 3 (2 mL) was added. The reaction mixture was stirred for 30 min, filtered and the solid was washed with water (50 mL). The crude product was purified by flash silica chromatography using 0-30% MeOH in DCM to yield 5-[4-[(2-ethyl-3-oxo-4H- Quinoa Lin-6-yl) methyl] piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example]

[11] , 93.0 mg, 41.6%). 1H NMR (500 MHz, DMSO-d 6) 1.22 (3H, t), 2.52 - 2.60 (4H, m), 2.73 - 2.85 (5H, m), 3.30 (4H, m, overlapping with water peak), 3.62 ( 2H, s), 7.22 - 7.31 (2H, m), 7.39 (1H, dd), 7.69 (1H, d), 7.83 (1H, d), 8.23 ​​- 8.31 (1H, m), 8.39 (1H, br d ), 12.13 - 12.36 (1H, m); m / z (ES +) [M+H] +=407.

[0210]

[0211] [ , Synthesis example , ] [ , 12 , ] : 5-[4-[(2-Ethyl-3-oxo-4H-quinone Phenyl-6-yl)methyl]piperyl-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide

[0212] Add 7-(bromomethyl)-3-ethylquine Lin-2(1H)-one ( [intermediate]

[30] , [] 150 mg, 0.56 mmol) to 6-fluoro-N-methyl-5-piper-1-yl-pyridine-2-carboxamide ( [intermediate]

[23] , 60 mg, 0.25 mmol) and DIPEA (0.270 mL, 1.55 mmol) in NMP (2 mL). The resulting mixture was stirred at 80°C for 2 hours. Solvent was removed under reduced pressure. By preparative HPLC (column: XBridge Shield RP18 OBD column, 5um, 19x150 mm; mobile phase A: water (10 MMOL / L NH 4HCO 3,0.1% NH 3.H 2O), mobile phase B: ACN; Flow rate: 20 mL / min; Gradient: 28% B to 38% B in 8 min; 254; 220 nm; RT: 8.02 min) to purify the crude product. Fractions containing the desired compound were evaporated to dryness to give 5-[4-[(2-ethyl-3-oxo-4H-quinone as a white solid Lin-6-yl)methyl]piperone-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide ( [synthesis example]

[12] , 9 mg, 42.9%). 1H NMR (400 MHz, CD 3OD) δ 1.33 (3H, t), 2.65 - 2.72 (4H, m), 2.87 - 2.95 (5H, m), 3.26 - 3.30 (4H, m), 3.71 (2H, s) , 7.33 - 7.41 (2H, m), 7.52 (1H, dd), 7.76 (1H, d), 7.90 (1H, dd); 19F NMR (376 MHz, CD 3OD) δ -73.40; m / z (ES + ) [M+H] +=425.

[0213]

[0214] [ , intermediate , ] [ , 43 , ] : 5-bromo-N,6-dimethylpyridinamide A 2 M solution of methylamine in THF (20 mL, 40.00 mmol) was added to methyl 5-bromo-6-methylpicolinate ( [intermediate]

[42] , 2.0 g, 8.69 mmol) and the resulting mixture was stirred at 80°C for 18 hours. Solvent was removed under reduced pressure. The crude product was purified by reverse phase chromatography with a gradient of 5 to 80% MeOH in water (0.1% NH4HCO3). The pure fractions were evaporated to dryness to give 5-bromo-N,6-dimethylpyridinamide ( [intermediate]

[43] , 1.5 g, 75%). 1H NMR (400 MHz, DMSO-d 6) δ 2.65 (3H, s), 2.82 (3H, d), 7.75 (1H, d), 8.17 (1H, d), 8.57 – 8.76 (1H, m); m / z (ES +) [M+H] +=229.

[0215] [ , intermediate , ] [ , 44 , ] : 4-(2-Methyl-6-(methylaminoformyl)pyridin-3-yl)piperone-1-carboxylate tertiary butyl Under nitrogen, 5-bromo-N,6-dimethylpyridinamide ( [intermediate]

[43] , 1.0 g, 4.37 mmol) added to tertiary-butylpiperone-1-carboxylate (0.894 g, 4.80 mmol), BINAP (0.272 g, 0.44 mmol), Pd(OAc) in toluene (20 mL) 2 (0.098 g, 0.44 mmol) and Cs 2CO 3 (3.56 g, 10.91 mmol). The resulting mixture was stirred at 80°C for 16 hours. Solvent was removed under reduced pressure. The crude product was purified by reverse phase chromatography with a gradient of 5 to 30% MeOH in water (0.4% HCO 2H). The pure fractions were evaporated to dryness to afford tert-butyl 4-(2-methyl-6-(methylaminoformyl)pyridin-3-yl)piperoxa-1-carboxylate as a brown solid ( [intermediate]

[44] , 1.2 g, 82%). 1H NMR (300 MHz, CD 3OD) δ 1.50 (9H, s), 2.58 (3H, s), 2.92 – 3.00 (7H, m), 3.62 (4H, m), 7.50 (1H, d), 7.88 (1H , d); m / z (ES +) [M+H] +=335.

[0216] [ , intermediate, ] [ , 45 , ] : N,6-Dimethyl-5-(piper 𠯤-1-yl)pyridinamide Add tertiary butyl 4-(2-methyl-6-(methylaminoformyl)pyridin-3-yl)piperone-1-carboxylate ( [intermediate]

[44] , 1.18 g, 3.53 mmol) to HCl in 1,4-bis in a 4 M solution of alkanes (10 mL, 329.15 mmol). The resulting mixture was stirred at room temperature for 1 hour. The precipitate was collected by filtration, washed with petroleum ether (5 mL x 2), Et2O (5 mL x 2), and dried under vacuum to give N,6-dimethyl-5-(piperidine as a yellow solid 𠯤-1-yl)pyridinamide ( [intermediate]

[45] , 0.77 g, 81%). 1H NMR (300 MHz, CD 3OD) δ 2.86 (3H, s), 3.02 (3H, s), 3.42 – 3.54 (8H, m), 8.29 (2H, d); m / z (ES +) [M+ H ] +=235.

[0217] [ , Synthesis example , ] [ , 13 , ] : 5-[4-[(2-Ethyl-3-oxo-4H-quinone phyllin-6-yl)methyl]piperone-1-yl]-N,6-dimethyl-pyridine-2-carboxamide

[0218] 7-(bromomethyl)-3-ethylquine Lin-2(1H)-one ( [intermediate]

[30] , 100 mg, 0.37 mmol) N,6-dimethyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[45] , 90 mg, 0.33 mmol) and DIPEA (0.36 mL, 2.05 mmol). The resulting mixture was stirred at 80°C for 2 hours. Solvent was removed under reduced pressure. By preparative HPLC (column: XBridge Prep OBD C18 column 30 x 150mm, 5um; mobile phase A: water (10 MMOL / L NH 4HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; gradient : 30% B to 40% B in 7 min; 254; 220 nm; RT: 6.43 min) to purify the crude product. Fractions containing the desired compound were evaporated to dryness to give 5-[4-[(2-ethyl-3-oxo-4H-quinone as an off-white solid Lin-6-yl) methyl] piper-1-yl]-N,6-dimethyl-pyridine-2-carboxamide ( [synthesis example]

[13] , 68.7 mg, 43.6%). 1H NMR (400 MHz, CD 3OD) δ 1.33 (3H, t), 2.55 (3H, s), 2.71 (4H, s), 2.87 – 2.99 (5H, m), 3.05 (4H, t), 3.73 (2H , s), 7.35 (1H, s), 7.38 (1H, d), 7.49 (1H, d), 7.77 (1H, d), 7.87 (1H, d); m / z (ES+) [M+H] +=421.

[0219]

[0220] [ , intermediate , ] [ , 47 , ] : 6-Chloro-5-(piperyl 𠯤-1-yl) methyl picolinate Piperidine (1.0 g, 11.61 mmol) was added to methyl 6-chloro-5-fluoropicolinate ( [intermediate]

[46] , 1.0 g, 5.28 mmol). The resulting mixture was stirred at 80°C for 18 hours. Solvent was removed under reduced pressure. The crude product was purified by reverse phase chromatography with a gradient of 5 to 60% MeCN in water (0.1% NH4HCO3). The pure fractions were evaporated to dryness to give methyl 6-chloro-5-(piperone-1-yl)picolinate as a red oil ( [intermediate]

[47] , 1.28 g, 95%). 1H NMR (400 MHz, DMSO-d 6) δ 2.81 – 2.91 (4H, m), 3.04 - 3.08 (4H, m), 3.85 (3H, s), 7.61 (1H, d), 8.00 (1H, d) (NH protons not shown); m / z (ES +) [M+H] +=256.

[0221] [ , intermediate , ] [ , 48 , ] : 6-Chloro-N-methyl-5-(piper 𠯤-1-yl)pyridinamide A 2 M solution of methylamine in THF (40 mL, 80.00 mmol) was added to methyl 6-chloro-5-(piper-1-yl)picolinate ( [intermediate]

[47] , 1.26 g, 4.93 mmol). The resulting mixture was stirred at 80°C for 18 hours. Solvent was removed under reduced pressure. The crude product was purified by reverse phase chromatography with a gradient of 5 to 60% MeCN in water (0.1% NH4HCO3). Evaporation of the pure fractions to dryness afforded 6-chloro-N-methyl-5-(piperone-1-yl)pyridinamide ( [intermediate]

[48] , 1.12 g, 89%). 1H NMR (300 MHz, DMSO-d 6) δ 2.79 (3H, d), 2.85 - 2.89 (4H, m), 2.97 - 3.02 (4H, m), 7.63 (1H, d), 7.94 (1H, d) , 8.45 (1H, q) (pipera-NH proton not shown); m / z (ES +) [M+H] +=255.

[0222] [ , Synthesis example , ] [ , 14 , ] : 6-chloro-5-[4-[(2-ethyl-3-oxo-4H-quinone phyllin-6-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-formamide

[0223] 7-(bromomethyl)-3-ethylquine Lin-2(1H)-one ( [intermediate]

[30] , 200 mg, 0.75 mmol) was added to 6-chloro-N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[48] , 100 mg, 0.39 mmol) and DIPEA (0.358 mL, 2.05 mmol). The resulting mixture was stirred at 80°C for 2 hours. Solvent was removed under reduced pressure. By preparative HPLC (column: XBridge Prep OBD C18 column 30×150 mm 5um; mobile phase A: water (10 MMOL / L NH 4HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; gradient : 30% B to 40% B in 8 min; 254; 220 nm; RT: 7.3 min) to purify the crude product. Fractions containing the desired compound were evaporated to dryness to give 6-chloro-5-[4-[(2-ethyl-3-oxo-4H-quinone as a white solid Lin-6-yl) methyl] piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example]

[14] , 52.6 mg, 30.4%). 1HNMR (400 MHz, CD 3OD) δ 1.33 (3H, t), 2.71 (4H, s), 2.87 – 2.96 (5H, m), 3.23 (4H, s), 3.73 (2H, s), 7.33 – 7.41 ( 2H, m), 7.62 (1H, d), 7.77 (1H, d), 8.00 (1H, d); m / z (ES +) [M+H] +=441.

[0224]

[0225] [ , intermediate , ] [ , 50 , ] : 7-bromo-3-(trifluoromethyl)quinone Lin-2(1H)-one 4-bromobenzene-1,2-diamine ( [intermediate]

[49] , 0.9 g, 4.81 mmol) was added to methyl 3,3,3-trifluoro-2-oxopropionate (0.9 g, 5.77 mmol) in toluene (10 mL). The resulting mixture was stirred at 100°C for 60 minutes. Solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography with a gradient of 0 to 50% EtOAc in petroleum ether. Evaporation of the pure fractions to dryness afforded 7-bromo-3-(trifluoromethyl)quinone as an off-white solid Lin-2(1H)-one and 6-bromo-3-(trifluoromethyl)quinone Regioisomeric mixture (regioisomeric mixture) of lin-2(1H)-one ( [intermediate] [50+] [intermediate]

[51] , 1.28 g, 45.4%). Mixture of positional isomers separated, uninterpreted 1H NMR spectrum; m / z (ES +) [M+H] +=295.

[0226] [ , intermediate , ] [ , 52 , ] : 7-(hydroxymethyl)-3-(trifluoromethyl)quinone Lin-2(1H)-one Add Pd(Ph 3P) 4 (0.3 g, 0.26 mmol) to 7-bromo-3-(trifluoromethyl)quinone Lin-2(1H)-one and 6-bromo-3-(trifluoromethyl)quinone Lin-2(1H)-one ( [intermediate] [50+] [intermediate]

[51] , 1.2 g, 2.05 mmol) and (tributylstannyl)methanol (1.2 g, 3.74 mmol) in 1,4-bis a mixture of alkanes (40 mL). The resulting mixture was stirred at 100°C under nitrogen for 18 hours. Solvent was removed under reduced pressure. The crude product was purified by reverse phase chromatography with a gradient of 5 to 50% MeCN in water (0.1% HCO 2H). Evaporation of the pure fractions to dryness afforded 7-(hydroxymethyl)-3-(trifluoromethyl)quinone as an off-white solid Lin-2(1H)-one ( [intermediate]

[52] , 0.32 g, 64.0%). 1H NMR (300 MHz, DMSO-d 6,) δ 4.63 (2H, d), 5.52 (1H, t), 7.30 (1H, dd), 7.38 (1H, d), 7.83 (1H, d), 13.05 ( 1H, s); m / z (ES +) [M+H] +=245.

[0227] [ , Synthesis example , ] [ , 15 , ] : N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinone Lin-6-yl] methyl] piper-1-yl] pyridin-2-formamide

[0228] A solution of 33% HBr in AcOH (3 mL, 18.23 mmol) was added to 7-(hydroxymethyl)-3-(trifluoromethyl)quinone Lin-2(1H)-one ( [intermediate]

[52] , 111 mg, 0.45 mmol). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. DIEA (0.5 mL, 2.86 mmol) and N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[13] , 100 mg, 0.45 mmol) was added to the mixture listed above in NMP (3 mL). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. By preparative HPLC (column: XBridge Prep OBD C18 column, 30×150 mm 5um; mobile phase A: water (10 MMOL / L NH 4HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; Gradient: 22 B to 32 B in 7 min; 254; 220 nm; RT: 5.77) Purify the crude product. Fractions containing the desired compound were evaporated to dryness to give N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinone as a white solid Lin-6-yl] methyl] piper-1-yl] pyridine-2-formamide ( [synthesis example]

[15] , 44.0 mg, 21.71%). 1H NMR (400 MHz, DMSO-d 6) δ 2.55 - 2.62 (m, 4H), 2.78 (d, 3H), 3.34 - 3.38 (t, 4H), 3.69 (s, 2H), 7.34 - 7.44 (m, 3H), 7.80 - 7.91 (m, 2H), 8.27 (d, 1H), 8.36 - 8.41 (m, 1H), 12.97 (s, 1H); 19F NMR (376 MHz, DMSO-d 6) δ -68.36; m / z (ES +) [M+H] +=447.

