Combination of an Antibody-Drug Conjugate and a PARP1 Selective Inhibitor

Abstract

There are provided pharmaceutical products for the combined administration of an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor. The anti-HER2 antibody-drug conjugate is an antibody-drug conjugate in which a drug linker represented by the following formula (where A represents the position of linkage to the antibody) is conjugated to the anti-HER2 antibody via a thioether bond. There are also provided therapeutic uses and methods in which the antibody-drug conjugate and the PARP1 selective inhibitor are administered in combination to a subject: Formula (I)

Description

[Technical Field]

[0001] This disclosure relates to a pharmaceutical product for administration in combination with a specific antibody-drug conjugate and a selective PARP1 inhibitor, the specific antibody-drug conjugate having an anti-tumor drug conjugated to an anti-HER2 antibody via a linker structure. This disclosure also relates to therapeutic use and methods in which the specific antibody-drug conjugate and the selective PARP1 inhibitor are administered in combination to a subject. [Background Technology]

[0002] The poly(ADP-ribose) polymerase (PARP) family of enzymes plays an important role in many cellular 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, for example, in WO 2004 / 080976.

[0003] PARP1 and PARP2 are extensively studied PARPs due to their roles in DNA damage repair. PARP1 is activated by DNA breakage and catalyzes the addition of the poly(ADP-ribose) (PAR) chain to the target protein. This post-translational modification (called PARylation) mediates the recruitment of additional DNA repair factors to the DNA damage site. After completing this recruitment task, PARP auto-PARylation triggers the release of bound PARP from the DNA, allowing the use of other DNA repair proteins to complete the repair. Therefore, PARP binding to the damaged site, its catalytic activity, and its eventual release from the DNA are all important steps in cancer cells' response to DNA damage induced by chemotherapy and radiotherapy (Bai P. Biology of poly(ADP-ribose) polymerases: the factotums of cell maintenance. Mol Cell 2013; 38: 947-58).

[0004] Inhibition of PARP family enzymes has been used as a strategy to selectively kill cancer cells by inactivating complementary DNA repair pathways. Numerous preclinical and clinical studies have demonstrated that tumor cells bearing detrimental alterations to BRCA1 or BRCA2 (key tumor suppressor proteins involved in the repair of double-stranded DNA breaks (DSBs) via homologous recombination (HR)) are selectively sensitive to small-molecule inhibitors of the PARP family of DNA repair enzymes. These tumors have defective homologous recombination repair (HRR) pathways, and their survival depends on the function of PARP enzymes. Although PARP inhibitor therapy primarily targets BRCA-mutated cancers, PARP inhibitors have been clinically tested in non-BRCA-mutated tumors exhibiting homologous recombination deficiency (HRD) (Turner N, Tutt A, Ashworth A. Hallmarks of 'BRCAness' in sporadic cancers. Nat Rev Cancer 2004; 4: 8149).

[0005] It is believed that PARP inhibitors with increased selectivity for PARP1 may have improved efficacy and reduced toxicity compared to non-selective PARP inhibitors. It is also believed that strong selective inhibition of PARP1 will lead to PARP1 capture on DNA, which results in DNA double-strand breaks (DSBs) by causing the replication fork to collapse in S phase. PARP1-DNA capture is also believed to be an effective mechanism for selectively killing tumor cells with HRD.

[0006] Antibody-drug conjugates (ADCs), consisting of cytotoxic drugs conjugated with antibodies, can selectively deliver drugs to cancer cells and are therefore expected to cause drug accumulation within cancer cells and kill them (Ducry, L. et al., Bioconjugate Chem. (2010) 21, 5-13; Alley, SC et al., Current Opinion in Chemical Biology (2010) 14, 529-537; Damle NK Expert Opin. Biol. Ther. (2004) 4, 1445-1452; Senter PD 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 an antibody targeting HER2 and a derivative of eczematidine (Ogitani Y. et al., Clinical Cancer Research (2016) 22(20), 5097-5108; Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). Delutec-trastuzumab ( DS-8201 has demonstrated significant clinical efficacy in HER2-expressing solid tumors, including breast cancer, gastric cancer, colorectal cancer, and non-small cell lung cancer. Notably, DS-8201 has shown promising activity in HER2-low tumors within these indications. There is a need to identify combination partners of DS-8201 to enhance efficacy, increase the durability of treatment response, improve patient tolerability, and / or reduce dose-dependent toxicity.

[0008] Although antibody-drug conjugates (such as dlutecan-trastuzumab) and PARP1 inhibitors have therapeutic potential, no literature has been published describing test results demonstrating the superior efficacy of combining antibody-drug conjugates and selective PARP1 inhibitors.

[0009] Therefore, there is still a need for improved therapeutic compositions and methods that can enhance the efficacy of existing cancer therapies, increase the durability of treatment responses, improve patient tolerability, and / or reduce dose-dependent toxicity. [Summary of the Invention]

[0010] The antibody-drug conjugates used in this disclosure (including anti-HER2 antibody-drug conjugates of derivatives of the topoisomerase I inhibitor ezetezine, as components) have been shown to exhibit excellent antitumor effects in the treatment of certain cancers, such as breast and gastric cancer, when administered alone. Furthermore, PARP1 inhibitors have been shown to exhibit antitumor activity in the treatment of certain cancers. However, there is a need to provide drugs and treatments that can achieve excellent antitumor effects in cancer treatment, such as enhanced efficacy, increased durability of treatment response, and / or reduced dose-dependent toxicity.

[0011] This disclosure provides a pharmaceutical product that demonstrates excellent antitumor efficacy in cancer treatment when administered in combination with an anti-HER2 antibody-drug conjugate and a selective PARP1 inhibitor. This disclosure also provides therapeutic uses and methods in which the anti-HER2 antibody-drug conjugate and the selective PARP1 inhibitor are administered in combination to a subject.

[0012] Specifically, this disclosure relates to the following[1] through

[54] :

[0013] [1] A pharmaceutical product comprising an antiHER2 antibody-drug conjugate and a PARP1 selective inhibitor for combined administration, wherein the antiHER2 antibody-drug conjugate is an antibody-drug conjugate in which a drug-linker represented by the following formula is conjugated to an antiHER2 antibody via a thioether bond:

[0014]

[0015] Where A represents the binding site with the antibody;

[0016] [2] The pharmaceutical product as described in [1], wherein the PARP1 selective inhibitor is a compound represented by the following formula (I):

[0017]

[0018] in:

[0019] X 1 and X 2 Each is independently selected from N and C(H).

[0020] X 3 Independently selected from N and C(R) 4 ), where R 4 It is H or fluorine.

[0021] R 1 It is C 1-4 Alkyl or C 1-4 fluoroalkyl,

[0022] R 2 Independently selected from H, halogenated, C 1-4 Alkyl and C 1-4 Fluoroalkyl groups, and

[0023] R 3 Is it H or C? 1-4 alkyl,

[0024] or its pharmaceutically acceptable salt

[0025] The conditions are:

[0026] When X 1 When it is N, then X 2 It is C(H), and X 3 It is C(R) 4 ),

[0027] When X 2 When it is N, then X 1 =C(H), and X 3 It is C(R) 4 ),and

[0028] When X 3 When it is N, then X 1 and X 2 All are C(H):

[0029] [3] The pharmaceutical product as described in [2], wherein, in formula (I), R 3 It is C 1-4 alkyl;

[0030] [4] The pharmaceutical product as described in [3], wherein, in formula (I), R 3 It is methyl;

[0031] [5] The pharmaceutical product as described in any one of [2] to [4], wherein, in formula (I), R 1 It is ethyl;

[0032] [6] The pharmaceutical product as described in [1], wherein the PARP1 selective inhibitor is a compound represented by the following formula (Ia):

[0033]

[0034] in

[0035] R 1 It is C 1-4 alkyl,

[0036] R 2 Selected from H, halogenated, C 1-4 Alkyl and C 1-4 fluoroalkyl,

[0037] R 3 Is it H or C? 1-4 Alkyl, and

[0038] R 4 It's H.

[0039] Or its pharmaceutically acceptable salt;

[0040] [7] The pharmaceutical product as described in [6], wherein, in formula (Ia), R 2 It is H or halogenated;

[0041] [8] The pharmaceutical product as described in [6], wherein, in formula (Ia), R 1 It is ethyl, R 2 Selected from H, chlorine, and fluorine, and R 3 It is methyl;

[0042] [9] The drug product as described in [1], wherein the PARP1 selective inhibitor is AZD5305, also known as AZ14170049, is represented by the following formula:

[0043]

[0044] Or its pharmaceutically acceptable salt;

[0045]

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

[0046]

[11] The pharmaceutical product as described in any one of [1] to [9], wherein the antiHER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region consisting of an amino acid sequence represented by amino acid residues 1 to 120 of SEQ ID NO: 9 [= SEQ ID NO: 1], and the light chain comprising a light chain variable region consisting of an amino acid sequence represented by amino acid residues 1 to 107 of SEQ ID NO: 2;

[0047]

[12] The pharmaceutical product as described in any one of [1] to [9], wherein the antiHER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence represented by SEQ ID NO: 1 and the light chain comprising the amino acid sequence represented by SEQ ID NO: 2;

[0048]

[13] The pharmaceutical product as described in any one of [1] to [9], wherein the antiHER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence represented by SEQ ID NO: 11 [= amino acid residues 1 to 449 of SEQ ID NO: 1], and the light chain comprising the amino acid sequence represented by SEQ ID NO: 2;

[0049]

[14] The pharmaceutical product as described in any one of [1] to

[13] , wherein the anti-HER2 antibody-drug conjugate is represented by the following formula:

[0050]

[0051] Where 'antibody' indicates an antiHER2 antibody conjugated to a drug-linker via a thioether bond, and n indicates the average number of drug-linker units conjugated to each antibody molecule in the antibody-drug conjugate, where n is in the range of 7 to 8;

[0052]

[15] The pharmaceutical product as described in any one of [1] to

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

[0053]

[16] The pharmaceutical product as described in any one of [1] to

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

[0054]

[17] The pharmaceutical product as described in any one of [1] to

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

[0055]

[18] The pharmaceutical product as described in any one of [1] to

[17] , wherein the product is used to treat cancer;

[0056]

[19] The pharmaceutical product as described in

[18] , wherein the cancer is selected from at least one of the following: breast cancer, gastric 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, hepatocellular carcinoma, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma and melanoma;

[0057]

[20] The pharmaceutical product as described in

[19] , wherein the cancer is breast cancer;

[0058]

[21] The pharmaceutical product as described in

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

[0059]

[22] The pharmaceutical product as described in

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

[0060]

[23] The pharmaceutical product as described in

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

[0061]

[24] The pharmaceutical product as described in

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

[0062]

[25] The pharmaceutical product as described in

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

[0063]

[26] The pharmaceutical product as described in

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

[0064]

[27] The pharmaceutical product as described in

[18] , wherein the cancer is gastric cancer;

[0065]

[28] The pharmaceutical product as described in

[18] , wherein the cancer is colorectal cancer;

[0066]

[29] The pharmaceutical product as described in

[18] , wherein the cancer is lung cancer;

[0067]

[30] The pharmaceutical product as described in

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

[0068]

[31] The pharmaceutical product as described in

[18] , wherein the cancer is pancreatic cancer;

[0069]

[32] The pharmaceutical product as described in

[18] , wherein the cancer is ovarian cancer;

[0070]

[33] The pharmaceutical product as described in

[18] , wherein the cancer is prostate cancer;

[0071]

[34] The pharmaceutical product as described in

[18] , wherein the cancer is kidney cancer;

[0072]

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

[17] , which is used to treat cancer;

[0073]

[36] For use in pharmaceutical products as described in

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

[19] to

[34] ;

[0074]

[37] Use of an antiHER2 antibody-drug conjugate or a PARP1 selective inhibitor in the preparation of a medicament for the treatment of cancer by combination administration of the antiHER2 antibody-drug conjugate and the PARP1 selective inhibitor, wherein the antiHER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] .

[0075]

[38] The use as described in

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

[19] to

[34] ;

[0076]

[39] As described in

[37] or

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

[0077]

[40] Uses as described in

[37] or

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

[0078]

[41] An antiHER2 antibody-drug conjugate for the treatment of cancer in combination with a PARP1 selective inhibitor, wherein the antiHER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] ;

[0079]

[42] For use in antiHER2 antibody-drug conjugates as described in

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

[19] to

[34] ;

[0080]

[43] For use of antiHER2 antibody-drug conjugates as described in

[41] or

[42] , wherein the use comprises sequential administration of the antiHER2 antibody-drug conjugate and the PARP1 selective inhibitor;

[0081]

[44] For use of antiHER2 antibody-drug conjugates as described in

[41] or

[42] , wherein the use comprises simultaneous administration of the antiHER2 antibody-drug conjugate and a PARP1 selective inhibitor;

[0082]

[45] An antiHER2 antibody-drug conjugate for cancer treatment of a subject, wherein the treatment comprises administering to the subject separately, sequentially or simultaneously i) the antiHER2 antibody-drug conjugate and ii) a PARP1 selective inhibitor, wherein the antiHER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] ;

[0083]

[46] A PARP1 selective inhibitor in combination with an antiHER2 antibody-drug conjugate for cancer treatment, wherein the antiHER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] ;

[0084]

[47] For use of a PARP1 selective inhibitor as described in

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

[19] to

[34] ;

[0085]

[48] ​​For use as described in

[46] or

[47] , wherein the use comprises sequential administration of an antiHER2 antibody-drug conjugate and a PARP1 selective inhibitor;

[0086]

[49] For use as described in

[46] or

[47] , wherein the use comprises the simultaneous administration of an antiHER2 antibody-drug conjugate and a PARP1 selective inhibitor;

[0087]

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

[15] ;

[0088]

[51] A method of treating cancer, comprising administering to a subject in need a combination of an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor as defined in any of [1] to

[15] ;

[0089]

[52] The method as described in

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

[19] to

[34] ;

[0090]

[53] The method as described in

[51] or

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

[0091]

[54] The method as described in

[51] or

[52] , wherein the method comprises the simultaneous administration of an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor.

[0092] [Disclosed beneficial effects]

[0093] This disclosure provides pharmaceutical products in which an anti-HER2 antibody-drug conjugate and a selective PARP1 inhibitor are administered in combination. The anti-HER2 antibody-drug conjugate is an antitumor drug conjugated to an anti-HER2 antibody via a linker structure. This disclosure also provides therapeutic uses and methods in which the specific antibody-drug conjugate and the selective PARP1 inhibitor are administered in combination to a subject. Therefore, this disclosure can provide pharmaceuticals and treatments that can achieve excellent antitumor effects in cancer treatment. [Attached Image Description]

[0094] [ Figure 1 ] Figure 1 This is a diagram showing the amino acid sequence (SEQ ID NO: 1) of the heavy chain of the anti-HER2 antibody.

[0095] [ Figure 2 ] Figure 2 This is a diagram showing the amino acid sequence (SEQ ID NO: 2) of the light chain of the anti-HER2 antibody.

[0096] [ Figure 3 ] Figure 3 This 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]).

[0097] [ Figure 4 ] Figure 4 This is a diagram showing the amino acid sequence of the heavy chain CDRH2 (SEQ ID NO: 4 [= amino acid residues 51 to 58 of SEQ ID NO: 1]).

[0098] [ Figure 5 ] Figure 5 This is a diagram showing the amino acid sequence of the heavy chain CDRH3 (SEQ ID NO: 5 [= amino acid residues 97 to 109 of SEQ ID NO: 1]).

[0099] [ Figure 6 ] Figure 6 This 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]).

[0100] [ Figure 7 ] Figure 7 This is a diagram showing the amino acid sequence (SEQ ID NO: 7 [= amino acid residues 50 to 56 of SEQ ID NO: 2]) containing the light chain CDRL2 (SAS).

[0101] [ Figure 8 ] Figure 8 This 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]).

[0102] [ Figure 9 ] Figure 9 This is a diagram showing the amino acid sequence of the variable region of the heavy chain (SEQ ID NO: 9 [= amino acid residues 1 to 120 of SEQ ID NO: 1]).

[0103] [ Figure 10 ] Figure 10 This is a diagram showing the amino acid sequence of the variable region of the light chain (SEQ ID NO: 10 [=amino acid residues 1 to 107 of SEQ ID NO: 2]).

[0104] [ Figure 11 ] Figure 11 This 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]).

[0105] [ Figure 12A and 12B ] Figure 12A and 12B This is a diagram showing the combination matrix obtained by high-throughput screening of DS-8201 in cell lines with high HER2 expression by combining AZD5305 (AZ14170049; a selective inhibitor of PARP1).

[0106] [ Figure 13A and 13B ] Figure 13A and 13B This is a diagram showing the combination matrix obtained by high-throughput screening of DS-8201 and AZD5305 in cell lines with low HER2 expression.

[0107] [ Figure 14 ] Figure 14 This is a graph showing the combined Emax and Loewe synergistic scores in cell lines treated with a combination of DS-8201 and AZD5305.

[0108] [ Figure 15A and 15B ] Figure 15A and 15B This is a diagram showing the combination matrix used to combine DS-8201 with AZD5305 in cell lines with low or high HER2 expression.

[0109] [ Figure 16A and 16B ] Figure 16A and 16B The X-ray diffraction pattern and representative DSC trace of synthetic example 4, form A, are shown respectively.

[0110] [ Figure 17 ] Figure 17 This graph shows the tumor volume after in vivo treatment with DS-8201 or AZD5305 alone, or in combination with DS-8201 and AZD5305. The dashed line indicates the end of the AZD5305 dosing period.

[0111] [ Figure 18A , 18B and 18C] Figure 18A , 18B18C is a diagram showing the combination matrix obtained by high-throughput screening of DS-8201 and AZD5305 in NSCLC cell lines with low or high HER2 expression.

[0112] [ Figure 19A , 19B and 19C] Figure 19A , 19B 19C is a diagram showing the combination matrix obtained by high-throughput screening of DS-8201 and AZD5305 in urinary tract cancer cell lines with HER2 mutant expression.

[0113] To make this disclosure easier to understand, some terms are first defined. Further definitions are listed throughout the detailed explanation.

[0114] Before describing this disclosure in detail, it should be understood that this disclosure is not limited to specific compositions or method steps, as these compositions or method steps can vary. As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include the plural references. The term “a” (or “an”) and the terms “one or more” and “at least one” are used interchangeably herein.

[0115] Furthermore, “and / or” as used herein is considered to be a specific disclosure of each of the two specified features or components, with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” is intended herein to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as 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).

[0116] Unless otherwise defined, 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 edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide general dictionary notes for those skilled in the art regarding many of the terms used in this disclosure.

[0117] Units, prefixes, and symbols are represented in a form accepted by their International System of Units (SI). Numerical ranges include the numbers that define the range.

[0118] It should be understood that, in all cases where this article uses the language “contains” to describe aspects, other similar aspects described as “composed of” and / or “mainly composed of” are also provided.

[0119] The terms “inhibition,” “block,” and “repression” are used interchangeably herein and refer to any statistically significant reduction in biological activity, including complete blockage of activity. For example, “inhibition” can refer to a reduction in biological activity of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Cell proliferation can be determined using techniques recognized in the art that measure the rate of cell division, and / or the fraction of cells undergoing cell division in the cell population, and / or the rate of cell loss from the cell population due to terminal differentiation or cell death (e.g., thymidine incorporation).

[0120] The term "subject" refers to any animal (e.g., a mammal) that is to become a recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably in this document with respect to human subjects.

[0121] The term "pharmaceutical product" refers to a formulation whose form allows for the biological activity of the active ingredients, either as a composition containing all the active ingredients (for simultaneous administration) or as a combination of individual compositions each containing at least one, but not all, of the active ingredients (for sequential or simultaneous administration), and which does not contain any additional components that would have unacceptable toxicity to the subject 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 sequentially, with time intervals between administrations. The time interval may be, for example, less than 24 hours, preferably less than 6 hours, and more preferably less than 2 hours.

[0122] Terms such as “treating” or “treatment or to treat” or “alleviating or to alleviate” refer to (1) therapeutic measures that cure, alleviate, reduce symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) preventive or preventative measures that prevent and / or slow the development of a targeted pathological condition or disorder. Therefore, those requiring treatment include those who already have the disorder; those who are predisposed to have the disorder; and those among them who require prevention of the disorder. In some respects, if a patient demonstrates, for example, a general, partial, or transient remission of a certain type of cancer, then the subject’s cancer has been successfully “treated” according to the methods disclosed herein.

[0123] The terms “cancer,” “tumor,” “cancerous,” and “malignant” refer to or describe a physiological condition in mammals characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, 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, hepatocellular carcinoma, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma. Cancer includes hematologic malignancies such as acute myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, as well as solid tumors such as breast cancer, lung cancer, neuroblastoma, and colon cancer.

[0124] As used herein, the term "cytotoxic agent" is broadly defined and refers to a substance that inhibits or prevents cellular function and / or causes cell destruction (cell death), and / or exerts an antitumor / antiproliferative effect. For example, cytotoxic agents directly or indirectly inhibit the development, maturation, or spread of neoplastic tumor cells. The term also includes such agents that cause only an inhibitory effect on cell growth and not merely a cytotoxic effect. The term includes chemotherapeutic agents, along with other HER2 antagonists, anti-angiogenic agents, tyrosine kinase inhibitors, protein kinase A inhibitors, members of the cytokine family, radioactive isotopes, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin.

[0125] The term "chemotherapeutic agent" is a subset of the term "cytotoxic agent," which includes natural or synthetic chemical compounds.

[0126] According to the methods or uses disclosed herein, the compounds disclosed may be administered to patients to promote an active therapeutic response to cancer. The term "active therapeutic response" for cancer treatment refers to improvement in symptoms associated with the disease. For example, improvement in the disease can be characterized as a complete response. The term "complete response" means the absence of clinically detectable disease and normal results for any prior tests. Alternatively, improvement in the disease can be categorized as a partial response. "Active therapeutic response" encompasses a reduction or inhibition of cancer progression and / or duration, a reduction or improvement in cancer severity, and / or improvement in one or more of its symptoms resulting from the administration of the compounds disclosed herein. In a particular aspect, such terms refer to one, two, three, or more of the following outcomes following the administration of the compounds disclosed:

[0127] (1) Stabilization, reduction or elimination of cancer cell populations;

[0128] (2) Stabilization or reduction of cancer growth;

[0129] (3) Impaired cancer formation;

[0130] (4) Eradication, removal, or control of primary, regional, and / or metastatic cancers;

[0131] (5) Mortality rate decreased;

[0132] (6) No disease, no recurrence, no progression, and / or an increase in overall survival, duration or rate;

[0133] (7) Increase in response rate, duration of response, or number of patients in response or remission;

[0134] (8) Decrease in hospitalization rate,

[0135] (9) Reduced hospital stay

[0136] (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

[0137] (11) An increase in the number of patients in remission.

[0138] (12) Otherwise, the number of adjuvant therapies (such as chemotherapy or hormone therapy) required to treat cancer is reduced.

[0139] Clinical response can be assessed using screening techniques such as PET, magnetic resonance imaging (MRI), X-ray imaging, computed tomography (CT) scans, flow cytometry or fluorescence activated cell sorting (FACS) analysis, histology, macroscopic pathology, and blood chemistry, including but not limited to changes detectable by ELISA, RIA, chromatography, etc. In addition to these positive treatment responses, subjects undergoing treatment may experience beneficial effects such as improvement in disease-related symptoms.

[0140] The alkyl group and part are either straight-chain or branched, for example, C 1-8 Alkyl, C 1-6 Alkyl, C 1-4 Alkyl or C 5-6 Alkyl groups. 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.

[0141] A fluoroalkyl group is an alkyl group in which one or more H atoms are replaced by one or more fluorine atoms, such as C. 1-8 fluoroalkyl, C 1-6 fluoroalkyl, C 1-4 fluoroalkyl or C 5-6 Fluoroalkyl groups. Examples include fluoromethyl (CH2F.), difluoromethyl (CHF2-), trifluoromethyl (CF3-), 2,2,2-trifluoroethyl (CF3CH2-), 1,1-difluoroethyl (CH3CHF2-), 2,2-difluoroethyl (CHF2CH2-), and 2-fluoroethyl (CH2FCH2-).

[0142] Halogenation refers to fluorine, chlorine, bromine, and iodine. In one embodiment, halogenation is fluorine or chlorine.

[0143] 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 condition to be treated (e.g., provide a positive clinical response). The effective amount of an active ingredient used in a pharmaceutical product will vary depending on the specific condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent treatment, one or more specific active ingredients used, one or more specific pharmaceutically acceptable excipients / carriers utilized, and similar factors within the knowledge and expertise of the attending physician. In particular, the effective amount of a compound used in combination with an antibody-drug conjugate for the treatment of cancer is an amount sufficient to symptomatically relieve cancer symptoms in warm-blooded animals such as humans, slow cancer progression, or reduce the risk of deterioration in patients with cancer symptoms.

