Antibody-drug conjugates and PARP1 selective inhibitor combinations

Abstract

A pharmaceutical product is provided for the administration of an anti-HER2 antibody-drug conjugate in combination with a PARP1 selective inhibitor. The anti-HER2 antibody-drug conjugate has the following formula: [Formula 1] JPEG2023545096000101.jpg78170 wherein A represents the attachment position to the antibody, is conjugated to the anti-HER2 antibody via a thioether bond. Therapeutic uses and methods are also provided in which the antibody-drug conjugate and a PARP1-selective inhibitor are administered in combination to a subject.

Description

[Technical Field]

[0001] This disclosure relates to a pharmaceutical product for the administration of a specific antibody-drug conjugate having an antitumor agent conjugated to an anti-HER2 antibody via a linker structure in combination with a PARP1 selective inhibitor, and to therapeutic uses and methods of administering the specific antibody-drug conjugate and the PARP1 selective inhibitor in combination to a target. [Background technology]

[0002] The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a crucial role in several cellular processes, including 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 explained, for example, in International Publication No. 2004 / 080976.

[0003] PARP1 and PARP2 are the most widely studied PARPs in terms of their role in DNA damage repair. PARP1 is activated by DNA damage cleavage and catalyzes the attachment of a poly(ADP-ribose) (PAR) chain to a target protein. This post-translational modification, known as PARization, mediates the recruitment of additional DNA repair factors to the DNA damage. Once this recruitment is complete, auto-PARization of PARP triggers the release of bound PARP from the DNA, allowing it to approach other DNA repair proteins and complete the repair. Thus, the binding of PARP to the damage site, its catalytic activity, and its final release from DNA are all crucial steps in cancer cells' response to DNA damage caused by chemotherapy and radiotherapy (Bai P. Biology of poly(ADP-ribose)polymerases: the factotums of cell maintenance. Mol Cell 2015;58:947-58).

[0004] Inhibition of PARP family enzymes has been used as a strategy to selectively kill cancer cells by inactivating complementary DNA repair pathways. Multiple preclinical and clinical studies have demonstrated that tumor cells with adverse mutations in BRCA1 or BRCA2, key tumor suppressor proteins involved in homologous recombination (HR) double-strand break (DSB) repair, are selectively sensitive to small molecule inhibitors of the PARP family of DNA repair enzymes. Such tumors lack homologous recombination repair (HRR) pathways and their survival depends on the function of PARP enzymes. While PARP inhibitor therapy primarily targets BRCA-mutated cancers, PARP inhibitors are also being clinically tested in non-BRCA-mutated tumors, i.e., tumors exhibiting homologous recombination deficiency (HRD) (Turner N, Tutt A, Ashworth A. Hallmarks of ''BRCAness' in sporadic cancers. Nat Rev Cancer 2004;4:814-9).

[0005] PARP inhibitors with improved selectivity for PARP1 are thought to have improved efficacy and reduced toxicity compared to non-selective PARP inhibitors. Furthermore, selective and strong inhibition of PARP1 may lead to PARP1 trapping on DNA, resulting in DNA double-strand breaks (DSBs) due to the disruption of the S-phase replication fork. PARP1-DNA trapping may also be an effective mechanism for selectively killing tumor cells with HRD.

[0006] Antibody-drug conjugates (ADCs), which consist of cytotoxic drugs conjugated to antibodies, can selectively deliver drugs to cancer cells and are therefore expected to kill cancer cells by causing drug accumulation within 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 consists of a HER2-targeted antibody, trastuzumab deruxtecan, a derivative of exatecan (Ogitani Y. et al., Clinical Cancer Research (2016) 22(20), 5097-5108; Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). Trastuzumab deruxtecan (Enhertu®, DS-8201) has shown significant clinical efficacy in HER2-expressing solid tumors, including breast cancer, gastric cancer, colorectal cancer, and non-small cell lung cancer. Significantly, DS-8201 has demonstrated promising activity in HER2-low-expressing tumors in the above indications. There is a need to identify combination partners for DS-8201 to enhance efficacy, improve the duration of the therapeutic response, improve patient tolerability, and / or reduce dose-dependent toxicity.

[0008] Despite the therapeutic potential of antibody-drug conjugates such as trastuzumab deruxtecan and PARP1 inhibitors, no published literature has documented study results demonstrating the superior efficacy of the combined use of antibody-drug conjugates and PARP1 selective inhibitors.

[0009] Therefore, there remains a need for improved therapeutic compositions and methods that can enhance the efficacy of existing cancer treatments, improve the duration of the therapeutic response, improve patient tolerance, and / or reduce dose-dependent toxicity. [Overview of the Initiative]

[0010] The antibody-drug conjugate used in this disclosure (an anti-HER2 antibody-drug conjugate containing a derivative of the topoisomerase I inhibitor exatecan as a component) has been shown to exhibit excellent antitumor effects when administered alone in the treatment of certain cancers such as breast cancer and gastric cancer. Furthermore, PARP1 inhibitors have been shown to exhibit antitumor effects in the treatment of certain cancers. However, there is a need to provide drugs and treatments that can achieve superior antitumor effects in the treatment of cancer, such as enhanced efficacy, prolonged duration of treatment response, and / or dose-dependent reduction of toxicity.

[0011] This disclosure provides a pharmaceutical product that can demonstrate outstanding antitumor efficacy in the treatment of cancer through the administration of an anti-HER2 antibody-drug conjugate combined with a PARP1 selective inhibitor. This disclosure also provides therapeutic uses and methods in which the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are administered in combination to a target.

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

[54] . [1] A pharmaceutical product comprising an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor for combined administration, wherein the anti-HER2 antibody-drug conjugate has the following formula: [ka] A drug-drug conjugate is a pharmaceutical product in which a drug-linker represented by (wherein A represents the attachment site to the antibody) is conjugated to an anti-HER2 antibody via a thioether linkage; [2] The PARP1 selective inhibitor is a compound represented by the following formula (I): [Chemical formula] (In the formula, X and X 2 are each independently selected from N and C(H), X 3 is independently selected from N and C(R 4 )(where R 4 is H or fluoro), R 1 is C 1~4 alkyl or C 1~4 fluoroalkyl, R 2 is independently selected from H, halo, C 1~4 alkyl, and C 1~4 fluoroalkyl, R 3 is H or C 1~4 alkyl), or a pharmaceutically acceptable salt thereof, (provided that when X 1 is N, X 2 is C(H), and X 3 is C(R 4 ), when 2 X 1 is N, X 3 is C(H), and X 4 is C(R when 3 X<​​​​​​​​​​​​​​[5] In equation (I), R 1 A pharmaceutical product according to any one of [2] to [4], wherein is ethyl; [6] PARP1 selective inhibitors are given by the following formula (Ia): [ka] (In the formula, R 1 However, C 1~4 It is alkyl, R 2 However, H, Haro, C 1~4 Alkyl and C 1~4 Selected from fluoroalkyl groups, R 3 However, H or C 1~4 It is alkyl, R 4 Compounds represented by (where H is present), or a pharmaceutically acceptable salt thereof, the pharmaceutical product described in [1]; [7] In equation (Ia), R 2 The pharmaceutical product described in [6] is H or halo; [8] In equation (Ia), R 1 is ethyl, and R 2 R is selected from H, chloro, and fluoro. 3 The pharmaceutical product described in [6], wherein is methyl; [9] The PARP1 selective inhibitor is AZD5305, also known as AZ14170049, and is given by the following formula: [ka] or a pharmaceutically acceptable salt thereof, the pharmaceutical product described in [1];