[0229]

[0230] [ , Synthesis example , ] [ , 16 , ] : 6-Chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinone Lin-6-yl] methyl] piper-1-yl] pyridin-2-formamide

[0231] AcOH (3 mL, 18.23 mmol) in 33% HBr was added to 7-(hydroxymethyl)-3-(trifluoromethyl)quinone Lin-2(1H)-one ( [intermediate]

[52] , 43.1 mg, 0.18 mmol). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. Add DIPEA (0.5 mL, 2.86 mmol) and 6-chloro-N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[48] , 45 mg, 0.18 mmol) to the mixture listed above in NMP (5 mL). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. By preparative HPLC (column: XBridge Prep OBD C18 column, 30×150 mm 5um; mobile phase A: water (10 MMOL / L NH 4HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; Gradient: 10 B to 50 B in 7 min; 254; 220 nm; RT: 6.75) Purify the crude product. Fractions containing the desired compound were evaporated to dryness to give 6-chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H- Quinoa Lin-6-yl] methyl] piper-1-yl] pyridine-2-formamide ( [synthesis example]

[16] , 22.00 mg, 25.9%). 1H NMR (400 MHz, DMSO-d 6) δ 2.56 - 2.64 (s, 4H), 2.79 (d, 3H), 3.09 - 3.17 (m, 4H), 3.71 (s, 2H), 7.36 - 7.42 (m, 2H), 7.67 (d, 1H), 7.88 (d, 1H), 7.94 (d, 1H), 8.39 - 8.44 (m, 1H), 12.89 (s, 1H); 19F NMR (376 MHz, DMSO) δ - 68.41; m / z (ES+) [M+H] +=481.

[0232]

[0233] [ , Synthesis example , ] [ , 17 , ] : 6-fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinone Lin-6-yl] methyl] piper-1-yl] pyridin-2-formamide

[0234] 33% HBr (3 mL, 55.25 mmol) in AcOH was added to 7-(hydroxymethyl)-3-(trifluoromethyl)quinone Lin-2(1H)-one ( [intermediate]

[52] , 102 mg, 0.42 mmol). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. 6-fluoro-N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[23] , 100 mg, 0.42 mmol) and DIPEA (0.5 mL, 2.86 mmol) were added to the mixture listed above in NMP (5 mL). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. By preparative HPLC (column: XBridge Prep OBD C18 column, 30×150 mm 5um; mobile phase A: water (10 MMOL / L NH 4HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; Gradient: 15 B to 40 B in 8 min; 254; 220 nm; RT: 7.2) Purify the crude product. Fractions containing the desired compound were evaporated to dryness to give 6-fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H- Quinoa Lin-6-yl] methyl] piper-1-yl] pyridine-2-formamide ( [synthesis example]

[17] , 66.0 mg, 33.9%). 1H NMR (400 MHz, DMSO-d 6) δ 2.55 - 2.69 (m, 4H), 2.77 (d, 3H), 3.15 - 3.23 (m, 4H), 3.69 (s, 2H), 7.33 - 7.46 (m, 2H), 7.58 (dd, 1H), 7.78 – 7.93 (m, 2H), 8.37 - 8.42 (m, 1H), 12.99 (s, 1H); 19F NMR (376 MHz, DMSO-d 6) δ -68.36, -72.52; m / z (ES +) [M+H] +=465.

[0235]

[0236] [ , intermediate , ] [ , 54 , ] : Methyl 2-aminovalerate hydrochloride SOCl (17 mL, 232.94 mmol) was added dropwise to 2-aminovaleric acid ( [intermediate]

[53] , 10.0 g, 85.36 mmol). The resulting mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to give methyl 2-aminovalerate hydrochloride as a white solid ( [intermediate]

[54] , 15.78 g, 110%). 1H NMR (DMSO-d 6, 400 MHz) δ 0.88 (3H, t), 1.19 – 1.51 (2H, m), 1.67 – 1.83 (2H, m), 3.74 (3H, s), 3.89 - 3.93 (1H, m), 8.64 (3H, s); m / z (ES+) [M+H] +=132.

[0237] [ , intermediate , ] [ , 55 , ] : methyl 4-(1-methoxy-1-pentoxypent-2-ylamino)-3-nitrobenzoate Sodium bicarbonate (20.0 g, 238.08 mmol) was added to methyl 2-aminovalerate hydrochloride in THF ( [intermediate]

[54] , 15.57 g, 92.88 mmol) and methyl 4-fluoro-3-nitrobenzoate (9.0 g, 45.19 mmol) (160 mL). The resulting mixture was stirred at room temperature for 18 hours. Solvent was removed under reduced pressure. The reaction mixture was diluted with EtOAc (150 mL), washed successively with water (100 mL x 1), saturated NaHCO 3 (100 mL x 1) and saturated brine (100 mL x 1). The organic layer was dried over Na2SO4, filtered and evaporated to give methyl 4-(1-methoxy-1-oxopent-2-ylamino)-3-nitrobenzoate as a yellow oil ( [intermediate]

[55] , 14.09 g, 100%). 1H NMR (400 MHz, DMSO-d 6) δ 0.89 (3H, t), 1.26 – 1.41 (2H, m), 1.84 – 1.94 (2H, m), 3.73 (3H, s), 3.83 (3H, s) , 4.68 - 4.75 (1H, m), 7.12 (1H, d), 8.00 (1H, d), 8.60 (1H, d), 8.63 (1H, d); m / z (ES+) [M+H] + =311.

[0238] [ , intermediate , ] [ , 56 , ] : 3-oxo-2-propyl-1,2,3,4-tetrahydroquinone Methyl phen-6-carboxylate Pd(OH)2 / C (20% wt, 1.58 g, 2.25 mmol) was added to 4-((1-methoxy-1-oxopent-2-yl)amine in MeOH (300 mL) Base)-3-Nitrobenzoic acid methyl ester ( [intermediate]

[55] , 14.05 g, 45.28 mmol). The resulting mixture was stirred at room temperature under H2 for 30 h. The reaction mixture was filtered. The precipitate was washed with DMF (100 mL) and the filtrate was evaporated to dryness to give crude product. The crude product was washed with DCM (10 mL) and dried under vacuum to give 3-oxo-2-propyl-1,2,3,4-tetrahydroquinone as a white solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[56] , 9.12 g, 81%). 1H NMR (400 MHz, DMSO-d 6) δ 0.87 (3H, t), 1.32 – 1.46 (2H, m), 1.57 - 1.64 (2H, m), 3.74 (3H, s), 3.88 - 3.93 (1H, m), 6.70 (1H, d), 6.83 (1H, d), 7.32 (1H, d), 7.40 (1H, dd), 10.38 (1H, s); m / z (ES +) [M+H] +=249.

[0239] [ , intermediate , ] [ , 57 , ] : 3-oxo-2-propyl-3,4-dihydroquinone Methyl phen-6-carboxylate Add DDQ (9.42 g, 41.50 mmol) to 1,4-di 3-oxo-2-propyl-1,2,3,4-tetrahydroquinoline of alkanes (200 mL) Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[56] , 9.12 g, 36.73 mmol). The resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with saturated NaHCO 3 (200 mL). The resulting mixture was stirred at room temperature for 0.5 hours. The precipitate was collected by filtration, washed with water (1000 mL) and dried under vacuum to give 3-oxo-2-propyl-3,4-dihydroquinone as an off-white solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[57] , 7.86 g, 87%). 1H NMR (400 MHz, DMSO-d 6) δ 0.98 (3H, t), 1.68 - 1.80 (2H, m), 2.75 - 2.83 (2H, m), 3.89 (3H, s), 7.73 - 7.85 (2H, m), 7.88 (1H, d), 12.45 (1H, s); m / z (ES +) [M+H] +=247.

[0240] [ , intermediate , ] [ , 58 , ] : 7-(hydroxymethyl)-3-propylquinoline Lin-2(1H)-one At 0°C, a 1 M solution of DIBAL-H in THF (100 mL, 100.00 mmol) was added dropwise to 3-oxo-2-propyl-3,4-dihydroquinone in THF (200 mL). Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[57] , 7.81 g, 31.71 mmol). The resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with MeOH (5 mL) and saturated aqueous monopotassium monosodium tartrate tetrahydrate (20 mL), and the organic layer was evaporated to give 7-(hydroxymethyl)-3-propylquinoline as a white solid. Lin-2(1H)-one ( [intermediate]

[58] , 1.2 g, 17.34%). 1H NMR (400 MHz, DMSO-d 6) δ 0.97 (3H, t), 1.36 - 1.77 (2H, m), 2.71 - 2.79 (2H, m), 4.59 (2H, s), 5.39 (1H, s) , 7.18 (1H, dd), 7.27 (1H, d), 7.65 (1H, d), 12.30 (1H, s); m / z (ES +) [M+H] +=219.

[0241] [ , intermediate , ] [ , 59 , ] : 7-(bromomethyl)-3-propylquinoline Lin-2(1H)-one 33% HBr (74.6 µl, 1.37 mmol) in AcOH was added to 7-(hydroxymethyl)-3-propylquinoline Lin-2(1H)-one ( [intermediate]

[58] , 300 mg, 1.37 mmol). The resulting mixture was stirred at 80°C for 1 hour. The solvent was removed under reduced pressure to afford 7-(bromomethyl)-3-propylquinoline as a brown solid Lin-2(1H)-one ( [intermediate]

[59] , 600 mg, 155%) (the crude product was impure and contained AcOH and other impurities. The product was used in the next step without further purification. 1H NMR spectrum was unclear and uninterpreted; m / z (ES + ) [M+H] +=282.

[0242] [ , Synthesis example , ] [ , 18 , ] : N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinone phyllin-6-yl)methyl]piperone-1-yl]pyridine-2-formamide

[0243] Add DIPEA (200 µL, 1.15 mmol) to 7-(bromomethyl)-3-propylquine in NMP (3 mL) Lin-2(1H)-one ( [intermediate]

[59] , 200 mg, 0.71 mmol) and N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[13] , 80 mg, 0.36 mmol). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. By preparative HPLC (column: XBridge Shield RP18 OBD column, 19 x 250mm, 10um; mobile phase A: water (10 MMOL / L NH 4HCO 3 , 0.1% NH 3.H 2O), mobile phase B: ACN ; flow rate: 20 mL / min; gradient: 38 B to 50 B in 7 min; 254 / 220 nm; RT: 6.20) to purify the crude product. Fractions containing the desired compound were evaporated to dryness to give N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinone as a white solid Lin-6-yl) methyl] piper-1-yl] pyridine-2-formamide ( [synthesis example]

[18] , 71.0 mg, 46.5%). 1H NMR (400 MHz,DMSO-d 6) δ 0.97 (3H, t), 1.66 - 1.80 (2H, m), 2.55 - 2.61 (4H, m), 2.73 - 2.85 (5H, m), 3.33 - 3.40 ( 4H, m), 3.62 (2H, s), 7.19 – 7.31 (2H, m), 7.40 (1H, dd), 7.68 (1H, d), 7.83 (1H, d), 8.27 (1H, d), 8.35 - 8.45 (1H, m), 12.26 (1H, s); m / z (ES+) [M+H] +=421.

[0244]

[0245] [ , Synthesis example , ] [ , 19 , ] : 6-Chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoline phyllin-6-yl)methyl]piperone-1-yl]pyridine-2-formamide

[0246] Add DIPEA (200 µL, 1.15 mmol) to 7-(bromomethyl)-3-propylquinoline in NMP (3 mL) Lin-2(1H)-one ( [intermediate]

[59] , 200 mg, 0.71 mmol) and 6-chloro-N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[48] , 80 mg, 0.31 mmol). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. By preparative HPLC (column: XBridge Shield RP18 OBD column, 19 x 250 mm, 10 um; mobile phase A: water (0.1% HCO 2H), mobile phase B: ACN; flow rate: 20 mL / min; gradient : 18 B to 30 B in 7 min; 254 / 220 nm; RT: 5.93) to purify the crude product. Fractions containing the desired compound were evaporated to dryness to give 6-chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinone as a white solid Lin-6-yl) methyl] piper-1-yl] pyridine-2-formamide ( [synthesis example]

[19] , 52.0 mg, 36.4%). 1H NMR (400 MHz, DMSO-d 6) δ 0.97 (3H, t), 1.66 - 1.79 (2H, m), 2.55 - 2.65 (4H, m), 2.71 - 2.85 (5H, m), 3.06 - 3.12 ( 4H, m), 3.64 (2H, s), 7.20 – 7.32 (2H, m), 7.64 - 7.72 (2H, m), 7.94 (1H, d), 8.40 - 8.50 (1H, m), 12.27 (1H, s);m / z (ES +) [M+H] +=455.

[0247]

[0248] [ , Synthesis example , ] [ , 20 , ] : 6-fluoro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoline phyllin-6-yl)methyl]piperone-1-yl]pyridine-2-formamide

[0249] Add DIPEA (500 µl, 2.86 mmol) to 7-(bromomethyl)-3-propylquinoline in NMP (3 mL) Lin-2(1H)-one ( [intermediate]

[59] , 200 mg, 0.71 mmol) and 6-fluoro-N-methyl-5-(piper-1-yl)pyridinamide, 2 HCl ( [intermediate]

[23] , 100 mg, 0.32 mmol). The resulting mixture was stirred at 80°C for 1 hour. Solvent was removed under reduced pressure. By preparative HPLC (column: SunFire C18 OBD Prep column, 100 Å, 5 µm, 19 mm x 250 mm; mobile phase A: water (0.1% HCO 2H), mobile phase B: ACN; flow rate: 25 mL / min; Gradient: 10 B to 20 B in 13 min; 254 / 220 nm; RT: 12.13) Purify the crude product. Fractions containing the desired compound were evaporated to dryness to give 6-fluoro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinone as a white solid Lin-6-yl) methyl] piper-1-yl] pyridine-2-formamide ( [synthesis example]

[20] , 71.0 mg, 50.4%). 1H NMR (400 MHz, DMSO-d 6) δ 0.97 (3H, t), 1.66 - 1.78 (2H, m), 2.54 - 2.60 (4H, m), 2.71 - 2.83 (5H, m), 3.14 - 3.25 ( 4H, m), 3.62 (2H, s), 7.19 – 7.33 (2H, m), 7.57 (1H, dd), 7.68 (1H, d), 7.85 (1H, dd), 8.37 – 5.43 (1H, m) , 12.27 (1H, s); 19F NMR (376 MHz, DMSO-d 6) δ -72.51; m / z (ES +) [M+H] +=439.