[0144] In this specification, unless otherwise stated, the term "pharmaceutically acceptable" as used herein means those compounds, materials, compositions, and / or dosage forms that, to a reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications (in proportion to a reasonable benefit / risk ratio).

[0145] It should be understood that compounds having formula (I) can form stable, pharmaceutically acceptable acid salts or base salts, and in such cases, it may be appropriate to administer the compound as a salt. Examples of acid addition salts include acetates, adipates, ascorbic acid salts, benzoates, benzenesulfonates, bicarbonates, bisulfates, butyrates, camphorates, camphorsulfonates, choline, citrates, cyclohexylaminosulfonates, diethylenediamine, ethanesulfonates, fumarates, glutamates, glycolates, hemisulfates, 2-hydroxyethylsulfonates, heptanates, hexanoates, hydrochlorides, hydrobromides, hydroiodates, hydroxymaleates, lactates, malates, maleates, methanesulfonates, meglumine, 2-naphthalenesulfonates, nitrates, oxalates, dihydroxynaphthalate, persulfates, phenylacetic acid salts, phosphates, hydrogen phosphates, picrates, neopentanoates, propionates, quinates, salicylates, stearates, succinates, aminosulfonates, aminobenzenesulfonates, sulfates, tartrates, toluenesulfonates (p-toluenesulfonates), trifluoroacetates, and undecanoates. Although other salts can be used, such as in the separation or purification of products, non-toxic, physiologically acceptable salts are preferred.

[0146] These salts can be formed by conventional means, such as by reacting the free basic form of the product with an equivalent of one or more suitable acids in a solvent or medium in which the salt is insoluble or in a solvent (e.g., a solvent in which water has been removed in a vacuum), by freeze-drying, or by exchanging the anion of an existing salt for another anion on a suitable ion exchange resin.

[0147] Compounds having formula (I) may have more than one chiral center, and it should be understood that this application covers all individual stereoisomers, enantiomers, and diastereomers, and mixtures thereof. Therefore, it should be understood that where a compound having formula (I) exists in an optically active or racemic form by means of one or more asymmetric carbon atoms, this application includes any such optically active or racemic form having the aforementioned activity as defined herein. This application covers all such stereoisomers having the activity defined herein.

[0148] Therefore, throughout this specification, when referring to compounds having formula (I), it should be understood that the term "compound" includes diastereomers, mixtures of diastereomers, and enantiomers that are PARP1 inhibitors.

[0149] It should also be understood that certain compounds having formula (I) and their pharmaceutical salts can exist in both solvated and non-solvated forms (e.g., hydrated and anhydrous forms). It should be understood that the compounds referred to herein encompass all such solvated forms. For clarity, this includes both solvation of the free form of the compound (e.g., aqueous) and solvation of the salts of the compound (e.g., aqueous).

[0150] Some compounds having formula (I) can be crystalline and may have more than one crystalline form. It should be understood that this disclosure covers any crystalline or amorphous form or mixture thereof that has PARP1 selective inhibitory activity. It is generally 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).

[0151] Formula (I) as described herein is intended to cover all isotopes of its constituent atoms. For example, H (or hydrogen) includes any isotopic form of hydrogen, including 1 H, 2 H(D), and 3 H(T); C includes any isotopic form of carbon, including 12 C 13 C, and 14 C; O includes any isotopic form of oxygen, including 16 O、 17 O, and 18 O; N includes any isotopic form of nitrogen, including 13 N、 14 N, and 15 N; F includes any isotopic form of fluorine, including 19 F and 18F; etc. On one hand, compounds having formula (I) include isotopes of the atoms covered herein, the amounts of which correspond to their naturally occurring abundance. However, in some cases, it may be desirable to enrich one or more atoms of a specific isotope that would normally exist in lower abundance. For example, under normal circumstances... 1 H is present in abundance greater than 99.98%; however, on the other hand, compounds having any of the chemical formulas presented herein can be enriched at one or more sites where H is present. 2 H or 3 H. In another respect, when a compound having any of the chemical formulas presented herein is enriched with a radioactive isotope (e.g., 3 H and 14 In case C), the compound can be used for drug and / or substrate tissue distribution assays. It should be understood that this application covers all such isotopic forms.

Detailed Implementation Methods

[0152] Preferred methods for implementing this disclosure are described below. The embodiments described below are merely examples illustrating typical embodiments of this disclosure and are not intended to limit the scope of this disclosure.

[0153] 1. Antibody-drug conjugates

[0154] The antibody-drug conjugates used in this disclosure are antibody-drug conjugates in which the drug-linker represented by the following formula is conjugated to an anti-HER2 antibody via a thioether bond.

[0155]

[0156] Where A represents the binding site with the antibody.

[0157] In this disclosure, the portion of the antibody-drug conjugate consisting of a linker and a drug is referred to as the "drug-linker." The drug-linker is attached to a thiol group (in other words, the sulfur atom of a cysteine ​​residue) formed at interchain disulfide bond sites in the antibody (two sites between the heavy chains and two sites between the heavy and light chains).

[0158] The drug-connector disclosed herein includes eczemac (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]indolazino[1,2-b]quinoline-10,13-dione, (also represented by the chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-10,13(9H,15H)-dione), which is a topoisomerase I inhibitor, as a component. Ecinotecan is a camptothecin derivative with antitumor activity, represented by the following formula:

[0159]

[0160] The anti-HER2 antibody-drug conjugate used in this disclosure can also be represented by the following formula:

[0161]

[0162] Here, the drug-linker is conjugated to the anti-HER2 antibody (“antibody-”) via a thioether bond. The meaning of 'n' is the same as that of the so-called drug-to-antibody ratio (DAR), indicating the average number of drug-linker units conjugated to each antibody molecule.

[0163] After migrating into cancer cells, the anti-HER2 antibody-drug conjugate used in this disclosure is cleaved at the linker site to release a compound represented by the following formula:

[0164]

[0165] It is speculated that this compound is the original source of the antitumor activity of the antibody-drug conjugate used in this disclosure, and it has been confirmed to have topoisomerase I inhibitory activity (Ogitani Y. et al., Clinical Cancer Research, Oct 2016, 15; 22(20): 5097-5108, Epub March 29, 2016).

[0166] The anti-HER2 antibody-drug conjugates used in this disclosure are known to exhibit a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). The bystander effect operates through a process in which the antibody-drug conjugates used in this disclosure are internalized in cancer cells expressing the target, and the released compounds also exert antitumor effects on cancer cells present in their vicinity that do not express the target. This bystander effect demonstrates superior antitumor efficacy even when the anti-HER2 antibody-drug conjugates are used in combination with a PARP1 selective inhibitor according to this disclosure.

[0167] 2. Antibodies in antibody-drug conjugates

[0168] The anti-HER2 antibody in the antibody-drug conjugate used in this disclosure can be derived from any species, and is preferably derived from human, rat, mouse, or rabbit anti-HER2 antibodies. In cases where the antibody is derived from a species other than human, it is preferable to use well-known techniques to chimeric or humanize it. The anti-HER2 antibody can be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody.

[0169] The antibody in the antibody-drug conjugate used in this disclosure is preferably an anti-HER2 antibody that has the property of targeting cancer cells, and is preferably an antibody that has, for example, the property of recognizing cancer cells, the property of binding to cancer cells, the property of internalization in cancer cells, and / or the property of killing cells against cancer cells.

[0170] The binding activity of anti-HER2 antibodies to cancer cells can be confirmed by flow cytometry. The internalization of antibodies into cancer cells can be confirmed by: (1) observation of the antibody incorporated into cells under a fluorescence microscope using a secondary antibody (fluorescently labeled) bound to a therapeutic antibody (Cell Death and Differentiation, 2008) 15, 751-761); (2) measurement of the fluorescence intensity in incorporated cells using a secondary antibody (fluorescently labeled) bound to a therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004); or (3) a Mab-ZAP assay using an immunotoxin bound to a therapeutic antibody, wherein the toxin is released after incorporation to inhibit cell growth (Bio Techniques, 28: 162-165, January 2000). As an immunotoxin, a recombinant complex of diphtheria toxin catalytic domain and protein G can be used.

[0171] The antitumor activity of antiHER2 antibodies can be confirmed in vitro by measuring their inhibitory activity against cell growth. For example, cancer cell lines overexpressing HER2, the target protein of the antibody, can be cultured, and the antibody can be added to the culture system at different concentrations to measure its inhibitory activity against lesion formation, colony formation, and globular growth. Antitumor activity can be confirmed in vivo, for example, by administering the antibody to nude mice with transplanted cancer cell lines that highly express the target protein and measuring changes in the cancer cells.

[0172] Since the compounds conjugated in the antiHER2 antibody-drug conjugate exert antitumor effects, it is preferred, but not essential, that the antiHER2 antibody itself should possess antitumor activity. For the antitumor compound to exert its cytotoxic activity against cancer cells specifically and selectively, it is important and preferred that the antiHER2 antibody possess the property of internalization to migrate into cancer cells.

[0173] The anti-HER2 antibody in the antibody-drug conjugate used in this disclosure can be obtained by methods known in the art. For example, the antibody disclosed herein can be obtained using methods commonly practiced in the art, which involve immunizing an animal with an antigenic peptide and collecting and purifying the antibodies produced in vivo. The source of the antigen is not limited to humans, and the animal can be immunized with antigens derived from non-human animals such as mice, rats, etc. In this case, the cross-reactivity of the antibody binding to the obtained heterologous antigen with the human antigen can be tested to screen for antibodies suitable for human diseases.

[0174] Alternatively, according to methods known in the art, antibody-producing cells that produce antibodies against the antigen are fused with myeloma cells (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; and Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, New York (1980)) to establish a hybridoma from which monoclonal antibodies can then be obtained.

[0175] Antigens can be obtained by genetically engineering host cells to produce genes encoding antigen proteins. Specifically, a vector that allows antigen gene expression is prepared and transferred into host cells, thereby expressing the gene. The expressed antigen can then be purified. Antibodies can also be obtained by immunizing animals with the genetically engineered antigen-expressing cells or cell lines expressing the antigen described above.

[0176] The anti-HER2 antibody in the antibody-drug conjugate used in this disclosure is preferably a recombinant antibody obtained by artificial modification to reduce its heteroantigenicity to humans, such as a chimeric antibody or a humanized antibody, or preferably an antibody having only the gene sequence of an antibody derived from a human, i.e., a human antibody. These antibodies can be produced using known methods.

[0177] As chimeric antibodies, examples can be given of antibodies in which the variable region and constant region are derived from different species, such as chimeric antibodies in which the variable region of a mouse or rat antibody is linked to the constant region of a human antibody (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0178] Examples of humanized antibodies include antibodies obtained by integrating only the complementarity-determining region (CDR) of a heterologous antibody into a human antibody (Nature [Nature] (1986) 321, pp. 522-525), antibodies obtained by transplanting a portion of the amino acid residues of the heterologous antibody framework and the CDR sequence of the heterologous antibody into a human antibody via a CDR transplantation method (WO 90 / 07861), and antibodies humanized using a gene transformation mutagenesis strategy (US Patent No. 5821337).

[0179] As examples of human antibodies, one can cite antibodies produced by mice that produce human antibodies, which possess human chromosome segments containing the heavy and light chains of the human antibody (see Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects, Vol. 10, pp. 69-73 (edited by Kitagawa, Y., Matsuda, T. and Iijima, S.), Kluwer Academic). Publishers [Kruwell Academic Publishers], 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] (2000) 97, pp. 722-727, etc.). Alternatively, examples can be given of antibodies obtained by phage display, which are selected from human antibody libraries (see Wormstone, I.M. et al., Investigative Ophthalmology & Visual Science

[2002] 43(7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics

[2002] 1(2), pp. 189-203; Siriwardena, D. et al., Ophthalmology

[2002] 109(3), pp. 427-431, etc.).

[0180] This disclosure also includes modified variants of the anti-HER2 antibody in the antibody-drug conjugates used in this disclosure. A modified variant is a variant obtained through chemical or biological modification of the antibody according to this disclosure. Examples of chemically modified variants include variants comprising a chemical moiety linked to an amino acid backbone, variants comprising a chemical moiety linked to an N- or O-linked carbohydrate chain, etc. Examples of biologically modified variants include variants obtained through post-translational modifications (such as N- or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, or methionine oxidation), and variants by adding a methionine residue to the N-terminus through expression in prokaryotic host cells. Furthermore, antibodies labeled to enable the detection or separation of the antibody or antigen according to this disclosure, such as enzyme-labeled antibodies, fluorescently labeled antibodies, and affinity-labeled antibodies, are also included in the meaning of modified variants. Such modified variants of the antibody according to this disclosure can be used to improve antibody stability and blood retention, reduce its antigenicity, detect or separate the antibody or antigen, etc.

[0181] Furthermore, by modulating the modification (glycosylation, deglycosylation, etc.) of the glycan linked to the antibody according to this disclosure, it is possible to enhance antibody-dependent cytotoxic activity. Techniques for modulating the glycan modification of antibodies are known from disclosures in WO 99 / 54342, WO00 / 61739, WO 02 / 31140, WO 2007 / 133855, WO 2013 / 120066, etc. However, this technique is not limited thereto. The anti-HER2 antibody according to this disclosure also includes an antibody in which the glycan modification is modulated.

[0182] It is known that antibodies produced in cultured mammalian cells have a lysine residue deletion at the C-terminus of the heavy chain (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that antibodies produced in cultured mammalian cells have a two-amino acid residue (glycine and lysine) deletion at the C-terminus of the heavy chain, and are amidated with a new trans-proline residue at the C-terminus (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector functions (complement activation, antibody-dependent cytotoxicity, etc.) of the antibody. Therefore, the anti-HER2 antibody disclosed herein also includes antibodies and functional fragments of antibodies modified in this way, and also includes deletion variants in which one or two amino acids are missing at the C-terminus of the heavy chain, variants obtained by amidation of the deletion variants (e.g., heavy chains in which the C-terminal proline residue has been amidated), etc. The types of deletion variants with a deletion at the C-terminus of the heavy chain of the anti-HER2 antibody according to this disclosure are not limited to those described above, as long as antigen-binding affinity and effector function are preserved. The two heavy chains constituting the antibody according to this disclosure can be one type selected from the group consisting of the full-length heavy chain and the deletion variants described above, or a combination of two types selected therefrom. The ratio of the amount of each deletion variant can be affected by the type of mammalian cells cultured to produce the anti-HER2 antibody according to this disclosure; however, examples of antibodies in which one amino acid residue at the C-terminus of the two heavy chains of the antibody according to this disclosure is missing are preferred.

[0183] As isotypes of the anti-HER2 antibody according to this disclosure, examples may include IgG (IgG1, IgG2, IgG3, IgG4), and IgG1 or IgG2 may be cited as preferred.

[0184] In this disclosure, the term "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (human epidermal growth factor receptor type 2; ErbB-2), and preferably has the activity of internalization in HER2-expressing cells by binding to HER2.

[0185] Examples of anti-HER2 antibodies include trastuzumab (US Patent No. 5,821,337) and pertuzumab (WO 01 / 00245), with trastuzumab being a preferred example.

[0186] 3. Production of antibody-drug conjugates

[0187] The pharmaceutical linker intermediate used to produce the anti-HER2 antibody-drug conjugate according to this disclosure is represented by the following formula:

[0188]

[0189] The drug-connector intermediate can be represented by the chemical name N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]glycyl-glycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycamide, and can be referenced in WO 2014 / 057687, WO 2015 / 098099, WO 2015 / 115091, WO 2015 / 155998, WO The description in 2019 / 044947 is used for production.

[0190] The anti-HER2 antibody-drug conjugate used in this disclosure can be produced by reacting the aforementioned drug-connector intermediate with an anti-HER2 antibody having a thiol group (also known as a mercapto group).

[0191] Thiol-containing anti-HER2 antibodies can be obtained by methods known in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, 456-493, Academic Press (1996)). For example, thiol-containing anti-HER2 antibodies with partially or completely reduced interchain disulfides can be obtained by reacting each interchain disulfide in the antibody with a reducing agent such as tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) in a buffer solution containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA).

[0192] Furthermore, by using 2 to 20 molar equivalents of a drug-linker intermediate on each antiHER2 antibody with a thiol group, antiHER2 antibody-drug conjugates in which each antibody molecule is conjugated to 2 to 8 drug molecules can be produced.

[0193] The average number of conjugated drug molecules per antiHER2 antibody molecule of the produced antibody-drug conjugate can be determined, for example, by a calculation method based on UV absorbance measurements of the antibody-drug conjugate and its conjugated precursor at two wavelengths of 280 nm and 370 nm (UV method) or a quantitative calculation method based on HPLC measurements of fragments obtained by treating the antibody-drug conjugate with a reducing agent (HPLC method).

[0194] The descriptions in WO 2014 / 057687, WO 2015 / 098099, WO 2015 / 115091, WO 2015 / 155998, WO 2017 / 002776, and WO 2018 / 212136 can be used to calculate the conjugation between anti-HER2 antibodies and drug-linker intermediates, as well as the average number of conjugated drug molecules per antibody molecule in antibody-drug conjugates.

[0195] In this disclosure, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate according to this disclosure is an anti-HER2 antibody.

[0196] The anti-HER2 antibody is preferably an antibody comprising the following heavy and light chains: the heavy chain comprises CDRH1 consisting of amino acid sequences of amino acid residues 26 to 33 of SEQ ID NO: 1, CDRH2 consisting of amino acid sequences of amino acid residues 51 to 58 of SEQ ID NO: 1, and CDRH3 consisting of amino acid sequences of amino acid residues 97 to 109 of SEQ ID NO: 1; the light chain comprises CDRL1 consisting of amino acid sequences of amino acid residues 27 to 32 of SEQ ID NO: 2, CDRL2 consisting of amino acid sequences of amino acid residues 50 to 52 of SEQ ID NO: 2, and CDRL3 consisting of amino acid sequences of amino acid residues 89 to 97 of SEQ ID NO: 2; and more preferably an antibody comprising the following heavy and light chains: the heavy chain comprises a heavy chain variable region consisting of amino acid sequences of amino acid residues 1 to 120 of SEQ ID NO: 1; the light chain comprises a light chain variable region consisting of amino acid sequences of amino acid residues 1 to 107 of SEQ ID NO: 2; and even more preferably comprises a heavy chain variable region consisting of amino acid sequences of amino acid residues 1 to 120 of SEQ ID NO: 1; and even more preferably comprises a heavy chain variable region consisting of amino acid sequences of amino acid residues 1 to 120 of SEQ ID NO: 1; and even more preferably comprises a heavy chain variable region consisting of amino acid sequences of amino acid residues 1 to 120 of SEQ ID NO: 2 ... An antibody comprising a heavy chain consisting of the amino acid sequence represented by NO:1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:2, or an antibody comprising a heavy chain consisting of amino acid residues 1 to 449 of SEQ ID NO:1 and a light chain consisting of all amino acid residues 1 to 214 of SEQ ID NO:2.

[0197] In the anti-HER2 antibody-drug conjugate, the average number of drug-linker units conjugated to each antibody molecule is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0198] The anti-HER2 antibody-drug conjugates used in this disclosure can be produced with reference to the descriptions in WO 2015 / 115091, etc.

[0199] In a preferred embodiment, the anti-HER2 antibody-drug conjugate is dlutecan-trastuzumab (DS-8201).

[0200] 4. Selective PARP1 inhibitors

[0201] In this disclosure, the term "PARP1 selective inhibitor" refers to a PARP inhibitor that exhibits selectivity for PARP1 relative to other PARP family members (such as PARP2, PARP3, PARP5a, and PARP6), advantageously relative to PARP2, preferably at least 10-fold selectivity relative to PARP2, and more preferably at least 100-fold selectivity relative to PARP2. Examples of preferred PARP1 selective inhibitors may include those disclosed herein.

[0202] Examples of PARP1 selective inhibitors that may be used according to this disclosure include azaquinolone compounds having formula (I). The azaquinolone compounds having formula (I) described herein exhibit unexpectedly higher selectivity for PARP1 than other PARP family members such as PARP2, PARP3, PARP5a, and PARP6. Advantageously, the compounds having formula (I) described herein possess low hERG activity. It is well known that blockade of cardiac ion channels encoded by the human ether-à-gogo-associated gene (hERG) is a risk factor for drug discovery and development, and that hERG blockade can lead to safety issues such as arrhythmias.

[0203] 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):

[0204]

[0205] in:

[0206] X 1 and X 2 Each is independently selected from N and C(H).

[0207] X 3Independently selected from N and C(R) 4 ), where R 4 It is H or fluorine.

[0208] R 1 It is C 1-4 Alkyl or C 1-4 Fluoroalkyl groups (preferably ethyl groups),

[0209] R 2 Independently selected from H, halogenated, C 1-4 Alkyl and C 1-4 Fluoroalkyl groups, and

[0210] R 3 Is it H or C? 1-4 Alkyl (preferably C) 1-4 Alkyl groups, more preferably methyl groups,

[0211] or its pharmaceutically acceptable salt

[0212] The conditions are:

[0213] When X 1 When it is N, then X 2 It is C(H), and X 3 It is C(R) 4 ),

[0214] When X 2 When it is N, then X 1 =C(H), and X 3 It is C(R) 4 ),and

[0215] When X 3 When it is N, then X 1 and X 2 All are C(H).

[0216] In one embodiment, the PARP1 selective inhibitor used in this disclosure is a compound having formula (Ia):

[0217]

[0218] in

[0219] R 1 It is C 1-4 Alkyl, R 2 Selected from H, halogenated, C 1-4 Alkyl and C 1-4 fluoroalkyl groups (preferably selected from difluoromethyl, trifluoromethyl, and methyl, or H or halogenated), R 3 Is it H or C? 1-4 Alkyl, and R 4It is H. In compounds having formula (Ia), R is preferred. 1 It is ethyl, R 2 Selected from H, chlorine and fluorine, R 3 It is methyl, and R 4 It's H.

[0220] In another embodiment, the PARP1 selective inhibitor used in this disclosure is a compound having formula (Ib):

[0221]

[0222] in

[0223] R 1 It is C 1-4 Alkyl, R 2 It is H or halogenated, and R 3 Is it H or C? 1-4 Alkyl group. In compounds having formula (Ib), R is preferred. 1 It is an ethyl group, R2 is selected from H, chlorine and fluorine, and R 3 It is a methyl group.

[0224] In another embodiment, the PARP1 selective inhibitor used in this disclosure is a compound having formula (Ic):

[0225]

[0226] in

[0227] R 1 It is C 1-4 Alkyl or C 1-4 fluoroalkyl, R 2 Independently selected from H, halogenated, C 1-4 Alkyl and C 1-4 fluoroalkyl,

[0228] R 3 Is it H or C? 1-4 Alkyl, and R 4 It is H or fluorine.

[0229] In another embodiment, the PARP1 selective inhibitor is a compound having formula (Ic), wherein:

[0230] R 1 Independently selected from ethyl, n-propyl, trifluoromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2-fluoroethyl, and 2,2,2-trifluoroethyl; R 2 Independently selected from H, methyl, ethyl, trifluoromethyl, difluoromethyl, fluoromethyl, fluorine, and chlorine; R 3 It is H or methyl, and R 4 It's H.

[0231] In another embodiment, the PARP1 selective inhibitor is a compound having formula (I) or formula (Ia), (Ib) or (Ic) that has selectivity for PARP1 relative to PARP2, preferably having at least 10 times selectivity for PARP1 relative to PARP2, and more preferably having at least 100 times selectivity for PARP1 relative to PARP2.

[0232] In other embodiments, the PARP1 selective inhibitors used in this disclosure are compounds selected from the following:

[0233] 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyl-7-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0234] 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthid-7-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridin-2-carboxamide,

[0235] 6-Chloro-5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthid-7-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0236] 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyl-3-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0237] 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridin-2-carboxamide,

[0238] 6-Chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0239] 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide

[0240] 6-Ethyl-5-[4-[(2-Ethyl-3-oxo-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0241] 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide,

[0242] 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0243] 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0244] 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridin-2-carboxamide,

[0245] 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridin-2-carboxamide,

[0246] 6-Chloro-5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0247] N-Methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxalo-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide,

[0248] 6-Chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxalo-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide,

[0249] 6-Fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxalo-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide,

[0250] N-Methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide,

[0251] 6-Chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide,

[0252] 6-Fluoro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide,

[0253] 5-[4-[(2-ethyl-7-fluoro-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridin-2-carboxamide,

[0254] 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0255] 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0256] 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-6-fluoro-N-methylpyridin-2-carboxamide,

[0257] 5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0258] 6-Fluoro-5-[4-[[2--(2-fluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0259] N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxalo-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide, and

[0260] 6-Fluoro-N-methyl-5-(4-((3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinoxalo-6-yl)methyl)piperazin-1-yl)pyridineamide,

[0261] or its pharmaceutically acceptable salt

[0262] In another embodiment, the PARP1 selective inhibitor used in this disclosure is a compound selected from the following:

[0263] 6-(difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide,

[0264] 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide,

[0265] 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridin-2-carboxamide, and

[0266] N-Ethyl-5-[4-[(7-Ethyl-6-oxo-5H-1,5-naphthyl-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide,

[0267] Or its pharmaceutically acceptable salt.