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

[11] A pharmaceutical product according to any one of [1] to [9], wherein the anti-HER2 antibody comprises a heavy chain containing a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 9 (= amino acid residues 1 to 120 of SEQ ID NO: 1) and a light chain containing a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 10 (= amino acid residues 1 to 107 of SEQ ID NO: 2);

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

[13] A pharmaceutical product according to any one of [1] to [9], wherein the anti-HER2 antibody comprises 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 represented by SEQ ID NO: 2;

[14] The anti-HER2 antibody-drug conjugate is expressed by the following formula: [ka] (In the formula, "antibody" refers to an anti-HER2 antibody conjugated to a drug-linker via a thioether bond, and n refers to the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate, where n is in the range of 7 to 8.) A pharmaceutical product as described in any one of [1] to

[13] , represented by [1];

[15] A pharmaceutical product according to any one of [1] to

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

[16] A pharmaceutical product according to 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 co-administration;

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

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

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

[17] , wherein the product is for the treatment of cancer;

[19] The pharmaceutical product according to

[18] , wherein the cancer is at least one selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, head and neck cancer, gastroesophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, uterine carcinoma, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma;

[20] The drug product described in

[19] , wherein the cancer is breast cancer;

[21] Breast cancer with a HER2 status score of IHC 3+, as described in

[20] ;

[22] The drug product described in

[20] , which is breast cancer with HER2 low expression;

[23] Breast cancer having an IHC 2+ HER2 status score, as described in

[20] ;

[24] Breast cancer with an IHC 1+ HER2 status score, as described in

[20] ;

[25] Pharmacopoeia according to

[20] , in which breast cancer has an IHC > 0 and a HER2 status score < 1+;

[26] The drug product described in

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

[27] The drug product described in

[18] , wherein the cancer is gastric cancer;

[28] The drug product described in

[18] , wherein the cancer is colorectal cancer;

[29] The drug product described in

[18] , wherein the cancer is lung cancer;

[30] The pharmaceutical product described in

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

[31] The drug product described in

[18] , wherein the cancer is pancreatic cancer;

[32] The drug product described in

[18] , wherein the cancer is ovarian cancer;

[33] The drug product described in

[18] , wherein the cancer is prostate cancer;

[34] The drug product described in

[18] , wherein the cancer is kidney cancer;

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

[17] for use in the treatment of cancer;

[36] A pharmaceutical product for use as described in

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

[19] to

[34] ;

[37] Use of an anti-HER2 antibody-drug conjugate or a PARP1 selective inhibitor in the manufacture of a drug for administering in combination an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor for the treatment of cancer, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] ;

[38] Cancer is defined as in any one of

[19] -

[34] , as used in

[37] ;

[39] Use according to

[37] or

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

[40] Use according to

[37] or

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

[41] Anti-HER2 antibody-drug conjugate for use in combination with a PARP1 selective inhibitor in the treatment of cancer, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] ;

[42] Cancer is defined as any one of

[19] -

[34] , an anti-HER2 antibody-drug conjugate for use as described in

[41] ;

[43] An anti-HER2 antibody-drug conjugate for use as described in

[41] or

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

[44] An anti-HER2 antibody-drug conjugate for use as described in

[41] or

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

[45] Anti-HER2 antibody-drug conjugate for use in the treatment of cancer in a subject, wherein the treatment comprises separate, sequential or concurrent administration to the subject of i) the anti-HER2 antibody-drug conjugate and ii) a PARP1 selective inhibitor, wherein the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor are as defined in any one of [1] to

[15] ;

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

[15] ;

[47] A PARP1 selective inhibitor for use as described in

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

[19] to

[34] ;

[48] ​​A PARP1 selective inhibitor for use as described in

[46] or

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

[49] A PARP1 selective inhibitor for use as described in

[46] or

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

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

[15] ;

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

[15] ;

[52] Cancer is defined as any one of

[19] -

[34] , in the manner described in

[51] ;

[53] The method according to

[51] or

[52] , comprising sequentially administering an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor; and

[54] The method according to

[51] or

[52] , wherein the method comprises administering an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor simultaneously. [Effects of the Invention]

[0013] This disclosure provides a pharmaceutical product in which an anti-HER2 antibody-drug conjugate having an antitumor drug conjugated to an anti-HER2 antibody via a linker structure is administered in combination with a PARP1 selective inhibitor, as well as therapeutic uses and methods for administering a specific antibody-drug conjugate and a PARP1 selective inhibitor in combination to a target. Accordingly, this disclosure may provide agents and therapies that can achieve higher antitumor effects in cancer treatment. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 is a diagram showing the amino acid sequence (SEQ ID NO: 1) of the heavy chain of the anti-HER2 antibody. [Figure 2] Figure 2 is a diagram showing the amino acid sequence of the light chain of the anti-HER2 antibody (SEQ ID NO: 2). [Figure 3] Figure 3 is a diagram showing the amino acid sequence of heavy chain CDRH1 (SEQ ID NO: 3 [= amino acid residues 26-33 of SEQ ID NO: 1]). [Figure 4] Figure 4 is a diagram showing the amino acid sequence of the heavy chain CDRH2 (Sequence ID 4 [= amino acid residues 51-58 of Sequence ID 1]). [Figure 5] Figure 5 is a diagram showing the amino acid sequence of heavy chain CDRH3 (SEQ ID NO: 5 [= amino acid residues 97-109 of SEQ ID NO: 1]). [Figure 6] Figure 6 is a diagram showing the amino acid sequence of the light chain CDRL1 (SEQ ID NO: 6 [= amino acid residues 27-32 of SEQ ID NO: 2]). [Figure 7] Figure 7 is a diagram showing the amino acid sequence (SAS) of the light chain CDRL2 (SEQ ID NO: 7 [= amino acid residues 50-56 of SEQ ID NO: 2]). [Figure 8] Figure 8 is a diagram showing the amino acid sequence of the light chain CDRL3 (SEQ ID NO: 8 [= amino acid residues 89-97 of SEQ ID NO: 2]). [Figure 9] Figure 9 is a diagram showing the amino acid sequence of the heavy chain variable region (SEQ ID NO: 9 [= amino acid residues 1-120 of SEQ ID NO: 1]). [Figure 10] Figure 10 is a diagram showing the amino acid sequence of the light chain variable region (SEQ ID NO: 10 [= amino acid residues 1-107 of SEQ ID NO: 2]). [Figure 11] Figure 11 is a diagram showing the amino acid sequence of the heavy chain (SEQ ID NO: 11 [= amino acid residues 1-449 of SEQ ID NO: 1]). [Figures 12A-12B]Figures 12A and 12B show combination matrices obtained using a high-throughput screen in cell lines with high HER2 expression, combining DS-8201 with AZD5305 (AZ14170049; a PARP1 selective inhibitor). [Figures 13A-13B] Figures 13A and 13B show combination matrices obtained using a high-throughput screen combining DS-8201 with AZD5305 in cell lines with low HER2 expression. [Figure 14] Figure 14 is a chart showing the combined Emax and Loewe synergy scores for cell lines treated with DS-8201 in combination with AZD5305. [Figures 15A-15B] Figures 15A and 15B are diagrams showing combination matrices for combining DS-8201 with AZD5305 in cell lines with low or high HER2 expression. [Figures 16A-16B] Figures 16A and 16B show the X-ray diffraction pattern and representative DSC trace of Synthesis Example 4 Form A, respectively. [Figure 17] Figure 17 is a chart showing tumor volume for in vivo treatment with DS-8201 or AZD5305 alone, or with DS-8201 in combination with AZD5305. The dotted line indicates the end of the AZD5305 administration period. [Figure 18A-18C] Figures 18A, 18B, and 18C show combination matrices obtained using high-throughput screening of DS-8201 combined with AZD5305 in NSCLC cell lines with low or high HER2 expression. [Figures 19A-19C] Figures 19A, 19B, and 19C show combination matrices obtained using a high-throughput screen combining DS-8201 with AZD5305 in urinary tract cancer cell lines expressing HER2 mutants. [Modes for carrying out the invention]