[0250]

[0251] [ , intermediate , ] [ , 61 , ] : Methyl 2-aminobutyrate hydrochloride SOCl (17 mL, 232.94 mmol) was added dropwise to 2-aminobutyric acid ( [intermediate]

[60] , 10.0 g, 96.97 mmol). The resulting mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to give methyl 2-aminobutyrate hydrochloride as a white solid ( [intermediate]

[61] , 14.84 g, 100%). 1H NMR (400 MHz, DMSO-d 6) δ 0.91 (3H, t), 1.75 – 1.95 (2H, m), 3.73 (3H, s), 3.93 (1H, t), 8.72 (3H, s); m / z (ES +) [M+H] +=118.

[0252] [ , intermediate , ] [ , 62 , ] : Methyl 2-fluoro-4-(1-methoxy-1-oxobutan-2-ylamino)-5-nitrobenzoate Add DIPEA (4.02 mL, 23.03 mmol) to methyl 2,4-difluoro-5-nitrobenzoate (1.0 g, 4.61 mmol) and methyl 2-aminobutyrate in NMP (10 mL) Salt ( [intermediate]

[61] , 0.707 g, 4.61 mmol). The resulting mixture was stirred at rt for 5 hours. The crude product was purified by reverse phase chromatography with a gradient of 5 to 80% MeCN in water (0.1% NH 4 HCO 3 ). The pure fractions were evaporated to dryness to give methyl 2-fluoro-4-(1-methoxy-1-oxobutan-2-ylamino)-5-nitrobenzoate as a black solid ( [intermediate]

[62] , 1.2 g, 83%). 1H NMR (400 MHz, DMSO-d 6) δ 0.88 (3H, t), 1.78 – 2.03 (2H, m), 3.75 (3H, s), 3.83 (3H, s), 4.73 – 4.80 (1H, m) , 7.06 (1H, d), 8.66 – 8.72 (2H, m); m / z (ES +) [M+H] +=315.

[0253] [ , intermediate , ] [ , 63 , ] : 2-ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinone Methyl phen-6-carboxylate Under hydrogen, methyl 2-fluoro-4-((1-methoxy-1-oxobutan-2-yl)amino)-5-nitrobenzoate ( [intermediate]

[62] , 1.15 g, 3.66 mmol) was added to 20 wt% Pd(OH) 2 (500 mg, 0.71 mmol) in MeOH (300 mL) and ethyl acetate (50 mL). The resulting mixture was stirred at room temperature for 3 days. The reaction is not complete. The reaction mixture was filtered. Evaporation of the organic layer gave the crude product, 2-ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinone, as a brown gum Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[63] , 0.780 g, 85%). The crude product was used directly in the next step without further purification. The crude product was not clear and the 1HNMR spectrum was uninterpreted; m / z (ES+) [M+H]+=253.

[0254] [ , intermediate , ] [ , 64 , ] : 2-ethyl-7-fluoro-3-oxo-3,4-dihydroquinone Methyl phen-6-carboxylate 2-Ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinone Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[63] , 760 mg, 3.01 mmol) was added to DDQ (821 mg, 3.62 mmol) in DCM (20 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction is complete. The resulting mixture was concentrated under reduced pressure to obtain a brown solid. Sat. aq. NaHCO 3 (10 mL) was added to the solid and stirred at room temperature for 1 h. The precipitate was filtered and rinsed with additional aqueous NaHCO 3 (10 mL x 5). The solid was dried under vacuum to give 2-ethyl-7-fluoro-3-oxo-3,4-dihydroquinone as a brown solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[64] , 750 mg, 99%). 1H NMR (300 MHz, DMSO-d 6) δ 1.20 (3 H, t), 2.82 (2 H, q), 3.87 (3 H, s), 7.65 (1 H, d), 7.76 (1 H, d ), 12.42 (1 H, s); m / z (ES +) [M+H] +=251.

[0255] [ , intermediate , ] [ , 65 , ] : 3-ethyl-6-fluoro-7-(hydroxymethyl)quinone Lin-2(1H)-one A 1 M solution of diisobutylaluminum hydride in THF (15.35 mL, 15.35 mmol) was added in portions to 2-ethyl-7-fluoro-3-oxo-3,4-di Hydroquinone Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[64] , 640 mg, 2.56 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction is complete. The reaction mixture was quenched at 0 °C with saturated aqueous potassium sodium tartrate (20 mL) and MeOH (10 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered and washed with THF (50 mL x 3). The organic layer was evaporated to dryness to give crude product. The crude product was purified by reverse phase chromatography with a gradient of 5 to 60% MeOH in water (0.4% HCO 2H). Evaporation of the pure fractions to dryness afforded 3-ethyl-6-fluoro-7-(hydroxymethyl)quinone as an off-white solid Lin-2(1H)-one ( [intermediate]

[65] , 110 mg, 19.37%). 1H NMR (400 MHz, DMSO-d 6) δ 1.21 (3H, t), 2.80 (2H, q), 4.63 (2H, d), 5.49 (1H, t), 7.41 (1H, d), 7.49 (1H , d), 12.36 (1H, s); m / z (ES +) [M+H] +=223.

[0256] [ , Synthesis example , ] [ , 21 , ] : 5-[4-[(2-Ethyl-7-fluoro-3-oxo-4H-quinone Phenyl-6-yl)methyl]piperyl-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide

[0257] 3-Ethyl-6-fluoro-7-(hydroxymethyl)quinone Lin-2(1H)-one ( [intermediate]

[65] , 50 mg, 0.23 mmol) was added to 33% HBr in AcOH (2 mL, 12.15 mmol). The resulting mixture was stirred at 80°C for 2 hours. The reaction mixture was evaporated under vacuum to give 7-(bromomethyl)-3-ethyl-6-fluoroquine Lin-2(1H)-one (crude product). The product was used directly in the next step without further purification. Add DIPEA (0.196 mL, 1.13 mmol) to 7-(bromomethyl)-3-ethyl-6-fluoroquine in NMP (2 mL) Lin-2(1H)-one and 6-fluoro-N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[23] , 70 mg, 0.29 mmol). The resulting mixture was stirred at 80°C for 2 hours. The resulting mixture was analyzed by preparative HPLC (column: Sunfire prep C18 column, 30 x 150 mm, 5 um; mobile phase A: water (0.1% HCO 2H), mobile phase B: ACN; flow rate: 60 mL / min ; gradient: 10 B to 35 B in 8 min; 254 / 220 nm; RT: 7.37) for purification. Fractions containing the desired compound were evaporated to dryness to give 5-[4-[(2-ethyl-7-fluoro-3-oxo-4H-quinone as an off-white solid Lin-6-yl)methyl]piperone-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide ( [synthesis example]

[21] , 55.0 mg, 53.7%). 1H NMR(400 MHz, DMSO-d 6) δ 1.21 (3H, t), 2.61 (4H, m), 2.73 – 2.85 (5H, m), 3.18 (4H, m), 3.68 (2H, s), 7.38 (1H, d), 7.51 – 7.61 (2H, m), 7.84 (1H, dd), 8.13 (0.29H, s), 8.38 (1H, m), 12.29 (1H, s); 19F NMR (376 MHz, DMSO-d 6) δ -72.53, -124.31; m / z (ES+) [M+H] +=443.

[0258]

[0259] [ , intermediate , ] [ , 67 , ] : methyl 4-(3-hydroxy-1-methoxy-1-oxobutan-2-ylamino)-3-nitrobenzoate Add DIPEA (8.77 mL, 50.22 mmol) to methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and methyl 2-amino-3-hydroxybutyrate in DMF (20 mL) Hydrochloride( [intermediate]

[66] , 2.04 g, 12.05 mmol). The resulting mixture was stirred at rt for 16 hours. The reaction mixture was diluted with EtOAc (100 mL), and washed sequentially with saturated aqueous NH 4 Cl (100 mL×1), and brine (100 mL×4). The organic layer was dried over Na2SO4, filtered and evaporated to give the desired product as a yellow solid 4-((3-hydroxy-1-methoxy-1-oxobutan-2-yl)amino)- Methyl 3-nitrobenzoate ( [intermediate]

[67] , 2.9 g, 92%). 1H NMR (400 MHz, DMSO-d 6) δ 1.15 – 1.27 (3H, m), 3.64 – 3.74 (3H, m), 3.83 (3H, s), 4.08 – 4.44 (1H, m), 4.61 – 4.72 ( 1H, m), 5.39 – 5.60 (1H, m), 7.03 – 7.15 (1H, m), 7.90 – 8.03 (1H, m), 8.62 – 8.69 (1H, m), 8.73 – 8.89 (1H, m); m / z (ES+) [M+H] +=313.

[0260] [ , intermediate , ] [ , 68 , ] : 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinone Methyl phen-6-carboxylate Under hydrogen, 20% Pd(OH)2 / C (0.648 g, 0.92 mmol) was added to 4-((3-hydroxy-1-methoxy-1-oxobutane) in MeOH (300 mL) -2-yl)amino)-3-nitrobenzoic acid methyl ester ( [intermediate]

[67] , 2.88 g, 9.22 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction is complete. The reaction mixture was filtered through celite. Evaporation of the organic layer gave 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinone as a gray solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[68] , 2.290 g, 99%). 1H NMR (400 MHz, DMSO-d 6) δ 1.07 (3H, m), 2.81 (1H, d), 3.72 (1H, m), 3.74 (3H, s), 4.78 (1H, d), 6.70 – 6.86 (2H, m), 7.27 (1H, d), 7.37 (1H, dd), 10.38 (1H, d); m / z (ES +) [M+H] +=251.

[0261] [ , intermediate , ] [ , 69 , ] : 2-(1-hydroxyethyl)-3-oxo-3,4-dihydroquinone Methyl phen-6-carboxylate DDQ (2.265 g, 9.98 mmol) was added to 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinone in DCM (100 mL) Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[68] , 2.27 g, 9.07 mmol). The resulting mixture was stirred at room temperature for 1 hour. The reaction is complete. The reaction mixture was concentrated under reduced pressure to obtain a brown solid. Sat. aq. NaHCO 3 (100 mL) was added to the solid and stirred at room temperature for 1 h. The precipitate was filtered and rinsed with additional aq NaHCO 3 solution (30 mL x 3). The solid was dried under vacuum to give 2-(1-hydroxyethyl)-3-oxo-3,4-dihydroquinone as a gray solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[69] , 2.24 g, 99%). 1H NMR (400 MHz, DMSO-d 6 ) δ 1.40 (3H, d), 3.88 (3H, s), 4.94 (1H, q), 7.69 (1H, dd), 7.77 (1H, d), 7.90 (1H , d) (2 protons not shown); m / z (ES +) [M+H] +=249.

[0262] [ , intermediate , ] [ , 70 , ] [ , : , ] 2-Acetyl-3-oxo-3,4-dihydroquinone Methyl phen-6-carboxylate Add Dess-Martin periodinane (2.56 g, 6.04 mmol) to 2-(1-hydroxyethyl)-3-oxo-3,4-dihydroquinone in DCM (30 mL) Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[69] , 1.0 g, 4.03 mmol). The resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was evaporated to give crude product. The crude product was purified by reverse phase chromatography with a gradient of 5 to 30% MeCN in water (0.4% HCO 2H). Evaporation of the pure fractions to dryness afforded 2-acetyl-3-oxo-3,4-dihydroquinone as a pale yellow solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[70] , 0.62 g, 62.5%). 1H NMR (400 MHz, DMSO-d 6) δ 2.58 (3H, s), 3.91 (3H, s), 7.84 (1H, dd), 7.91 – 8.03 (2H, m), 12.86 (1H, s); m / z (ES +) [M+H] +=247.

[0263] [ , intermediate , ] [ , 71 , ] : 2-(1,1-difluoroethyl)-3-oxo-3,4-dihydroquinone Methyl phen-6-carboxylate BAST (1.35 mL, 7.31 mmol) was added to 2-acetyl-3-oxo-3,4-dihydroquinone in DCM (20 mL) Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[70] , 600 mg, 2.44 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was evaporated to give crude product. The crude product was purified by reverse phase chromatography with a gradient of 5 to 30% MeCN in water (0.4% HCO 2H). Evaporation of the pure fractions to dryness afforded 2-(1,1-difluoroethyl)-3-oxo-3,4-dihydroquinone as an off-white solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[71] , 174 mg, 26.6%). 1H NMR (400 MHz, DMSO-d 6 ) δ 2.07 (3H, t), 3.91 (3H, s), 7.84 (1H, dd), 7.92 – 7.99 (2H, m), 12.90 (1H, s); 19F NMR (376 MHz, DMSO-d 6) δ -93.26; m / z (ES +) [M+H] +=269.

[0264] [ , intermediate , ] [ , 72 , ] : 3-(1,1-difluoroethyl)-7-(hydroxymethyl)quinone Lin-2(1H)-one A 1M solution of diisobutylaluminum hydride in THF (2.39 mL, 2.39 mmol) was added to 2-(1,1-difluoroethyl)-3-oxo in THF (50 mL) at 0 °C. -3,4-dihydroquinone Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[71] , 160 mg, 0.60 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous potassium sodium tartrate (3 mL) and MeOH (1 mL) at 0 °C. The resulting mixture was stirred for 1 hour. The reaction mixture was filtered and washed with THF (10 mL x 3). The organic layer was evaporated to give the crude product, 3-(1,1-difluoroethyl)-7-(hydroxymethyl)quinone Lin-2(1H)-one ( [intermediate]

[72] , 120 mg, 84%). The product was used directly in the next step without further purification. 1H NMR (400 MHz, DMSO-d 6 ) δ 2.06 (3H, t), 4.63 (2H, s), 5.47 (1H, s), 7.26 (1H, dd), 7.35 (1H, d), 7.78 (1H , d), 12.75 (1H, br s); m / z (ES+) [M+H] +=241.