[0268] In a preferred embodiment, the PARP1 selective inhibitor used in this disclosure is the compound AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide) represented by the following formula:

[0269]

[0270] Or its pharmaceutically acceptable salt.

[0271] 5. Combinations of antibody-drug conjugates and PARP1 selective inhibitors

[0272] In the first combination embodiment disclosed herein, the anti-HER2 antibody-drug conjugate combined with the PARP1 selective inhibitor is an antibody-drug conjugate wherein the drug-linker represented by the following formula is conjugated to the anti-HER2 antibody via a thioether bond:

[0273]

[0274] Where A represents the binding site with the antibody.

[0275] In another combination embodiment, the anti-HER2 antibody-drug conjugate as defined above for the first combination embodiment is combined with a PARP1 selective inhibitor, which is a compound represented by formula (I):

[0276]

[0277] in:

[0278] X 1 and X 2 Each is independently selected from N and C(H).

[0279] X 3 Independently selected from N and C(R) 4 ), where R 4 It is H or fluorine.

[0280] R 1 It is C 1-4 Alkyl or C 1-4 fluoroalkyl,

[0281] R 2 Independently selected from H, halogenated, C 1-4 Alkyl and C1-4 Fluoroalkyl groups, and

[0282] R 3 Is it H or C? 1-4 alkyl,

[0283] or its pharmaceutically acceptable salt

[0284] The conditions are:

[0285] When X 1 When it is N, then X 2 It is C(H), and X 3 It is C(R) 4 ),

[0286] When X 2 When it is N, then X 1 =C(H), and X 3 It is C(R) 4 ),and

[0287] When X 3 When it is N, then X 1 and X 2 All are C(H).

[0288] 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 It is C 1-4 alkyl.

[0289] In another combination embodiment, an anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, wherein, in formula (I), R 3 It is a methyl group.

[0290] In another combination embodiment, an anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, wherein, in formula (I), R 1 It is an ethyl group.

[0291] In another combination embodiment, an anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor, which is a compound represented by formula (Ia):

[0292]

[0293] in

[0294] R 1 It is C 1-4 alkyl,

[0295] R 2 Selected from H, halogenated, C 1-4 Alkyl and C 1-4 fluoroalkyl,

[0296] R 3 Is it H or C? 1-4 Alkyl, and

[0297] R 4 It's H.

[0298] Or its pharmaceutically acceptable salt.

[0299] In another combination embodiment, an anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, wherein, in formula (Ia), R 2 It is H or halogenated.

[0300] In another combination embodiment, an anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, wherein, in formula (Ia), R 1 It is ethyl, R 2 Selected from H, chlorine, and fluorine, and R 3 It is a methyl group.

[0301] 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:

[0302]

[0303] Or its pharmaceutically acceptable salt.

[0304] In each of the above combined embodiments, the anti-HER2 antibody comprises a heavy chain and a light chain. The heavy chain comprises CDRH1, CDRH2, and CDRH3, consisting of the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 5. The light chain comprises CDRL1, CDRL2, CDRL2, and CDRL3, consisting of the amino acid sequence represented by amino acid residues 1 to 3 of SEQ ID NO: 7. In another embodiment of each of the above combined embodiments, the anti-HER2 antibody comprises a heavy chain and a light chain. The heavy chain comprises a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 9, and 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 combined embodiments, the anti-HER2 antibody comprises a heavy chain and a light chain. The heavy chain consists of the amino acid sequence represented by SEQ ID NO: 1, and the light chain consists of the amino acid sequence represented by SEQ ID NO: 2. In another embodiment of each of the above combined embodiments, the anti-HER2 antibody comprises a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 11 and the light chain consisting of the amino acid sequence represented by SEQ ID NO: 2.

[0305] In the particularly preferred combination embodiments disclosed herein, the anti-HER2 antibody-drug conjugate is dlutecan-trastuzumab (DS-8201), and the PARP1 selective inhibitor is a compound represented by the following formula:

[0306] Also known as AZD5305.

[0307] 6. Uses and methods of treatment combination

[0308] The following describes the pharmaceutical product and therapeutic uses and methods of administration, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are administered in combination according to this disclosure.

[0309] The pharmaceutical products and therapeutic uses and methods disclosed herein may be characterized in that the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are contained as active ingredients in different formulations and are administered simultaneously or at different times, or are characterized in that the antibody-drug conjugate and the PARP1 selective inhibitor are contained as active ingredients in a single formulation and administered.

[0310] In the pharmaceutical products and treatments disclosed herein, the single PARP1 selective inhibitor used herein may be administered in combination with an anti-HER2 antibody-drug conjugate, or two or more different PARP1 selective inhibitors may be administered in combination with an antibody-drug conjugate.

[0311] The pharmaceutical products and treatments disclosed herein can be used to treat cancer, and are preferably used to treat at least one cancer selected from the group consisting of: breast cancer (including triple-negative breast cancer and intraluminal breast cancer), gastric cancer (also known as gastric adenocarcinoma), colorectal cancer (also known as colorectal cancer, and 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), esophagogastric junction adenocarcinoma, biliary tract cancer (including bile duct cancer), Paget's disease, pancreatic cancer, ovarian cancer, and uterine cancer. Sarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma, and more preferably 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.

[0312] The presence or absence of the HER2 tumor marker can be determined by, for example, by collecting tumor tissue from cancer patients to prepare formalin-fixed paraffin-embedded (FFPE) specimens and testing the specimens for gene products (proteins), such as by immunohistochemistry (IHC), flow cytometry, or Western blotting; or by performing gene transcription tests, such as by in situ hybridization (ISH), quantitative PCR (q-PCR), or microarray analysis; or by collecting cell-free circulating tumor DNA (ctDNA) from cancer patients and testing the ctDNA using methods such as next-generation sequencing (NGS).

[0313] The pharmaceutical products and treatments disclosed herein can be used for cancers that express HER2, which may be cancers that overexpress HER2 (high or intermediate) or cancers that express HER2 at low levels.

[0314] In this disclosure, the term "cancer overexpressing HER2" is not particularly limited, provided that it is recognized by those skilled in the art as cancer overexpressing HER2. Preferred examples of cancer overexpressing HER2 may include cancers with a HER2 expression score of 3+ in IHC assays and cancers with a HER2 expression score of 2+ in IHC assays and HER2 expression identified as positive in in situ hybridization (ISH). The in situ hybridization methods disclosed herein include fluorescence in situ hybridization (FISH) and two-color in situ hybridization (DISH).

[0315] In this disclosure, the term "cancer with low HER2 expression" is not particularly limited, as long as it is considered by those skilled in the art to be cancer with low HER2 expression. Preferred examples of cancer with low HER2 expression may include cancers with a HER2 expression score of 2+ in IHC and a HER2 expression score of 1+ in in situ hybridization, and cancers with a HER2 expression score of 1+ in IHC.

[0316] There are no particular limitations on the methods used to score HER2 expression levels by IHC or to determine HER2 expression as positive or negative by in situ hybridization, provided that they are acceptable to those skilled in the art. Examples of such methods may include those described in the 4th edition of the HER2 testing guidelines for breast cancer (developed by the Japanese Pathology Board for optimal use of HER2 in breast cancer).

[0317] The cancer (especially in relation to breast cancer treatment) can be breast cancer that overexpresses HER2 (high or intermediate) or low expresses HER2, or triple-negative breast cancer, and / or can have a HER2 status score of IHC 3+, IHC 2+, IHC 1+ or IHC > 0 and < 1+.

[0318] The pharmaceutical products and treatments disclosed herein are preferably used in mammals, but more preferably in humans.

[0319] The antitumor efficacy of the drug products and treatments disclosed herein can be demonstrated by: transplanting cancer cells into test animals to establish a model and measuring the reduction in tumor volume or the extension of life through the application of the drug products and treatments disclosed herein. Then, the efficacy of the combination of the antibody-drug conjugates used in this disclosure and the PARP1 selective inhibitor can be demonstrated by comparing the antitumor efficacy of single administration of the antibody-drug conjugates used in this disclosure with that of the PARP1 selective inhibitor.

[0320] The antitumor efficacy of the drug products and treatments disclosed herein can be confirmed in clinical trials using any of the assessment methods such as the Responsive Evaluation Criteria for Solid Tumors (RECIST), WHO assessment methods, Macdonald assessment methods, weight measurement, and other methods, and can be determined based on indices such as complete response (CR), partial response (PR), disease progression (PD), objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), and overall survival (OS).

[0321] By using the methods described above, it can be demonstrated that the drug products and treatment methods disclosed herein are superior to existing drug products and treatment methods for cancer treatment in terms of anti-tumor efficacy.

[0322] The drug products and treatments disclosed herein can delay the development of cancer cells, inhibit their growth, and further kill cancer cells. These effects can relieve cancer patients of cancer-related symptoms or improve their quality of life (QOL), and achieve therapeutic effects by sustaining their lives. Even if the drug products and treatments disclosed herein cannot kill cancer cells, they can still achieve higher QOL for cancer patients and longer survival by inhibiting or controlling the growth of cancer cells.

[0323] It is expected that the pharmaceutical products disclosed herein will exert their therapeutic effects by being applied to patients as a systemic therapy and also by being applied topically to cancerous tissue.

[0324] In another aspect, the pharmaceutical products and treatment methods disclosed herein provide for use as adjuncts in cancer treatment with ionizing radiation or other chemotherapy agents. For example, in the treatment of cancer, the treatment may include administering a therapeutically effective amount of the pharmaceutical product to a subject requiring treatment, simultaneously or sequentially with ionizing radiation or other chemotherapy agents.

[0325] The pharmaceutical products and treatments disclosed herein can be used as adjuvant chemotherapy in conjunction with surgery. The pharmaceutical products disclosed herein can be administered prior to surgery for the purpose of reducing tumor size (referred to as preoperative adjuvant chemotherapy or neoadjuvant therapy), or can be administered after surgery for the purpose of preventing tumor recurrence (referred to as postoperative adjuvant chemotherapy or adjuvant therapy).

[0326] In another respect, the pharmaceutical products disclosed herein can be used to treat cancers lacking homologous recombination (HR)-dependent DNA DSB repair activity. The HR-dependent DNA DSB repair pathway repairs double-strand breaks (DSBs) in DNA via homologous mechanisms to rebuild continuous DNA helices (KK Khanna and SP Jackson, Nat. Genet. [Nature Genetics] 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), and XRCC3 (NM_007068). RAD52 (NM_002879), RAD54L (NM_003579), RAD54B (NM_012415), BRCA1 (NM_007295), BRCA2 (NM_000059), RAD50 (NM_005732), MRE11A (NM_005590), and NBS1 (NM_002485) are all involved in the HR-dependent DNA DSB repair pathway. Other proteins involved include regulators such as EMSY (Hughes-Davies et al., Cell, 115, pp. 523-535). The HR component is also described in Wood et al., Science, 291, 1284-1289 (2001). Cancer lacking HR-dependent DNA DSB repair may comprise, or be composed of, one or more cancer cells that have a reduced or eliminated capacity to repair DNA DSBs via this pathway relative to normal cells; 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 may be eliminated in one or more cancer cells in an individual with cancer lacking HR-dependent DNA DSB repair. Components of the HR-dependent DNA DSB repair pathway are well characterized in the art (see, for example, Wood et al., Science, 291, 1284-1289 (2001)) and include the components listed above.

[0327] In some embodiments, cancer cells may have a BRCA1 and / or BRCA2 defective phenotype, meaning that BRCA1 and / or BRCA2 activity is reduced or eliminated in the cancer cells. Cancer cells with this phenotype have BRCA1 and / or BRCA2 defects, meaning that the expression and / or activity of BRCA1 and / or BRCA2 can be reduced or eliminated in the cancer cells, for example, through mutations or polymorphisms in the encoding nucleic acids, or through amplification, mutation, or polymorphism in genes encoding regulatory factors (e.g., the EMSY gene encoding a BRCA2 regulatory factor) (Hughes-Davies et al., Cell, 115, 523-535). BRCA1 and BRCA2 are known tumor suppressor factors, and their wild-type alleles are frequently lost in tumors of heterozygous carriers (Jasin M., Oncogene, 21(58), 8981-93 (2002); Tutt et al., Trends in Molecular Medicine, 8(12), 571-6 (2002)). The association between BRCA1 and / or BRCA2 mutations and breast cancer is well characterized in the field (Radice, PJ, Exp Clin Cancer Res., 21(3 Supplement), 9-12 (2002)). Amplification of the EMSY gene, which encodes the BRCA2 binding factor, is also known to be associated with breast and ovarian cancer. Carriers of mutations in BRCA1 and / or BRCA2 also have a higher risk of developing certain cancers, including breast, ovarian, pancreatic, prostate, hematologic, gastrointestinal, and lung cancers. In some embodiments, an individual is heterozygous for one or more variations (e.g., mutations and polymorphisms) in BRCA1 and / or BRCA2 or their regulators. Detection of variations in BRCA1 and BRCA2 is well known in the art and is described, for example, in EP 699754, EP 705903, Neuhausen, SL and Ostrander, EA, Genet. Test, 1, 75-83 (1992); Chappnis, PO and Foulkes, WO, Cancer TreatRes, 107, 29-59 (2002); Janatova M. et al., Neoplasia, 50(4), 246-505 (2003); Jankarkova, N., Ceska Gynekol., 68(1), 11-6 (2003). The amplification of the BRCA2 binding factor EMSY was described in Hughes-Davies et al., Cell, 115, 523-535.

[0328] Cancer-related 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) peptides.

[0329] The pharmaceutical products disclosed herein can be administered, containing at least one pharmaceutically suitable ingredient. Depending on the dosage, administration concentration, etc., of the antibody-drug conjugate and PARP1 selective inhibitor used in this disclosure, the pharmaceutically suitable ingredient can be appropriately selected and applied from formulation additives and the like commonly used in the art. The anti-HER2 antibody-drug conjugate used in this disclosure can be administered, for example, as a pharmaceutical product containing buffers such as histidine buffers, mediators such as sucrose and trehalose, and surfactants such as polysorbate 80 and 20. The pharmaceutical products containing antibody-drug conjugates used in this disclosure are preferably used as injections, more preferably as aqueous injections or lyophilized injections, and even more preferably as lyophilized injections. In the case where the pharmaceutical product containing the anti-HER2 antibody-drug conjugate used in this disclosure is an aqueous injection, the aqueous injection is preferably diluted with a suitable diluent and then administered as an intravenous infusion. Examples of diluents may include dextran solution and physiological saline, with dextran solution being a preferred example, and a 5% dextran solution being a more preferred example. In the case of a lyophilized injection, the required amount of the lyophilized injection pre-dissolved in water for injection can preferably be diluted with a suitable diluent and then administered as an intravenous infusion. Examples of diluents may include dextran solution and physiological saline, with dextran solution being a preferred example, and a 5% dextran solution being a more preferred example.

[0330] Examples of routes of administration suitable for administering the pharmaceutical products disclosed herein may include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes, with intravenous administration being preferred.

[0331] The anti-HER2 antibody-drug conjugate used in this disclosure can be administered to humans at intervals ranging from 1 day to 180 days, preferably at intervals of one week, two weeks, three weeks, or four weeks, and more preferably at intervals of three weeks. The anti-HER2 antibody-drug conjugate used in this disclosure can be administered at doses ranging from about 0.001 mg / kg to 100 mg / kg per administration, and preferably at doses ranging from 0.8 mg / kg to 12.4 mg / kg per administration. For example, the anti-HER2 antibody-drug conjugate can be administered every three weeks at doses of 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, and preferably at doses of 5.4 mg / kg or 6.4 mg / kg per three weeks.

[0332] PARP1 selective inhibitors can be administered at appropriate doses via any suitable route of administration. The required dose for therapeutic treatment of a specific disease state will necessarily vary depending on the subject being treated, the route of administration, and the severity of the disease being treated. Further information on routes of administration and dosage regimens can be found in Comprehensive Medicinal Chemistry, Volume 5, Chapter 25.3 (Editorial Board Chair Corwin Hansch), Pergamon Press, 1990.

[0333] Compounds having formula (I) or pharmaceutically acceptable salts thereof are typically administered orally in pharmaceutical formulations comprising the active ingredient or a pharmaceutically acceptable salt or solvation thereof, or a solvation of such salt, in a pharmaceutically acceptable dosage form. Depending on the disorder to be treated and the patient, the composition may be administered at different doses.

[0334] Pharmaceutical formulations of compounds having formula (I) described above can be prepared for oral administration, particularly in tablet or capsule form, and particularly relate to techniques aimed at providing drug release targeting the colon (Patel, MM Expert Opin. Drug Deliv [Expert Opinion on Drug Delivery] 2011, 8(10), 1247-1258).

[0335] The pharmaceutical formulations of the compounds having formula (I) described above can be conveniently administered in unit dosage forms and can be prepared by any method known in the pharmaceutical field, such as that described in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA. (1985).

[0336] Pharmaceutical formulations of compounds having formula (I) suitable for oral administration may contain one or more physiologically compatible carriers and / or excipients and may be in solid or liquid form. Tablets and capsules may be prepared using binders, fillers, lubricants, and / or surfactants (such as sodium lauryl sulfate). Liquid compositions may contain conventional additives such as suspending agents, emulsifiers, and / or preservatives. Liquid compositions may be encapsulated, for example, in gelatin to provide unit dosage forms. Solid oral dosage forms include tablets, two-piece hard-shell capsules, and soft elastic gelatin (SEG) capsules. Such two-piece hard-shell capsules may be prepared, for example, by filling a compound having formula (I) into a gelatin or hydroxypropyl methylcellulose (HPMC) shell.

[0337] Dry-shell formulations of compounds having formula (I) typically contain about 40% to 60% w / w gelatin, about 20% to 30% plasticizer (such as glycerin, sorbitol, or propylene glycol), and about 30% to 40% water. Other materials, such as preservatives, dyes, opacifiers, and flavorings, may also be present. Liquid filler materials include solid pharmaceutical products that have been dissolved, solubilized, or dispersed (using suspending agents such as beeswax, hydrogenated castor oil, or polyethylene glycol 4000) or liquid pharmaceutical products in a combination of one or more mediators (such as mineral oil, vegetable oil, triglycerides, glycols, polyols, and surfactants).

[0338] When used for therapeutic treatment in humans, a suitable daily dose of a compound having formula (I) or a pharmaceutically acceptable salt thereof is about 0.0001-100 mg / kg body weight. Oral formulations are preferred, particularly tablets or capsules, which can be formulated by methods known to those skilled in the art to provide a dose of the active compound in the range of 0.1 mg to 1000 mg.

[0339] [Example]

[0340] This disclosure is described in light of the examples illustrated below. However, this disclosure is not limited to these examples. Furthermore, it should not be interpreted in a restrictive manner.

[0341] Synthetic examples of PARP1 selective inhibitors

[0342] Examples 1 to 32 of the synthesis of PARP1 selective inhibitors described below are as described in Examples 1 to 32 of WO 2021 / 013735.

[0343] General experimental conditions

[0344] Unless otherwise specified, use a Bruker 300MHz, 400MHz, or 500MHz spectrometer, at 27°C. 1 1H NMR spectra; chemical shifts are expressed in parts per million (ppm, δ units) and referenced to residual solvent. 1H isotopes (CHCl3: 7.24 ppm; CHDCl2: 5.32 ppm; CD3S(=O)CD2H: 2.49 ppm). Coupling constants are given in Hertz (Hz). Splitting modes describe apparent multiplicity and are designated as s (singleton), d (doublet), t (triplet), q (quartet), m (multiplet), and brs (broad peak). LC-MS was performed using a Waters UPLC equipped with a Waters SQD mass spectrometer or a Shimadzu LC-20AD, LC-20XR, or LC-30AD equipped with a Shimadzu 2020 mass spectrometer. Unless otherwise specified, the reported molecular ion corresponds to [M+H]+; for molecules with multiple isotopic modes (Br, Cl, etc.), unless otherwise specified, the reported values ​​are those obtained for the lowest isotopic mass.

[0345] The following rapid chromatography method was used: [from Biotage] TM SP1 TM On the purification system, from ISCO Rf or on Gilson from Thermo Fisher Scientific, using positive silicon dioxide FLASH+ TM (40M, 25M or 12M) or SNAp TM KP-Sil columns (340, 100, 50, or 10), Agela's fast column silica-CS column, straight-phase rapid chromatography using C18 fast columns, or standard rapid chromatography. Typically, all solvents used are commercially available and analytical grade. Anhydrous solvents are routinely used for the reactions. The phase separators used in these examples are... Phase separation column. The intermediates and examples listed below are named using ACD / Name 12.01 from Advanced Chemistry Development, Inc. (ACD / Laboratory). Starting materials were obtained from commercial sources or prepared via literature routes.

[0346] X-ray powder diffraction (XRPD) analysis

[0347] XRPD analysis was performed using a Bruker D8 diffractometer, which is commercially available from Bruker AXS Inc. TM(Madison, Wisconsin). XRPD spectroscopy was obtained by mounting a sample of the material to be analyzed (approximately 10 mg) on ​​a single-silicon crystal wafer scaffold (e.g., a Bruker silicon zero-background X-ray diffraction sample holder) and spreading the sample into a thin layer using a microscope slide. The sample was rotated at 30 rpm (to improve counting statistics) and irradiated with X-rays at a wavelength of 1.5406 Å (i.e., approximately 1.54 Å) generated by a long, thin copper focusing tube operating at 40 kV and 40 mA. The sample was exposed for 1 second in 0.02° 2-θ increments (continuous scan mode) in θ-θ mode within the 2-θ range from 5° to 40°. The run time for D8 was 15 min.

[0348] The XRPD 2θ value can vary within a reasonable range, for example, within ±0.2°, and the XRPD intensity may vary when measured on substantially the same crystal form for a variety of reasons, including, for example, preferred orientation. The principles of XRPD are described in publications such as Giacovazzo, C. et al. (1995), Fundamentals of Crystallography, Oxford University Press; Jenkins, R. and Snyder, RL (1996), Introduction to X-Ray Powder Diffractometry, John Wiley & Sons, New York; and Klug, HP & Alexander, LE (1974), X-ray Diffraction Procedures, John Wiley and Sons, New York.

[0349] DSC analysis

[0350] For samples prepared according to standard methods, use samples obtainable from TA Q SERIES (Newcastle, Delaware) TM DSC analysis was performed using a Q1000 DSC calorimeter. The sample (approximately 2 mg) was weighed into an aluminum sample pan and transferred to the DSC. The instrument was purged with nitrogen at 50 mL / min, and data were collected between 22°C and 300°C using a dynamic heating rate of 10°C / min. The thermal data were analyzed using standard software (e.g., from TA). The analysis was performed using the general version (v.4.5A).

[0351] The following abbreviations were used: AcOH = acetic acid; aq = aqueous; BAST = bis(2-methoxyethyl)aminosulfur trifluoride; Boc2O = ditert-butyl dicarbonate; Boc = tert-butoxycarbonyl; CDCl3 = deuterated chloroform; CD3OD = deuterated methanol; CH3NO2 = nitromethane; DCE = 1,2-dichloroethane; DCM = dichloromethane; DEA = diethylamine; DEAD = diethyl azodicarbonate; Des-Martin periodoyl alkyl = 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benzyl-3-(1H) - Ketone; DIPEA = N,N-diisopropylethylamine; DMAP = 2,6-dimethylaminopyridine; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DMSO-d6 = deuterated dimethyl sulfoxide; DPPA = diphenyl azidophosphate; dppf = 1,1′-bis(biphenylphosphino)ferrocene; DIAD = di-isopropyl(E)-diazepine-1,2-dicarboxylate; DSC = differential scanning calorimetry; DTAD = di-tert-butyl(E)-diazepine-1,2-dicarboxylate; ee = enantiomer excess; eq.= Equivalent; ESI = Electrospray ionization; Et2O = Diethyl ether; EtOAc or EA = Ethyl acetate; EtOH = Ethanol; FA = Formic acid; Grubbs catalyst (1,3-di-leucoylimidazoline-2-ylidene)(tricyclohexylphosphine) ruthenium dichloride; h = hour; HATU = (dimethylamino)-N,N-dimethyl(3-oxo-1H-[1,2,3]triazolo[4,5-b]pyridyl)methyliminoonium hexafluorophosphate; HCl = Hydrochloric acid; H2O2 = Hydrogen peroxide; HP = High pressure; IPA = Isopropanol; LC = Liquid chromatography; LiClO4 = Lithium perchlorate; mmol = millimole; mCPBA = m-chloroperoxybenzoic acid; MeOH = methanol; min = minutes; MeCN or CH3CN = acetonitrile; MeNO2 = nitromethane; MS = mass spectrometry; NMP = N-methyl-2-pyrrolidone; NMR = nuclear magnetic resonance; Pd / C = palladium on carbon; Pd2dba3 = tris(diphenylmethyleneacetone)dipalladium(0); PdCl2(dppf) = 1,1′-bis(di-tert-butylphosphino)ferrocene palladium dichloride; PE = petroleum ether; PPh3 = triphenylphosphine; rt = room temperature; Rt or RT = retention time; Ruphos Pd G3 = (2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate; sat = saturated; SFC = supercritical fluid chromatography; T3P = 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphacyclohexane 2,4,6-trioxide; TBTU = 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisoureaium tetrafluoroborate; TFA = trifluoroacetic acid; THF = tetrahydrofuran; TLC = Thin-layer chromatography; TMS = Trimethylsilyl; Xantphos = 4,5-bis(biphenylphosphino)-9,9-dimethylxanthanyl; 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 hydrogenate; NH4HCO3 = Ammonium bicarbonate; BINAP = 2,2′-bis(biphenylphosphino)-1,1′-binaphthyl.