[0015] To make this disclosure easier to understand, certain terms are defined first. Additional definitions are provided throughout the detailed description.

[0016] Before describing this disclosure in detail, it should be understood that this disclosure is not limited to any particular composition or method or process and is therefore modifiable. Where used herein and in the appended claims, unless explicitly indicated by the context, the singular forms “one (a),” “one (an),” and “it” refer to multiple subjects. The terms “one (a)” (or “one (an)”) and “one or more” and “at least one” may be used interchangeably herein.

[0017] Furthermore, as used herein, “and / or” should be considered to be a specific disclosure of each of two specific features or components, with or without the presence of other features or components. Accordingly, the term “and / or” as used in phrases such as “A and / or B” herein shall include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, when the term “and / or” is used in phrases such as “A, B and / or C,” it is intended to encompass each of the following embodiments: 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).

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would be commonly understood by those skilled in the art in the field to which this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide those skilled in the art with many general dictionaries of the terms used herein.

[0019] Units, prefixes, and symbols are expressed in the forms recognized by these International System of Units (SI). Numerical ranges include the numbers that define that range.

[0020] Whereever an embodiment is described in this specification with the word “includes,” it should be understood that other embodiments similar in respects are also provided, which are always described using the terms “consisting of” and / or “essentially consisting of.”

[0021] The terms “inhibit,” “block,” and “suppress” are used synonymously herein and refer to any statistically significant reduction in biological activity (including complete blockade). For example, “inhibit” may refer to a reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of biological activity. Cell proliferation can be assayed using techniques accepted in the art to measure the rate of cell division and / or the percentage of cells undergoing cell division within a cell population and / or the rate of cell loss from the cell population due to terminal differentiation or cell death (e.g., thymidine uptake).

[0022] The term "subject" refers to any animal (e.g., mammal) that is a recipient of a particular treatment, including but not limited to humans, non-human primates, and rodents. Typically, the terms "subject" and "patient" are used interchangeably herein in relation to human subjects.

[0023] The term "pharmaceutical product" refers to a formulation that enables the biological activity of the active ingredients, either as a composition containing all of the active ingredients (for simultaneous administration) or as a combination of individual compositions, each containing at least one of the active ingredients but not all of them (combination preparation) (for sequential or simultaneous administration), and that does not contain additional components that are unacceptably toxic to the target to which the product is administered. Such a product may be sterile. "Simultaneous administration" means that the active ingredients are administered simultaneously. "Sequential administration" means that the active ingredients are administered one by one in any order, with time intervals between each administration. The time interval may be, for example, less than 24 hours, preferably less than 6 hours, and more preferably less than 2 hours.

[0024] The terms “to treat,” “to cure,” “to treat,” or “to alleviate,” or “to alleviate,” refer to both (1) therapeutic measures that cure, slow, reduce, and / or halt the progression of the symptoms of a diagnosed condition or disease, and (2) prophylactic or preventative measures that prevent and / or slow the onset of a target condition or disease. Therefore, those who require treatment include those who already have the disease; those who are prone to developing the disease; and those who should be prevented from developing the disease. In some embodiments, if a patient exhibits, for example, complete, partial, or transient remission of a particular type of cancer, the cancer in question is considered “treated” without issue according to the methods of this disclosure.

[0025] The terms “cancer,” “tumor,” “cancerous,” and “malignant” typically 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, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, head and neck cancer, esophagogastric junction adenocarcinoma, biliary tract cancer, Paget’s disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, uterine carcinoma, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma. Cancers include hematological malignancies such as acute myeloid leukemia, multiple myeloma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, and solid tumors such as breast cancer, lung cancer, neuroblastoma, and colon cancer.

[0026] As used herein, the term “cytotoxic agent” is broadly defined to mean a substance that inhibits or prevents the function of cells and / or causes cell destruction (cell death) and / or exerts an antineoplastic / antiproliferative effect. For example, a cytotoxic agent directly or indirectly prevents the development, maturation, or spread of neoplastic tumor cells. The term also includes agents that cause only a cell proliferation inhibitory effect and not a mere cytotoxic effect. The term includes chemotherapeutic agents as defined below, as well as other HER2 antagonists, anti-angiogenic agents, tyrosine kinase inhibitors, protein kinase A inhibitors, members of cytokine families, radioisotopes, and toxins, such as enzymatically active toxins of bacterial, fungal, plant, or animal origin.

[0027] The term "chemotherapeutic agent" is a subset of the term "cytotoxic agent," which includes natural or synthetic compounds.

[0028] The methods or uses of the present disclosure may enable the administration of the compounds of the present disclosure to a patient to promote a positive therapeutic response to cancer. In the context of cancer treatment, the term “positive therapeutic response” refers to an improvement in the symptoms associated with the disease. For example, improvement in the disease may be characterized as a complete response. The term “complete remission” refers to the absence of clinically detectable disease, normalized from any prior test results. Alternatively, improvement in the disease may also be classified as partial remission. “Positive therapeutic response” encompasses a reduction or inhibition of cancer progression and / or duration, a reduction or improvement in cancer severity and / or improvement of one or more of its symptoms resulting from the administration of the compounds of the present disclosure. In specific embodiments, such terms refer to one, two, three or more outcomes following the administration of the compounds of the present disclosure: (1) Stabilization, reduction, or elimination of cancer cell populations; (2) Stabilization or reduction of cancer growth; (3) Reduced cancer formation; (4) Eradication, removal, or control of primary, localized, and / or metastatic cancer; (5) Reduction of mortality rate; (6) Increased duration or rate of disease-free, relapse-free, progression-free and / or overall survival; (7) Increase in response rate, duration of response, or number of patients who respond or go into remission; (8) Decrease in hospitalization rate, (9) Shortening of hospital stay, (10) The size of the tumor is maintained and does not increase or increases by less than 10%, preferably less than 5%, preferably less than 4%, preferably less than 2%, and (11) Increase in the number of patients in remission. (12) A reduction in the number of adjuvant agents (e.g., chemotherapy agents or hormone therapy agents) that would otherwise be needed to treat the cancer.