[0265] [ , Synthesis example , ] [ , 22 , ] : 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinone Lin-6-yl] methyl] piper-1-yl]-N-methyl-pyridine-2-formamide

[0266] 3-(1,1-difluoroethyl)-7-(hydroxymethyl)quinone Lin-2(1H)-one ( [intermediate]

[72] , 60 mg, 0.25 mmol) was added to 33% HBr in acetic acid (2 mL, 12.15 mmol). The resulting mixture was stirred at 80°C for 2 hours. The reaction mixture was evaporated under vacuum to give 7-(bromomethyl)-3-(1,1-difluoroethyl)quinone Lin-2(1H)-one (crude product). The product was used directly in the next step without further purification. Add DIPEA (0.218 mL, 1.25 mmol) to 7-(bromomethyl)-3-(1,1-difluoroethyl)quinone in NMP (3 mL) Lin-2(1H)-one (crude product) and N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[13] , 60 mg, 0.27 mmol). The resulting mixture was stirred at 80°C for 1 hour. The reaction mixture was concentrated and analyzed by preparative HPLC (column: XBridge Shield RP18 OBD column, 30 x 150 mm, 5um; mobile phase A: water (0.05% NH 3 H 2 O), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 13 B to 33 B in 7 min; 254; 220 nm; RT: 5.70) for purification. Fractions containing the desired compound were evaporated to dryness to give 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinone as a yellow solid Lin-6-yl] methyl] piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example]

[22] , 47.8 mg, 43.2%). 1H NMR (400 MHz, DMSO-d 6) δ 2.06 (3H, t), 2.52 - 2.62 (4H, m), 2.78 (3H, d), 3.30 - 3.40 (4H, m), 3.67 (2H, s) , 7.32 - 7.42 (3H, m), 7.80 - 7.86 (2H, m), 8.27 (1H, d), 8.34 - 8.42 (1H, m), 12.70 (1H, s); 19F NMR (376 MHz, DMSO- d 6) δ -92.74; m / z (ES +) [M+H] +=443.

[0267]

[0268] [ , intermediate , ] [ , 74 , ] : methyl 4-(4,4-difluoro-1-methoxy-1-oxobutan-2-ylamino)-3-nitrobenzoate DIPEA (8.77 mL, 50.22 mmol) was added to methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and 2-amino-4,4-difluorobutyl in DMF (20 mL) Acid methyl ester hydrochloride ( [intermediate]

[73] , 2.0 g, 10.55 mmol). The resulting mixture was stirred at 40°C for 8 hours. The reaction mixture was diluted with EtOAc (100 mL), washed successively with saturated NH 4 Cl (100 mL×1) and brine (100 mL×4). The organic layer was dried over Na2SO4, filtered and evaporated to give the desired product as 4-((4,4-difluoro-1-methoxy-1-oxobutan-2-yl)amine as a yellow solid Base)-3-Nitrobenzoic acid methyl ester ( [intermediate]

[74] , 2.5 g, 74.9%). 1H NMR (300 MHz, DMSO-d 6) δ 2.50 – 2.76 (2H, m), 3.71 (3H, s), 3.82 (3H, s), 4.95 (1H, q), 6.22 (1H, tt), 7.18 (1H, d), 7.99 (1H, dd), 8.63 (1H, d), 8.66 (1H, d); m / z (ES+) [M+H] +=333.

[0269] [ , intermediate , ] [ , 75 , ] : 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinone Methyl phen-6-carboxylate Under hydrogen, 20% Pd(OH)2 / C (0.465 g, 0.66 mmol) was added to the 4-((4,4-difluoro-1-methoxy-1-side) in MeOH (300 mL) Oxybut-2-yl)amino)-3-nitrobenzoic acid methyl ester ( [intermediate]

[74] , 2.2 g, 6.62 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered through celite. The filtrate was evaporated to give 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinone as a yellow solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[75] , 1.64 g, 92%). 1H NMR (400 MHz, DMSO-d 6) δ 2.24 - 2.32 (2H, m), 3.76 (3H, s), 4.10 - 4.18 (1H, m), 6.27 (1H, tt), 6.73 (1H, d) , 6.89 (1H, s), 7.37 (1H, d), 7.44 (1H, dd), 10.58 (1H, s); m / z (ES +) [M+H] +=271.

[0270] [ , intermediate , ] [ , 76 , ] : 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinone Methyl phen-6-carboxylate DDQ (1.478 g, 6.51 mmol) was added to 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinone in DCM (100 mL) Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[75] , 1.6 g, 5.92 mmol). The resulting mixture was stirred at room temperature for 3 hours. The resulting mixture was removed under reduced pressure to obtain a brown solid. Aq NaHCO 3 saturated solution (100 mL) was added to the solid and stirred at room temperature for 1 h. The precipitate was filtered and rinsed with additional aq NaHCO 3 solution (30 mL x 3). The solid was dried under vacuum to give 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinone as an off-white solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[76] , 1.58 g, 99%). 1H NMR (400 MHz, DMSO-d 6) δ 3.46 (2H, td), 3.90 (3H, s), 6.57 (1H, t), 7.79 – 7.92 (3H, m), 12.68 (1H, s); m / z (ES +) [M+H] +=269.

[0271] [ , intermediate , ] [ , 77 , ] : 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinone Lin-2(1H)-one At 0°C, a 1 M solution of diisobutylaluminum hydride in THF (22.37 mL, 22.37 mmol) was added in portions to 2-(2,2-difluoroethyl)-3- Pendantoxy-3,4-dihydroquinone Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[76] , 1.0 g, 3.73 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched at 0 °C with saturated aqueous potassium sodium tartrate (20 mL) and MeOH (10 mL). The resulting mixture was stirred for 1 hour. The reaction mixture was filtered and washed with THF (30 mL x 3). Evaporation of the organic layer gave 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinone as a red solid (crude product). Lin-2(1H)-one (0.72 g, 80 %). The crude product was purified by reverse phase chromatography with a gradient of 5 to 60% MeOH in water (0.4% HCO 2H). The pure fractions were evaporated to dryness to give 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinone as a red solid Lin-2(1H)-one ( [intermediate]

[77] , 500 mg, 69.4%). 1H NMR (300 MHz, DMSO-d 6 ) δ 3.42 (2H, td), 4.61 (2H, s), 5.42 (1H, brs), 6.56 (1H, tt), 7.23 (1H, dd), 7.32 (1H , d), 7.71 (1H, d), 12.55 (1H, s); m / z (ES+) [M+H] +=241.

[0272] [ , intermediate , ] [ , 78 , ] : 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinone Phenyl-6-carbaldehyde Add Dess-Martin periodinane (530 mg, 1.25 mmol) to 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinone in DCM (5 mL) Lin-2(1H)-one ( [intermediate]

[77] , 200 mg, 0.83 mmol). The resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was evaporated to give crude product. The crude product was purified by reverse phase chromatography with a gradient of 5 to 30% MeCN in water (0.4% HCO 2H). The pure fractions were evaporated to dryness to give 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinone as a yellow solid Phyloline-6-carbaldehyde ( [intermediate]

[78] , 160 mg, 81%). 1H NMR (400 MHz, DMSO-d 6) δ 3.47 (2H, td), 6.58 (1H, tt), 7.77 - 7.85 (2H, m), 7.90 - 7.98 (1H, m), 10.09 (1H, s) , 12.79 (1H, s); m / z (ES +) [M+H] +=239.

[0273] [ , Synthesis example , ] [ , 23 , ] : 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinone Lin-6-yl] methyl] piper-1-yl]-N-methyl-pyridine-2-formamide

[0274] Titanium isopropoxide (65.6 mg, 0.23 mmol) was added to 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinone in THF (2 mL) Phyloline-6-carbaldehyde ( [intermediate]

[78] , 55 mg, 0.23 mmol) and N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[13] , 60 mg, 0.23 mmol). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetyloxyborohydride (196 mg, 0.92 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with MeOH (0.1 mL). The reaction mixture was evaporated to obtain the crude product, which was analyzed by preparative HPLC (column: XBridge Shield RP18 OBD column, 30 x 150mm, 5um; mobile phase A: water (0.05% NH 3 H 2 O), mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 13 B to 33 B in 7 min; 254; 220 nm; RT: 5.70) for purification. Fractions containing the desired compound were evaporated to dryness to give 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinone as a yellow solid Lin-6-yl] methyl] piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example]

[23] , 8.76 mg, 8.57%). 1H NMR (400 MHz, DMSO-d 6) δ 2.56 (4H, m), 2.78 (3H, d), 3.32 - 3.48 (6H, m), 3.64 (2H, s), 6.55 (1H, tt), 7.27 - 7.33 (2H, m), 7.39 (1H, dd), 7.73 (1H, d), 7.83 (1H, d), 8.26 (1H, d), 8.37 (1H, m), 12.49 (1H, s); 19F NMR (376 MHz, DMSO-d 6) δ -114.29; m / z (ES +) [M+H] +=443.

[0275]

[0276] [ , Synthesis example , ] [ , 24 , ] : 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinone Phylin-6-yl]methyl]piperone-1-yl]-6-fluoro-N-methyl-pyridine-2-formamide

[0277] Titanium isopropoxide (59.7 mg, 0.21 mmol) was added to 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinone in THF (2 mL) Phyloline-6-carbaldehyde ( [intermediate]

[78] , 50 mg, 0.21 mmol) and 6-fluoro-N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[23] , 50.0 mg, 0.21 mmol). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetyloxyborohydride (178 mg, 0.84 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour. The reaction is complete. The reaction mixture was quenched with MeOH (0.1 mL). The reaction mixture was evaporated to give crude product. By preparative HPLC (column: Sunfire prep C18 column, 30 x 150, 5 um; mobile phase A: water (0.1% HCO 2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: in 2B to 27B in 7 min; 254 / 220 nm; RT: 6.78) to purify the crude product. Fractions containing the desired compound were evaporated to dryness to give 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinone as a yellow solid Lin-6-yl] methyl] piper-1-yl] -6-fluoro-N-methyl-pyridine-2-formamide ( [synthesis example]

[24] , 21.72 mg, 22.13%). 1H NMR (400 MHz, DMSO-d 6) δ 2.54 – 2.61 (4H, m), 2.76 (3H, d), 3.14 – 3.22 (4H, m), 3.41 (2H, td), 3.64 (2H, s) , 6.39 – 6.71 (1H, m), 7.26 – 7.33 (2H, m), 7.57 (1H, dd), 7.73 (1H, d), 7.82 – 7.86 (1H, m), 8.13 (0.16H, s), 8.37 (1H, m), 12.49 (1H, s); 19F NMR (376 MHz, DMSO-d 6) δ -72.52, -114.29; m / z (ES +) [M+H] +=461.

[0278]

[0279] [ , intermediate , ] [ , 80 , ] : methyl 4-(4-fluoro-1-methoxy-1-oxobutan-2-ylamino)-3-nitrobenzoate Add DIPEA (8.77 mL, 50.22 mmol) to methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and methyl 2-amino-4-fluorobutyrate in DMF (20 mL) Hydrochloride( [intermediate]

[79] , 1.81 g, 10.55 mmol). The resulting mixture was stirred at 40°C for 8 hours. The reaction mixture was diluted with EtOAc (100 mL), and washed sequentially with saturated NH 4 Cl (100 mL×1) and brine (100 mL×4). The organic layer was dried over Na2SO4, filtered and evaporated to give the desired product as a yellow solid 4-((4-fluoro-1-methoxy-1-oxobutan-2-yl)amino)- Methyl 3-nitrobenzoate ( [intermediate]

[80] , 2.5 g, 79%). 1H NMR (300 MHz, DMSO-d 6) δ 2.25 – 2.35 (1H, m), 2.35 – 2.45 (1H, m), 3.71 (3H, s), 3.82 (3H, s), 4.36 – 4.58 (1H, m), 4.56 – 4.74 (1H, m), 4.84 (1H, q), 7.14 (1H, d), 7.99 (1H, dd), 8.63 (1H, d), 8.67 (1H, d); m / z (ES+) [M+H]+=315.

[0280] [ , intermediate , ] [ , 81 , ] : 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinone Methyl phen-6-carboxylate Under hydrogen, 20% Pd(OH)2 / C (0.547 g, 0.78 mmol) was added to 4-((4-fluoro-1-methoxy-1-oxobutane) in MeOH (300 mL) -2-yl)amino)-3-nitrobenzoic acid methyl ester ( [intermediate]

[80] , 2.45 g, 7.80 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction is complete. The reaction mixture was filtered through celite. Evaporation of the filtrate gave 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinone as a gray solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[81] , 1.9 g, 97%). 1H NMR (400 MHz, DMSO-d 6) δ1.91 – 2.19 (2H, m), 3.75 (3H, s), 4.03 (1H, m), 4.49 – 4.73 (2H, m), 6.73 (1H, d ), 6.91 (1H, d), 7.35 (1H, d), 7.42 (1H, dd), 10.46 (1H, s); m / z (ES +) [M+H] +=253.

[0281] [ , intermediate , ] [ , 82 , ] : 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinone Methyl phen-6-carboxylate DDQ (1.83 g, 8.07 mmol) was added to 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinone in DCM (100 mL) Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[81] , 1.85 g, 7.33 mmol). The resulting mixture was stirred at room temperature for 3 hours. The resulting mixture was removed under reduced pressure to obtain a brown solid. Aq. NaHCO 3 saturated solution (100 mL) was added to the solid and stirred at room temperature for 1 h. The precipitate was filtered and rinsed with additional aq NaHCO 3 solution (30 mL x 3). The solid was dried under vacuum to give 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinone as a gray solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[82] , 1.8 g, 98%). 1H NMR (400 MHz, DMSO-d 6 ) δ 3.23 (2H, dt), 3.89 (3H, s), 4.90 (2H, dt), 7.76 – 7.85 (2H, m), 7.88 (1H, d), 12.55 (1H, s);m / z (ES +) [M+H] +=251.

[0282] [ , intermediate , ] [ , 83 , ] : 3-(2-fluoroethyl)-7-(hydroxymethyl)quinone Lin-2(1H)-one A 1 M solution of diisobutylaluminum hydride in THF (15.99 mL, 15.99 mmol) was added portionwise to 2-(2-fluoroethyl)-3-oxo in THF (100 mL) at 0 °C. -3,4-dihydroquinone Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[82] , 1.0 g, 4.00 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched at 0 °C with saturated aqueous potassium sodium tartrate (20 mL) and MeOH (10 mL). The resulting mixture was stirred for 1 hour. The reaction mixture was filtered and washed with THF (30 mL x 3). The organic layer was evaporated to give crude product. The crude product was purified by reverse phase chromatography with a gradient of 5 to 60% MeOH in water (0.4% HCO 2H). Evaporation of the pure fractions to dryness afforded 3-(2-fluoroethyl)-7-(hydroxymethyl)quinone as a brown solid Lin-2(1H)-one ( [intermediate]

[83] , 0.49 g, 55.2%). 1H NMR (300 MHz, DMSO-d 6 ) δ 3.20 (2H, dt), 4.60 (2H, d), 4.90 (2H, dt), 5.41 (1H, t), 7.21 (1H, dd), 7.30 (1H , d), 7.68 (1H, d), 12.42 (1H, s); m / z (ES+) [M+H] +=223.