[0352] Synthesis of starting materials and intermediates

[0353]

[0354] Intermediate 2: 7-bromo-3-ethyl-1H-1,6-naphthid-2-one

[0355] At 0 °C, butyryl chloride (0.143 mL, 1.37 mmol) was added dropwise to a stirred solution of 4-amino-6-bromo-pyridin-3-carboxaldehyde (intermediate 1250 mg, 1.24 mmol), DIPEA (1.086 mL, 6.22 mmol), and DMAP (30.4 mg, 0.25 mmol) in CH2Cl2 (5 mL). The resulting solution was stirred at rt for 4 h. More than 2 eq of butyryl chloride was added and the reaction was continued for another 24 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to give a crude product. 1.5 mL of MeOH was added and the solid (product) was filtered off and washed with 1 mL of MeOH to give 7-bromo-3-ethyl-1H-1,6-naphthid-2-one (intermediate 2, 167 mg, 53.1%) as a white solid.

[0356] ¹H NMR (DMSO-d6) 1.17 (3H, t), 245–2.50 (2H, m, overlapping with 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.

[0357] Intermediate 3: 3-Ethyl-7-vinyl-1H-1,6-naphthid-2-one

[0358] PdCl2 (dppf) (37.6 mg, 0.05 mmol) was added to a stirred mixture of 7-bromo-3-ethyl-1H-1,6-naphthid-2-one (intermediate 2, 130 mg, 0.51 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxane (0.105 mL, 0.62 mmol), and K2CO3 (213 mg, 1.54 mmol) in 1,4-dioxane (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 give a crude product. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 20% MeOH in DCM). The product fraction was concentrated under reduced pressure and dried to give 3-ethyl-7-vinyl-1H-1,6-naphthid-2-one as a yellow solid (intermediate 3, 93 mg, 90%).

[0359] 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.

[0360] Intermediate 4: 3-Ethyl-2-oxo-1H-1,6-naphthyl-7-carboxaldehyde

[0361] Osmium tetroxide (0.024 mL, 3.00 μmol) in H₂O was added to a solution of 3-ethyl-7-vinyl-1H-1,6-naphthid-2-one (intermediate 3, 30 mg, 0.15 mmol), 2,6-dimethylpyridine (0.035 mL, 0.30 mmol), and sodium periodate (128 mg, 0.60 mmol) in THF (1 mL) / water (0.200 mL) and stirred overnight at rt. The reaction mixture was diluted with water and extracted with ethyl acetate, and the filtrate was concentrated to dryness. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 15% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 3-ethyl-2-oxo-1H-1,6-naphthid-7-carboxaldehyde (intermediate 4, 24.00 mg, 79%) as a pale yellow foam.

[0362] ¹H NMR (DMSO-d6) 1.20 (3H, t), 2.55–2.62 (2H, m, overlapping with 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.

[0363] Intermediate 5: 3-Ethyl-7-(hydroxymethyl)-1H-1,6-naphthid-2-one

[0364] Sodium borohydride (61.4 mg, 1.62 mmol) was slowly added to a stirred solution of 3-ethyl-2-oxo-1H-1,6-naphthid-7-carboxaldehyde (intermediate 4, 82 mg, 0.41 mmol) in methanol (2 mL) at 0 °C, and the resulting mixture was stirred at room temperature for 1 h. Methanol was removed under vacuum, and the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 35% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 3-ethyl-7-(hydroxymethyl)-1H-1,6-naphthid-2-one (intermediate 5, 68.0 mg, 82%) as a pale yellow solid.

[0365] ¹H NMR (500 MHz, DMSO-d6): 1.18 (3H, t), 2.52–2.55 (2H, m, overlapping with 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.

[0366] Intermediate 6: 7-(bromomethyl)-3-ethyl-1H-1,6-naphthid-2-one

[0367] At 0 °C, CBr4 (928 mg, 2.80 mmol) was added to a stirred solution of 3-ethyl-7-(hydroxymethyl)-1H-1,6-naphthid-2-one (intermediate 5,381 mg, 1.87 mmol) and triphenylphosphine (734 mg, 2.80 mmol) in CH2Cl2 (18.656 mL), and the resulting solution was stirred at 0 °C for 2 hours. The reaction was concentrated, and the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 15% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 7-(bromomethyl)-3-ethyl-1H-1,6-naphthid-2-one (intermediate 6,386 mg, 77%) as a white solid (containing triphenylphosphine oxide, which was difficult to separate). This compound was used in the next step without further purification.

[0368] m / z(ES + [M] + =267.

[0369] Synthetic Example 1: 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthid-7-yl)methyl]piperazin-1-yl]-N-methyl pyridine-2-carboxamide

[0370]

[0371] At 20°C, DIPEA (0.059 mL, 0.34 mmol) was added to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,6-naphthidium-2-one (intermediate 6, 30 mg, 0.11 mmol) and N-methyl-5-piperazin-1-ylpyridin-2-carboxamide 2HCl (intermediate 13, 42.8 mg, 0.15 mmol) in 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 subjected to reversed-phase chromatography (RediSep Rf). The product fraction was further purified (C18, 0% to 90% acetonitrile in water, 0.1% NH4OH as an additive). The product fraction was concentrated under reduced pressure and dried to give 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthid-7-yl)methyl]piperazin-1-yl]-N-methyl-pyridin-2-carboxamide as a pale yellow solid (Synthetic Example 1, 23.60 mg, 51.7%).

[0372] ¹H NMR (500 MHz, 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, brdd), 7.77–7.88 (2H, m), 8.29 (1H, br d), 8.40 (tH, br d), 8.75 (1H, s), 11.60–12.11 (1H, m); m / z (ES) + [M+H] + =407.

[0373] Synthetic Example 2: 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthid-7-yl)methyl]piperazin-1-yl]-6-fluoro- N-Methylpyridine-2-carboxamide

[0374]

[0375] At 20 °C, DIPEA (0.082 mL, 0.47 mmol) was added to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,6-naphthid-2-one (intermediate 6, 25 mg, 0.09 mmol) and 6-fluoro-N-methyl-5-piperazin-1-ylpyridin-2-carboxamide HCl (intermediate 23, 28.3 mg, 0.10 mmol) in acetonitrile (2 mL). The resulting solution was stirred at 70 °C for 2 hours. The solvent was removed under vacuum. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 20% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthid-7-yl)methyl]piperazin-1-yl]-6-fluoro-N-methyl-pyridin-2-carboxamide as a pale yellow solid (Synthetic Example 2, 17.00 mg, 42.8%).

[0376] ¹H NMR (500 MHz, DMSO-d⁶): 1.18 (3H, t), 2.52–2.55 (2H, m, overlapping with 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.

[0377] Synthetic Example 3: 6-O-5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthid-7-yl)methyl]piperazin-1-yl]- N-Methylpyridine-2-carboxamide

[0378]

[0379] At 20 °C, DIPEA (0.082 mL, 0.47 mmol) was added to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,6-naphthyl-2-one (intermediate 6, 25 mg, 0.09 mmol) and 6-chloro-N-methyl-5-piperazin-1-ylpyridin-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. The solvent was removed under vacuum. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 20% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 6-chloro-5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthid-7-yl)methyl]piperazin-1-yl]-N-methyl-pyridin-2-carboxamide as a white solid (Synthetic Example 3, 19.20 mg, 46.5%).

[0380] ¹H NMR (500 MHz, DMSO-d₆): 1.18 (3H, t), 2.53 (2H, m, overlapping with 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.

[0381]

[0382] Intermediate 8: Ethyl 6-formyl-5-nitro-pyridine-3-carboxylate

[0383] A mixture of ethyl 6-methyl-5-nitro-pyridine-3-carboxylate (intermediate 7, 10 g, 47.58 mmol) and selenium dioxide (7.92 g, 71.36 mmol) in 1,4-dioxane (50 mL) was stirred at 110 °C for 20 h. The reaction mixture was cooled to room temperature, filtered through a diatomaceous earth pad, and the diatomaceous earth was washed with ethyl acetate. The combined filtrates were concentrated, and the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 70% ethyl acetate in hexane). The product fraction was concentrated under reduced pressure to give ethyl 6-formyl-5-nitro-pyridine-3-carboxylate (intermediate 8, 9.70 g, 91%) as a brown oil. 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.

[0384] Intermediate 9: Ethyl 6-[(E)-2-ethoxycarbonylbut-1-enyl]-5-nitropyridine-3-carboxylate (EZ isomer) (a mixture of substances)

[0385] Ethyl 2-(diethoxyphosphoryl)butyrate (60.8 g, 240.89 mmol) was added dropwise to a stirred solution of sodium hydride (9.63 g, 240.89 mmol) in anhydrous THF (100 mL) at 0 °C using a feeding funnel to give a gray mixture. The resulting mixture was stirred at 0 °C for 10 min and heated to room temperature over 10 min, then stirred at 40 °C for 5 min. The reaction mixture was cooled to -78 °C, and then a solution of ethyl 6-formyl-5-nitro-pyridine-3-carboxylate (intermediate 8, 22.5 g, 100.37 mmol) in 100 mL THF was slowly added to the cooled reaction mixture. The mixture was quenched with saturated NH4Cl solution and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4, filtered, and concentrated to give a crude product. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 50% ethyl acetate in hexane). The product fraction was concentrated under reduced pressure to give ethyl 6-[(E)-2-ethoxycarbonylbut-1-enyl]-5-nitro-pyridine-3-carboxylate (intermediate 9, 24.30 g, 75%) (a 1:1 mixture of E / Z isomers) as a yellow oil. 1HNMR (500MHz, 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.

[0386] Intermediate 10: Ethyl 7-ethyl-6-oxo-7,8-dihydro-5H-1,5-naphthyl-3-carboxylate

[0387] A mixture of ethyl 6-[(E)-2-ethoxycarbonylbut-1-enyl]-5-nitro-pyridine-3-carboxylate (a 1:1 mixture of E / Z isomers) (intermediate 9, 3.75 g, 11.63 mmol) and Pd / C (1.857 g, 1.75 mmol) (10%) in ethanol (30 mL) was degassed, filled with H2 (gas bag), and the reaction was stirred overnight at room temperature under H2 atmosphere. The mixture was filtered through a diatomaceous earth bed, and the diatomaceous earth bed was washed with ethanol. After concentration, 4 M HCl in dioxane (15 mL) was added to the resulting residue, and the mixture was stirred at room temperature for 30 min. The mixture was diluted with diethyl ether, and the solid was filtered off, washed with diethyl ether, and dried under vacuum to give ethyl 7-ethyl-6-oxo-7,8-dihydro-5H-1,5-naphthyl-3-carboxylate (intermediate 10, 2.260 g, 78%) as a white solid. 1H NMR(500MHz, 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.

[0388] Intermediate 11: Ethyl 7-ethyl-6-oxo-5H-1,5-naphthyl-3-carboxylate

[0389] Ethyl 7-ethyl-6-oxo-7,8-dihydro-5H-1,5-naphthyl-3-carboxylate (intermediate 10, 2.26 g, 9.10 mmol) was dissolved in 1,4-dioxane (40 mL), DDQ (2.273 g, 10.01 mmol) was added, and the mixture was stirred under reflux for 3 h. The solvent was removed under reduced pressure, a saturated NaHCO3 solution was added, and the residue was stirred at room temperature for 1 h. The solid was filtered off and washed with water followed by 10 mL of diethyl ether. The resulting solid was dried under vacuum to give ethyl 7-ethyl-6-oxo-5H-1,5-naphthyl-3-carboxylate (intermediate 11, 1.738 g, 78%) as a light brown solid.

[0390] 1H NMR (500MHz, 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.

[0391] Intermediate 12: 3-Ethyl-7-(hydroxymethyl)-1H-1,5-naphthid-2-one

[0392] Lithium aluminum hydride (2 M, in THF (29.2 mL, 58.47 mmol)) was added dropwise to ethyl 7-ethyl-6-oxo-5H-1,5-naphthyl-3-carboxylate (intermediate l1, 7.2 g, 29.24 mmol) in tetrahydrofuran (150 mL) over a nitrogen period of 45 minutes at 0 °C. The resulting mixture was stirred at 0 °C for 1.5 hours. The reaction mixture was quenched dropwise by adding 1 M aq HCl (29 mL). The reaction mixture was concentrated and the solid was diluted with water (approximately 150 mL) and 29 mL of 1 M HCl solution to give a yellow suspension. The solid was collected by filtration, washed with water and diethyl ether, and dried to produce a 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. The solid was resuspended in a methanol / DCM mixture and this procedure was repeated 5 times to remove most of the product from the mixture. The combined filtrates were then concentrated to about 100 ml, and the solid was collected by filtration, washed with diethyl ether, and dried under vacuum to give 3-ethyl-7-(hydroxymethyl)-1H-1,5-naphthid-2-one (intermediate 12, 4.35 g, 72.8%) as a yellow solid. 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.

[0393] Synthetic Example 4: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl pyridine-2-carboxamide

[0394]

[0395] At 0 °C, thionyl chloride (6.41 mL, 88.14 mmol) was added dropwise to a suspension of 3-ethyl-7-(hydroxymethyl)-1,5-naphthidin-2(1H)-one (intermediate 12, 3 g, 14.69 mmol) and N,N-dimethylformamide (0.114 mL, 1.47 mmol) in CH2Cl2 (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-naphthidin-2-one (intermediate 17).

[0396] At 20 °C, DIPEA (12.83 mL, 73.45 mmol) was added to a stirred solution of 7-(chloromethyl)-3-ethyl-1H-1,5-naphthid-2-one (intermediate 17, crude, from above), potassium iodide (0.488 g, 2.94 mmol), and N-methyl-5-piperazin-1-ylpyridin-2-carboxamide 2HCl (intermediate 13, 4.31 g, 14.69 mmol) in acetonitrile (50.00 mL). The resulting solution was stirred at 80 °C for 2 hours. The solvent was removed under vacuum. The crude material was diluted with water, alkalized with an aqueous solution of NaHCO3, and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to give the crude product. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 15% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide as a grayish-white partially crystalline solid (Synthetic 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.

[0397]

[0398] Intermediate 14: 7-(bromomethyl)-3-ethyl-1H-1,5-naphthid-2-one

[0399] At 0 °C, CBr4 (219 mg, 0.66 mmol) was added to a stirred solution of 3-ethyl-7-(hydroxymethyl)-1H-1,5-naphthid-2-one (intermediate 12, 90 mg, 0.44 mmol) and triphenylphosphine (173 mg, 0.66 mmol) in CH2Cl2 (4 mL). The resulting solution was stirred at 0 °C for 2 hours. The reaction was concentrated under vacuum, and the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 15% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 7-(bromomethyl)-3-ethyl-1H-1,5-naphthid-2-one (intermediate 14, 84 mg, 71.4%) (containing triphenylphosphine oxide, which was difficult to separate). This compound was used in the next step without further purification.

[0400] m / z(ES + [M] + =267.

[0401] Synthetic Example 5: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-6-fluoro- N-Methylpyridine-2-carboxamide

[0402]

[0403] At 20 °C, DIPEA (0.082 mL, 0.47 mmol) was added to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,5-naphthidin-2-one (intermediate 14, 25 mg, 0.09 mmol) and 6-fluoro-N-methyl-5-piperazin-1-yl-pyridin-2-carboxamide, 2HCl (intermediate 23, 32.0 mg, 0.10 mmol) in acetonitrile (2 mL). The resulting solution was stirred at 70 °C for 2 hours. The solvent was removed under vacuum. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 20% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-6-fluoro-N-methyl-pyridine-2-carboxamide (synthetic example 5, 13.00 mg, 33%) as a pale yellow solid. ¹H NMR (500 MHz, 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.

[0404] Synthetic Example 6: 6-Chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]- N-Methylpyridine-2-carboxamide

[0405]

[0406] At 20 °C, DIPEA (0.082 mL, 0.47 mmol) was added to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,5-naphthidium-2-one (intermediate 14, 25 mg, 0.09 mmol) and 6-chloro-N-methyl-5-piperazin-1-yl-pyridin-2-carboxamide (intermediate 48, 26.2 mg, 0.10 mmol) in acetonitrile (2 mL). The resulting solution was stirred at 70 °C for 2 hours. The solvent was removed under vacuum. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 20% MeOH in DCM). The product fraction was concentrated under reduced pressure to give 6-chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridin-2-carboxamide as a pale yellow solid (Synthetic Example 6, 19.80 mg, 48.0%). ¹H NMR (500 MHz, 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.

[0407]

[0408] Intermediate 16: Methyl 5-piperazine-1-ylpyridine-2-carboxylate

[0409] HCl (4.67 mL, 18.67 mmol) in dioxane was added to a stirred solution of tert-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 15,600 mg, 1.87 mmol) in MeOH (1 mL), and the resulting solution was stirred at rt for 18 hours. The solvent was removed under vacuum to give methyl 5-piperazine-1-ylpyridin-2-carboxylate 2HCl (intermediate 16,543 mg, 99%) as a pale yellow solid.

[0410] 1H NMR (500MHz, 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.

[0411] Intermediate 18; methyl 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]pyridine 2-Pyridine carbamate

[0412] At 20 °C, DIPEA (944 μl, 5.40 mmol) was added to a stirred solution of 7-(chloromethyl)-3-ethyl-1H-1,5-naphthid-2-one HCl (intermediate 17, 200 mg, 0.77 mmol), sodium iodide (11.57 mg, 0.08 mmol), and methyl 5-piperazin-1-ylpyridine-2-carboxylate, 2HCl (intermediate 16, 250 mg, 0.85 mmol) in acetonitrile (6774 μl). The resulting solution was stirred at 80 °C for 3 hours. The solvent was removed under vacuum, and 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 methyl 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxylate as a grayish-white solid (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, brs), 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.

[0413] Synthetic Example 7: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]pyridine- 2-Formamide

[0414]

[0415] Ammonia (4 mL, 28.00 mmol) from methanol was added to methyl 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxylate (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, and the solid was filtered off and washed with 2 mL of methanol to give 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide as a light brown solid (synthetic example 7, 88 mg, 90%). ¹H NMR (500 MHz, 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.

[0416]

[0417] Intermediate 20: Methyl 5-bromo-6-fluoro-pyridine-2-carboxylate

[0418] An oven-dried flask was filled with methyl 5-bromopyridine-2-carboxylate (intermediate 19, 6 g, 27.77 mmol) in acetonitrile (60 mL). Silver(II) fluoride (14.18 g, 97.21 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was filtered through filter paper and washed with DCM. The filtrate was concentrated to give a pale brown solid. The residue was suspended in a mixture of DCM and saturated NH4Cl solution, and the white suspension was filtered off. The organic layers were separated, and the aqueous layer was extracted with DCM (100 mL x 2). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 25% EtOAc in hexane). The product fraction was concentrated to dryness under reduced pressure to give methyl 5-bromo-6-fluoropyridine-2-carboxylate (intermediate 20, 5.98 g, 90% yield). 1 ¹H NMR (500 MHz, chloroform-d) 4.01 (3H, s), 7.93 (1H, d), 8.15 (1H, t); m / z (ES) + [M] + =234.

[0419] Intermediate 21: tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate

[0420] A mixture of tert-butylpiperazine-1-carboxylate (13.11 g, 70.41 mmol), methyl 5-bromo-6-fluoropyridine-2-carboxylate (intermediate 20, 10.985 g, 46.94 mmol), RuphosPd-G3 (2.5 g, 2.99 mmol), and Cs₂CO₃ (38 g, 116.63 mmol) in 1,4-dioxane (200 mL) was stirred overnight under N₂. 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 Na₂SO₄, filtered, and concentrated. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 100% EtOAc in hexane). The product fraction was concentrated to dryness under reduced pressure to give a yellow solid of tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (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.

[0421] Intermediate 22: tert-butyl 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate

[0422] tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 21, 12.49 g, 36.80 mmol) in methylamine (120 mL, 36.80 mmol, 33 wt%, in ethanol) was stirred at rt for 24 hr (sealed tube). The solvent was removed under reduced pressure. The residue was dissolved in DCM and filtered through a silica gel bed and washed with ethyl acetate. The filtrate was concentrated and dried under vacuum to give tert-butyl 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 22, 12.45 g, 100%) as a yellow solid. 1H NMR (500MHz, 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.

[0423] Intermediate 23: 6-Fluoro-N-methyl-5-piperazin-1-yl-pyridine-2-carboxamide

[0424] At 0 °C, HCl (4 M, in dioxane, 100 mL, 400.00 mmol) was added to a solution of tert-butyl 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 22, 12.5 g, 36.94 mmol) in 1,4-dioxane (50 mL). The reaction was stirred for 5 h, during which time the temperature was raised to room temperature to give a yellow suspension. The suspension was diluted with diethyl ether, the solid was filtered off and washed with diethyl ether. This solid was dried under vacuum to give 6-fluoro-N-methyl-5-piperazine-1-yl-pyridin-2-carboxamide 2HCl (intermediate 23, 11.42 g, 99%) as a pale yellow solid. 1H NMR (500MHz, DMSO-d6) δppm 2.8 (d, J=4.6Hz, 3H) 3.3 (br s, 4H) 3.4 (br d, J=4.4Hz, 4H) 7.6-7.7 (m, 1H) 7.9 (d, J=8.1Hz, 1H) 8.4 (br d, J=4.4Hz, 1H) 9.0-9.3 (m, 2H); m / z (ES + [M+H] + =239

[0425]

[0426] Intermediate 15: tert-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate

[0427] Ruphos Pd G3 (4.07 g, 4.86 mmol) was added to a degassed mixture of methyl 5-bromopyridine-2-carboxylate (intermediate 19, 30 g, 138.87 mmol), piperazine-1-carboxylate tert-butyl ester (27.2 g, 145.81 mmol), and Cs₂CO₃ (90 g, 277.73 mmol) in 1,4-dioxane (200 mL), and the mixture was stirred at 110 °C under a N₂ atmosphere for 6 h. The mixture was then cooled to room temperature, diluted with water, and extracted with ethyl acetate (150 mL x 3). The combined organic layers were dried over anhydrous Na₂SO₄ and filtered. 3-(diethylenetriamino)propyl-functionalized silica gel (12 g, 1.3 mmol / g loading) was added to this filtrate, and the mixture was stirred at rt for 1 hr. The mixture was filtered, and the filtrate was concentrated to approximately 100 mL. The crystalline yellow solid was filtered off, washed with diethyl ether, and dried under vacuum to give tert-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperazine-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.

[0428] Intermediate 24: tert-butyl 4- / 6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate

[0429] Methylamine (100 mL, 1155.26 mmol, 40% in water) was added to a solution of tert-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 15, 36 g, 112.02 mmol) in MeOH (100 mL), and the reaction was stirred at room temperature for 4 h to give a white suspension. The mixture was concentrated, and the residue was partitioned between saturated NH4Cl solution and DCM to separate the layers. The aqueous layer was extracted with DCM, the organic layers were combined, washed with brine, dried over Na2SO4, filtered, and concentrated to give tert-butyl 4-[6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 24, 35.9 g, 100%) as a yellow solid. 1H NMR (500MHz, 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.