[0029] Clinical response can be evaluated using the following: screening techniques, e.g., PET, magnetic resonance imaging (MRI) scans, X-ray imaging, computed tomography (CT) scans, flow cytometry or fluorescence-activated cell sorting (FACS) analysis, histological examination, macroscopic findings, and blood chemistry tests, e.g., ELISA, RIA, chromatography, and similar methods, for detecting changes. In addition to these positive therapeutic responses, patients receiving treatment may also experience beneficial effects of improvement in disease-related symptoms.

[0030] The alkyl group and alkyl moiety are linear or branched chains, for example, C 1~8 Alkyl, C 1~6 Alkyl, C 1~4 Alkyl or C 5~6 It is an alkyl group. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl, for example, methyl or n-hexyl.

[0031] A fluoroalkyl group is an alkyl group in which one or more H atoms are substituted with one or more fluoro atoms, for example, C 1~8 Fluoroalkyl, C 1~6 Fluoroalkyl, C 1~4 Fluoroalkyl or C 5~6 These are fluoroalkyl compounds. 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-).

[0032] Halo refers to fluoro, chloro, bromo, and iodine. In one embodiment, halo is fluoro or chloro.

[0033] As used herein, the term “effective dose” means the amount of compound or composition that is sufficient to significantly and positively alter (e.g., provide a positive clinical response) the symptoms and / or conditions to be treated. The effective dose of the active ingredient used in a pharmaceutical product will vary within the knowledge and expertise of the physician, depending on the specific condition being treated, the severity of the condition, the duration of treatment, the nature of the concurrent therapy, the specific active ingredient used, the specific pharmaceutically acceptable excipients / carriers used, and similar factors. Specifically, the effective dose of the compound of formula (I) used in combination with an antibody-drug conjugate for the treatment of cancer is the amount that is sufficient for this combination to alleviate symptoms in warm-blooded animals (e.g., humans), to alleviate cancer symptoms, to slow the progression of cancer, or to reduce the risk of exacerbation in patients with cancer symptoms.

[0034] In this specification, unless otherwise specified, the term “pharmaceutically acceptable” as used herein means a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that has a reasonable risk-benefit ratio.

[0035] Compounds of formula (I) may form stable, pharmaceutically acceptable acidic or basic salts, in which case administration of the compound as a salt may be appropriate. Examples of acid addition salts include acetate, adipine, ascorbate, benzoate, benzenesulfonate, bicarbonate, bisulfate, butyrate, camphorate, camphorsulfonate, choline, citrate, cyclohexylsulfamate, diethylenediamine, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxyethylsulfonate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, and hydroxy Examples include maleate, lactate, malate, maleate, methanesulfonate, meglumine, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate, diphosphate, picrate, pivalate, propionate, quinate, salicylate, stearate, succinate, sulfamate, sulfanilate, sulfate, tartrate, tosylate (p-toluenesulfonate), trifluoroacetate, and undecanoate. Non-toxic, physiologically acceptable salts are preferred, but other salts may be useful, for example, in the isolation or purification of the product.

[0036] These salts can be formed by conventional means, for example, by reacting the free base form of the product with one equivalent or more of a suitable acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, and the solvent is removed in a vacuum, by freeze-drying, or by exchanging the anions of the existing salt with other anions using a suitable ion-exchange resin.

[0037] The compounds of formula (I) may have two or more chiral centers, and naturally, this application encompasses all individual stereoisomers, enantiomers, and diastereoisomers, as well as mixtures thereof. Therefore, naturally, insofar as the compounds of formula (I) can exist as optically active or racemic compounds due to one or more chiral carbon atoms, this application includes in its definition any such optically active or racemic compounds having the above-described activity. This application encompasses all such stereoisomers having the activity defined herein.

[0038] Therefore, throughout this specification, when referring to the compound of formula (I), the term "compound" naturally includes diastereoisomers, mixtures of diastereoisomers, and enantiomers, which are PARP1 inhibitors.

[0039] Naturally, a compound of a particular formula (I) and its pharmaceutically acceptable salts can exist in solvated and non-solvated forms, e.g., hydrated and anhydrous forms. Naturally, the compounds described herein encompass all such solvated forms. For clarification, this includes both the solvated (e.g., hydrated) form of the free form of the compound and the solvated (hydrated) form of the salt of the compound.

[0040] Some compounds of formula (I) may be crystalline and may have two or more crystalline forms. It should be understood that this disclosure encompasses any crystalline or amorphous form, or mixtures thereof, that have PARP1 selective inhibitory activity. Crystalline materials are generally known to be analyzed using conventional techniques such as X-ray powder diffraction (XRPD) analysis and differential scanning calorimetry (DSC).

[0041] Formula (I) described herein shall include all isotopes of its constituent atoms. For example, H (or hydrogen) 1 H, 2 H(D), and 3 It includes all isotopic forms of hydrogen, such as H(T); C is 12 C, 13 C, and14 It contains all isotopic forms of carbon, such as C; O is, 16 O, 17 O and 18 It includes all isotopic forms of oxygen such as O; N is, 13 N, 14 N and 15 It includes all isotopic forms of nitrogen, such as N; F is, 19 F and 18 This includes all isotopic forms of fluorine, such as F; and so on. In one embodiment, the compound of formula (I) contains isotopes of the atoms it comprises in amounts corresponding to their natural abundances. However, in certain cases, it may be desirable to enrich one or more atoms of specific isotopes that normally exist in smaller amounts. For example, 1 H is usually present in amounts exceeding 99.98%, but in one embodiment, the compounds of any of the formulas shown herein are present at one or more positions where H is present. 2 H or 3 H may be enriched. In another embodiment, a compound of any of the formulas shown herein may be a radioisotope, for example, 3 H and 14 When rich in C, the compound may be useful in drug and / or substrate tissue distribution assays. Naturally, this application encompasses all such isotopic forms.

[0042] Description of the Embodiment Preferred modes for carrying out the Disclosure are described below. The embodiments described below are provided only to illustrate examples of typical embodiments of the Disclosure and are not intended to limit the scope of the Disclosure.

[0043] 1. Antibody-drug conjugates The antibody-drug conjugate used in this disclosure is given by the following formula: [ka] This is an antibody-drug conjugate in which a drug-linker represented by (wherein A represents the attachment site to the antibody) is conjugated to an anti-HER2 antibody via a thioether linkage.

[0044] In this disclosure, a substructure consisting of a linker and a drug in an antibody-drug conjugate is referred to as a "drug-linker." The drug-linker is connected to thiol groups (in other words, sulfur atoms of cysteine ​​residues) formed at interchain disulfide bond sites in the antibody (two sites between heavy chains and two sites between heavy and light chains).

[0045] The drug-linker of this disclosure contains exatecan (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]indolidino[1,2-b]quinoline-10,13-dione, (also represented as 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]indolidino[1,2-b]quinoline-10,13(9H,15H)-dione) as a component, which is a topoisomerase I inhibitor. Exatecan is given by the following formula: [ka] It is a camptothecin derivative with antitumor effects, represented by [the formula shown].

[0046] The anti-HER2 antibody-drug conjugate used in this disclosure can also be represented by the following formula. [ka]

[0047] In this specification, a drug-linker is conjugated to an anti-HER2 antibody ("antibody-") via a thioether linkage. The meaning of n is the same as what is called the average number of conjugated drug molecules (DAR; drug-antibody ratio), referring to the average number of units of drug-linker conjugated per antibody molecule.