[0283] [ , intermediate , ] [ , 84 , ] : 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinone Phenyl-6-carbaldehyde Add Dess-Martin periodinane (229 mg, 0.54 mmol) to 3-(2-fluoroethyl)-7-(hydroxymethyl)quinone in DCM (3 mL) Lin-2(1H)-one ( [intermediate]

[83] , 100 mg, 0.45 mmol). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated to give crude product. The crude product was purified by reverse phase chromatography with a gradient of 5 to 30% MeCN in water (0.4% HCO 2H). The pure fractions were evaporated to dryness to give 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinone as a yellow solid Phyloline-6-carbaldehyde ( [intermediate]

[84] , 93 mg, 94%). 1H NMR (300 MHz, DMSO-d 6) δ 3.20 – 3.28 (2H, m), 4.90 (2H, dt), 7.74 – 7.80 (2H, m), 7.91 (1H, d), 10.06 (1H, s) , 12.66 (1H, s); m / z (ES+) [M+H] +=221.

[0284] [ , Synthesis example , ] [ , 25 , ] : 5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinone Lin-6-yl] methyl] piper-1-yl]-N-methyl-pyridine-2-formamide

[0285] Titanium isopropoxide (64.5 mg, 0.23 mmol) was added to 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinone in THF (3 mL) Phyloline-6-carbaldehyde ( [intermediate]

[84] , 50 mg, 0.23 mmol) and N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[13] , 50.0 mg, 0.23 mmol). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetyloxyborohydride (192 mg, 0.91 mmol) was added. The resulting mixture was stirred at room temperature for 2 hours. Repeat this step in another batch and combine the two batches for purification. The combined reaction mixture was analyzed by preparative HPLC (column: XBridge Prep OBD C18 column, 30 × 150mm 5um; mobile phase A: water (10 MMOL / L NH 4HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 20 B to 35 B in 7 min; 254 / 210 nm; RT: 6.38) for purification. Fractions containing the desired compound were evaporated to dryness to give 5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinone as a white solid Lin-6-yl] methyl] piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example]

[25] , 4.83 mg, 2.54%). 1H NMR (400 MHz, DMSO-d 6) δ 2.53 – 2.59 (4H, m), 2.78 (3H, d), 3.17 (1H, t), 3.23 (1H, t), 3.32 – 3.38 (4H, m) , 3.63 (2H, s), 4.83 (1H, t), 4.95 (1H, t), 7.25 - 7.32 (2H, m), 7.39 (1H, dd), 7.71 (1H, d), 7.83 (1H, d ), 8.26 (1H, d), 8.37 (1H, d), 12.36 (1H, s); 19F NMR (376 MHz, DMSO-d 6) δ -217.70; m / z (ES +) [M+H] +=425.

[0286]

[0287] [ , Synthesis example , ] [ , 26 , ] : 6-fluoro-5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinone Lin-6-yl] methyl] piper-1-yl]-N-methyl-pyridine-2-formamide

[0288] Titanium isopropoxide (90 mg, 0.32 mmol) was added to 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinone in THF (3 mL) Phyloline-6-carbaldehyde ( [intermediate]

[84] , 70 mg, 0.32 mmol) and 6-fluoro-N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[23] , 76 mg, 0.32 mmol). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetyloxyborohydride (269 mg, 1.27 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with MeOH (0.1 mL). The reaction mixture was evaporated to give crude product. By preparative HPLC (column: XBridge Prep OBD C18 column, 30 × 150mm 5um; mobile phase A: water (10 MMOL / L NH 4HCO 3 ), mobile phase B: ACN; flow rate: 60 mL / min; gradient : 28 B to 35 B in 8 min; 254 / 210 nm; RT: 7) Purify the crude product. Fractions containing the desired compound were evaporated to dryness to give crude product. The crude product was analyzed by preparative HPLC (column: Xselect CSH OBD column 30*150mm 5um, n; mobile phase A: water (0.1% HCO 2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 5 B to 20 B in 7 min; 254; 220 nm; RT: 6.83) for further purification. Fractions containing the desired compound were evaporated to dryness to give 6-fluoro-5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinone as a yellow solid Lin-6-yl] methyl] piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example]

[26] , 3.79 mg, 2.65%). 1H NMR (400 MHz, DMSO-d 6) δ 2.55 – 2.60 (4H, m), 2.76 (3H, d), 3.14 – 3.25 (6H, m), 3.63 (2H, s), 4.89 (2H, dt) , 7.24 – 7.31 (2H, m), 7.57 (1H, dd), 7.70 (1H, d), 7.84 (1H, d), 8.24 (0.174H, s), 8.38 (1H, d), 12.37 (1H, s); 19F NMR (376 MHz, DMSO-d 6) δ -72.51, -217.71; (ES +) [M+H] +=443.

[0289]

[0290] [ , intermediate , ] [ , 86 , ] : methyl 3-nitro-4-(4,4,4-trifluoro-1-methoxy-1-oxobutan-2-ylamino)benzoate DIPEA (8.77 mL, 50.22 mmol) was added to methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and 2-amino-4,4,4-tris in DMF (20 mL) Methyl fluorobutyrate hydrochloride ( [intermediate]

[85] , 2.2 g, 10.55 mmol). The resulting mixture was stirred at 50°C for 10 hours. The reaction mixture was diluted with EtOAc (100 mL), and washed successively with saturated aqueous NH 4 Cl (100 mL×1) and brine (100 mL×4). The organic layer was dried over Na2SO4, filtered and evaporated to give the desired product as a yellow solid 3-nitro-4-((4,4,4-trifluoro-1-methoxy-1-oxo But-2-yl) amino) methyl benzoate ( [intermediate]

[86] , 3.0 g, 85%). 1H NMR (400 MHz, DMSO-d 6) δ 2.99 – 3.28 (2H, m), 3.73 (3H, s), 3.84 (3H, s), 5.18 (1H, td), 7.28 (1H, d), 8.01 (1H, dd), 8.65 (1H, d), 8.71 (1H, d); m / z (ES +) [M+H] +=351.

[0291] [ , intermediate , ] [ , 87 , ] : 3-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinone Methyl phen-6-carboxylate Under hydrogen, 20% Pd(OH)2 / C (0.601 g, 0.86 mmol) was added to 3-nitro-4-((4,4,4-trifluoro-1- Methoxy-1-oxobutan-2-yl)amino)methyl benzoate ( [intermediate]

[86] , 3.0 g, 8.57 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered through celite. The filtrate was evaporated to dryness to give 3-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinone as an off-white solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[87] , 2.3 g, 93%). 1H NMR (400 MHz, DMSO-d 6) δ 2.64 – 2.83 (2H, m), 3.76 (3H, s), 4.32 - 4.37 (1H, m), 6.78 (1H, d), 6.90 (1H, d) , 7.37 (1H, d), 7.43 (1H, dd), 10.64 (1H, s); m / z (ES +) [M+H] +=289.

[0292] [ , intermediate , ] [ , 88 , ] : 3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinone Methyl phen-6-carboxylate DDQ (1.975 g, 8.70 mmol) was added to 3-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinone in DCM (100 mL) Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[87] , 2.28 g, 7.91 mmol). The resulting mixture was stirred at room temperature for 3 hours. The resulting mixture was removed under reduced pressure to obtain a brown solid. Aq. NaHCO 3 saturated solution (100 mL) was added to the solid and stirred at room temperature for 1 h. The precipitate was filtered and rinsed with additional aqueous NaHCO 3 (30 mL x 3). The solid was dried under vacuum to give 3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinone as a brown solid Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[88] , 2.2 g, 97%). 1H NMR (400 MHz, DMSO-d 6) δ 3.88 – 3.98 (5H, m), 7.81 (1H, dd), 7.86 – 7.94 (2H, m), 12.75 (1H, s); m / z (ES + ) [M+H] +=287.

[0293] [ , intermediate , ] [ , 89 , ] : 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinoline Lin-2(1H)-one At 0°C, a 1 M solution of diisobutylaluminum hydride in THF (20.96 mL, 20.96 mmol) was added in batches to 3-oxo-2-(2,2,2- Trifluoroethyl)-3,4-dihydroquinone Phyloline-6-carboxylic acid methyl ester ( [intermediate]

[88] , 1.0 g, 3.49 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched at 0 °C with saturated aqueous potassium sodium tartrate (20 mL) and MeOH (10 mL). The resulting mixture was stirred for 1 hour. The reaction mixture was filtered and washed with THF (30 mL x 3). Evaporation of the organic layer gave an off-white solid, which was purified by flash silica chromatography with a gradient of 5 to 55% MeOH in water (0.4% HCO 2H). Evaporation of the pure fractions to dryness afforded 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinone as a yellow solid Lin-2(1H)-one ( [intermediate]

[89] , 650 mg, 72.2%). 1H NMR (300 MHz, DMSO-d 6 ) δ 3.88 (2 H, q), 4.62 (2H, d), 5.45 (1H, t), 7.24 (1H, dd), 7.33 (1H, d), 7.73 ( 1H, d), 12.62 (1H, s); m / z (ES +) [M+H] +=259.

[0294] [ , Synthesis example , ] [ , 27 , ] : N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinone Lin-6-yl] methyl] piper-1-yl] pyridin-2-formamide

[0295] 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinone Lin-2(1H)-one ( [intermediate]

[89] , 50 mg, 0.19 mmol) was added to 33% HBr in AcOH (2 mL, 12.15 mmol). The resulting mixture was stirred at 80°C for 2 hours. The reaction mixture was evaporated under vacuum to give 7-(bromomethyl)-3-(2,2,2-trifluoroethyl)quinone Lin-2(1H)-one (crude product). The product was used directly in the next step without further purification. Add DIPEA (0.169 mL, 0.97 mmol) to 7-(bromomethyl)-3-(2,2,2-trifluoroethyl)quinone in NMP (2 mL) Lin-2(1H)-one (crude product) and N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[13] , 50 mg, 0.23 mmol). The resulting mixture was stirred at 80°C for 1 hour. The reaction mixture was concentrated and analyzed by preparative HPLC (column: Sunfire prep C18 column, 30 x 150, 5 um; mobile phase A: water (0.1% HCO 2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 10 B to 25 B in 7 min; 254 / 220 nm; RT: 6.57) for purification. Fractions containing the desired compound were evaporated to dryness to give N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)- 4H-quinone Lin-6-yl] methyl] piper-1-yl] pyridine-2-formamide ( [synthesis example]

[27] , 41.5 mg, 46.6%). 1H NMR (400 MHz, DMSO-d 6) δ 2.56 (4H, m), 2.78 (3H, d), 3.35 (4H, m), 3.65 (2H, s), 3.88 (2H, q), 7.29 – 7.42 (3H, m), 7.79 (2H, m), 8.25 – 8.30 (1H, m), 8.38 (1H, m),12.60 (1H, br s); 19F NMR (376 MHz, DMSO-d 6) δ - 61.53; m / z (ES +) [M+H] +=461.

[0296]

[0297] [ , Synthesis example , ] [ , 28 , ] : 6-fluoro-N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoline Lin-6-yl] methyl] piper-1-yl] pyridin-2-formamide

[0298] 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinone Lin-2(1H)-one ( [intermediate]

[89] , 60 mg, 0.23 mmol) was added to 33% HBr in AcOH (2 mL, 12.15 mmol). The resulting mixture was stirred at 80°C for 2 hours. The reaction mixture was evaporated under vacuum to give 7-(bromomethyl)-3-(2,2,2-trifluoroethyl)quinone Lin-2(1H)-one (crude product). The product was used directly in the next step without further purification. Add DIPEA (0.203 mL, 1.16 mmol) to 7-(bromomethyl)-3-(2,2,2-trifluoroethyl)quinone in NMP (2 mL) Lin-2(1H)-one (crude product) and 6-fluoro-N-methyl-5-(piper-1-yl)pyridinamide ( [intermediate]

[23] , 60 mg, 0.25 mmol). The resulting mixture was stirred at 80°C for 2 hours. The resulting mixture was analyzed by preparative HPLC (column: Sunfire prep C18 column, 30 x 150, 5um; mobile phase A: water (0.1% HCO 2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient : 12 B to 30 B in 7 min; 254 / 220 nm; RT: 6.25) for purification. Fractions containing the desired compound were evaporated to dryness to give 6-fluoro-N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoro Ethyl)-4H-quinone Lin-6-yl] methyl] piper-1-yl] pyridine-2-formamide ( [synthesis example]

[28] , 49.0 mg, 43.3%). 1H NMR (400 MHz, DMSO-d 6) δ 2.53 - 2.63 (4H, m), 2.76 (3H, d), 3.15 - 3.22 (4H, m), 3.65 (2H, s), 3.88 (2H, q) , 7.28 - 7.35 (2H, m), 7.57 (1H, dd), 7.76 (1H, d), 7.84 (1H, dd), 8.17 (0.185H, s), 8.38 (1H, m), 12.57 (1H, s); 19F NMR (376 MHz, DMSO-d 6) δ -61.54, -72.52; m / z (ES +) [M+H] +=479.

[0299] [ , Synthesis example , ] [ , 29 , ] : 6-(difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperidine-1-yl]-N-methyl-pyridine-2-formamide

[0300] Add DIPEA (330 µl, 1.89 mmol) to 7-(chloromethyl)-3-ethyl-1,5- Pyridine-2(1H)-one, HCl ( [intermediate]

[17] , 70 mg, 0.27 mmol), sodium iodide (4.05 mg, 0.03 mmol) and 6-(difluoromethyl)-N-methyl-5-piper-1-yl-pyridine-2-methyl Amide, 2HCl ( [intermediate]

[41] , 102 mg, 0.30 mmol) in acetonitrile (2.4 mL) and stirred the resulting solution at 50°C for 3 hours. The solvent was removed under vacuum and 50 mL of water was added followed by 3 mL of sat NaHCO 3 . The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by flash silica chromatography with a gradient of 0 to 30% MeOH in DCM. The product fractions were concentrated to dryness under reduced pressure to give 6-(difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridine-3-yl)methyl]piper-1-yl]-N-methyl-pyridine-2-formamide ( [synthesis example]

[29] , 52.0 mg, 42%). 1H NMR (500MHz, DMSO-d 6) 1.19 (3H, t), 2.54 - 2.58 (2H, m), 2.63 (4H, br s), 2.84 (3H, d), 3.03 (4H, br t), 3.68 (2H, s), 7.14 (1H, t), 7.62 (1H, d), 7.76 (1H, s), 7.86 (1H, d), 8.10 (1H, d), 8.32 - 8.45 (2H, m), 11.86 (1H, s); m / z (ES +) [M+H] +=457.