[0430] Intermediate 13: Formate N-methyl-5-piperazin-1-ylpyridine-2-carboxamide

[0431] HCl (4M, in dioxane, 150 mL, 600.00 mmol) was added to a suspension of tert-butyl-4-[6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 24, 35.9 g, 112.05 mmol) in MeOH (50 mL), and the resulting orange suspension was stirred at rt for 4 hr. Approximately 80 mL of solvent was removed under reduced pressure, and the mixture was diluted with diethyl ether and hexane (200 mL, 1 / 1). The solid was collected by filtration, washed with hexane, dried, and dried under vacuum to give N-methyl-5-piperazine-1-yl-pyridin-2-carboxamide 2HCl salt (intermediate 13, 37.0 g, 100%) as a yellow solid. 1HNMR (500MHz, 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

[0432]

[0433] Intermediate 26: Methyl 4-(1-methoxycarbonylpropylamino)-3-nitrobenzoate

[0434] Sodium bicarbonate (27.0 g, 321.39 mmol) was added in portions to a stirred mixture of methyl 4-fluoro-3-nitrobenzoate (intermediate 25, 16 g, 80.35 mmol) and methyl 2-aminobutyrate HCl (14.81 g, 96.42 mmol) in THF (100 mL). The reaction mixture was stirred overnight at room temperature. The reaction was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with a saturated aqueous solution of NaHCO3, dried over MgSO4, and concentrated to dryness to give methyl 4-(1-methoxycarbonylpropylamino)-3-nitrobenzoate (intermediate 26, 22.86 g, 96%) as a bright yellow solid. 1HNMR (500MHz, DMSO-d6)0.91 (3H, t), 1.75-2.12 (2H, m), 3.75 (3H, s), 3.8 5 (3H, s), 4.63-4.82 (1H, m), 7.15 (1H, d), 8.00 (1H, dd), 8.52-8.76 (2H, m).

[0435] Intermediate 27: Methyl 2-ethyl-3-oxo-2,4-dihydro-1H-quinoxaline-6-carboxylate

[0436] Pd / C (4.15 g, 3.90 mmol) was added in portions to a stirred solution of methyl 4-(1-methoxycarbonylpropylamino)-3-nitrobenzoate (intermediate 26, 23.1 g, 77.97 mmol) in MeOH (300 mL), and the resulting slurry was stirred at room temperature under H2 atmosphere 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 onto diatomaceous earth and washed with 50 mL of DMF (the material has very low solubility in organic solvents such as MeOH / DCM / EtOAc). The filtrate was concentrated in Genevac to give methyl 2-ethyl-3-oxo-2,4-dihydro-1H-quinoxaloline-6-carboxylate (intermediate 27, 15.80 g, 87%) as a gray solid. The material was analyzed by NMR and used directly 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,t d), 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.

[0437] Intermediate 28: Methyl 2-ethyl-3-oxo-4H-quinoxaline-6-carboxylate

[0438] DDQ (15.87 g, 69.92 mmol) was added to a suspension of methyl 2-ethyl-3-oxo-2,4-dihydro-1H-quinoxaloline-6-carboxylate (intermediate 27, 15.6 g, 66.59 mmol) in 1,4-dioxane (150 mL). The reaction mixture was stirred overnight at room temperature. The mixture was slowly added to a saturated aqueous solution of NaHCO3 (about 500 mL) and stirred at room temperature for 20 min. The precipitate was filtered, washed with water (100 mL), and dried to yield methyl 2-ethyl-3-oxo-4H-quinoxaloline-6-carboxylate (intermediate 28, 11.40 g, 73.7%) as a grayish-white solid. 1H NMR (500MHz, 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.

[0439] Intermediate 29: 3-Ethyl-7-(hydroxymethyl)-1H-quinoxaloline-2-one

[0440] Lithium aluminum hydride (2M, in THF (49.1 mL, 98.17 mmol)) was added dropwise to a slurry of methyl 2-ethyl-3-oxo-4H-quinoxaline-6-carboxylate (intermediate 28, 11.4 g, 49.09 mmol) in tetrahydrofuran (350 mL) over a nitrogen atmosphere at 0 °C for 50 minutes. The resulting mixture was stirred at 0 °C for 1.5 hours. At 0 °C, the reaction mixture was slowly poured into a 1M aqueous HCl solution (300 mL). The reaction mixture was extracted with ethyl acetate (approximately 300 mL x 2) followed by extraction with DCM / methanol (5:1) (150 mL x 3). The combined organic layers were concentrated to 300 mL and diluted with diethyl ether (200 mL) to give a suspension. The solid was collected by filtration, washed with diethyl ether, and dried under vacuum to yield 3-ethyl-7-(hydroxymethyl)-1H-quinoxalin-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.

[0441] Intermediate 30: 7-(bromomethyl)-3-ethyl-1H-quinoxaloline-2-one

[0442] Hydrogen bromide (60 mL, 48 wt%, in water) was added to 3-ethyl-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 29, 7.8 g, 38.19 mmol) (producing a clear brown solution), and the mixture was stirred at 80 °C for 8 hours. The reaction mixture was cooled to room temperature and poured into 150 mL of ice water to give a grayish-white precipitate. The solid was filtered under vacuum, washed with water followed by diethyl ether, and dried to give 7-(bromomethyl)-3-ethyl-1H-quinoxalin-2-one (intermediate 30, 11.10 g, 84%) as a pale yellow solid with a purity of 80%. 1H NMR (500MHz, 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.

[0443]

[0444] Intermediate 32: tert-butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate

[0445] A mixture of tert-butylpiperazine-1-carboxylate (intermediate 31, 2.57 g, 13.80 mmol), methyl 6-bromo-5-fluoro-pyridin-2-carboxylate (1.9 g, 8.12 mmol), and potassium carbonate (1.459 g, 10.55 mmol) in DMF (20 mL) was stirred at 110 °C for 5 h, and LCMS indicated complete conversion. The mixture was cooled to rt, 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 Na₂SO₄, filtered, and concentrated. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 50% EtOAc in hexane). The product fraction was concentrated to dryness under reduced pressure to give tert-butyl-4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 32, 2.200 g, 67.7%) as a pale yellow solid. 1H NMR (500MHz, 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.

[0446] Intermediate 33: tert-butyl 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate

[0447] A sealed pressure vessel was filled with tert-butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (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, with LCMS indicating complete conversion. The mixture was concentrated, and the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 80% EtOAc in hexane). The product fraction was concentrated to dryness under reduced pressure to give tert-butyl 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 33, 2.200 g, 100%) as a white solid. 1H NMR (500MHz, 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.

[0448] Intermediate 34: tert-butyl 4- / 6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate

[0449] A mixture of tert-butyl 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 33, 200 mg, 0.50 mmol), tributyl(vinyl)stanane (0.161 mL, 0.55 mmol), and a second-generation XPhos Pd cycle (19.71 mg, 0.03 mmol) in 1,4-dioxane (5 mL) was stirred at 100 °C under N2 for 2.5 hr, with LCMS indicating complete conversion. The mixture was diluted with DCM, washed with saturated NH4Cl, and the organic layer was dried (anhydrous Na2SO4), filtered, and concentrated. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 80% EtOAc in hexane). The product fraction was concentrated to dryness under reduced pressure to give tert-butyl 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate as a white solid (intermediate 34, 174 mg, 100%). m / z (ES + [M+H] + =347

[0450] Intermediate 35: tert-butyl-4-[2-ethyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate

[0451] Pd / C (53.5 mg, 0.05 mmol) (10 wt% dry weight, wet loading) was added to a solution of tert-butyl 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate (intermediate 34, 174 mg, 0.50 mmol) in MeOH (6 mL). The flask was degassed and refilled with H2 (gas bag). The mixture was stirred overnight at rt. LCMS indicated that the reaction was incomplete. More Pd / C (53.5 mg, 0.05 mmol) was added, and the resulting mixture was stirred at rt under H2 atmosphere for 5 hr. The mixture was filtered through a diatomaceous earth pad, washed with methanol, and the filtrate was concentrated to dryness to produce tert-butyl 4-[2-ethyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 35, 172 mg, 98%) as a colorless residue. 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.

[0452] Intermediate 36: 6-Ethyl-N-methyl-5-piperazine-1-,yl-pyridine-2-carboxamide

[0453] A mixture of tert-butyl 4-[2-ethyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 35, 172 mg, 0.49 mmol) in HCl (4 M, in dioxane, 8 mL, 32.00 mmol) was stirred at rt 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-piperazine-1-yl-pyridin-2-carboxamide 2HCl (intermediate 36, 159 mg, 100%) as a pale yellow solid. 1H NMR (500MHz, 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) s); m / z(ES + [M+H] + =249.

[0454] Synthetic Example 8: 6-Ethyl-5-[4-[(2-Ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]- N-Methylpyridine-2-carboxamide

[0455]

[0456] DIPEA (0.203 mL, 1.17 mmol) was added to a suspension of 6-ethyl-N-methyl-5-piperazin-1-ylpyridin-2-carboxamide 2HCl (intermediate 36, 75 mg, 0.23 mmol) and 7-(bromomethyl)-3-ethyl-1H-quinoxalin-2-one (intermediate 30, 69.3 mg, 0.23 mmol) in acetonitrile (3 mL). The resulting mixture was stirred at 60 °C for 3 h, and LCMS indicated complete conversion. The mixture was cooled to rt, concentrated, and the residue was purified on a Gilson reversed-phase column (eluted with 0% to 95% ACN / water / 0.1% TFA, run for 15 min, and collected from 5 to 9 min). The fraction containing the product was concentrated, and the residue was then dissolved in methanol and DCM. 300 mg of tetraalkylammonium bicarbonate was polymerized (40-90 mesh, 2.5-3.5 mmol / g), and the mixture was stirred at rt for 10 min. 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 produce 6-ethyl-5-[4-[(2-ethyl-3-oxo-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide as a pale yellow solid (Synthetic Example 8, 60.0 mg, 59.1%). 1H NMR (500MHz, 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.

[0457]

[0458] Intermediate 37: tert-butyl-4-[6-(methylcarbamoyl)-2-(trifluoromethyl)-3-pyridyl]piperazine-1-methyl esters

[0459] At room temperature, trimethyl(trifluoromethyl)silane (0.247 mL, 1.67 mmol) was added to a well-stirred mixture of silver fluoride (I) (176 mg, 1.39 mmol) in DMF (2 mL). The mixture was stirred for 20 min, followed by the addition of copper powder (133 mg, 2.09 mmol). The reaction mixture turned blue after stirring for 4 h (indicating the formation of CuCF3). Tert-butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 33, 150 mg, 0.38 mmol) was added to the mixture, and the resulting dark mixture was stirred at 90 °C for 18 h to give 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 and then with brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 70% EtOAc in hexane). The product fraction was concentrated to dryness under reduced pressure to give tert-butyl 4-[6-(methylcarbamoyl)-2-(trifluoromethyl)-3-pyridyl]piperazine-1-carboxylate as a yellow residue (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.

[0460] Intermediate 38: N-methyl-5-piperazin-1-yl-6-(trifluoromethyl)pyridine-2-carboxamide

[0461] A mixture of tert-butyl 4-[6-(methylcarbamoyl)-2-(trifluoromethyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 37, 146 mg, 0.38 mmol) in HCl (4 M, in dioxane, 8 mL, 32.00 mmol) was stirred at rt for 2 hr. LCMS indicated complete conversion. The solvent was concentrated to a volume of 2 mL, and the mixture was diluted with diethyl ether / hexane (15 mL, 5 / 1). The solid was filtered off and dried under vacuum to give N-methyl-5-piperazine-1-yl-6-(trifluoromethyl)pyridin-2-carboxamide 2HCl (intermediate 38, 127 mg, 94%) as a pink solid. 1H NMR (500MHz, DMSO-d6) 2.83 (3H, d), 3.21 (8H, brs), 8.09 (1H, d), 8.23 ​​(1H, d), 8.46 (1H, br d), 9.08 (2H, br d); m / z (ES + [M+H]+ =289.

[0462] Synthetic Example 9: 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methyl- 6-(trifluoromethyl)pyridine-2-carboxamide

[0463]

[0464] DIPEA (0.121 mL, 0.69 mmol) was added to a suspension of N-methyl-5-piperazin-1-yl-6-(trifluoromethyl)pyridin-2-carboxamide 2HCl (intermediate 38, 50 mg, 0.14 mmol) and 7-(bromomethyl)-3-ethylquinoxalin-2(1H)-one (intermediate 30, 46.2 mg, 0.14 mmol) in acetonitrile (3 mL), and the mixture was stirred at 60 °C for 3 hr. The mixture was cooled to rt, concentrated, and the residue was purified on a Gilson reversed-phase column (eluted with 0% to 95% ACN / water / 0.1% TFA). The fraction containing the product was concentrated at room temperature. The residue was then dissolved in methanol and DCM, followed by the addition of 250 mg of tetraalkylammonium bicarbonate polymer-linked (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 mixture of water / CH3CN and lyophilized to dryness to give 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide as a white solid (Synthetic Example 9, 40.0 mg, 60.9%). 1H NMR (500MHz, 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.

[0465]

[0466] Intermediate 39: tert-butyl 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate

[0467] Osmium tetroxide (0.050 mL, 6.35 μmol) in H₂O was added to a solution of tert-butyl-4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate (intermediate 34, 110 mg, 0.32 mmol), 2,6-dimethylpyridine (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 overnight at rt to give a yellow suspension. LCMS and TLC indicated complete conversion. The reaction mixture was diluted with water and extracted with ethyl acetate. After concentration, the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 100% EtOAc in hexane). The product fraction was concentrated to dryness under reduced pressure to give tert-butyl 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate as a yellow solid (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.

[0468] Intermediate 40: tert-butyl-4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-methyl esters

[0469] The tert-butyl 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 39.99 mg, 0.28 mmol) in CH2Cl2 (2 mL) was cooled to 0 °C, DAST (0.710 mL, 0.71 mmol) (1 M, in DCM) was added, and the resulting mixture was stirred at room temperature for 3 hr. TLC and LCMS indicated complete conversion. The reaction was quenched dropwise with saturated NaHCO3 solution and extracted with DCM. The combined organic compounds were dried over anhydrous Na2SO4, filtered, and concentrated to give a crude product. The resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 100% EtOAc in hexane). The product fraction was concentrated to dryness under reduced pressure to give tert-butyl 4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate as a grayish-white solid (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.

[0470] Intermediate 41: 6-(difluoromethyl)-N-methyl-5-piperazin-1-yl-pyridine-2-carboxamide

[0471] The mixture of tert-butyl 4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 40, 92 mg, 0.25 mmol) in HCl 4M of 1,4-dioxane (6 ml, 24.00 mmol) was stirred at rt for 1.5 hr, giving an orange suspension. The mixture was diluted with diethyl ether, filtered, and the solid was redissolved in methanol and concentrated to dryness to give 6-(difluoromethyl)-N.-methyl-5-piperazine-1-yl-pyridin-2-carboxamide 2HCl (intermediate 41, 56.0 mg, 65.7%) as an orange solid. 1H NMR (500MHz, 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.

[0472] Synthetic Example 10: 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazine [1-azinyl]-N-methylpyridine-2-carboxamide

[0473]

[0474] DIPEA (0.127 mL, 0.73 mmol) was added to a suspension of 6-(difluoromethyl)-N-methyl-5-piperazin-1-yl-pyridin-2-carboxamide 2HCl (intermediate 41, 50 mg, 0.15 mmol) and 7-(bromomethyl)-3-ethylquinoxalin-2(1H)-one (intermediate 30, 48.6 mg, 0.15 mmol) in acetonitrile (3 mL). The resulting mixture was stirred at 60 °C for 3 h, and LCMS indicated complete conversion. The mixture was concentrated, and the residue was purified on a Gilson reversed-phase column (eluted with 0% to 95% ACN / water / 0.1% TFA). The fraction containing the product was concentrated at room temperature. The residue was then dissolved in methanol and DCM, followed by the addition of 250 mg of tetraalkylammonium bicarbonate polymer-linked (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 mixture of water / CH3CN and lyophilized to dryness to give 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide (synthetic example 10, 50.0 mg, 75%) as a yellow solid. 1H NMR (500MHz, 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.

[0475]

[0476] Synthetic Example 11: 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl / -N-methyl pyridine-2-carboxamide

[0477]

[0478] Add 7-(bromomethyl)-3-ethylquinoxalin-2(1H)-one (intermediate 30, 0.147 g, 0.55 mmol) and N-methyl-5-piperazin-1-ylpyridin-2-carboxamide, along with 2HCl (intermediate 13, 0.161 g, 0.55 mmol) to a 20 mL vial. Seal the vial, evacuate it, and refill it with N2. Add acetonitrile (3 mL) and DIPEA (0.481 mL, 2.75 mmol) to the vial and place it in a heating block preheated to 70 °C. Stir the reaction mixture at the same temperature for 2 hours and cool to room temperature. Reduce the volume of the reaction mixture to 1 / 3 of its initial volume under vacuum and add 2 mL of NaHCO3 aqueous solution. Stir the reaction mixture for 30 min, filter, and wash the solid with 50 mL of water. The crude product was purified by rapid silica chromatography (using 0%-30% MeOH in DCM) to produce 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide as a pale yellow solid (Synthetic Example 11, 93.0 mg, 41.6%). 1 H NMR (500MHz, DMSO-d6) 1.22 (3H, t), 2.52-2.60 (4H, m), 2.73-2.85 (5H, m), 3.30 (4H, m, overlapped 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.

[0479]

[0480] Synthetic Example 12: 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-6-fluoro- N-Methylpyridine-2-carboxamide

[0481]

[0482] 7-(bromomethyl)-3-ethylquinoxalin-2(1H)-one (intermediate 30, 150 mg, 0.56 mmol) was added to 6-fluoro-N-methyl-5-piperazin-1-ylpyridin-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. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge Shield RP18 OBD column, 5 μm, 19 x 150 mm; mobile phase A: water (10 mmol / L NH4HCO3, 0.1% NH3·H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 28% B to 38% B over 8 min; 254; 220 nm; RT: 8.02 min). The fraction containing the desired compound was evaporated to dryness to provide 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methyl-pyridine-2-carboxamide as a white solid (Synthetic Example 12, 9 mg, 42.9%). 1 H NMR (400MHz, CD3OD) δ1.33 (3H, t), 2.65-2.72 (4H, m), 2.87-2.95 (5H, m), 3.26-3.3 0(4H, m), 3.71(2H, s), 7.33-7.41(2H, m), 7.52(1H, dd), 7.76(1H, d), 7.90(1H, dd); 19 F NMR (376MHz, CD3OD) δ-73.40; m / z (ES + [M+H] + =425.

[0483]

[0484] Intermediate 43: 5-Bromo-N,6-Dimethylpyridineamide

[0485] A 2M solution of methylamine in THF (20 mL, 40.00 mmol) was added to methyl 5-bromo-6-methylpyridinecarboxylate (intermediate 42, 2.0 g, 8.69 mmol), and the resulting mixture was stirred at 80 °C for 18 hours. The solvent was removed under reduced pressure. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 80% MeOH in water (0.1% NH4HCO3)). The purified fraction was evaporated to dryness to give 5-bromo-N,6-dimethylpyridineamide (intermediate 43, 1.5 g, 75%) as a pale yellow solid. 1H NMR (400MHz, DMSO-d6) δ2.65 (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.

[0486] Intermediate 44: tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate Under nitrogen, 5-bromo-N,6-dimethylpyridine amide (intermediate 43, 1.0 g, 4.37 mmol) was added to tert-butylpiperazine-1-carboxylate (0.894 g, 4.80 mmol), BINAP (0.272 g, 0.44 mmol), Pd(OAc)₂ (0.098 g, 0.44 mmol), and Cs₂CO₃ (3.56 g, 10.91 mmol) in toluene (20 mL). The resulting mixture was stirred at 80 °C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 30% MeOH in water (0.4% HCO₂H)). The pure fraction was evaporated to dryness to give tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate as a brown solid (intermediate 44, 1.2 g, 82%). 1 H NMR (300MHz, CD3OD) δ1.50 (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.

[0487] Intermediate 45: N,6-Dimethyl-5-(piperazin-1-yl)pyridine amide

[0488] Tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate (intermediate 44, 1.18 g, 3.53 mmol) was added to a 4 M HCl solution in 1,4-dioxane (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) and Et2O (5 mL x 2), and dried under vacuum to give N,6-dimethyl-5-(piperazine-1-yl)pyridine amide (intermediate 45, 0.77 g, 81%) as a yellow solid. 1 H NMR (300MHz, CD3OD) δ2.86 (3H, s), 3.02 (3H, s), 3.42-3.54 (8H, m), 8.29 (2H, d); m / z (ES + [M+H]+ =235.

[0489] Synthetic Example 13: 5-[4-](2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N,6-trisyl] Methylpyridine-2-carboxamide

[0490]

[0491] 7-(bromomethyl)-3-ethylquinoxalin-2(1H)-one (intermediate 30, 100 mg, 0.37 mmol) was added to N,6-dimethyl-5-(piperazin-1-yl)pyridine amide (intermediate 45, 90 mg, 0.33 mmol) and DIPEA (0.36 mL, 2.05 mmol) in NMP (2 mL). The resulting mixture was stirred at 80 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge Prep OBD C18 column 30 x 150 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 30% B to 40% B over 7 min; 254 nm; 220 nm; RT: 6.43 min). The fraction containing the desired compound was evaporated to dryness to give 5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N,6-dimethyl-pyridine-2-carboxamide as a grayish-white solid (Synthetic Example 13, 68.7 mg, 43.6%). 1 H NMR (400MHz, CD3OD) δ1.33 (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.

[0492]

[0493] Intermediate 47: Methyl 6-chloro-5-(piperazin-1-yl)pyridine carboxylate

[0494] Piperazine (1.0 g, 11.61 mmol) was added to methyl 6-chloro-5-fluoropyridine carboxylate (intermediate 46, 1.0 g, 5.28 mmol) in MeCN (30 mL). The resulting mixture was stirred at 80 °C for 18 hours. The solvent was removed under reduced pressure. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 60% MeCN in water (0.1% NH4HCO3)). The purified fraction was evaporated to dryness to give methyl 6-chloro-5-(piperazin-1-yl)pyridine carboxylate (intermediate 47, 1.28 g, 95%) as a red oil. 1 ¹H NMR (400 MHz, DMSO-d⁶) 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.

[0495] Intermediate 48: 6-Chloro-N-methyl-5-(piperazin-1-yl)pyridine amide

[0496] A 2M solution of methylamine in THF (40 mL, 80.00 mmol) was added to methyl 6-chloro-5-(piperazin-1-yl)pyridinecarboxylate (intermediate 47, 1.26 g, 4.93 mmol). The resulting mixture was stirred at 80 °C for 18 hours. The solvent was removed under reduced pressure. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 60% MeCN in water (0.1% NH4HCO3)). The purified fraction was evaporated to dryness to give 6-chloro-N-methyl-5-(piperazin-1-yl)pyridine amide (intermediate 48, 1.12 g, 89%) as a pale yellow oil. 1 ¹H NMR (300 MHz, DMSO-d⁶) 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) (piperazine – no NH proton shown); m / z (ES) + [M+H] + =255.

[0497] Synthetic Example 14: 6-Chloro-5-[4-[(2-ethyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]- N-Methylpyridine-2-carboxamide

[0498]

[0499] 7-(bromomethyl)-3-ethylquinoxalin-2(1H)-one (intermediate 30, 200 mg, 0.75 mmol) was added to 6-chloro-N-methyl-5-(piperazin-1-yl)pyridine amide (intermediate 48, 100 mg, 0.39 mmol) and DIPEA (0.358 mL, 2.05 mmol) in NMP (2 mL). The resulting mixture was stirred at 80 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 30% B to 40% B over 8 min; 254 nm; 220 nm; RT: 7.3 min). The fraction containing the desired compound was evaporated to dryness to provide 6-chloro-5-[4-[(2-ethyl-3-oxo-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide as a white solid (Synthetic Example 14, 52.6 mg, 30.4%). 1 HNMR (400MHz, CD3OD) δ1.33 (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.

[0500]

[0501] Intermediate 50; 7-bromo-3-(trifluoromethyl)quinoxaline-2(1H)-one

[0502] 4-Bromophenyl-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 min. The solvent was removed under reduced pressure. The crude product was purified by rapid silica chromatography (elution gradient of 0% to 50% EtOAc in petroleum ether). The purified fraction was evaporated to dryness to give a regiomeric mixture of 7-bromo-3-(trifluoromethyl)quinoxalin-2(1H)-one and 6-bromo-3-(trifluoromethyl)quinoxalin-2(1H)-one (intermediate 50 + intermediate 51, 1.28 g, 45.4%) as an off-white solid. The regiomeric mixture was separated, and the specific details are not provided. 1 H NMR spectrum; m / z (ES) + [M+H]+ =295.

[0503] Intermediate 52: 7-(hydroxymethyl)-3-(trifluoromethyl)quinoxaline-2(1H)-one

[0504] Pd(Ph3P)4 (0.3 g, 0.26 mmol) was added to a mixture of 7-bromo-3-(trifluoromethyl)quinoxalin-2(1H)-one and 6-bromo-3-(trifluoromethyl)quinoxalin-2(1H)-one (intermediate 50 + intermediate 51, 1.2 g, 2.05 mmol) and (tributyltinyl)methanol (1.2 g, 3.74 mmol) in 1,4-dioxane (40 mL). The resulting mixture was stirred at 100 °C under nitrogen for 18 hours. The solvent was removed under reduced pressure. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 50% MeCN in water (0.1% HCO2H)). The pure fraction was evaporated to dryness to give 7-(hydroxymethyl)-3-(trifluoromethyl)quinoxaline-2(1H)-one as a grayish-white solid (intermediate 52, 0.32 g, 64.0%). 1 H NMR (300MHz, DMSO-d6,) δ4.63 (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.