[0048] After being transferred into cancer cells, the anti-HER2 antibody-drug conjugate used in this disclosure is cleaved at the linker moiety, releasing a compound represented by the following formula. [ka]

[0049] This compound is presumed to be the source of the antitumor activity of the antibody-drug conjugate used in this disclosure and has been confirmed to have a topoisomerase I inhibitory effect (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15;22(20):5097-5108, Epub 2016 Mar 29).

[0050] 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). This bystander effect is exerted throughout the entire process, in which the antibody-drug conjugates used in this disclosure are internalized in cancer cells expressing the target, and subsequently, the released compound exerts an antitumor effect on surrounding cancer cells that do not express the target. This bystander effect is also evident when the anti-HER2 antibody-drug conjugates are used in combination with a PARP1 selective inhibitor as described in this disclosure, resulting in a superior antitumor effect.

[0051] 2. Antibodies in antibody-drug conjugates The anti-HER2 antibody in the antibody-drug conjugate used in this disclosure may be derived from any species, preferably an anti-HER2 antibody derived from humans, rats, mice, or rabbits. If the antibody is derived from a species other than humans, it is preferably chimeric or humanized using well-known techniques. The anti-HER2 antibody may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody.

[0052] The antibody in the antibody-drug conjugate used in this disclosure is preferably an anti-HER2 antibody having properties that enable it to target cancer cells, and more preferably an antibody having, for example, the property of recognizing cancer cells, the property of binding to cancer cells, the property of internalizing within cancer cells, and / or cell-destructive activity against cancer cells.

[0053] The binding activity of anti-HER2 antibodies against cancer cells can be confirmed using flow cytometry. Internalization of antibodies into cancer cells can be confirmed using (1) an assay to visualize antibodies incorporated into cells under a fluorescence microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay to measure the fluorescence intensity of antibodies incorporated into cells using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay using an immunotoxin that binds to the therapeutic antibody and releases toxins to inhibit cell growth when incorporated into cells (Bio Techniques 28: 162-165, January 2000). As the immunotoxin, a recombinant complex protein of the diphtheria toxin catalytic domain and protein G can be used.

[0054] The antitumor activity of anti-HER2 antibodies can be confirmed in vitro by measuring their inhibitory activity on cell growth. For example, cancer cell lines that overexpress HER2 as the antibody's target protein are cultured, and the antibody is added to the culture system at various concentrations to measure its inhibitory activity on lesion formation, colony formation, and globule proliferation. Alternatively, antitumor activity can be confirmed in vivo by administering the antibody to nude mice transplanted with cancer cell lines that express a large amount of the target protein and measuring the changes in the cancer cells.

[0055] Since anti-HER2 antibody-drug conjugates have compounds that exert antitumor effects bound to them, it is preferable, but not essential, that the anti-HER2 antibody itself has antitumor effects. For the purpose of specifically and selectively exerting the cytotoxicity of antitumor compounds against cancer cells, it is important and preferable that the anti-HER2 antibody has the property of being internalized and migrated into tumor cells.

[0056] The anti-HER2 antibodies in the antibody-drug conjugates used in this disclosure can be obtained by procedures known in the art. For example, the antibodies in this disclosure can be obtained using methods commonly practiced in the art, which involve immunizing animals with an antigenic polypeptide, and then recovering and purifying the antibodies produced in vivo. The antigen is not limited to human origin; animals can also be immunized with antigens derived from non-human animals such as mice and rats. In this case, the cross-reactivity of the obtained heterologous antigen with the human antigen can be tested to screen for antibodies applicable to human diseases.

[0057] Alternatively, antibody-producing cells that produce antibodies against an antigen can be fused with myeloma cells according to methods known in the art (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; and Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)) to establish a hybridoma from which monoclonal antibodies can be obtained.

[0058] Antigens can be obtained by genetically engineering host cells to produce genes encoding antigen proteins. Specifically, a vector capable of expressing the antigen gene is prepared and introduced into host cells to induce gene expression. The thus 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.

[0059] The anti-HER2 antibody in the antibody-drug conjugate used in this disclosure is preferably a recombinant antibody obtained by artificial modification for the purpose of reducing heteroantigenicity against humans, such as a chimeric antibody or a humanized antibody, or preferably an antibody derived from humans, i.e., an antibody having only the gene sequence of a human antibody. These antibodies can be produced using known methods.

[0060] Chimeric antibodies include antibodies derived from different species, such as those in which the variable region and constant region of an antibody are conjugated to the constant region of a human antibody (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0061] Examples of humanized antibodies include antibodies obtained by incorporating only the complementarity-determining region (CDR) of a heterologous antibody into a human-derived antibody (Nature (1986) 321, pp. 522-525), antibodies obtained by transplanting a portion of the amino acid residues of the framework of a heterologous antibody and the CDR sequence of the heterologous antibody into a human antibody using the CDR transplantation method (International Publication No. 90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Patent No. 5821337).

[0062] Examples of human antibodies include antibodies produced using human antibody-producing mice that possess human chromosome fragments containing the genes for the heavy and light chains of human antibodies (see Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, p. 722-727, etc.). As an alternative, examples include antibodies obtained by phage display, or antibodies selected from human antibody libraries (see Wormstone, I. et. al., Investigative Ophthalmology & Visual Science. (2002) 43(7), p. 2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1(2), p. 189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109(3), p. 427-431, etc.).

[0063] This disclosure also includes modified variants of the anti-HER2 antibody in the antibody-drug conjugate used herein. These modified variants refer to variants obtained by subjecting the antibody relating to this disclosure to chemical or biological modification. Examples of chemically modified variants include: variants involving the linking of chemical sites to an amino acid backbone; variants involving the linking of chemical sites to N-linked or O-linked carbohydrate chains; and biologically modified variants include: variants obtained by post-translational modification (e.g., N-linked or O-linked glycosylation, N-terminus or C-terminus treatment, deamidation, aspartic acid isomerization, or methionine oxidation); and variants in which a methionine residue is added to the N-terminus by expression in prokaryotic host cells. Furthermore, antibodies labeled to enable detection or isolation of the antibody or antigen relating to this disclosure (e.g., enzyme-labeled antibodies, fluorescently labeled antibodies, and affinity-labeled antibodies) are also included in the meaning of modified variants. Such modified variants of antibodies relating to this disclosure are useful for improving antibody stability and retention in the blood, reducing their antigenicity, detecting or isolating antibodies or antigens, and similar purposes.

[0064] Furthermore, antibody-dependent cytotoxic activity can be enhanced by regulating the modification (glycosylation, defucosylation, etc.) of the glycan linked to the antibody relating to this disclosure. Techniques for regulating the modification of antibody glycans are known, as disclosed in International Publication Nos. 99 / 54342, 00 / 61739, 02 / 31140, 2007 / 133855, and 2013 / 120066. However, this technique is not limited to these. Anti-HER2 antibodies relating to this disclosure also include antibodies in which glycan modification has been regulated.