[0301] [ , Synthesis example , ] [ , 30 , ] : 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperidine-1-yl]-N-methyl-6 (trifluoromethyl)pyridine-2-formamide

[0302] Add DIPEA (330 µl, 1.89 mmol) to 7-(chloromethyl)-3-ethyl-1,5- Pyridine-2(1H)-one, HCl ( [intermediate]

[17] , 70 mg, 0.27 mmol), sodium iodide (4.05 mg, 0.03 mmol) and N-methyl-5-piperone-1-yl-6-(trifluoromethyl)pyridine-2-formyl Amine, 2HCl ( [intermediate]

[38] , 107 mg, 0.30 mmol) in acetonitrile (2.4 mL) and stirred the resulting solution at 50°C for 3 hours. The solvent was removed under vacuum and 50 mL of water followed by 3 mL of sat NaHCO 3 was added. The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by flash silica chromatography with a gradient of 0 to 30% MeOH in DCM. The product fractions were concentrated to dryness under reduced pressure to give 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-6(trifluoromethyl)pyridine-2-carboxamide ( [synthesis example]

[30] , 58.0 mg, 45 %). 1H NMR (500MHz, DMSO-d 6) 1.19 (3H, t), 2.54 - 2.62 (6H, m), 2.83 (3H, d), 3.04 (4H, br t), 3.67 (2H, s), 7.62 ( 1H, d), 7.75 (1H, s), 8.04 (1H, d), 8.19 (1H, d), 8.31 - 8.48 (2H, m), 11.85 (1H, s); m / z (ES +) [ M+H] +=475.

[0303] [ , Synthesis example , ] [ , 31 , ] : 5-[4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperidin-1-yl]-N,6-dimethyl-pyridine-2-carboxamide

[0304] Add DIPEA (0.366 mL, 2.10 mmol) to 7-(bromomethyl)-3-ethyl-1,5- Pyridine-2(1H)-one ( [intermediate]

[14] , 80 mg, 0.30 mmol) and N,6-dimethyl-5-piperone-1-yl-pyridine-2-carboxamide, 2HCl ( [intermediate]

[45] , 101 mg, 0.33 mmol) in acetonitrile (2 mL) and stirred the resulting solution at 70°C for 3 hours. The solvent was removed under vacuum and 50 mL of water followed by 3 mL of sat NaHCO 3 was added. The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by flash silica chromatography with a gradient of 0 to 30% MeOH in DCM. The product fractions were concentrated to dryness under reduced pressure to give 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]-N,6-dimethyl-pyridine-2-formamide ( [synthesis example]

[31] , 36.0 mg, 29 %). 1H NMR (500 MHz, DMSO-d 6 ) 1.19 (3H, t), 2.50 (3H, s), 2.54 - 2.57 (2H, m), 2.57 - 2.64 (4H, m), 2.81 (3H, d), 2.96 (4H, br s), 3.68 (2H, s), 7.49 (1H, d), 7.63 (1H, d), 7.76 (1H, s), 7.80 (1H, d), 8.35 - 8.47 (2H, m ), 11.85 (1H, br s); m / z (ES +) [M+H] +=421.

[0305]

[0306] [ , intermediate , ] [ , 90 , ] : tertiary butyl 4-[6-(ethylaminoformyl)-3-pyridyl]piperone-1-carboxylate Ethylamine (7M, 7.78 mL, 15.56 mmol) in methanol was added to tert-butyl 4-(6-(methoxycarbonyl)pyridin-3-yl)piperoxo-1-carboxylate ( [intermediate]

[15] [,] 500 mg, 1.56 mmol) and stirred the resulting solution at 50°C for 18 hours. The solvent was removed under vacuum and the sample was further dried to give tert-butyl 4-[6-(ethylaminoformyl)-3-pyridyl]piperoxo-1-carboxylate ( [intermediate]

[90] , 0.495 g, 95%). 1H NMR (500MHz, DMSO-d6) 1.11 (3H, t), 1.43 (9H, s), 3.27 - 3.32 (6H, m), 3.44 - 3.52 (4H, m), 7.42 (1H, dd), 7.85 ( 1H, d), 8.28 (1H, d), 8.44 (1H, br t).

[0307] [ , intermediate , ] [ , 91 , ] : N-ethyl-5-piperyl-1-yl-pyridine-2-formamide slowly add to the HCl (0.473 mL, 15.58 mmol) of alkanes to tertiary butyl 4-(6-(ethylaminoformyl)pyridin-3-yl)piperone-1-carboxylate ( [intermediate]

[90] [,] 521 mg, 1.56 mmol) in a stirred solution of methanol (10 mL). The resulting solution was stirred at rt for 17 hours. The reaction was concentrated and the solids were dried to give N-ethyl-5-piperol-1-yl-pyridine-2-carboxamide, 2HCl ( [intermediate]

[91] ,421 mg,88 %); m / z (ES +) [M+H] +=235.

[0308] [ , Synthesis example , ] [ , 32 , ] : N-ethyl-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperidin-1-yl]pyridine-2-formamide []

[0309] Add DIPEA (0.320 mL, 1.83 mmol) to 7-(bromomethyl)-3-ethyl-1,5- Pyridine-2(1H)-one ( [intermediate]

[14] [,] 70 mg, 0.26 mmol) and N-ethyl-5-piperone-1-yl-pyridine-2-formamide, 2HCl ( [intermediate]

[91] [,] 89 mg, 0.29 mmol) in acetonitrile (2 mL) and stirred the resulting solution at 70°C for 3 hours. The solvent was removed under vacuum and 50 mL of water was added followed by 3 mL of sat NaHCO 3 . The mixture was extracted with ethyl acetate. After concentration, the crude product was purified by reverse phase chromatography (column: Xbrid C18) with a gradient of 20 to 50% MeCN in water (containing 0.2% NH4OH). The pure fractions were evaporated to dryness to give N-ethyl-5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]pyridine-2-formamide ( [synthesis example]

[32] , 28.0 mg, 25%). 1H NMR (500 MHz, DMSO-d 6) 1.10 (3H, t), 1.19 (3H, t), 2.52 - 2.55 (2H, m), 2.55 - 2.59 (4H, m), 3.26 - 3.30 (2H, m ), 3.34 (4H, br d), 3.66 (2H, s), 7.40 (1H, dd), 7.63 (1H, s), 7.76 (1H, s), 7.83 (1H, d), 8.27 (1H, d ), 8.36 - 8.46 (2H, m), 11.74 - 11.94 (1H, m); m / z (ES +) [M] +=420.

[0310] [ , Synthesis example , ] [ , 4- , ] [ , form , ] [ , A , ] [] In Synthesis Example 4, by evaporating the methanol / dichloromethane solution under reduced pressure, 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide. The crystalline material thus obtained was characterized as crystalline Form A in white color.

[0311] In the case of poor crystallinity, it can be obtained by suspending 20 mg of crude sample in 0.20 ml of water, methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, ethyl acetate or other solvents at ambient temperature or at 50°C. 1 day to obtain crystalline Form A.

[0312] Form A was analyzed by XRPD and the results are shown in Figure 16A and tabulated below: For the XRPD peak of Form A [angle] [(2 , θ , ±0.2 ] [°)] [strength] [(%)] 8.3 100.0 12.4 30.9 19.4 26.5 20.4 25.8 26.3 19.2 21.2 17.4 20.8 14.8 22.8 14.1 16.8 14.0 10.2 13.2 18.4 10.8 11.4 9.9 28.1 8.4 18.0 8.4 25.2 8.2 24.9 6.7 16.5 6.4 17.3 5.3 22.1 4.0 29.3 3.3 24.3 2.7 30.3 2.5 38.2 2.0 33.9 1.4 14.2 1.4 13.7 1.4 33.0 1.3 36.5 1.2 39.2 1.2

[0313] Form A is characterized by providing at least one of the following 2Θ values ​​measured using CuKα radiation: 8.3, 12.4, and 19.4°.

[0314] Form A was analyzed by thermal technique. DSC analysis indicated that Form A had a melting point starting at 254°C and peaking at 255°C. A representative DSC trace for Form A is shown in Figure 16B.

[0315] [biological assay] [(PARP1] [Selective Inhibitor] [)] The inhibitory properties of the PARP1 selective inhibitor compounds described herein can be determined using the following test procedure.

[0316] PARP Fluorescence Anisotropy Binding Assay [] The recombinant full-length 6HIS-tagged PARP1 protein was diluted to 6 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl, and incubated with an equal volume of 2 nM fluorescent probe for four hours ( The fluorescent probe was diluted in 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0317] The recombinant full-length PARP2 protein was diluted to 6 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl, and incubated with an equal volume of 2 nM fluorescent probe for four hours (the fluorescent Probes were diluted in 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0318] The recombinant full-length PARP3 protein was diluted to 100 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl, and incubated with an equal volume of 6 nM fluorescent probe for four hours (the fluorescent Probes were diluted in 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0319] The recombinant PARP5a binding domain was diluted to 160 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl, and incubated with an equal volume of 6 nM fluorescent probe for four hours (the fluorescent probe Needles were diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0320] The recombinant full-length GST-tagged PARP6 protein was diluted to 160 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl, and incubated with an equal volume of 6 nM fluorescent probe for four hours ( The fluorescent probe was diluted in 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl 2 , 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0321] Fluorescence anisotropy of probes upon protein binding was measured and the effect on anisotropy determined using a BMG Pherastar FS© in the presence of test compounds or solvent controls. % inhibition values ​​were calculated for different test compound concentrations and fitted to four parameter logarithmic plots to determine IC50 values. When necessary, compound Ki can be determined from IC50 values ​​using the Munson Rodbard equation, defined in Anal. Biochem. 1980 Sep 1;107(1):220-39, and based on correlation with Known KD of probes bound by relevant PARP proteins.

[0322] hERG electrophysiological assay Electrophysiological recordings from stably transfected CHO hKv11.1 cells were obtained using Nanion Syncropatch 768PE (all performed at RT). Add test compound, vehicle, or positive control in 6 compound plates, each at different concentrations, to allow cumulative doses (10 mM, 3.167 mM, 1 mM, 0.3167 mM, 0.1 mM , 0.03167 mM). Resuspend 600 l of compound in 90 µl of reference buffer (in mM, NaCl 80, KCL 4, CaCl 5, MgCl 1, NMDG Cl 60, D-glucose monohydrate 5, HEPES 10 (pH7.4 HCL , 298mOsm), the final compound concentrations were 39.6 µM, 13.2 µM, 4.4 µM, 1.46 µM, 0.48 µM, 0.16 µM. For each Nanion Syncropatch 768PE run, in the presence of extracellular solution (in mM, NaCl 80, KCL 4, The magnitude of the current in each cell was measured in the presence of CaCl 5 , MgCl 1 , NMDG Cl 60 , D-glucose monohydrate 5 , HEPES 10 (pH 7.4 HCL, 298 mOsm) and all experiments were performed using the Syncropatch liquid handling system. Liquid addition. Add 40 μL of external solution (in mM, HBPS, CaCl2, MgCl2 (pH7.4, NaOH)) to the 384-well porous media resistance recording chip, and perfuse the internal buffer (in mM, KF 130, KCl 20, MgCl 1, EGTA 10, HEPES 10, Escin 25 (all Sigma-Aldrich; pH 7.2–7.30, use 10 M KOH, 320 mOsm) to the bottom of the plate. At a density of 1e6 cells / ml, add 20 μL of cells maintained at ~9°C was dispensed into each well of the chip, followed by the addition of 20 μL of seal enhancer (in mM, NaCl 80, KCl 3, CaCl 10, HEPES 10, MgCl 1 (pH7.4 NaOH )). Perform a wash step, leaving a residual volume of 40 μL. Prior to the addition of the test compound, dispense 40 μL of reference buffer to establish a stable baseline, perform a 40 μL removal step after 3 min, and repeat. Pipette Dispense 40 µL of concentration 1 (0.16 µM) compound before removing 40 µL, and record 3 min exposure "on the fly". Repeat this step for an additional 5 subsequent compound discs to generate a cumulative curve analysis. All data are leak subtracted (leak subtract), 2 pulses to -80 mV 100 ms, 100 ms delay. The outward K+ current was then induced by a voltage step from the holding potential of -90 mV to +60 mV, each pulse at a frequency of 2 Hz for 15 s Pulse interval transmission.

[0323] PARP Proliferation Assay (4-Day Compound Dosing) Harvest DLD1 cells and BRCA2 (- / -) DLD1 cells with densities of 1.875E4 cells / ml and 6.25E4 cells / ml respectively in complete medium, use Multidrop Combi, and inoculate 40 µL / well into 384-well plates ( Greiner, Kremsmunster, Austria; 781090), and then incubated overnight at 37°C, 5% CO 2 . The next day (day 1), add sytox green (5ul, 2uM) and saponin (10ul, 0.25% stock solution) to the plate on day 0 using the Multidrop Combi, seal the plate with a black adhesive lid (adhesive lid), and place in Incubate >3 hr at RT. Cells were imaged using Cell Insight (Thermo Fisher) equipped with a 4x objective. Add the test compound using an Echo 555, place it in an incubator maintained at 37 °C, 5% CO, and incubate for 4 days. On day 5, add sytox green (5 ul, 2 uM) to the dish followed by saponin (10 ul, 0.25% stock solution), seal the dish using a black adhesive lid and incubate >3 hr at RT. Read all cells on Cell Insight with 4x objective. Proliferation rate in Genedata was determined by evaluating the total cell number output by Cell Insight for day 0 and day 5 plates.