[0505] Synthetic Example 15: N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxalin-6-yl]methyl]piperazine [Zinc-1-yl]pyridine-2-carboxamide

[0506]

[0507] A solution of 33% HBr in AcOH (3 mL, 18.23 mmol) was added to 7-(hydroxymethyl)-3-(trifluoromethyl)quinoxalin-2(1H)-one (intermediate 52, 111 mg, 0.45 mmol). The resulting mixture was stirred at 80 °C for 1 hour. The solvent was removed under reduced pressure. DIEA (0.5 mL, 2.86 mmol) and N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 13, 100 mg, 0.45 mmol) were added to the above mixture in NMP (3 mL). The resulting mixture was stirred at 80 °C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge Prep OBD C18 column, 30 × 150 mm 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 22B to 32B over 7 min; 254; 220 nm; RT: 5.77). The fraction containing the desired compound was evaporated to dryness to give N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaloline-6-yl]methyl]piperazin-1-yl]pyridin-2-carboxamide as a white solid (Synthetic Example 15, 44.0 mg, 21.71%). 1 H NMR (400MHz, DMSO-d6) δ2.55-2.62(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); 19 F NMR(376MHz, DMSO-d6)δ-68.36; m / z(ES + [M+H] + =447.

[0508]

[0509] Synthetic Example 16: 6-Chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxalin-6-yl]methyl [[ ...][[[[[[[[[[[[[][[[[[[[[[[][[[[[[[[][[[[[[[][[[[[[[[][[[[[][[[[[[[][[

[0510]

[0511] 33% HBr (3 mL, 18.23 mmol) in AcOH was added to 7-(hydroxymethyl)-3-(trifluoromethyl)quinoxaline-2(1H)-one (intermediate 52, 43.1 mg, 0.18 mmol). The resulting mixture was stirred at 80 °C for 1 hour. The solvent was removed under reduced pressure. DIPEA (0.5 mL, 2.86 mmol) and 6-chloro-N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 48, 45 mg, 0.18 mmol) were added to the above mixture in NMP (5 mL). The resulting mixture was stirred at 80 °C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge Prep OBD C18 column, 30 × 150 mm 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 10B to 50B over 7 min; 254 nm; 220 nm; RT: 6.75). The fraction containing the desired compound was evaporated to dryness to give 6-chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaloline-6-yl]methyl]piperazin-1-yl]pyridin-2-carboxamide as a grayish-white solid (Synthetic Example 16, 22.00 mg, 25.9%). 1 H NMR (400MHz, DMSO-d6) δ2.56-2.64 (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); 19 F NMR(376MHz, DMSO)δ-68.41; m / z(ES+)[M+H] + =481.

[0512]

[0513] Synthetic Example 17: 6-Fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxalin-6-yl]methyl [[ ...][[[[[[[[[[[[[][[[[[[[[[[][[[[[[[[][[[[[[[][[[[[[[[][[[[[][[[[[[[][[

[0514]

[0515] 33% HBr (3 mL, 55.25 mmol) in AcOH was added to 7-(hydroxymethyl)-3-(trifluoromethyl)quinoxaline-2(1H)-one (intermediate 52, 102 mg, 0.42 mmol). The resulting mixture was stirred at 80 °C for 1 hour. The solvent was removed under reduced pressure. 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 23, 100 mg, 0.42 mmol) and DIPEA (0.5 mL, 2.86 mmol) were added to the above mixture in NMP (5 mL). The resulting mixture was stirred at 80 °C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge Prep OBD C18 column, 30 × 150 mm 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 15B to 40B over 8 min; 254; 220 nm; RT: 7.2). The fraction containing the desired compound was evaporated to dryness to give 6-fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaloline-6-yl]methyl]piperazin-1-yl]pyridin-2-carboxamide as a white solid (Synthetic Example 17, 66.0 mg, 33.9%). 1 H NMR (400MHz, DMSO-d6) δ2.55-2.69(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); 19 F NMR (376MHz, DMSO-d6) δ-68.36, -72.52; m / z (ES + [M+H] + =465.

[0516]

[0517] Intermediate 54: Methyl 2-aminovalerate hydrochloride

[0518] At 0 °C, SOCl2 (17 mL, 232.94 mmol) was added dropwise to 2-aminovaleric acid (intermediate 53, 10.0 g, 85.36 mmol) in MeOH (200 mL). The resulting mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to give methyl 2-aminovaleric acid hydrochloride (intermediate 54, 15.78 g, 110%) as a white solid. 1H NMR (DMSO-d6, 400MHz) δ0.88 (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.

[0519] Intermediate 55: Methyl 4-(1-methoxy-1-oxopentane-2-ylamino)-3-nitrobenzene ester

[0520] Sodium bicarbonate (20.0 g, 238.08 mmol) was added to methyl 2-aminopentanoate hydrochloride (intermediate 54, 15.57 g, 92.88 mmol) and methyl 4-fluoro-3-nitrobenzene ester (9.0 g, 45.19 mmol) in THF (160 mL). The resulting mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure. The reaction mixture was diluted with EtOAc (150 mL) and washed successively with water (100 mL x 1), saturated NaHCO3 (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-oxopentane-2-ylamino)-3-nitrobenzene ester (intermediate 55, 14.09 g, 100%) as a yellow oil. 1 H NMR (400MHz, DMSO-d6) δ0.89 (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.

[0521] Intermediate 56: Methyl 3-oxo-2-propyl-1,2,3,4-tetrahydroquinoxaline-6-carboxylate

[0522] Pd(OH)₂ / C (20% wt, 1.58 g, 2.25 mmol) was added to methyl 4-((1-methoxy-1-oxopentane-2-yl)amino)-3-nitrobenzene ester (intermediate 55, 14.05 g, 45.28 mmol) in MeOH (300 mL). The resulting mixture was stirred at room temperature under H₂ for 30 hours. The reaction mixture was filtered. The precipitate was washed with DMF (100 mL) and the filtrate was evaporated to dryness to give a crude product. The crude product was washed with DCM (10 mL) and dried under vacuum to give methyl 3-oxo-2-propyl-1,2,3,4-tetrahydroquinoxaline-6-carboxylate ester (intermediate 56, 9.12 g, 81%) as a white solid.1 HNMR (400MHz, DMSO-d6) δ0.87 (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. 3 8(1H, s); m / z(ES) + [M+H] + =249.

[0523] Intermediate 57: Methyl 3-oxo-2-propyl-3,4-dihydroquinoxaline-6-carboxylate

[0524] DDQ (9.42 g, 41.50 mmol) was added to methyl 3-oxo-2-propyl-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 56, 9.12 g, 36.73 mmol) in 1,4-dioxane (200 mL). The resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with saturated NaHCO3 (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 methyl 3-oxo-2-propyl-3,4-dihydroquinoxaline-6-carboxylate (intermediate 57, 7.86 g, 87%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6) δ0.98 (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.

[0525] Intermediate 58: 7-(hydroxymethyl)-3-propylquinoxalin-2(1H)-one

[0526] At 0 °C, a 1 M solution of DIBAL-H in THF (100 mL, 100.00 mmol) was added dropwise to methyl 3-oxo-2-propyl-3,4-dihydroquinoxaline-6-carboxylate (intermediate 57, 7.81 g, 31.71 mmol) in THF (200 mL). The resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with MeOH (5 mL) and a saturated aqueous solution of potassium monosodium tartrate tetrahydrate (20 mL), and the organic layer was evaporated to give 7-(hydroxymethyl)-3-propylquinoxaline-2(1H)-one (intermediate 58, 1.2 g, 17.34%) as a white solid. 1H NMR (400MHz, DMSO-d6) δ0.97 (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.

[0527] Intermediate 59: 7-(bromomethyl)-3-propylquinoxalin-2(1H)-one

[0528] 33% HBr (74.6 μl, 1.37 mmol) in AcOH was added to 7-(hydroxymethyl)-3-propylquinoxalin-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 give 7-(bromomethyl)-3-propylquinoxalin-2(1H)-one (intermediate 59, 600 mg, 155%) as a brown solid (the crude product was impure and contained AcOH and other impurities). The product was used for the next step without further purification. 1 The H NMR spectrum is not clean and is not specified; m / z (ES) + [M+H] + =282.

[0529] Synthetic Example 18: N-methyl-5-[4-[(3-oxo-12-propyl-4H-quinoxalin-6-yl)methyl]piperazin-1-yl] Pyridine-2-carboxamide

[0530]

[0531] DIPEA (200 μL, 1.15 mmol) was added to 7-(bromomethyl)-3-propylquinoxalin-2(1H)-one (intermediate 59, 200 mg, 0.71 mmol) and N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 13, 80 mg, 0.36 mmol) in NMP (3 mL). The resulting mixture was stirred at 80 °C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge Shield RP18 OBD column, 19 x 250 mm, 10 μm; mobile phase A: water (10 mmol / L NH4HCO3, 0.1% NH3·H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 38 B to 50 B over 7 min; 254 / 220 nm; RT: 6.20). The fraction containing the desired compound was evaporated to dryness to give N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide as a white solid (Synthetic Example 18, 71.0 mg, 46.5%). 1 H NMR (400MHz, DMSO-d6) δ0.97 (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.

[0532]

[0533] Synthetic Example 19: 6-Chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxalo-6-yl)methyl]piperazine- 1-yl]pyridine-2-carboxamide

[0534]

[0535] DIPEA (200 μL, 1.15 mmol) was added to 7-(bromomethyl)-3-propylquinoxalin-2(1H)-one (intermediate 59, 200 mg, 0.71 mmol) and 6-chloro-N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 48, 80 mg, 0.31 mmol) in NMP (3 mL). The resulting mixture was stirred at 80 °C for 1 h. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge Shield RP18 OBD column, 19 x 250 mm, 10 μm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 18B to 30B over 7 min; 254 / 220 nm; RT: 5.93). The fraction containing the desired compound was evaporated to dryness to give 6-chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxalo-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide as a white solid (Synthetic Example 19, 52.0 mg, 36.4%). 1 H NMR (400MHz, DMSO-d6) δ0.97 (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.

[0536]

[0537] Synthetic Example 20: 6-Fluoro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxalo-6-yl)methyl]piperazine- 1-yl]pyridine-2-carboxamide

[0538]

[0539] DIPEA (500 μl, 2.86 mmol) was added to 7-(bromomethyl)-3-propylquinoxalin-2(1H)-one (intermediate 59, 200 mg, 0.71 mmol) and 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide 2HCl (intermediate 23, 100 mg, 0.32 mmol) in NMP (3 mL). The resulting mixture was stirred at 80 °C for 1 hour. The solvent was removed under reduced pressure. The crude product was passed through a preparative HPLC (column: SunFire C18 OBD Prep column). Purification was performed using a mobile phase A of 5 μm (19 mm x 250 mm), mobile phase B of water (0.1% HCO2H), mobile phase B of ACN, flow rate of 25 mL / min, gradient of 10 B to 20 B over 13 min (254 / 220 nm, RT: 12.13). The fraction containing the desired compound was evaporated to dryness to give 6-fluoro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxaloline-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide as a white solid (Synthetic Example 20, 71.0 mg, 50.4%). 1 H NMR (400MHz, DMSO-d6) δ0.97 (3H, t), 1.66-1.78 (2H, m), 2.54-2.60 (4H, m), 2.7l-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); 19 F NMR(376MHz, DMSO-d6)δ-72.51; m / z(ES + [M+H] + =439.

[0540]

[0541] Intermediate 61: Methyl 2-aminobutyrate hydrochloride

[0542] At 0 °C, SOCl2 (17 mL, 232.94 mmol) was added dropwise to 2-aminobutyric acid (intermediate 60, 10.0 g, 96.97 mmol) in MeOH (100 mL). The resulting mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to give methyl 2-aminobutyrate hydrochloride (intermediate 61, 14.84 g, 100%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ0.91 (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.

[0543] Intermediate 62: Methyl 2-fluoro-4-(1-methoxy-1-oxobutane-2-ylamino)-5-benzoate

[0544] DIPEA (4.02 mL, 23.03 mmol) was added to methyl 2,4-difluoro-5-nitrobenzene (1.0 g, 4.61 mmol) and methyl 2-aminobutyrate hydrochloride (intermediate 61, 0.707 g, 4.61 mmol) in NMP (10 mL). The resulting mixture was stirred at room temperature for 5 hours. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 80% MeCN in water (0.1% NH4HCO3)). The purified fraction was evaporated to dryness to give methyl 2-fluoro-4-(1-methoxy-1-oxobutane-2-ylamino)-5-nitrobenzene (intermediate 62, 1.2 g, 83%) as a black solid. 1 H NMR (400MHz, DMSO-d6) δ0.88 (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.

[0545] Intermediate 63: Methyl 2-ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate

[0546] Under hydrogen conditions, methyl 2-fluoro-4-((1-methoxy-1-oxobutan-2-yl)amino)-5-nitrobenzene ester (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 was incomplete. The reaction mixture was filtered. The organic layer was evaporated to give a crude product, methyl 2-ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate ester (intermediate 63, 0.780 g, 85%), as a brown gel. This crude product was used directly in the next step without further purification. The crude product was not clean and its properties were not described. 1 HNMR spectrum; m / z (ES) + [M+H] + =253.

[0547] Intermediate 64: Methyl 2-ethyl-7-fluoro-3-oxo-3,4-dihydroquinoxaline-6-carboxylate

[0548] Methyl 2-ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (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 was complete. The resulting mixture was concentrated under reduced pressure to obtain a brown solid. A saturated aqueous solution of NaHCO3 (10 mL) was added to the solid and stirred at room temperature for 1 hour. The precipitate was filtered and washed with another aqueous solution of NaHCO3 (10 mL x 5). The solid was dried under vacuum to give methyl 2-ethyl-7-fluoro-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 64,750 mg, 99%) as a brown solid. 1 H NMR (300MHz, DMSO-d6) δ1.20 (3H, t), 2.82 (2H, q), 3.87 (3H, s), 7.65 (1H, d), 7.76 (1H, d), 12.42 (1H, s); m / z (ES + [M+H] + =251.

[0549] Intermediate 65: 3-Ethyl-6-fluoro-7-(hydroxymethyl)quinoxaline-2(1H)-one

[0550] A 1M solution of diisobutylaluminum hydride in THF (15.35 mL, 15.35 mmol) was added fractionally to methyl 2-ethyl-7-fluoro-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 64, 640 mg, 2.56 mmol) in THF (300 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction was complete. The reaction mixture was quenched at 0 °C with 20 mL of saturated sodium potassium tartrate aqueous solution and 10 mL of MeOH. 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 the crude product. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 60% MeOH in water (0.4% HCO2H)). The pure fraction was evaporated to dryness to give 3-ethyl-6-fluoro-7-(hydroxymethyl)quinoxaline-2(1H)-one as a grayish-white solid (intermediate 65, 110 mg, 19.37%). 1 H NMR (400MHz, DMSO-d6) δ1.21 (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.

[0551] Synthetic Example 21: 5-[4-[(2-ethyl-7-fluoro-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]- 6-Fluoro-N-methylpyridine-2-carboxamide

[0552]

[0553] 3-Ethyl-6-fluoro-7-(hydroxymethyl)quinoxalin-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-fluoroquinoxalin-2(1H)-one (crude product). This product was used directly in the next step without further purification. DIPEA (0.196 mL, 1.13 mmol) was added to 7-(bromomethyl)-3-ethyl-6-fluoroquinoxalin-2(1H)-one and 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 23, 70 mg, 0.29 mmol) in NMP (2 mL). The resulting mixture was stirred at 80 °C for 2 hours. The resulting mixture was purified by preparative HPLC (column: Sunfire prep C18 column, 30x150mm, 5µm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 10B to 35B over 8 min; 254 / 220 nm; RT: 7.37). The fraction containing the desired compound was evaporated to dryness to give 5-[4-[(2-ethyl-7-fluoro-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridin-2-carboxamide as a grayish-white solid (Synthetic Example 21, 55.0 mg, 53.7%). 1 HNMR (400MHz, DMSO-d6) δ1.21 (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); 19 F NMR(376MHz, DMSO-d6)δ-72.53,-124.31; m / z(ES+)[M+H] + =443.

[0554]

[0555] Intermediate 67: Methyl 4-(3-hydroxy-1-methoxy-1-oxobutane-2-ylamino)-3-nitrobenzene ester

[0556] DIPEA (8.77 mL, 50.22 mmol) was added to methyl 4-fluoro-3-nitrobenzene (2.0 g, 10.04 mmol) and methyl 2-amino-3-hydroxybutyrate hydrochloride (intermediate 66, 2.04 g, 12.05 mmol) in DMF (20 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with EtOAc (100 mL) and washed successively with saturated NH4Cl aqueous solution (100 mL x 1) and brine (100 mL x 4). The organic layer was dried over Na2SO4, filtered, and evaporated to give the desired product, methyl 4-((3-hydroxy-1-methoxy-1-oxobutane-2-yl)amino)-3-nitrobenzene (intermediate 67, 2.9 g, 92%), as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ1.15-1.27(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.

[0557] Intermediate 68: Methyl 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate

[0558] Under hydrogen conditions, 20% Pd(OH)₂ / C (0.648 g, 0.92 mmol) was added to methyl 4-((3-hydroxy-1-methoxy-1-oxobutane-2-yl)amino)-3-nitrobenzene ester (intermediate 67, 2.88 g, 9.22 mmol) in MeOH (300 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction was complete. The reaction mixture was filtered through diatomaceous earth. The organic layer was evaporated to give methyl 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate ester (intermediate 68, 2.290 g, 99%) as a gray solid. 1 H NMR (400MHz, DMSO-d6) δ1.07 (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.

[0559] Intermediate 69: Methyl 2-(1-hydroxyethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate

[0560] DDQ (2.265 g, 9.98 mmol) was added to methyl 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 68, 2.27 g, 9.07 mmol) in DCM (100 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain a brown solid. A saturated aqueous solution of NaHCO3 (100 mL) was added to the solid and stirred at room temperature for 1 hour. The precipitate was filtered and washed with another aqueous solution of NaHCO3 (30 mL x 3). The solid was dried under vacuum to give methyl 2-(1-hydroxyethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 69, 2.24 g, 99%) as a gray solid. 1 H NMR (400MHz, DMSO-d6) δ1.40 (3H, d), 3.88 (3H, s), 4.94 (1H, q), 7.69 (1H, dd), 7.77 (1H, d), 7.90 (1H, d) (2protons are not shown); m / z (ES + [M+H] + =249.

[0561] Intermediate 70: Methyl 2-acetyl-3-oxo-3,4-dihydroquinoxaline-6-carboxylate

[0562] Des Martin periodane (2.56 g, 6.04 mmol) was added to methyl 2-(1-hydroxyethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 69, 1.0 g, 4.03 mmol) in DCM (30 mL). The resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was evaporated to give a crude product. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 30% MeCN in water (0.4% HCO2H)). The purified fraction was evaporated to dryness to give methyl 2-acetyl-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 70, 0.62 g, 62.5%) as a pale yellow solid. 1 H NMR (400MHz, DMSO-d6) δ2.58 (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.

[0563] Intermediate 71: Methyl 2-(1,1-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate

[0564] BAST (1.35 mL, 7.31 mmol) was added to methyl 2-acetyl-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 70, 600 mg, 2.44 mmol) in DCM (20 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was evaporated to give a crude product. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 30% MeCN in water (0.4% HCO2H)). The purified fraction was evaporated to dryness to give methyl 2-(1,1-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 71, 174 mg, 26.6%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6) δ2.07 (3H, t), 3.91 (3H, s), 7.84 (1H, dd), 7.92-7.99 (2H, m), 12.90 (1H, s); 19 F NMR(376MHz, DMSO-d6)δ-93.26; m / z(ES + [M+H] + =269.

[0565] Intermediate 72: 3-(1,1-difluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one

[0566] At 0 °C, a solution of 1 M diisobutylaluminum hydride in THF (2.39 mL, 2.39 mmol) was added to methyl 2-(1,1-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 71, 160 mg, 0.60 mmol) in THF (50 mL). The resulting mixture was stirred at room temperature for 16 hours. At 0 °C, the reaction mixture was quenched with a saturated aqueous solution of sodium potassium tartrate (3 mL) and MeOH (1 mL). 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)quinoxaline-2(1H)-one (intermediate 72, 120 mg, 84%). This product was used directly in the next step without further purification. 1 H NMR (400MHz, DMSO-d6) δ2.06 (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.

[0567] Synthetic Example 22: 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazine-1- [N-methylpyridine-2-carboxamide]

[0568]

[0569] 3-(1,1-difluoroethyl)-7-(hydroxymethyl)quinoxalin-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)quinoxalin-2(1H)-one (crude product). This product was used directly in the next step without further purification. DIPEA (0.218 mL, 1.25 mmol) was added to 7-(bromomethyl)-3-(1,1-difluoroethyl)quinoxalin-2(1H)-one (crude product) and N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 13, 60 mg, 0.27 mmol) in NMP (3 mL). The resulting mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated and purified by preparative HPLC (column: XBridge Shield RP18 OBD column, 30x150 mm, 5 μm; mobile phase A: water (0.05% NH3H2O), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 13B to 33B over 7 min; 254; 220 nm; RT: 5.70). The fraction containing the desired compound was evaporated to dryness to give 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide as a yellow solid (Synthetic Example 22, 47.8 mg, 43.2%). 1 H NMR (400MHz, DMSO-d6) δ2.06 (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); 19 F NMR(376MHz, DMSO-d6)δ-92.74; m / z(ES + [M+H] + =443.

[0570]

[0571] Intermediate 74: Methyl 4-(4,4-difluoro-1-methoxy-1-oxobutane-2-ylamino)-3-nitrobenzene

[0572] DIPEA (8.77 mL, 50.22 mmol) was added to methyl 4-fluoro-3-nitrobenzene (2.0 g, 10.04 mmol) and methyl 2-amino-4,4-difluorobutyrate hydrochloride (intermediate 73, 2.0 g, 10.55 mmol) in DMF (20 mL). The resulting mixture was stirred at 40 °C for 8 hours. The reaction mixture was diluted with EtOAc (100 mL) and washed successively with saturated NH4Cl (100 mL x 1) and brine (100 mL x 4). The organic layer was dried over Na2SO4, filtered, and evaporated to give the desired product, methyl 4-((4,4-difluoro-1-methoxy-1-oxobutan-2-yl)amino)-3-nitrobenzene (intermediate 74, 2.5 g, 74.9%), as a yellow solid. 1 H NMR (300MHz, DMSO-d6) δ2.50-2.76 (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.

[0573] Intermediate 75: Methyl 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate

[0574] Under hydrogen conditions, 20% Pd(OH)₂ / C (0.465 g, 0.66 mmol) was added to methyl 4-((4,4-difluoro-1-methoxy-1-oxobutane-2-yl)amino)-3-nitrobenzene ester (intermediate 74, 2.2 g, 6.62 mmol) in MeOH (300 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered through diatomaceous earth. The filtrate was evaporated to give methyl 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate ester (intermediate 75, 1.64 g, 92%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ2.24-2.32(2H, m), 3.76(3H, s), 4.10-4.18(1H, m), 6.27(1 H, 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.

[0575] Intermediate 76: Methyl 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate

[0576] DDQ (1.478 g, 6.51 mmol) was added to methyl 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 75, 1.6 g, 5.92 mmol) in DCM (100 mL). The resulting mixture was stirred at room temperature for 3 hours. The resulting mixture was removed under reduced pressure to obtain a brown solid. A saturated aqueous solution of NaHCO3 (100 mL) was added to the solid and stirred at room temperature for 1 hour. The precipitate was filtered and washed with another aqueous solution of NaHCO3 (30 mL x 3). The solid was dried under vacuum to give methyl 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 76, 1.58 g, 99%) as an off-white solid. 1 H NMR (400MHz, DMSO-d6) δ3.46 (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.

[0577] Intermediate 77: 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one

[0578] At 0 °C, a 1 M solution of diisobutylaluminum hydride in THF (22.37 mL, 22.37 mmol) was added in portions to methyl 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 76, 1.0 g, 3.73 mmol) in THF (100 mL). The resulting mixture was stirred at room temperature for 16 hours. At 0 °C, the reaction mixture was quenched with a saturated aqueous solution of sodium potassium 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 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one (0.72 g, 80%) as a red solid (crude product). The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 60% MeOH in water (0.4% HCO2H)). The purified fraction was evaporated to dryness to give 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one (intermediate 77,500 mg, 69.4%) as a red solid. 1H NMR (300MHz, DMSO-d6) δ3.42(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.