[0065] It is known that antibodies produced in cultured mammalian cells lack a lysine residue at the carboxyl terminus of the heavy chain (Journal of Chromatography A, 705:129-134 (1995)), and that two amino acid residues (glycine and lysine) are deleted at the carboxyl terminus of the heavy chain of antibodies produced in cultured mammalian cells, with a newly located proline residue at this carboxyl terminus being amidated (Analytical Biochemistry, 360:75-83 (2007)). However, such deletions and modifications of the heavy chain do not affect the antigen-binding affinity or effector function (complement activation, antibody-dependent cytotoxicity, etc.) of this antibody. Accordingly, the anti-HER2 antibodies according to this disclosure include antibodies subjected to such modifications, as well as functional fragments of such antibodies, and include deletion variants in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, variants obtained by amidation of deletion variants (e.g., heavy chains in which a proline residue at the carboxyl terminus is amidated), and similar variants. The types of deletion variants having a deletion at the carboxyl terminus of the heavy chain of the anti-HER2 antibody according to this disclosure are not limited to the above variants, as long as antigen-binding affinity and effector function are preserved. The two heavy chains constituting the antibody according to this disclosure may be one selected from the group consisting of a full-length heavy chain and the deletion variants described above, or a combination of two selected from these. The ratio of the amounts of each deletion variant may be affected by the type of cultured mammalian cells producing the anti-HER2 antibody according to this disclosure and the culture conditions, but an antibody in which one amino acid residue at the carboxyl terminus is deleted in both heavy chains of the antibody according to this disclosure can be exemplified as a preferred example.

[0066] Examples of isotypes of anti-HER2 antibodies relating to this disclosure include, for example, IgG (IgG1, IgG2, IgG3, IgG4), with IgG1 or IgG2 being exemplified as preferred.

[0067] 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 internal distribution activity in HER2-expressing cells upon binding to HER2.

[0068] Examples of anti-HER2 antibodies include trastuzumab (U.S. Patent No. 5821337) and pertuzumab (International Publication No. 01 / 00245), with trastuzumab being a preferred example.

[0069] 3. Manufacturing of antibody-drug conjugates The drug-linker intermediate for use in the manufacture of anti-HER2 antibody-drug conjugates relating to this disclosure is given by the following formula: [ka] It is represented by [this].

[0070] The drug-linker intermediate has the chemical name N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]glycylglycyl-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] It may be expressed as indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide and may be prepared by reference to descriptions in International Publication Nos. 2014 / 057687, 2015 / 098099, 2015 / 115091, 2015 / 155998, and 2019 / 044947, among others.

[0071] The anti-HER2 antibody-drug conjugates used in this disclosure can be prepared by reacting the above-mentioned drug-linker intermediate with an anti-HER2 antibody having a thiol group (also known as a sulfidyl group).

[0072] Anti-HER2 antibodies containing sulfidyl groups can be obtained by methods well known in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, by using 0.3 to 3 molar equivalents of a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for each interchain disulfide in the antibody and reacting it with the antibody in a buffer containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), an anti-HER2 antibody containing sulfidyl groups with interchain disulfides that are partially or completely reduced can be obtained.

[0073] Furthermore, by using 2 to 20 molar equivalents of a drug-linker intermediate per anti-HER2 antibody containing a sulfidyl group, anti-HER2 antibody-drug conjugates can be created in which 2 to 8 drug molecules are conjugated to each antibody molecule.

[0074] The average number of conjugated drug molecules per anti-HER2 antibody molecule in the prepared antibody-drug conjugate can be determined, for example, by a method based on the measurement of UV absorption of the antibody-drug conjugate and its conjugation reaction precursor at two wavelengths, 280 nm and 370 nm (UV method), or by a method based on quantification through HPLC measurement of fragments obtained by treating the antibody-drug conjugate with a reducing agent (HPLC method).

[0075] The calculation of the average number of conjugated drug molecules per antibody molecule in the conjugation of anti-HER2 antibodies with drug-linker intermediates and antibody-drug conjugates can be performed by referring to the descriptions in International Publication Nos. 2014 / 057687, 2015 / 098099, 2015 / 115091, 2015 / 155998, 2017 / 002776, and 2018 / 212136, among others.

[0076] 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 described herein is an anti-HER2 antibody.

[0077] The anti-HER2 antibody preferably comprises a heavy chain containing CDRH1 consisting of amino acid sequences 26-33 of SEQ ID NO: 1, CDRH2 consisting of amino acid sequences 51-58 of SEQ ID NO: 1, and CDRH3 consisting of amino acid sequences 97-109 of SEQ ID NO: 1, and a light chain containing CDRL1 consisting of amino acid sequences 27-32 of SEQ ID NO: 2, CDRL2 consisting of amino acid sequences 50-52 of SEQ ID NO: 2, and CDRL3 consisting of amino acid sequences 89-97 of SEQ ID NO: 2. An antibody comprising an antibody, and more preferably an antibody comprising a heavy chain comprising a heavy chain variable region consisting of amino acid residues 1 to 120 of SEQ ID NO: 1, and a light chain comprising a light chain variable region consisting of amino acid residues 1 to 107 of SEQ ID NO: 2, and even more preferably an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID 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.

[0078] The average number of conjugated drug-linker units per antibody molecule in an anti-HER2 antibody-drug conjugate 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.

[0079] The anti-HER2 antibody-drug conjugates used in this disclosure may be prepared by referring to the descriptions in International Publication No. 2015 / 115091, etc.

[0080] In a preferred embodiment, the anti-HER2 antibody-drug conjugate is trastuzumab deruxtecan (DS-8201).

[0081] 4. PARP1 selective inhibitors In this disclosure, the term “PARP1 selective inhibitor” means a PARP inhibitor that exhibits selectivity for PARP1 over other PARP family members such as PARP2, PARP3, PARP5a, and PARP6, and more preferably selectivity for PARP1 over PARP2, preferably at least 10-fold selectivity for PARP1 over PARP2, and more preferably at least 100-fold selectivity for PARP1 over PARP2. Preferred examples of PARP1 selective inhibitors may include those disclosed herein.

[0082] Examples of PARP1 selective inhibitors that can be used in accordance with this disclosure include azaquinolone compounds of formula (I). The azaquinolone compounds of formula (I) described herein exhibit remarkably high selectivity for PARP1 over other PARP family members such as PARP2, PARP3, PARP5a, and PARP6. Advantageously, the compounds of formula (I) described herein have low hERG activity. It is well known that blockade of cardiac ion channels encoded by the human delayed-rectifier potassium ion channel gene (hERG) is a risk factor in drug discovery and development, and that blockade of hERG can cause safety issues such as cardiac arrhythmias.

[0083] Therefore, in preferred embodiments of the PARP1 selective inhibitor used in this disclosure, the PARP1 selective inhibitor is given by the following formula (I): [ka] (In the formula, X 1 and X 2 However, each is independently selected from N and C(H), X 3is, independently, selected from N and C(R 4 )(where R 4 is H or fluoro), R 1 is C 1~4 alkyl or C 1~4 fluoroalkyl (preferably ethyl), R 2 is, independently, selected from H, halo, C 1~4 alkyl, and C 1~4 fluoroalkyl, R 3 is H or C 1~4 alkyl (preferably C 1~4 alkyl, more preferably methyl)), a compound represented by or a pharmaceutically acceptable salt thereof (provided that when X 1 is N, X 2 is C(H) and X 3 is C(R 4 )), when X 2 is N, X 1 is C(H) and X 3 is C(R 4 )), when X 3 is N, X 1 and X 2 are both C(H)).