[0324] Synthesis example serial number PARP1 IC50 (µM) PARP2 IC50 (µM) PARP3 IC50 (µM) PARP5a IC50 (µM) PARP6 IC50 (µM) BRCA2 - / - DLD-1 prolif 4d IC50 (µM) WT DLD-1 prolif 4d IC50 (µM) hERG IC50 (µM) [1] 0.003 1.7 4 >100 34 0.010 >30 >40 [2] 0.004 0.88 9.9 20 14 0.008 >30 >40 [3] 0.005 1.3 12 >100 14 0.004 >30 22 [4] 0.004 >1.5 4.7 >100 19 >0.017 >30 >40 [5] 0.002 0.65 7.1 >100 23 0.006 >30 >40 [6] 0.003 0.84 9.3 >100 8.2 0.006 >30 >40 [7] 0.002 1.3 2.6 94 22 4.14 [8] 0.003 11 55 93 18 0.011 >19 >40 [9] 0.009 22 >100 >100 47 0.010 17 >40

[10] 0.005 17 48 56 26 0.006 >30 >40

[11] 0.005 4 13 >100 22 0.184 >30 >40

[12] 0.004 1.6 19 89 11 0.008 >30 >40

[13] 0.007 8.5 30 >100 30 0.005 >26 >40

[14] 0.004 2.9 30 50 11 0.006 >30 >40

[15] 0.011 3.6 35 >100 80 0.090 >30 >40

[16] 0.007 3.3 74 61 31 0.018 >22 >40

[17] 0.007 1.7 96 >100 59 0.020 >30 >40

[18] 0.031 17 >100 >100 >29 4.90 >30 5.2

[19] 0.015 >100 >100 >100 >29 0.015 >30 21

[20] 0.014 28 >100 >100 >100 0.016 >24 38

[21] 0.004 9.5 >100 >100 33 0.016 >30 >40

[22] 0.006 1 2.6 26 16 0.012 >30 >40

[23] 0.004 4.4 60 60 >100 4.2 36

[24] 0.003 5.1 >100 93 >100 0.010 14 37

[25] 0.002 6 43 >100 >100 >25 >40

[26] 0.005 6.7 >100 >100 >100 0.005 23 >40

[27] 0.007 16 >100 71 >100 10.3 >10 26

[28] 0.006 14 >100 >29 >100 0.027 >30 >40

[29] 0.004 6.1 9.9 >100 14 0.007 >30 >40

[30] 0.003 7.6 4.5 >100 10 0.004 >30 >40

[31] 0.005 3.7 2.6 >100 28 >40

[32] 0.003 2.1 1.9 >100 10 >40

[0325] Example 1: Production of antibody-drug conjugates According to the production method described in WO2015 / 115091 and using an anti-HER2 antibody (comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 11 (amino acid residues 1 to 449 of SEQ ID NO: 1) and light chain antibodies consisting of all amino acid residues 1 to 214 of SEQ ID NO:2), producing a drug-linker represented by the following formula via a thioether bond with Anti-HER2 Antibody Conjugated Anti-HER2 Antibody-Drug Conjugate (DS-8201: Trastuzumab Delutecan), Wherein A represents the linking position with the antibody. The DAR of the antibody-drug conjugate was 7.7 or 7.8.

[0326] Example 2: Production of PARP1 Selective Inhibitors According to the production method described herein, the PARP1 selective inhibitor of formula (I) was prepared. Specifically, 5-[4-[(7-ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide: (AZD5305).

[0327] Embodiment 3: antitumor test Antibody-drug conjugate DS-8201 (trastuzumab delutecan (Enhertu®)) with PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H -1,5- combination of pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide)

[0328] method: A high-throughput combinatorial screening was performed, in which 27 breast cancer cell lines with different HER2 expression and one gastric cell line with high HER2 expression were treated with the combination of DS-8201 and AZD5305 (PARP1 selective inhibitor) (Table 1).

[0329] Table 1 cell line HER2 status cancer type NCI-N87 Amp (high) Stomach SKBR3 Amp (high) the breasts AU565 Amp (high) the breasts HCC1569 Amp (High) the breasts HCC1187 Low the breasts HCC1954 Amp (High) the breasts KPL-4 Amp (High) the breasts HCC38 Amp (Low) the breasts MCF7 (ER+) Del (null) the breasts MDA-MB-157 Low the breasts HCC1419 Amp (high) the breasts ZR-75-30 Amp (high) the breasts ZR-75-1 (ER+) Low the breasts HCC1395 Low the breasts BT474 (ER+) Amp (high) the breasts EFM-19 (ER+) Low the breasts HCC1937 null the breasts BT-549 Del (null) the breasts MDA-MB-453 Low the breasts MDA-MB-361 Amp (Low) the breasts CAL-51 Low the breasts MDA-MB-468 Amp (Low) the breasts T-47D (ER+) Low the breasts HCC1143 Low the breasts JIMT1 Amp (Low) the breasts CAMA1 (ER+) Low the breasts MDA-MB-231 Low the breasts HCC70 Low the breasts

[0330] The readout for this screening is the 7-day CellTiter-Glo Cell Viability Assay [Determinate], performed as a 6 x 6 dose-response matrix (5-point log serial dilutions for DS-8201 and half-log serial dilutions for AZD5305). The maximum concentration of AZD5305 is 3 µM and the maximum concentration of DS-8201 is 10 µg / ml. In addition, trastuzumab and exitecan (DNA topoisomerase I inhibitor) were also screened in parallel with AZD5305 to help unravel the mechanism of action of the effective combination. Combination activity was assessed based on the combination of ΔEmax and Loewe synergy score.

[0331] result: Results for HER2-high cell lines (KPL4, NCI-N87, SKBR3, HCC1954, HCC1569, AU565) are shown in Figures 12A and 12B and Table 2, as well as for HER2-low cell lines (MDA-MB-468, MDA-MB-157, The results for HCC1187, T47D, HCC38) are shown in FIGS. 13A and 13B and Table 3.

[0332] Figures 12A and 13A show matrices of measured cell viability signals. The X-axis represents drug A (DS-8201), and the Y-axis represents drug B (AZD5305). Values ​​in boxes represent the ratio of cells treated with drugs A+B to DMSO control at day 7. All values ​​are normalized to day 0 cell viability values. Values ​​between 0 and 100 represent percent growth inhibition, and values ​​above 100 represent cell death.

[0333] Figures 12B and 13B show the Loewe excess matrix. Values ​​in boxes represent excess values ​​calculated by the Lewey additivity model.

[0334] Tables 2 and 3 show the HSA synergy and Lewey additivity scores: Table 2 [cell line] KPL4 NCI-N87 SKBR3 HCC1954 HCC1569 AU565 HSA Synergy Score 68.2 70.95 20.33 9.9 38.6 32.77 Lowell Synergy Score 68.2 70.95 20.33 9.9 38.6 32.77 table 3 [cell line] MDA-MB-468 MDA-MB-157 HCC1187 T47D HCC38 HSA Synergy Score 11.6 7.04 52.7 12.33 8.9 Lowell Synergy Score 11.6 6.5 52.7 12.33 8.8

[0335] Remark: If two compounds act on the same molecular target by the same mechanism, Le Weber dose additivity predicts the expected response. It calculates additivity based on the assumption of zero interactions between compounds, and it is independent of the nature of the dose-response relationship.

[0336] HAS (Highest Single Agent) [Berenbaum 1989] quantifies the higher of two single compound effects at corresponding concentrations. The combination effect is compared to the effect of each single agent at the concentrations used in the combination. Exceeding the highest single-dose effect indicates synergy. HSA does not require compounds to affect the same target.

[0337] Excess matrix: For each well in the concentration matrix, compare the measured or fitted value to the predicted non-synergistic value for each concentration pair. Predicted values ​​are determined by the selected model. Differences between predicted and observed values ​​may indicate synergy or antagonism and are shown in the excess matrix. Excess matrix values ​​are summarized by the combined fraction of excess volume and synergy fraction.

[0338] Figure 14 shows combined Emax and Levey synergy scores in various cell lines treated with the combination of DS-8201 and AZD5305.

[0339] As can be seen from Figures 12A and 12B and Table 2, AZD5305 interacts synergistically with DS-8201 and also increases cell death in HER2+ breast and gastric cell lines. As can be seen from Figures 13A and 13B and Table 3, AZD5305 interacted synergistically with DS-8201 and also increased HER2-low breast cancer cell lines at Emax (3 µM AZD5305 and 10 µg / ml DS-8201). cell death. As can be seen from Figure 14, in 11 cell lines including HER2-low breast cancer cell lines, combination treatment with DS-8201 and AZD5305 resulted in high combined Emax (>100) and Levey synergy scores (>5).

[0340] Embodiment 4: antitumor test Antibody-drug conjugate DS-8201 (trastuzumab delutecan (Enhertu®)) with PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H -1,5- combination of pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide)

[0341] method: Cells grown under the respective conditions were seeded in 96-well plates at an optimal density to allow linear proliferation during the assay period (4 to 8 days). Immediately after inoculation, the indicated compounds were administered to the cells in a total volume of 200 µL / well and placed in an incubator. Combinations were performed as a 6 x 8 concentration-response matrix per combination. At the endpoint, cells were fixed in 2% PFA for 20 min at room temperature. To obtain cell numbers at the beginning of treatment, use an additional dish per experiment and fix after cells attach. Cells were then permeabilized in 0.5% Triton-X100 in PBS for 10 minutes. After washing with PBS, cells were blocked in 5% FBS in PBS for 1 h at room temperature and incubated with primary antibodies in 5% FBS + 0.05% triton overnight at 4 °C. After washing 3 times in PBS, cells were incubated with secondary antibodies in 5% FBS + 0.05% triton containing Hoechst33258 for 1 hour at room temperature. After washing 3 times in PBS, scan the cells using a Cellinsight instrument with a 10x objective and 9 fields / well. Images were analyzed for cell counts using Columbus based on nuclear Hoechst staining. Use the total cell count / well to calculate the relative growth in each well compared to the solvent control. To calculate the synergy score, growth inhibition data were analyzed using the Combinefit software (Di Veroli, G.Y., et al., Combenefit: an interactive platform for the analysis and visualization of drug combinations. Bioinformatics, 2016,

[32] (18):p. 2866-8).

[0342] result: Results for a HER2 high cell line (KPL4) and two HER2 low cell lines (JIMT1, MDA-MB-468) are shown in Figures 15A and 15B.

[0343] Figure 15A shows a cell count matrix with drug A (DS-8201) on the Y axis and drug B (AZD5305) on the X axis. Values ​​in boxes represent relative total cell (nuclei) counts as a percentage of DMSO vehicle control.

[0344] Figure 15B shows a matrix where the Y-axis represents Drug A (DS-8201), the X-axis represents Drug B (AZD5305), and the values ​​in the boxes represent the calculated Levey synergy scores.

[0345] The results in Examples 3 and 4 demonstrate that selective PARP1 inhibition with AZD5305 enhances the antitumor efficacy of DS-8201 in both high and low HER2 expressing cell lines in vitro. In Example 3, the combination of AZD5305 and DS-8201 was effective in five HER2+ breast cancer cell lines, one HER2+ gastric cancer cell line (Figure 12A, 12B, 14 and Table 2) and five HER2-low breast cancer cell lines (Figure 13A, 13B and 14 and Table 3) show the combination benefit. In Example 4, the combination of AZD5305 and DS-8201 showed synergistic activity in HER2-high (KPL4) and HER2-low (JIMT-1, MDA-MB-468) cell lines (Figure 15A and 15B).

[0346] Example 5: Anti-tumor test - in vivo Antibody-drug conjugate DS-8201 (trastuzumab delutecan (Enhertu®)) with PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H -1,5- combination of pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide)

[0347] method: Female nude mice (Charles River) aged 5-8 weeks were used and acclimatized 7 days before entering the study. 1×10 7 NCI-N87 tumor cells (1:1 in Matrigel) were implanted subcutaneously in the flank of female nude mice. When tumors reached approximately 150 mm, tumors of similar size were randomly assigned to treatment groups, as shown in Table 4:

[0348] Table 4 [deal with] [dosage] [Injection path] [Dosing schedule] [(28] [sky] [)] medium ---- IV + PO Single dose + QD DS-8201 3mg / kg IV single dose DS-8201 1 mg / kg IV single dose AZD5305 1 mg / kg PO QD DS-8201 + AZD5305 1 mg / kg or 3 mg / kg + 1 mg / kg IV + PO Single dose + QD PO: oral administration (per os) QD: Dosing once a day (quaque die)

[0349] The compound dose for each animal was calculated based on the individual body weight on the day of dosing. DS8201 and AZD5305 were administered on the same day, and DS8201 was administered about 1 hour after AZD5305 PO administration. DS-8201 was administered as a single dose of 1 mg / kg or 3 mg / kg on day 1, and AZD5305 was administered at 1 mg / kg QD for 28 days. Dosing duration is 28 days.

[0350] [DS-8201] [At] [3 mg / kg] [and] [1 mg / kg] [concoctions of] The dosing solution of DS-8201 was diluted to 0.6 mg / ml on the day of dosing by diluting DS-8201 stock solution (20.1 mg / ml) in 25 mM histidine buffer, 9% sucrose (pH5.5) and 0.2 mg for 3 mg / kg and 1 mg / kg dosing solutions, respectively. Each dosing solution was mixed thoroughly using a pipette prior to administration via IV injection at a dosing volume of 5 ml / kg.

[0351] [AZD5305] [At] [1 mg / kg] [the concoction] In order to prepare a 1 mg / kg dosing solution, AZD5305 was prepared at a concentration of 0.1 mg / ml, which resulted in a dosing volume of 10 ml / kg for PO dosing. A total of 49 ml of vehicle is required. Add an amount of 15 μl 1M HCl to the compound and mix well by vortexing. Add a volume of 1 ml sterile water to the Eppendorf tube and mix well with the compound using a pellet pestle. Sonicate the compound for approximately 5 min, then transfer the contents to a glass vial. Rinse any residual compound in the Eppendorf tube using a volume of 1 ml sterile water and transfer it to a glass vial. The remaining volume of sterile water (37.2 ml; 80% of the total vehicle volume) was added to the glass vial and mixed well using a magnetic stirrer. The pH of the dosing solution was adjusted to pH 3.74, then the remaining vehicle (9.772ml sterile water) was added to the glass bottle and mixed thoroughly using a magnetic stirrer. Keep the dosing solution away from light, and take a small part for dosing every day. Store all remaining dosing solutions in the refrigerator for up to 7 days. The final dosing matrix of 1 mg / kg AZD5305 was a clear solution.

[0352] [Measurement] Tumor growth inhibition (TGI) was calculated as follows: TGI%={1-(MTV Treated / MTV Control)}*100 where MTV = mean tumor volume.

[0353] Statistical significance was assessed on the day of final measurement using a one-tailed t-test of (log(relative tumor volume)=log(final volume / starting volume)) compared to vehicle control.

[0354] [result] Tumor volumes treated with DS8201 or AZD5305 alone or in combination with DS-8201 and AZD5305 are shown in FIG. 17 . Data represent the change in tumor volume over time for the treatment groups. The dotted line in Figure 17 represents the end of the dosing period. See Table 4 above for complete dosage and schedule information. Values ​​shown are mean±SEM; initially n=10 for vehicle-treated mice and n=8 for all other treatment groups.