[0579] Intermediate 78: 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxaldehyde

[0580] Des-Martin periodane (530 mg, 1.25 mmol) was added to 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinoxalin-2(1H)-one (intermediate 77, 200 mg, 0.83 mmol) in DCM (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was evaporated to give a crude product. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 30% MeCN in water (0.4% HCO2H)). The purified fraction was evaporated to dryness to give 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxalin-6-carboxaldehyde (intermediate 78, 160 mg, 81%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ3.47 (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.

[0581] Synthetic Example 23: 5-[4-[[2-(2,2-trifluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazine-1- [-N-methylpyridine-2-carboxamide]

[0582]

[0583] Titanium isopropoxy (65.6 mg, 0.23 mmol) was added to 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaloline-6-carboxaldehyde (intermediate 78, 55 mg, 0.23 mmol) and N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 13, 60 mg, 0.23 mmol) in THF (2 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (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 give a crude product, which was purified by preparative HPLC (column: XBridgeShield RP18 OBD column, 30x150 mm, 5 μm; mobile phase A: water (0.05% NH3H2O), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 13B to 33B over 7 min; 254; 220 nm; RT: 5.70). The fraction containing the desired compound was evaporated to dryness to give 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide as a yellow solid (Synthetic Example 23, 8.76 mg, 8.57%). 1 H NMR (400MHz, DMSO-d6) δ2.56 (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); 19 F NMR(376MHz, DMSO-d6)δ-114.29; m / z(ES + [M+H] + =443.

[0584]

[0585] Synthetic Example 24: 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazine-1- 1,6-Fluoro-N-methylpyridine-2-carboxamide

[0586]

[0587] Titanium isopropoxy (59.7 mg, 0.21 mmol) was added to 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaloline-6-carboxaldehyde (intermediate 78, 50 mg, 0.21 mmol) and 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 23, 50.0 mg, 0.21 mmol) in THF (2 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (178 mg, 0.84 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour. The reaction was complete. The reaction mixture was quenched with MeOH (0.1 mL). The reaction mixture was evaporated to give the crude product. The crude product was purified by preparative HPLC (column: Sunfire prep C18 column, 30x150, 5µm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 2B to 27B over 7 min; 254 / 220 nm; RT: 6.78). The fraction containing the desired compound was evaporated to dryness to give 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-6-fluoro-N-methylpyridin-2-carboxamide (Synthetic Example 24, 21.72 mg, 22.13%) as a yellow solid. 1 HNMR (400MHz, DMSO-d6) δ2.54-2.61 (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); 19 F NMR (376MHz, DMSO-d6) δ-72.52, -114.29; m / z (ES + [M+H] + =461.

[0588]

[0589] Intermediate 80: Methyl 4-(4-fluoro-1-methoxy-1-oxobutane-2-ylamino)-3-nitrobenzene ester

[0590] DIPEA (8.77 mL, 50.22 mmol) was added to methyl 4-fluoro-3-nitrobenzene (2.0 g, 10.04 mmol) and methyl 2-amino-4-fluorobutyrate hydrochloride (intermediate 79, 1.81 g, 10.55 mmol) in DMF (20 mL). The resulting mixture was stirred at 40 °C for 8 hours. The reaction mixture was diluted with EtOAc (100 mL) and washed successively with saturated NH4Cl (100 mL x 1) and brine (100 mL x 4). The organic layer was dried over Na2SO4, filtered, and evaporated to give the desired product, methyl 4-((4-fluoro-1-methoxy-1-oxobutan-2-yl)amino)-3-nitrobenzene (intermediate 80, 2.5 g, 79%), as a yellow solid. 1 H NMR (300MHz, DMSO-d6) δ2.25-2.35(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.

[0591] Intermediate 81: Methyl 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate

[0592] Under hydrogen conditions, 20% Pd(OH)₂ / C (0.547 g, 0.78 mmol) was added to methyl 4-((4-fluoro-1-methoxy-1-oxobutane-2-yl)amino)-3-nitrobenzene ester (intermediate 80, 2.45 g, 7.80 mmol) in MeOH (300 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction was complete. The reaction mixture was filtered through diatomaceous earth. The filtrate was evaporated to give methyl 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate ester (intermediate 81, 1.9 g, 97%) as a gray solid. 1 H NMR (400MHz, DMSO-d6) 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.

[0593] Intermediate 82: Methyl 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate

[0594] DDQ (1.83 g, 8.07 mmol) was added to methyl 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 81, 1.85 g, 7.33 mmol) in DCM (100 mL). The resulting mixture was stirred at room temperature for 3 hours. The resulting mixture was removed under reduced pressure to obtain a brown solid. A saturated aqueous solution of NaHCO3 (100 mL) was added to the solid, and the mixture was stirred at room temperature for 1 hour. The precipitate was filtered and washed with another aqueous solution of NaHCO3 (30 mL x 3). The solid was dried under vacuum to give methyl 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 82, 1.8 g, 98%) as a gray solid. 1 H NMR (400MHz, DMSO-d6) δ3.23 (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.

[0595] Intermediate 83: 3-(2-fluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one

[0596] At 0 °C, a 1 M solution of diisobutylaluminum hydride in 15.99 mL (15.99 mmol) of THF was added fractionally to methyl 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 82, 1.0 g, 4.00 mmol) in 100 mL of THF. The resulting mixture was stirred at room temperature for 16 hours. At 0 °C, the reaction mixture was quenched with 20 mL of saturated sodium potassium tartrate aqueous solution and 10 mL of MeOH. The resulting mixture was stirred for 1 hour. The reaction mixture was filtered and washed with 30 mL x 3 of THF. The organic layer was evaporated to give the crude product. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 60% MeOH in water (0.4% HCO2H)). The pure fraction was evaporated to dryness to give 3-(2-fluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one as a brown solid (intermediate 83, 0.49 g, 55.2%). 1 H NMR (300MHz, DMSO-d6) δ3.20 (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.

[0597] Intermediate 84: 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxalo-6-carboxaldehyde

[0598] Des-Martin periodane (229 mg, 0.54 mmol) was added to 3-(2-fluoroethyl)-7-(hydroxymethyl)quinoxalin-2(1H)-one (intermediate 83, 100 mg, 0.45 mmol) in DCM (3 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated to give a crude product. The crude product was purified by reversed-phase chromatography (elution gradient of 5% to 30% MeCN in water (0.4% HCO2H)). The purified fraction was evaporated to dryness to give 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxalin-6-carboxaldehyde (intermediate 84, 93 mg, 94%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) δ3.20-3.28 (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.

[0599] Synthetic Example 25: 5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]- N-Methylpyridine-2-carboxamide

[0600]

[0601] Isopropoxytitanium (64.5 mg, 0.23 mmol) was added to 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaloline-6-carboxaldehyde (intermediate 84, 50 mg, 0.23 mmol) and N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 13, 50.0 mg, 0.23 mmol) in THF (3 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (192 mg, 0.91 mmol) was added. The resulting mixture was stirred at room temperature for 2 hours. This step was repeated in another batch, and the two batches were combined for purification. The combined reaction mixture was purified by preparative HPLC (column: XBridge Prep OBD C18 column, 30 × 150 mm 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 20 B to 35 B over 7 min; 254 / 210 nm; RT: 6.38). The fraction containing the desired compound was evaporated to dryness to give 5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide as a white solid (Synthetic Example 25, 4.83 mg, 2.54%). 1 H NMR (400MHz, DMSO-d6) δ2.53-2.59(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); 19 F NMR(376MHz, DMSO-d6)δ-217.70; m / z(ES + [M+H] + =425.

[0602]

[0603] Synthetic Example 26: 6-Fluoro-5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazine- 1-yl]-N-methylpyridine-2-carboxamide

[0604]

[0605] Titanium isopropoxy (90 mg, 0.32 mmol) was added to 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaloline-6-carboxaldehyde (intermediate 84, 70 mg, 0.32 mmol) and 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 23, 76 mg, 0.32 mmol) in THF (3 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (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 the crude product. The crude product was passed through a preparative HPLC system (column: XBridge Prep OBDC18 column, 30×150mm 5µm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 28B to 35B over 8 min; 254 / 210 nm; RT: 7). The fraction containing the desired compound was evaporated to dryness to obtain the crude product. The crude product was further passed through a preparative HPLC system (column: Xselect CSH OBD column, 30×150mm). Purification was performed using a mobile phase A of 5 μm, n; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 5B to 20B over 7 min; 254; 220 nm; RT: 6.83. The fraction containing the desired compound was evaporated to dryness to give 6-fluoro-5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxaloline-6-yl]methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide as a yellow solid (Synthetic Example 26, 3.79 mg, 2.65%). 1 H NMR (400MHz, DMSO-d6) δ2.55-2.60 (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); 19 F NMR(376MHz, DMSO-d6)δ-72.51,-217.71; (ES + [M+H] + =443.

[0606]

[0607] Intermediate 86: Methyl 3-nitro-4-(4, 4 ， 4 -trifluoro-1-methoxy-1-oxobutane-2-ylamino)benzoic acid ester

[0608] DIPEA (8.77 mL, 50.22 mmol) was added to methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and methyl 2-amino-4,4,4-trifluorobutyrate hydrochloride (intermediate 85, 2.2 g, 10.55 mmol) in DMF (20 mL). 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 NH4Cl (100 mL x 1) and brine (100 mL x 4). The organic layer was dried over Na2SO4, filtered, and evaporated to give the desired product, methyl 3-nitro-4-((4,4,4-trifluoro-1-methoxy-1-oxobutane-2-yl)amino)benzoate (intermediate 86, 3.0 g, 85%), as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ2.99-3.28 (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.

[0609] Intermediate 87: Methyl 3-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinoxaline-6-carboxylate Under hydrogen conditions, 20% Pd(OH)₂ / C (0.601 g, 0.86 mmol) was added to methyl 3-nitro-4-((4,4,4-trifluoro-1-methoxy-1-oxobutane-2-yl)amino)benzoate (intermediate 86, 3.0 g, 8.57 mmol) in MeOH (300 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered through diatomaceous earth. The filtrate was evaporated to dryness to give methyl 3-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 87, 2.3 g, 93%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6) δ2.64-2.83 (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.

[0610] Intermediate 88: Methyl 3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinoxaline-6-carboxylate

[0611] DDQ (1.975 g, 8.70 mmol) was added to methyl 3-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 87, 2.28 g, 7.91 mmol) in DCM (100 mL). The resulting mixture was stirred at room temperature for 3 hours. The resulting mixture was removed under reduced pressure to obtain a brown solid. A saturated aqueous solution of NaHCO3 (100 mL) was added to the solid, and the mixture was stirred at room temperature for 1 hour. The precipitate was filtered and washed with another aqueous solution of NaHCO3 (30 mL x 3). The solid was dried under vacuum to give methyl 3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinoxaline-6-carboxylate (intermediate 88, 2.2 g, 97%) as a brown solid. 1 H NMR (400MHz, DMSO-d6) δ3.88-3.98 (5H, m), 7.81 (1H, dd), 7.86-7.94 (2H, m), 12.75 (1H, s); m / z (ES + [M+H] + =287.

[0612] Intermediate 89: 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinoxaline-2(1H)-one

[0613] At 0 °C, a 1 M solution of diisobutylaluminum hydride in THF (20.96 mL, 20.96 mmol) was added in portions to methyl 3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinoxaline-6-carboxylate (intermediate 88, 1.0 g, 3.49 mmol) in THF (100 mL). The resulting mixture was stirred at room temperature for 16 hours. At 0 °C, the reaction mixture was quenched with a saturated aqueous solution of sodium potassium 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 a grayish-white solid, which was purified by rapid silica chromatography (elution gradient of 5% to 55% MeOH in water (0.4% HCO2H)). The pure fraction was evaporated to dryness to give 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinoxaline-2(1H)-one as a yellow solid (intermediate 89,650 mg, 72.2%). 1 H NMR (300MHz, DMSO-d6) δ3.88 (2H, 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.

[0614] Synthetic Example 27: N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxalin-6-yl]methyl [[ ...][[[[[[[[[[[[[][[[[[[[[[[][[[[[[[[][[[[[[[][[[[[[[[][[[[[][[[[[[[][[

[0615]

[0616] 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinoxaline-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)quinoxaline-2(1H)-one (crude product). This product was used directly in the next step without further purification. DIPEA (0.169 mL, 0.97 mmol) was added to 7-(bromomethyl)-3-(2,2,2-trifluoroethyl)quinoxaline-2(1H)-one (crude product) and N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 13, 50 mg, 0.23 mmol) in NMP (2 mL). The resulting mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated and purified by preparative HPLC (column: Sunfire prep C18 column, 30x150, 5 μm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 10B to 25B over 7 min; 254 / 220 nm; RT: 6.57). The fraction containing the desired compound was evaporated to dryness to give N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxaloline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide as a grayish-white solid (Synthetic Example 27, 41.5 mg, 46.6%). 1 H NMR (400MHz, DMSO-d6) δ2.56 (4H, m), 2.78 (3H, d), 3.35 (4H, m), 3.65 (2H, s), 3.88 (2 H, 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); 19 F NMR(376MHz, DMSO-d6)δ-61.53; m / z(ES + [M+H] + =461.

[0617]

[0618] Synthetic Example 28: 6-Fluoro-N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxaline-6- [methyl]piperazine-1-yl]pyridine-2-carboxamide

[0619]

[0620] 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinoxalin-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)quinoxalin-2(1H)-one (crude product). This product was used directly in the next step without further purification. DIPEA (0.203 mL, 1.16 mmol) was added to 7-(bromomethyl)-3-(2,2,2-trifluoroethyl)quinoxalin-2(1H)-one (crude product) and 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide (intermediate 23, 60 mg, 0.25 mmol) in NMP (2 mL). The resulting mixture was stirred at 80 °C for 2 hours. The resulting mixture was purified by preparative HPLC (column: Sunfire prep C18 column, 30x150, 5 μm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 12B to 30B over 7 min; 254 / 220 nm; RT: 6.25). The fraction containing the desired compound was evaporated to dryness to give 6-fluoro-N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxaloline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide as a grayish-white solid (Synthetic Example 28, 49.0 mg, 43.3%). 1 H NMR (400MHz, DMSO-d6) δ2.53-2.63(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); 19 F NMR (376MHz, DMSO-d6) δ-61.54, -72.52; m / z (ES + [M+H] + =479.

[0621] Synthetic Example 29: 6-(difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazine [1-azinyl]-N-methylpyridine-2-carboxamide

[0622]

[0623] At 20 °C, DIPEA (330 μl, 1.89 mmol) was added to a stirred solution of 7-(chloromethyl)-3-ethyl-1,5-naphthidium-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-piperazin-1-yl-pyridin-2-carboxamide 2HCl (intermediate 41, 102 mg, 0.30 mmol) in acetonitrile (2.4 mL), and the resulting solution was stirred at 50 °C for 3 hours. The solvent was removed under vacuum and 50 mL of water was added, followed by 3 mL of saturated NaHCO3. The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 30% MeOH in DCM). The product fraction was concentrated under reduced pressure and dried to give 6-(difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide as a pale yellow solid (Synthetic Example 29, 52.0 mg, 42%). 1 H NMR (500MHz, DMSO-d6) 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.

[0624] Synthetic Example 30: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl 6-(trifluoromethyl)pyridine-2-carboxamide

[0625]

[0626] At 20 °C, DIPEA (330 μl, 1.89 mmol) was added to a stirred solution of 7-(chloromethyl)-3-ethyl-1,5-naphthidium-2(1H)-one HCl (intermediate 17, 70 mg, 0.27 mmol), sodium iodide (4.05 mg, 0.03 mmol), and N-methyl-5-piperazin-1-yl-6-(trifluoromethyl)pyridine-2-carboxamide 2HCl (intermediate 38, 107 mg, 0.30 mmol) in acetonitrile (2.4 mL), and the resulting solution was stirred at 50 °C for 3 hours. The solvent was removed under vacuum and 50 mL of water was added, followed by 3 mL of saturated NaHCO3. The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 30% MeOH in DCM). The product fraction was concentrated under reduced pressure and dried to give 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide as a pale yellow solid (Synthetic Example 30, 58.0 mg, 45%). 1 H NMR (500MHz, DMSO-d6) 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.

[0627] Synthetic Example 31: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N,6- Dimethylpyridine-2-carboxamide

[0628]

[0629] At 20 °C, DIPEA (0.366 mL, 2.10 mmol) was added to a stirred solution of 7-(bromomethyl)-3-ethyl-1,5-naphthidium-2(1H)-one (intermediate 14, 80 mg, 0.30 mmol) and N,6-dimethyl-5-piperazin-1-yl-pyridin-2-carboxamide 2HCl (intermediate 45, 101 mg, 0.33 mmol) in acetonitrile (2 mL), and the resulting solution was stirred at 70 °C for 3 hours. The solvent was removed under vacuum and 50 mL of water was added, followed by 3 mL of saturated NaHCO3. The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by rapid silica chromatography (elution gradient of 0% to 30% MeOH in DCM). The product fraction was concentrated under reduced pressure and dried to give 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N,6-dimethyl-pyridine-2-carboxamide as a pale yellow solid (Synthetic Example 31, 36.0 mg, 29%). 1 H NMR (500MHz, DMSO-d6) 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.

[0630]

[0631] Intermediate 90: tert-butyl 4-[6-(ethylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate

[0632] Ethylamine (7 M, 7.78 mL, 15.56 mmol) in methanol was added to a solution of tert-butyl 4-(6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate (intermediate 15, 500 mg, 1.56 mmol), and the resulting solution was stirred at 50 °C for 18 hours. The solvent was removed under vacuum, and the sample was further dried to give tert-butyl 4-[6-(ethylcarbamoyl)-3-pyridyl]piperazine-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).

[0633] Intermediate 91; N-Ethyl-5-piperazin-1-ylpyridine-2-carboxamide

[0634] HCl (0.473 mL, 15.58 mmol) in dioxane was slowly added to a stirred solution of tert-butyl 4-(6-(ethylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate (intermediate 90, 521 mg, 1.56 mmol) in methanol (10 mL). The resulting solution was stirred at room temperature for 17 hours. The reaction was concentrated and the solid was dried to give N-ethyl-5-piperazine-1-yl-pyridin-2-carboxamide 2HCl (intermediate 91, 421 mg, 88%); m / z (ES + [M+H] + =235.

[0635] Synthetic Example 32: N-ethyl-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazine-1- [Pyridine-2-carboxamide]

[0636]

[0637] At 20 °C, DIPEA (0.320 mL, 1.83 mmol) was added to a stirred solution of 7-(bromomethyl)-3-ethyl-1,5-naphthid-2(1H)-one (intermediate 14, 70 mg, 0.26 mmol) and N-ethyl-5-piperazin-1-yl-pyridin-2-carboxamide 2HCl (intermediate 91, 89 mg, 0.29 mmol) in acetonitrile (2 mL), and the resulting solution was stirred at 70 °C for 3 hours. The solvent was removed under vacuum and 50 mL of water was added, followed by 3 mL of saturated NaHCO3. The mixture was extracted with ethyl acetate. After concentration, the crude product was purified by reversed-phase chromatography (column: Xbrid C18, elution gradient: 20% to 50% MeCN in water (with 0.2% NH4OH)). The pure fraction was evaporated to dryness to give N-ethyl-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide as a white solid (Synthetic Example 32, 28.0 mg, 25%). 1HNMR (500MHz, DMSO-d6) 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.

[0638] Synthesis Example 4 - Form A

[0639] In Synthesis Example 4, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide was obtained as a partially crystalline solid by evaporating a methanol / dichloroethane solution under reduced pressure. The crystalline material thus obtained was characterized as crystalline form A.

[0640] In cases of poor crystallinity, crystalline form A can be obtained by suspending 20 mg of the crude sample in 0.20 ml of water, methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, ethyl acetate, or other solvents for one day at ambient temperature or at 50°C.

[0641] Form A is analyzed using XRPD, and the results are then... Figure 16A The following is a list of examples:

[0642] For XRPD peaks of form A

[0643]

[0644]

[0645] 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°.

[0646] Form A was analyzed using thermal techniques. DSC analysis showed that form A has a melting point that begins at 254°C and reaches its peak at 255°C. A representative DSC trace of form A is illustrated in [image / image / etc.]. Figure 16B middle.

[0647] Bioassay (PARP1 selective inhibitor)

[0648] The inhibitory properties of the PARP1 selective inhibitor compounds described herein can be determined using the following testing procedures.

[0649] PARP fluorescence anisotropic binding assay

[0650] The recombinant full-length 6-HIS-labeled PARP1 protein was diluted to 6 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl, and incubated for four hours with an equal volume of 2 nM fluorescent probe (diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0651] The recombinant full-length PARP2 protein was diluted to 6 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl, and incubated for four hours with an equal volume of 2 nM fluorescent probe (diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0652] The recombinant full-length PARP3 protein was diluted to 100 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl, and incubated for four hours with an equal volume of 6 nM fluorescent probe (diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0653] The recombinant PARP5a binding domain was diluted to 160 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl, and incubated for four hours with an equal volume of 6 nM fluorescent probe (diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0654] The recombinant full-length GST-labeled PARP6 protein was diluted to 160 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl, and incubated for four hours with an equal volume of 6 nM fluorescent probe (diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl). The final DMSO concentration of the probe was maintained below 1% (v / v).

[0655] When a test compound or solvent control is available, use BMG Pherastar. The fluorescence anisotropy of the probe upon protein binding was measured, and the effect of fluorescence on anisotropy was determined. The % inhibition value for different concentrations of the test compound was calculated and fitted to a four-parameter logarithmic plot to determine the IC50. 50 Value. If necessary, compound K i It can be used Analytical Biochemistry The Munson-Rodbard equation, defined on September 1, 1980; 107(1): 220-39, was developed by IC. 50 The value is determined, and it is based on the known K of the probe that binds to the relevant PARP protein. D .

[0656] hERG electrophysiological assay

[0657] Electrophysiological recordings (all performed on RT) were obtained from stably transfected CHO hKv11.1 cells using a Nanion Syncropatch 768PE plate. Test compounds, mediators, or positive controls were added to six compound plates (each at a different concentration) to allow for cumulative doses to the cells (10 mM, 3.167 mM, 1 mM, 0.3167 mM, 0.1 mM, 0.03167 mM). 600 μL of the compound was resuspended in 90 μL of reference buffer (in mM: NaCl 80, KCl 4, CaCl 5, MgCl 1, NMDG C160, D-glucose monohydrate 5, HEPES 10 (pH 7.4 HCl, 298 mOsm)) to final compound concentrations of 39.6 μM, 13.2 μM, 4.4 μM, 1.46 μM, 0.48 μM, and 0.16 μM. For each run of the Nanion Syncropatch 768PE, all liquid additions made using the Syncropatch liquid handling system were used to measure the current amplitude in each well in the presence of extracellular solution (in mM: NaCl 80, KCl 4, CaCl 5, MgCl 1, NMDG Cl 60, D-glucose monohydrate 5, HEPES 10 (pH 7.4 HCl, 298 mOsm)). 40 μL of external solution (in mM: HBPS, CaCl2, MgCl2 (pH 7.4, NaOH)) was added to a 384-well porous dielectric resistance recording chip, and internal buffer (in mM: KF 130, KCl2, MgCl2, EGTA 10, HEPES 10, Escin 25 (all from Sigma-Aldrich; pH 7.2-7.30, 320 mOsm) was perfused to the bottom of the plate. 20 μL of cells maintained at approximately 9°C were dispensed into each well of the chip at a density of 1e6 cells / ml, followed by the addition of 20 μL of sealing enhancer (in mM: NaCl 80, KCl 3, CaCl1 10, HEPES 10, MgCl1 (pH 7.4, NaOH)). 7.4 NaOH) Perform a washing step, leaving a residual volume of 40 μL. Before adding the test compound, dispense 40 μL of reference buffer to establish a stable baseline, followed by a 40 μL removal step after 3 min, repeating this step. Dispense 40 μL of concentration 1 (0.16 μM) compound, record the exposure "in real time" for 3 min, and then remove 40 μL. Repeat this step for 5 additional subsequent compound plates to generate cumulative curve analysis. All data were subtracted for leakage, 2 pulses to -80 mV for 100 ms, with a 100 ms delay.Then, the outward K+ current is induced from the holding potential of -90mV to +60mV through a voltage step, with each pulse transmitted at a frequency of 2Hz and a pulse interval of 15s.