[0084] In one embodiment, the PARP1 selective inhibitor used in the present disclosure is a compound of formula (Ia):

Chemical formula

[0085] In another embodiment, the PARP1 selective inhibitor used in the present disclosure is a compound of formula (Ib):

Chemical formula

[0086] In another embodiment, the PARP1 selective inhibitor used in the present disclosure is a compound of formula (Ic):

Chemical formula

[0087] In another embodiment, the PARP1 selective inhibitor is a compound of formula (Ic), where: R 1 However, these are independently selected from ethyl, n-propyl, trifluoromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2-fluoroethyl, and 2,2,2-trifluoroethyl; R 2 However, independently selected from H, methyl, ethyl, trifluoromethyl, difluoromethyl, fluoromethyl, fluoro, and chloro; R 3 is H or methyl, and R 4 H is H.

[0088] In another embodiment, the PARP1 selective inhibitor is a compound of formula (I), or a compound of formula (Ia), (Ib), or (Ic), having selectivity for PARP1 greater than PARP2, preferably at least 10 times selectivity for PARP1 greater than PARP2, and more preferably at least 100 times selectivity for PARP1 greater than PARP2.

[0089] In other embodiments, the PARP1 selective inhibitor used in this disclosure is a compound selected from the following: 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, 6-ethyl-5-[4-[(2-ethyl-3-oxo-4H-quinoline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide, 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Chloro-5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]pyridine-2-carboxamide, 6-Chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]pyridine-2-carboxamide, 6-Fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]pyridine-2-carboxamide, N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, 6-Chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, 6-Fluoro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, 5-[4-[(2-ethyl-7-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Fluoro-5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide, N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxalin-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide, and 6-fluoro-N-methyl-5-(4-((3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinoxalin-6-yl)methyl)piperazin-1-yl)picolylamide, or a pharmaceutically acceptable salt thereof.

[0090] In another embodiment, the PARP1 selective inhibitor used in the present disclosure is a compound selected from the following: 6-(difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthylridine-3-yl)methyl]piperazin-1-yl]-N-methyl-6(trifluoromethyl)pyridine-2-carboxamide, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl]-N,6-dimethyl-pyridine-2-carboxamide, and N-ethyl-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthylridine-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide, or a pharmaceutically acceptable salt thereof.

[0091] In a preferred embodiment, the PARP1 selective inhibitor used in the present disclosure is the compound AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide) represented by the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0092] 5. Combinations of antibody-drug conjugates and PARP1 selective inhibitors In the first combined embodiment of this disclosure, the anti-HER2 antibody-drug conjugate combined with a PARP1 selective inhibitor has a drug-linker of the following formula: [ka] (In the formula, A represents the attachment site to the antibody.) This is an antibody-drug conjugate in which a drug-linker represented by is conjugated to an anti-HER2 antibody via a thioether linkage.

[0093] In another combination embodiment, the anti-HER2 antibody-drug conjugate defined above for the first combination embodiment is given by the following formula (I): [ka] (In the formula, X 1 and X 2 These are independently selected from N and C(H), X 3 These are independently N and C(R 4 )(Here, R 4 (is selected from H or fluoro) R 1 However, C 1~4 Alkyl or C 1~4 It is a fluoroalkyl, R 2 However, independently, H, Haro, C 1~4 Alkyl and C 1~4 Selected from fluoroalkyl groups, R 3 However, H or C 1~4Compounds represented by (which are alkyl groups), Alternatively, it may be combined with a pharmaceutically acceptable salt of a PARP1 selective inhibitor. (however, X 1 When X is N, 2 is C(H) and X 3 is C(R 4 ) and X 2 When X is N, 1 is C(H) and X 3 is C(R 4 ) and X 3 When X is N, 1 and X 2 (All of these are C(H)).

[0094] In another combined embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, in formula (I), R 3 is C 1~4 It is alkyl.

[0095] In another combined embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, in formula (I), R 3 It is methyl.

[0096] In another combined embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, in formula (I), R 1 It is ethyl.

[0097] In another combined embodiment, the anti-HER2 antibody-drug conjugate as defined above is expressed by the following formula (Ia): [ka] (In the formula, R 1 However, C 1~4 It is alkyl, R 2 However, H, Haro, C 1~4 Alkyl and C 1~4 Selected from fluoroalkyl groups, R 3 However, H or C 1~4 It is alkyl, R 4 Compounds represented by (where H is present), Alternatively, it may be combined with a pharmaceutically acceptable salt thereof, such as a PARP1 selective inhibitor.

[0098] In another combined embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, in formula (Ia), R 2 This is H or halo.

[0099] In another combined embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor as defined above, in formula (Ia), R 1 is ethyl, and R 2 R is selected from H, chloro and fluoro, 3 It is methyl.

[0100] In another combined embodiment, the anti-HER2 antibody-drug conjugate as defined above is combined with a PARP1 selective inhibitor, wherein the PARP1 selective inhibitor is given by the following formula: [ka] AZD5305, represented by [formula], or a pharmaceutically acceptable salt thereof.

[0101] In each of the above combination embodiments, the anti-HER2 antibody includes a heavy chain comprising CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 3, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 4, and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain comprising CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 6, CDRL2 consisting of the amino acid sequence consisting of amino acid residues 1-3 of SEQ ID NO: 7, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 8. In each of the above combination embodiments, the anti-HER2 antibody includes a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 9 and a light chain comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 10. In each of the above combination embodiments, the anti-HER2 antibody includes a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 1 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 2. In each of the above combination embodiments, the anti-HER2 antibody includes a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 11 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 2.

[0102] In a particularly preferred combination embodiment of this disclosure, the anti-HER2 antibody-drug conjugate is trastuzumab deruxtecan (DS-8201), and the PARP1 selective inhibitor is given by the following formula: [ka] This is a compound represented by [formula], which is also identified as AZD5305.

[0103] 6. Combination therapy and treatment methods The pharmaceutical product and its therapeutic use and method are described below, in which the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor described herein are administered in combination.

[0104] The pharmaceutical products and therapeutic uses and methods of the present disclosure may be characterized by the inclusion of an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor as active ingredients in different formulations and administered simultaneously or at different times, or by the inclusion of an antibody-drug conjugate and a PARP1 selective inhibitor as active ingredients in a single formulation and administered.

[0105] In the pharmaceutical products and therapeutic methods of this disclosure, a single PARP1 selective inhibitor used in this disclosure 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.

[0106] The pharmaceutical products and therapeutic methods of this disclosure can be used to treat cancer, preferably breast cancer (including triple-negative breast cancer and luminal breast cancer), gastric cancer (also known as gastric adenocarcinoma), colorectal cancer (also known as colon and rectal cancer, including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, head and neck cancer (including salivary gland cancer and pharyngeal cancer), esophagogastric junction adenocarcinoma, biliary tract cancer (including bile duct cancer), Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumors, It can be used to treat at least one cancer selected from the group consisting of cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial carcinoma, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma multiforme, osteosarcoma, sarcoma, and melanoma, and more preferably, 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.