[0355] TGI responses (Day 41 TGI %) following treatment with DS8201 or AZD5305 alone or in combination with DS-8201 and AZD5305 in NCI-N87 xenografts are shown in Table 5:

[0356] table 5 [processing group] [TGI%] [Article]

[41] [sky] [p-] [value relative to] [medium] [significance] DS-8201 3 mg / kg 62% 0.00071 *** DS-8201 1 mg / kg 25% 0.025 †ns AZD5305 1 mg / kg 40% <0.0001 †ns DS-8201 1 mg / kg + AZD5305 1 mg / kg 55% <0.0001 ** DS-8201 3 mg / kg + AZD5305 1 mg / kg 90% <0.0001 **** †Not obvious

[0357] Monotherapy with DS-8201 3 mg / kg showed a TGI value of 62% on day 41 after treatment. It showed a TGI of 25% on day 41 after 1 mg / kg DS-8201 treatment. AZD5305 monotherapy achieved a TGI of 40% at day 41 post-treatment. Combination treatment of AZD5305 and DS-8201 at 1 mg / kg resulted in a TGI of 55% at 41 days post-treatment. Combination treatment with the higher DS-8201 3 mg / kg dose and AZD5305 achieved a significant TGI of 90% at day 41 after treatment and showed a better response than either therapy alone.

[0358] Treatment groups were generally well tolerated (two outlier animals were withdrawn from the study due to >15% body weight loss) and mean body weights for all treatment groups remained stable during the study.

[0359] Embodiment 6: Antibody-drug conjugate DS-8201 (trastuzumab delutecan (Enhertu ® )) combined with PARP1 selective inhibitor AZD5305 (5- [4-[(7-Ethyl-6-oxo-5H-1,5- Pyridin-3-yl)methyl]piperone-1-yl]-N-methyl-pyridine-2-carboxamide) combined dosage

[0360] method: High-throughput combinatorial screening was carried out, in which four lung cancer cell lines (Table 6) and one HER2 mutant cancer cell line (Table 7) with different HER2 expression were screened with the combination of DS-8201 and AZD5305.

[0361] Table 6 [cell line] [By Flow Cytometry] [in each cell] [HER2] [receptor] [IHC Dako H2L H-] [Fraction] [cancer type] HCC1171 146050±654 200 NSCLC adenocarcinoma NCIH1573 51090±165 100 NSCLC adenocarcinoma NCIH2170 2472499±21851 300 NSCLC squamous cell carcinoma Calu6 12935±200 0 NSCLC adenocarcinoma

[0362] Table 7 [cell line] [By Flow Cytometry] [in each cell] [HER2] [receptor] [HER2] [mutation] [cancer type] 5637 27497±14 p.S310F urethra

[0363] Screening readouts were 7-day CellTiter-Glo cell viability assays performed as a 6x6 dose-response matrix (DS-8201 and AZD5305, serially diluted in half-logs for each combination). The maximum concentration of AZD5305 is 3.33 or 10 µM, and the maximum concentration of DS-8201 is 100 µg / ml. Combination activity was assessed based on a combination of ΔEmax and Lewey synergy scores.

[0364] result: The results of HER2+, HER2 low, HER2 low / null NSCLC cell lines (HCC1171, NCIH1573, NCIH2170, Calu6) are shown in Figure 18A, 18B and 18C and Table 8, while the HER2 mutant cell line (5637) is shown in Figure 19A, 19B and 19C, and Table 9.

[0365] Figures 18A and 19A show matrices of measured cell viability signals. The X-axis represents drug A (DS-8201), and the Y-axis represents drug B (AZD5305). Values ​​in boxes represent the ratio of cells treated with drugs A+B to DMSO control at day 7. All values ​​were normalized to the cell viability value at day 0. Values ​​between 0 and 100 represent percent growth inhibition, and values ​​above 100 represent cell death.

[0366] Figures 18B and 19B show the Loewe excess matrix. Values ​​in boxes represent excess values ​​calculated by the Lewey additivity model.

[0367] Figures 18C and 19C show the HSA excess matrix. Values ​​in boxes represent excess values ​​calculated by the HAS (highest single dose) model.

[0368] Tables 8 and 9 show HAS synergy and Lewey additivity scores:

[0369] Table 8 [cell line] [(NSCLC)] [HCC1171] [NCIH1573] [NCIH2170] [Calu6] HSA Synergy Score 6.2 6.14 33.99 10.55 Loewy Synergy Score 6.2 5.4 33.99 10.55

[0370] Table 9 [cell line] [(] [urethra] [)]

[5637] HSA Synergy Score 11.2 Loewy Synergy Score 11.2

[0371] As can be seen from Figures 18A, 18B and 18C and Table 8, AZD5305 interacts synergistically with DS-8201, and also increases cell death, in the HER2+ cell line NCIH2170, at Emax (0.125 µM AZD5305 and 100 µg / ml DS-8201 -8201); in HER2-low cell line HCC1171, at (0.125 µM AZD5305 and 100 µg / ml DS-8201); and in HER2-low / null cell line Calu6, at Emax (1.25 µM AZD5305 and 100 µg / ml DS-8201 ). Combination activity was observed even in the absence or low activity of single agents. Although synergy was observed in the cell line NCIH1573, no cell death occurred.

[0372] From Figure 19A, 19B and 19C and Table 9, it can be seen that AZD5305 interacts synergistically with DS-8201 and also increases the expression of HER2 mutant cell line 5637 at Emax (1.25 µM AZD5305 and 100 µg / ml DS-8201). of cell death. Combination activity was observed even when AZD5305 was not active as a single agent.

[0373] The foregoing written description was considered sufficient to enable one of ordinary skill in the art to practice the specific embodiments. The foregoing specification and examples detail certain embodiments and describe the best mode contemplated by the inventors. It is to be understood, however, that no matter how detailed the foregoing may appear in context, specific embodiments may be implemented in various ways and that the claim includes any equivalents thereof.

[0374] Non-keyword text of sequence listing SEQ ID NO: 1 - Amino acid sequence of heavy chain of anti-HER2 antibody SEQ ID NO:2 - Amino acid sequence of light chain of anti-HER2 antibody SEQ ID NO:3 - Amino acid sequence of heavy chain CDRH1 [=amino acid residues 26 to 33 of SEQ ID NO:1] SEQ ID NO:4 - Amino acid sequence of heavy chain CDRH2 [=amino acid residues 51 to 58 of SEQ ID NO:1] SEQ ID NO:5 - Amino acid sequence of heavy chain CDRH3 [=amino acid residues 97 to 109 of SEQ ID NO:1] SEQ ID NO:6 - Amino acid sequence of light chain CDRL1 [=amino acid residues 27 to 32 of SEQ ID NO:2] SEQ ID NO:7 - Amino acid sequence comprising the amino acid sequence of the light chain CDRL2 (SAS) [=amino acid residues 50 to 56 of SEQ ID NO:2] SEQ ID NO:8 - Amino acid sequence of light chain CDRL3 [=amino acid residues 89 to 97 of SEQ ID NO:2] SEQ ID NO:9 - Amino acid sequence of heavy chain variable region [=amino acid residues 1 to 120 of SEQ ID NO:1] SEQ ID NO: 10 - Amino acid sequence of light chain variable region [= amino acid residues 1 to 107 of SEQ ID NO: 2] SEQ ID NO: 11 - Amino acid sequence of the heavy chain [= amino acid residues 1 to 449 of SEQ ID NO: 1]

[0375] none.

[0376]

Claims

1. A pharmaceutical product comprising an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor for combined administration, wherein the anti-HER2 antibody-drug conjugate is an antibody-drug conjugate in which a drug-linker represented by the following formula is bound to an anti-HER2 antibody via a thioether bond.

1. Wherein A represents the binding site with the antibody, and the PARP1 selective inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

1. Among them: X1 and X2 are each independently selected from N and C(H), X3 is independently selected from N and C(R4), wherein R4 is H or fluorine, R1 is C1-4 alkyl or C1-4 fluoroalkyl, R2 is independently selected from H, halogen, C1-4 alkyl, and C1-4 fluoroalkyl, and R3 is H or C1-4 alkyl, under the following conditions: when X1 is N, then X2 is C(H) and X3 is C(R4); when X2 is N, then X1 is C(H) and X3 is C(R4); and when X3 is N, then both X1 and X2 are C(H); wherein the anti-HER2 antibody is an antibody comprising the following heavy chain and light chain, the heavy chain comprising CDRH1 composed of the amino acid sequence represented by SEQ ID NO: 3, CDRH2 composed of the amino acid sequence represented by SEQ ID NO: 4, and the light chain composed of SEQ ID NO:

3. The light chain comprises CDRH3, which is composed of the amino acid sequence represented by NO:5, CDRL1, which is composed of the amino acid sequence represented by SEQ ID NO:6, CDRL2, which is composed of the amino acid sequence composed of amino acid residues 1 to 3 of SEQ ID NO:7, and CDRL3, which is composed of the amino acid sequence represented by SEQ ID NO:

8.

2. The pharmaceutical product of claim 1, wherein in formula (I), R3 is a C1-4 alkyl group.

3. The pharmaceutical product of claim 1, wherein in formula (I), R3 is methyl.

4. The pharmaceutical product of claim 1, wherein R1 in formula (I) is ethyl.

5. The pharmaceutical product of claim 1, wherein the PARP1 selective inhibitor is a compound represented by formula (Ia) or a pharmaceutically acceptable salt thereof.

5. Wherein R1 is a C1-4 alkyl group, R2 is selected from H, halogen group, C1-4 alkyl group and C1-4 fluoroalkyl group, R3 is H or C1-4 alkyl group and R4 is H.

6. The pharmaceutical product as claimed in claim 5, wherein in formula (Ia), R2 is H or a halogen group.

7. The pharmaceutical product of claim 5, wherein in formula (Ia), R1 is ethyl, R2 is selected from H, chloro and fluorine, and R3 is methyl.

8. The pharmaceutical product of claim 1, wherein the PARP1 selective inhibitor is AZD5305 or a pharmaceutically acceptable salt thereof, as represented by the following formula.

9. The pharmaceutical product of claim 1, wherein the anti-HER2 antibody is an antibody comprising the following heavy chain and light chain, wherein the heavy chain comprises a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 9, and the light chain comprises a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO:

10.

10. The pharmaceutical product of claim 1, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of an amino acid sequence represented by SEQ ID NO:

2.

11. The pharmaceutical product of claim 1, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 11 and a light chain consisting of an amino acid sequence represented by SEQ ID NO:

2.

12. The pharmaceutical product as claimed in claim 1, wherein the anti-HER2 antibody-drug conjugate is represented by the following formula:

12. Wherein “antibody” refers to an antiHER2 antibody that binds to a drug-linker via a thioether bond, and n represents the average number of drug-linker units bound to each antibody molecule in the antibody-drug conjugate, where n is in the range of 7 to 8.

13. The pharmaceutical product of claim 1, wherein the anti-HER2 antibody-drug conjugate is trastuzumab deruxtecan (DS-8201).

14. The pharmaceutical product of claim 1, wherein the product is a composition comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for simultaneous administration.

15. The pharmaceutical product of claim 1, wherein the product is a combination formulation comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor, for sequential or simultaneous administration.

16. The pharmaceutical product of any one of claims 1 to 15, wherein the product is used to treat cancer.

17. The pharmaceutical product of claim 16, wherein the cancer is selected from at least one of the following groups: breast cancer, stomach cancer, colorectal cancer, lung cancer, esophageal cancer, head and neck cancer, adenocarcinoma of the esophagogastric junction, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumor, digestive tract stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma.

18. The pharmaceutical product as claimed in claim 17, wherein the liver cancer is hepatocellular carcinoma.

19. The pharmaceutical product as claimed in claim 17, wherein the cancer is breast cancer.

20. The pharmaceutical product as claimed in claim 19, wherein the breast cancer has an IHC 3+ HER2 status score.

21. The pharmaceutical product as claimed in claim 19, wherein the breast cancer is HER2-hypoactive breast cancer.

22. The pharmaceutical product as claimed in claim 19, wherein the breast cancer has an IHC 2+ HER2 status score.

23. The pharmaceutical product as claimed in claim 19, wherein the breast cancer has an IHC 1+ HER2 status score.

24. The pharmaceutical product as claimed in claim 19, wherein the breast cancer has a HER2 status score of IHC > 0 and < 1+.

25. The pharmaceutical product as claimed in claim 19, wherein the breast cancer is triple-negative breast cancer.

26. The pharmaceutical product as claimed in claim 16, wherein the cancer is stomach cancer.

27. The pharmaceutical product as claimed in claim 16, wherein the cancer is colorectal cancer.

28. The pharmaceutical product as claimed in claim 16, wherein the cancer is lung cancer.

29. The pharmaceutical product as claimed in claim 28, wherein the lung cancer is non-small cell lung cancer.

30. The pharmaceutical product as claimed in claim 16, wherein the cancer is pancreatic cancer.

31. The pharmaceutical product as claimed in claim 16, wherein the cancer is ovarian cancer.

32. The pharmaceutical product as claimed in claim 16, wherein the cancer is prostate cancer.

33. The pharmaceutical product as claimed in claim 16, wherein the cancer is kidney cancer.

34. Use of an anti-HER2 antibody-drug conjugate or a PARP1 selective inhibitor in the manufacture of a medicament for treating cancer, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of claims 1 to 13.

35. As claimed in claim 34, wherein the cancer is selected from at least one of the following groups: breast cancer, stomach cancer, colorectal cancer, lung cancer, esophageal cancer, head and neck cancer, adenocarcinoma of the esophagogastric junction, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumor, digestive tract stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma.

36. As used in claim 35, wherein the liver cancer is hepatocellular carcinoma.

37. As used in claim 34, wherein the cancer is breast cancer.

38. As requested in claim 37, wherein the breast cancer has an IHC 3+ HER2 status score.

39. As claimed in claim 37, wherein the breast cancer is a HER2-hypoactive breast cancer.

40. As claimed in claim 37, wherein the breast cancer has an IHC 2+ HER2 status score.

41. As requested in claim 37, wherein the breast cancer has an IHC 1+ HER2 status score.

42. As used in claim 37, wherein the breast cancer has an IHC score of >0 and <1+ for HER2 status.

43. As used in claim 37, wherein the breast cancer is triple-negative breast cancer.

44. As used in claim 34, wherein the cancer is stomach cancer.

45. As claimed in claim 34, wherein the cancer is colorectal cancer.

46. ​​As used in claim 34, wherein the cancer is lung cancer.

47. As used in claim 46, wherein the lung cancer is non-small cell lung cancer.

48. As used in claim 34, wherein the cancer is pancreatic cancer.

49. As used in claim 34, wherein the cancer is ovarian cancer.

50. As claimed in claim 34, wherein the cancer is prostate cancer.

51. As claimed in claim 34, wherein the cancer is kidney cancer.

52. The use of any one of claims 34 to 51, wherein the drug is a composition comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor, for simultaneous administration.

53. The use of any one of claims 34 to 51, wherein the drug is a combination formulation comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor, for sequential or simultaneous administration.

Images