[0658] PARP proliferation assay (4-day compound administration)

[0659] DLD1 cells and BRCA2- / -)DLD1 cells were harvested in complete culture medium at densities of 1.875E4 cells / ml and 6.25E4 cells / ml, respectively. Using Multidrop Combi, they were seeded at 40 μL / well into 384-well plates (Greiner, Kremsmunster, Austria; 781090) and incubated overnight at 37°C and 5% CO2. On Day 1, sytox green (5 μL, 2 μM) and saponin (10 μL, 0.25% stock solution) were added to the Day 0 plates using Multidrop Combi. The plates were sealed with black adhesive caps and incubated at RT for >3 hours. Cell imaging was performed using a Cell Insight (Thermo Fisher Scientific) equipped with 4x objectives. The test compound was added using an Echo 555 incubator and placed in an incubator maintained at 37°C and 5% CO2 for 4 days. On day 5, Sytox Green (5 μL, 2 μM) was added to the plate, followed by saponin (10 μL, 0.25% stock solution). The plate was sealed with a black glue cap and incubated at RT for >3 hours. All cells were read on Cell Insight using a 4x objective. The proliferation rate in Genedata was determined by evaluating the total cell counts in the Cell Insight output for plates on days 0 and 5.

[0660]

[0661]

[0662] Example 1: Production of antibody-drug conjugates

[0663] According to the manufacturing method described in WO 2015 / 115091, and using an anti-HER2 antibody (comprising an 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 a light chain consisting of the amino acid sequence consisting of all amino acid residues 1 to 214 of SEQ ID NO: 2), an anti-HER2 antibody-drug conjugate (DS-8201: druticon-trastuzumab) is produced in which the drug-linker represented by the following formula is conjugated to the anti-HER2 antibody via a thioether bond.

[0664]

[0665] Where A represents the binding site with the antibody. The DAR of the antibody-drug conjugate is 7.7 or 7.8.

[0666] Example 2: Production of PARP1 selective inhibitors

[0667] According to the production method described herein, a PARP1 selective inhibitor having formula (I) is prepared. Specifically, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methylpyridin-2-carboxamide:

[0668]

[0669] It can be prepared according to the synthetic example 4 in this paper (Example 4 of WO 2021 / 013735).

[0670] Example 3: Anti-tumor test

[0671] Antibody-drug conjugate DS-8201 (Derlutecan-Trastuzumab) The combination of PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide).

[0672] method:

[0673] High-throughput combination screening was conducted, in which 27 breast cancer cell lines with different HER2 expression and one gastric cell line with high HER2 expression (Table 1) were treated with a combination of DS-8201 and AZD5305 (a selective inhibitor of PARP1).

[0674] Table 1

[0675]

[0676]

[0677] The readings displayed on the screen are from a 7-day CellTiter-Glo cell viability assay, performed using a 6x6 dose-response matrix (DS-8201 serially diluted 5-point logarithmically and AZD5305 serially diluted 0.5 logarithmically). The maximum concentration of AZD5305 was 3 μM and the maximum concentration of DS-8201 was 10 μg / ml. Furthermore, trastuzumab and eczemab (a DNA topoisomerase I inhibitor) were screened in parallel with AZD5305 to help deconvolve the mechanisms of action of effective combinations. Combination activity was evaluated based on ΔEmax and the Loewe co-score.

[0678] result:

[0679] Results of HER2 high-population cell lines (KPL4, NCI-N87, SKBR3, HCC1954, HCC1569, AU565) were obtained in Figure 12A and 12B And as shown in Table 2, and the results for HER2-low cell lines (MDA-MB-468, MDA-MB-157, HCC1187, T47D, HCC38) are in Figure 13A and 13B And as shown in Table 3.

[0680] Figure 12A and 13A A matrix showing the measured cell viability signals is presented. The X-axis represents drug A (DS-8201), and the Y-axis represents drug B (AZD5305). The values ​​in the boxes represent the ratio of cells treated with drug A+B to cells treated with the DMSO control on day 7. All values ​​are normalized to the cell viability values ​​of day 0. Values ​​between 0 and 100 represent % growth inhibition, and values ​​above 100 represent cell death.

[0681] Figure 12B and 13B The Loewe excess matrix is ​​shown. The values ​​in the boxes represent the excess values ​​calculated by the Loewe additive model.

[0682] Tables 2 and 3 show the HSA synergistic score and the Loewe additive score:

[0683] Table 2

[0684] cell lines KPL4 NCI-N87 SKBR3 HCC1954 HCC1569 AU565 HSA Collaborative Scoring 68.2 70.95 20.33 9.9 38.6 32.77 Loewe Collaborative Scoring 68.2 70.95 20.33 9.9 38.6 32.77

[0685] Table 3

[0686] cell lines MDA-MB-468 MDA-MB-157 HCC1187 T47D HCC38 HSA Collaborative Scoring 11.6 7.04 52.7 12.33 8.9 Loewe Collaborative Scoring 11.6 6.5 52.7 12.33 8.8

[0687] Notice:

[0688] If two compounds act on the same molecular target through the same mechanism, Loewe dose-additivity predicts the expected response. It calculates the additiveness based on the assumption of zero interaction between the compounds, and it is independent of the nature of the dose-response relationship.

[0689] HSA (Highest Single Agent) [Berenbaum 1989] quantifies the higher effect of two single compounds at their respective concentrations. The combined effect is compared to the effect of each single agent at the concentration used in the combination. Exceeding the highest single agent effect indicates synergy. HSA does not require the compounds to affect the same target.

[0690] Excess Matrix: For each well in the concentration matrix, the measured or fitted value is compared to the predicted non-synergistic value for each concentration pair. The predicted value is determined by the selected model. The difference between the predicted and observed values ​​may indicate synergistic or antagonistic effects and is displayed in the excess matrix. The excess matrix values ​​are summarized by combining the excess score and the synergistic score.

[0691] Figure 14 The combined Emax and Loewe scores are shown in different cell lines treated with a combination of DS-8201 and AZD5305. Figure 12A and 12B As shown in Table 2, AZD5305 and DS-8201 act synergistically and also increase cell death in HER2+ mammary and gastric cell lines. Figure 13A and 13B As shown in Table 3, AZD5305 and DS-8201 acted synergistically and also increased cell death in HER2-low breast cancer cell lines at Emax (3 μM AZD5305 and 10 μg / ml DS-8201). Figure 14 It can be seen that, among the eleven cell lines (including HER2 low-dose breast cancer cell lines), treatment with the combination of DS-8201 and AZD5305 resulted in high combined Emax (>100) and high Loewe synergistic score (>5).

[0692] Example 4: Anti-tumor test

[0693] Antibody-drug conjugate DS-8201 (Derlutecan-Trastuzumab) The combination of PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide).

[0694] method:

[0695] Cells grown under their respective conditions were seeded at optimal density in 96-well plates to allow linear proliferation throughout the assay period (4 to 8 days). Immediately after seeding, cells were administered the indicated compounds at a total volume of 200 μL / well and placed in an incubator. Combinations were performed using a 6x8 concentration-response matrix for each combination. At the endpoint, cells were fixed in 2% PFA at room temperature for 20 min. To obtain the initial cell count at the start of treatment, an additional plate was used for each experiment and fixed after cell attachment. Cells were then infiltrated in PBS containing 0.5% Triton-X100 for 10 min. After PBS washing, cells were blocked in PBS containing 5% FBS at RT for 1 h and incubated overnight at 4°C with primary antibody in 5% FBS + 0.05% Triton. After washing three times in PBS, cells were incubated at room temperature with secondary antibody in 5% FBS + 0.05% Triton containing Hoechst 33258 for 1 h. After washing three times in PBS, cells were scanned using a Cellinsight instrument with a 10x objective and 9 fields of view / well. Based on Hoechst staining of the cell nuclei, images were analyzed using Columbus for cell counts. Total cell count / well was used to calculate relative growth in each well compared to the solvent control. To calculate the synergistic score, growth inhibition data were analyzed using Combenefit software (Di Veroli, GY et al., Combenefit: an interactive platform for the analysis and visualization of drug combinations. Bioinformatics, 2016, 32(18): 2866-2868).

[0696] result:

[0697] Results for the HER2-high cell line (KPL4) and two HER2-low cell lines (JIMT1, MDA-MB-468) were presented in... Figure 15A and 15B As shown in the image.

[0698] Figure 15A The cell count matrix is ​​shown, where the Y-axis represents drug A (DS-8201) and the X-axis represents drug B (AZD5305). The values ​​in the boxes represent the percentage of total cell (nuclear) count relative to the DMSO-mediated control.

[0699] Figure 15BThe matrix is ​​shown, where the Y-axis represents drug A (DS-8201) and the X-axis represents drug B (AZD5305), and the values ​​in the boxes represent the calculated Loewe synergistic scores.

[0700] The results in Examples 3 and 4 demonstrate that selective PARP1 inhibition using 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 showed efficacy in five HER2+ breast cancer cell lines and one HER2+ gastric cancer cell line. Figure 12A , 12B (14 and Table 2) and five HER2-low breast cancer cell lines ( Figure 13A , 13B The combined benefits are shown in 14 and Table 3. In Example 4, the combination of AZD5305 and DS-8201 demonstrated synergistic activity in HER2 high (KPL4) and HER2 low (JIMT-1, MDA-MB-468) cell lines. Figure 15A and 15B ).

[0701] Example 5: Anti-tumor test - in vivo

[0702] Antibody-drug conjugate DS-8201 (Derlutecan-Trastuzumab) The combination of PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide).

[0703] method:

[0704] Female nude mice (Charles River) aged 5 to 8 weeks were used and acclimatized to the environment for 7 days before entering the study. 1x10 7 One NCI-N87 tumor cell (1:1 in matrix gel) was subcutaneously implanted into the flank of a female nude mouse. When the tumor reached approximately 150 mm... 3 Tumors of similar size were randomly assigned to treatment groups, as shown in Table 4:

[0705] Table 4

[0706]

[0707] PO: Oral administration (per os)

[0708] QD: Once daily (quaque die)

[0709] The compound dosage for each animal was calculated based on its individual body weight on the day of administration. DS-8201 and AZD5305 were administered on the same day, with DS-8201 administered approximately 1 hour after the AZD5305 PO administration. On day 1, DS-8201 was administered at a single dose of 1 mg / kg or 3 mg / kg, and AZD5305 was administered at a dose of 1 mg / kg QD for 28 days. The duration of administration was 28 days.

[0710] Preparations of DS-8201 at 3 mg / kg and 1 mg / kg

[0711] On the day of administration, DS-8201 dosing solutions were prepared by diluting the DS-8201 stock solution (20.1 mg / ml) to 0.6 mg / ml in 25 mM histidine buffer and 9% sucrose (pH 5.5), and by diluting the 3 mg / kg and 1 mg / kg dosing solutions to 0.2 mg / ml, respectively. Each dosing solution was thoroughly mixed using a pipette before administration via IV in a volume of 5 ml / kg.

[0712] 1 mg / kg of AZD5305 formulation

[0713] To prepare a 1 mg / kg dosing solution, AZD5305 at a concentration of 0.1 mg / ml was prepared, resulting in a PO dosing volume of 10 ml / kg. A total of 49 ml of media was required. 15 μl of 1M HCl was added to the compound and thoroughly mixed by vortexing. 1 ml of sterile water was added to an Eppendorf tube and thoroughly mixed with the compound using a pellet pestle. The compound was sonicated for approximately 5 minutes, and then the contents were transferred to a glass vial. Any residual compound in the Eppendorf tube was rinsed with 1 ml of sterile water and then transferred to a glass vial. The remaining volume of sterile water (37.2 ml; 80% of the total media volume) was added to the glass vial and thoroughly mixed using a magnetic stirrer. The pH of the dosing solution was adjusted to pH 3.74, and then the remaining media (9.772 ml of sterile water) was added to the glass vial and thoroughly mixed using a magnetic stirrer. The dosing solution was protected from light, and small aliquots were administered daily. Store all remaining dosing solution in a refrigerator for up to 7 days. The final dosing matrix for 1 mg / kg AZD5305 is a clear solution.

[0714] Measurement

[0715] Tumor growth inhibition (TGI) is calculated as follows:

[0716] TGI% = {1 - (MTV processed / MTV control)} * 100

[0717] Where MTV = mean tumor volume

[0718] On the day of final measurement, statistical significance was assessed using a one-tailed t-test of (log(relative tumor volume) = log(final volume / initial volume)) compared with a mediator control.

[0719] result

[0720] Tumor volume treated with DS-8201 or AZD5305 alone or in combination with DS-8201 and AZD5305 was within Figure 17 The data is shown below. The data represent the change in tumor volume over time in the treatment group. Figure 17 The dashed line in the table indicates the end of the dosing cycle. For complete dosage and schedule information, refer to Table 4 above. The values ​​shown are mean ± SEM; for mice treated with the vector, n = 10 initially, and for all other treatment groups, n = 8.

[0721] In NCI-N87 xenografts, the TGI response (day 41, TGI%) after treatment with DS-8201 or AZD5305 alone or in combination with DS-8201 and AZD5305 is shown in Table 5:

[0722] Table 5

[0723]

[0724] Not significant

[0725] On day 41 post-treatment, monotherapy with DS-8201 at 3 mg / kg showed a TGI of 62%. At 1 mg / kg, DS-8201 showed a TGI of 25% on day 41 post-treatment. Monotherapy with AZD5305 achieved a TGI of 40% on day 41 post-treatment. Combination therapy with AZD5305 at 1 mg / kg resulted in a TGI of 55% on day 41 post-treatment. Combination therapy with a higher dose (3 mg / kg) of DS-8201 and AZD5305 achieved a significant TGI of 90% on day 41 and showed a better response than either monotherapy.

[0726] The treatment groups were generally well tolerated (two outliers were removed from the study due to weight loss >15%) and the average weight of all treatment groups remained stable throughout the study.

[0727] Example 6:

[0728] In vitro, in HER2-high, HER2-low, and HER2-mutant cell lines, the antibody-drug conjugate DS-8201 (drugtecan-trastuzumab) was effective. Combination administration with the PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthid-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide).

[0729] method:

[0730] High-throughput combinatorial screening was performed, in which four lung cancer cell lines with different HER2 expression (Table 6) and HER2-mutant cancer cell lines (Table 7) were screened using a combination of DS-8201 and AZD5305.

[0731] Table 6

[0732]

[0733] Table 7

[0734]

[0735] The readings displayed on the screen are from a 7-day CellTiter-Glo cell viability assay, performed using a 6x6 dose-response matrix (DS-8201 and AZD5305 were each serially diluted semi-logarithmically for each combination). The maximum concentrations of AZD5305 were 3.33 μM or 10 μM and the maximum concentration of DS-8201 was 100 μg / ml. Combination activity was assessed based on the combination of ΔEmax and Loewe co-scoring.

[0736] result:

[0737] Results for HER2+, HER2-low, and HER2-low / null NSCLC cell lines (HCC1171, NCIH1573, NCIH2170, Calu6) were obtained in Figure 18A , 18B The results for HER2 mutant cell lines (5637) and 18C are shown in Table 8. Figure 19A , 19B And 19C and shown in Table 9.

[0738] Figure 18A and 19AA matrix showing the measured cell viability signals is presented. The X-axis represents drug A (DS-8201), and the Y-axis represents drug B (AZD5305). The values ​​in the boxes represent the ratio of cells treated with drug A+B to cells treated with the DMSO control on day 7. All values ​​are normalized to the cell viability values ​​of day 0. Values ​​between 0 and 100 represent % growth inhibition, and values ​​above 100 represent cell death.

[0739] Figure 18B and 19B The Loewe excess matrix is ​​shown. The values ​​in the boxes represent the excess values ​​calculated by the Loewe additive model.

[0740] Figure 18C and 19C The HSA excess matrix is ​​shown. The values ​​in the boxes represent the excess values ​​calculated by the HSA (Highest Single Agent) model.

[0741] Tables 8 and 9 show the HSA synergistic score and the Loewe additive score:

[0742] Table 8

[0743]

[0744] Table 9

[0745]

[0746] from Figure 18A , 18B As shown in Table 8 and 18C, AZD5305 synergistically interacts 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), in the HER2-low cell line HCC1171 at Emax (0.125 μM AZD5305 and 100 μg / ml DS-8201), and in the HER2-low / ineffective cell line Calu6 at Emax (1.25 μM AZD5305 and 100 μg / ml DS-8201). Combined activity was observed even when the activity of the individual agents was absent or low. Although synergy was observed in the NCIH1573 cell line, no cell death was observed.

[0747] from Figure 19A , 19BAs shown in Figure 19C and Table 9, AZD5305 and DS-8201 act synergistically and also increased cell death in the HER2 mutant cell line 5637 at Emax (1.25 μM AZD5305 and 100 μg / ml DS-8201). Combined activity was observed even when AZD5305 was inactive as a single agent.

[0748] The foregoing written specification is considered sufficient to enable those skilled in the art to practice these embodiments. The foregoing description and examples detail certain embodiments and describe the best mode expected by the inventors. However, it will be understood that, regardless of how detailed the foregoing may be herein, these embodiments can be practiced in many ways and the claims include any equivalents thereof.

[0749] Free text of sequence lists

[0750] SEQ ID NO: 1 - Amino acid sequence of the heavy chain of the anti-HER2 antibody

[0751] SEQ ID NO: 2 - Amino acid sequence of the light chain of the anti-HER2 antibody

[0752] The amino acid sequence of SEQ ID NO: 3-heavy chain CDRH1 [=amino acid residues 26 to 33 of SEQ ID NO: 1]

[0753] The amino acid sequence of SEQ ID NO: 4 - heavy chain CDRH2 [= amino acid residues 51 to 58 of SEQ ID NO: 1]

[0754] The amino acid sequence of SEQ ID NO: 5-heavy chain CDRH3 [=amino acid residues 97 to 109 of SEQ ID NO: 1]

[0755] The amino acid sequence of the light chain CDRL1 in SEQ ID NO: 6 [= amino acid residues 27 to 32 of SEQ ID NO: 2]

[0756] SEQ ID NO: 7 - Amino acid sequence containing the light chain CDRL2 (SAS) [= Amino acid residues 50 to 56 of SEQ ID NO: 2]

[0757] The amino acid sequence of the light chain CDRL3 in SEQ ID NO: 8 [= amino acid residues 89 to 97 of SEQ ID NO: 2]

[0758] SEQ ID NO: 9 - Amino acid sequence of the heavy chain variable region [= Amino acid residues 1 to 120 of SEQ ID NO: 1]

[0759] SEQ ID NO: 10 - Amino acid sequence of the light chain variable region [= Amino acid residues 1 to 107 of SEQ ID NO: 2]

[0760] SEQ ID NO: 11 - Amino acid sequence of the heavy chain [= Amino acid residues 1 to 449 of SEQ ID NO: 1] sequence list <110> AstraZeneca UK Limited and Daiichi Sankyo Co., Ltd. SANKYO COMPANY, LIMITED <120> Combination of antibody-drug conjugates and PARP1 selective inhibitors <130> PN838961WO <150> US63-089859 <151> 2020-10-09 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Heavy chain of humanized anti-HER2 antibody <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 2 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Light chain of humanized anti-HER2 antibody <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDRH1 of the heavy chain of humanized anti-HER2 antibody <400> 3 Gly Phe Asn Ile Lys Asp Thr Tyr 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDRH2 of the heavy chain of humanized anti-HER2 antibody <400> 4 Ile Tyr Pro Thr Asn Gly Tyr Thr 1 5 <210> 5 <211> 13 <212> PRT <213> Artificial sequence <220> <223> CDRH3 of the heavy chain of humanized anti-HER2 antibody <400> 5 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 1 5 10 <210> 6 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDRL1 of the light chain of humanized anti-HER2 antibody <400> 6 Gln Asp Val Asn Thr Ala 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> The CDRL2 sequence containing the light chain of the humanized anti-HER2 antibody. <400> 7 Ser Ala Ser Phe Leu Tyr Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDRL3 of the light chain of humanized anti-HER2 antibody <400> 8 Gln Gln His Tyr Thr Thr Pro Pro Thr 1 5 <210> 9 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Variable region of the heavy chain of humanized anti-HER2 antibody <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Variable region of the light chain of humanized anti-HER2 antibody <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 449 <212> PRT <213> Synthetic Sequence <220> <223> Heavy chain of humanized anti-HER2 antibody <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly

Claims

1. A pharmaceutical product comprising an anti-HER2 antibody-drug conjugate and a selective PARP1 inhibitor for combination administration to treat breast cancer, gastric cancer, lung cancer, or bladder cancer, wherein the anti-HER2 antibody-drug conjugate is an antibody-drug conjugate in which a drug-linker represented by the following formula is conjugated to an anti-HER2 antibody via a thioether bond: Where A represents the binding site with the antibody. The PARP1 selective inhibitor is a compound represented by the following formula (Ia): in R 1 is C 1-4 alkyl, R 2 selected from H, halo, C 1-4 alkyl, and C 1-4 fluoroalkyl, R 3 is H or C 1-4 alkyl, and R 4 is H, Or its pharmaceutically acceptable salt; and The anti-HER2 antibody is an antibody comprising a heavy chain and a light chain. The heavy chain comprises: 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 CDRH3 composed of the amino acid sequence represented by SEQ ID NO:

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

8. The anti-HER2 antibody is administered at a dose of 0.8-12.4 mg / kg per administration; and the PARP1 selective inhibitor is administered orally at a dose of 0.1 mg to 1000 mg.

2. The pharmaceutical product as described in claim 1, wherein, In formula (Ia), R 2 is H or halo.

3. The pharmaceutical product as described in claim 1, wherein, In formula (Ia), R 1 is ethyl, R 2 is selected from H, chloro and fluoro, and R 3 is methyl.

4. The pharmaceutical product of claim 1, wherein the PARP1 selective inhibitor is AZD5305 represented by the following formula: Or its pharmaceutically acceptable salt.

5. The pharmaceutical product according to any one of claims 1 to 4, wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 9, and the light chain comprising a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO:

10.

6. The pharmaceutical product according to any one of claims 1 to 4, wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:

2.

7. The pharmaceutical product according to any one of claims 1 to 4, wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 11, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:

2.

8. The pharmaceutical product according to any one of claims 1 to 4, wherein the anti-HER2 antibody-drug conjugate is represented by the following formula: Wherein 'antibody' refers to an antiHER2 antibody conjugated to a drug-linker via a thioether bond, and n indicates the average number of drug-linker units conjugated to each antibody molecule in the antibody-drug conjugate, wherein n is in the range of 7 to 8.

9. The pharmaceutical product according to any one of claims 1 to 4, wherein the anti-HER2 antibody-drug conjugate is dlutecan-trastuzumab (DS-8201).

10. The pharmaceutical product of any one of claims 1 to 4, wherein the product is a composition comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for simultaneous administration.

11. The pharmaceutical product of any one of claims 1 to 4, wherein the product is a combination formulation comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for sequential or simultaneous administration.

12. The pharmaceutical product of claim 1, wherein the cancer is breast cancer.

13. The pharmaceutical product of claim 12, wherein the breast cancer has an IHC 3+ HER2 status score.

14. The pharmaceutical product of claim 12, wherein the breast cancer is HER2-low expression breast cancer.

15. The pharmaceutical product of claim 12, wherein the breast cancer has an IHC 2+ HER2 status score.

16. The pharmaceutical product of claim 12, wherein the breast cancer has an IHC 1+ HER2 status score.

17. The pharmaceutical product of claim 12, wherein the breast cancer has a HER2 status score of IHC > 0 and < 1+.

18. The pharmaceutical product of claim 12, wherein the breast cancer is triple-negative breast cancer.

19. The pharmaceutical product of claim 1, wherein the cancer is gastric cancer.

20. The pharmaceutical product of claim 1, wherein the cancer is lung cancer.

21. The pharmaceutical product of claim 20, wherein the lung cancer is non-small cell lung cancer.

22. The pharmaceutical product of claim 1, wherein the cancer is bladder cancer.

23. Use of the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor in the preparation of a medicament for use in combination with the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor to treat breast cancer, gastric cancer, lung cancer, or bladder cancer, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of claims 1 to 9.

24. The use as described in claim 23, wherein the cancer is breast cancer.

25. The use as described in claim 24, wherein the breast cancer has an IHC 3+ HER2 status score.

26. The use as described in claim 24, wherein the breast cancer is HER2-low expressing breast cancer.

27. The use as described in claim 24, wherein the breast cancer has an IHC 2+ HER2 status score.

28. The use as described in claim 24, wherein the breast cancer has an IHC 1+ HER2 status score.

29. The use as described in claim 24, wherein the breast cancer has a HER2 status score of IHC > 0 and < 1+.

30. The use as described in claim 24, wherein the breast cancer is triple-negative breast cancer.

31. The use as described in claim 23, wherein the cancer is gastric cancer.

32. The use as described in claim 23, wherein the cancer is lung cancer.

33. The use as described in claim 32, wherein the lung cancer is non-small cell lung cancer.

34. The use as described in claim 23, wherein the cancer is bladder cancer.

35. The use as described in any one of claims 23 to 34, wherein the medicament is a composition comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for simultaneous administration.

36. The use as claimed in any one of claims 23 to 34, wherein the medicament is a combination formulation comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for sequential or simultaneous administration.

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