[0107] The presence or absence of the HER2 tumor marker can be determined, for example, by collecting tumor tissue from a cancer patient, preparing formalin-fixed paraffin-embedded (FFPE) test specimens, and subjecting the specimens to tests for gene products (proteins) using methods such as immunohistochemistry (IHC), flow cytometry, or Western blotting, or to tests for gene transcription using methods such as in situ hybridization (ISH), quantitative PCR (q-PCR), or microarray analysis; or by collecting cell-free circulating tumor DNA (ctDNA) from a cancer patient and subjecting the ctDNA to tests using methods such as next-generation sequencing (NGS).

[0108] The pharmaceutical products and therapeutic methods of this disclosure can be used for HER2-expressing cancers, which may be HER2-overexpressing cancers (high or moderate) or HER2-low expressing cancers.

[0109] In this disclosure, the term “HER2-overexpressing cancer” is not particularly limited insofar as it is recognized as such by those skilled in the art. Preferred examples of HER2-overexpressing cancers include cancers that score 3+ for HER2 expression in IHC and cancers that score 2+ for HER2 expression in IHC and are determined to be positive for HER2 expression in in situ hybridization (ISH). In situ hybridization methods in this disclosure include fluorescence in situ hybridization (FISH) and dichromatic in situ hybridization (DISH).

[0110] In this disclosure, the term “HER2-low-expressing cancer” is not particularly limited insofar as it is recognized as such by those skilled in the art. Preferred examples of HER2-low-expressing cancers include cancers that score 2+ for HER2 expression in IHC and are determined to be negative for HER2 expression in in situ hybridization, and cancers that score 1+ for HER2 expression in IHC.

[0111] Methods for scoring the degree of HER2 expression by IHC or determining whether HER2 expression is positive or negative by in situ hybridization are not particularly limited, as long as the methods are known to those skilled in the art. Examples of such methods include those described in the 4th edition of the guidelines for HER2 testing, breast cancer (edited by the Japanese Pathology Board for Optimal Use of HER2 for Breast Cancer).

[0112] Cancer (in particular, cancers related to the treatment of breast cancer) may be HER2-overexpressing (high or moderate) or low-expressing breast cancer, or triple-negative breast cancer, and / or may have an IHC status score of IHC 3+, IHC 2+, IHC 1+, or IHC>0 and <1+.

[0113] The pharmaceutical products and therapeutic methods of this disclosure may be used in mammals, but more preferably in humans.

[0114] The antitumor effects of the pharmaceutical products and therapeutic methods of this disclosure can be confirmed by preparing a model by transplanting cancer cells into test animals and measuring the reduction in tumor volume or the effect of extending life upon application of the pharmaceutical products and therapeutic methods of this disclosure. Next, the effects of the combined use of antibody-drug conjugates and PARP1 selective inhibitors used in this disclosure can be confirmed by comparing them with the antitumor effects of administering the antibody-drug conjugates and PARP1 selective inhibitors used in this disclosure alone.

[0115] The antitumor effects of the pharmaceutical products and therapeutic methods disclosed herein can be confirmed in clinical trials using any of the following evaluation methods: Response Evaluation Criteria in Solid Tumors (RECIST), WHO evaluation method, Macdonald evaluation method, or body weight measurement. These effects can be determined based on indicators 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).

[0116] By using the method described above, it is possible to confirm the superiority of the antitumor effects of the pharmaceutical products and therapeutic methods of this disclosure over existing pharmaceutical products and therapeutic methods related to cancer treatment.

[0117] The pharmaceutical products and therapeutic methods of this disclosure can delay the growth of cancer cells, inhibit their proliferation, and even kill cancer cells. These effects can provide therapeutic benefits by enabling cancer patients to be freed from cancer-induced symptoms, improving their quality of life (QOL), and prolonging their lives. Even if the pharmaceutical products and therapeutic methods of this disclosure do not achieve cancer cell killing, they can achieve longer survival and improve the QOL of cancer patients by inhibiting or controlling the proliferation of cancer cells.

[0118] The pharmaceutical products of this disclosure are expected to exert therapeutic effects when applied to patients as systemic therapy, and also when applied topically to cancerous tissue.

[0119] The pharmaceutical products and therapeutic methods of this disclosure, in another embodiment, offer use as adjuvants to cancer treatment using ionizing radiation or other chemotherapeutic agents. For example, in cancer treatment, the treatment may include administering a therapeutically effective amount of the pharmaceutical product to a subject in need of treatment, either simultaneously with or sequentially with ionizing radiation or other chemotherapeutic agents.

[0120] The pharmaceutical products and therapeutic methods of this disclosure can be used as adjuvant chemotherapy in combination with surgery. The pharmaceutical products of this disclosure may be administered before surgery to reduce tumor size (referred to as neoadjuvant chemotherapy or neoadjuvant therapy) or after surgery to prevent tumor recurrence (referred to as adjuvant chemotherapy or adjuvant therapy).

[0121] In a further embodiment, the pharmaceutical products of this disclosure may be used to treat cancers lacking homologous recombination (HR)-dependent DNA DSB repair activity. The HR-dependent DNA DSB repair pathway repairs DNA double-strand breaks (DSBs) and reforms continuous DNA helices by homologous mechanisms (KKKhanna and SPJackson, Nat. Genet. 27(3):247-254 (2001)). Components of the HR-dependent DNA DSB repair pathway include ATM (NM_000051), RAD51 (NM_002875), RAD51L1 (NM_002877), RAD51C (NM_002876), RAD51L3 (NM_002878), DMC1 (NM_007068), XRCC2 (NM_005431), XRCC3 (NM_005432), and RA Examples of proteins involved in the HR-dependent DNA DSB repair pathway include, but are not limited to, D52 (NM_002879), RAD54L (NM_003579), RAD54B (NM_012415), BRCA1 (NM_007295), BRCA2 (NM_000059), RAD50 (NM_005732), MRE11A (NM_005590), and NBS1 (NM_002485). Other proteins involved in the HR-dependent DNA DSB repair pathway include regulators such as EMSY (Hughes-Davies, et al., Cell, 115, pp523-535). The HR components are also described in Wood, et al., Science, 291, 1284-1289 (2001). Cancers lacking HR-dependent DNA DSB repair may contain or consist of one or more cancer cells that, compared to normal cells, have reduced or absent ability to repair DNA DSBs through the repair pathway. That is, the activity of the HR-dependent DNA DSB repair pathway may be reduced or absent in one or more cancer cells. The activity of one or more components of the HR-dependent DNA DSB repair pathway may be absent in one or more cancer cells of an individual with cancer lacking HR-dependent DNA DSB repair. The 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.

[0122] In some embodiments, cancer cells may have a BRCA1 and / or BRCA2 deficiency phenotype, that is, BRCA1 and / or BRCA2 activity is reduced or absent in cancer cells. Cancer cells with this phenotype may be deficient in BRCA1 and / or BRCA2. That is, the expression and / or activity of BRCA1 and / or BRCA2 may be reduced or absent in cancer cells, for example, by mutations or polymorphisms in the coding nucleic acid, or by amplification, mutation, or polymorphism in genes encoding regulatory factors (e.g., the EMSY gene encoding the BRCA2 regulator) (Hughes-Davies, et al., Cell, 115, 523-535). BRCA1 and BRCA2 are known tumor suppressors whose wild-type alleles are frequently lost in tumors of heterozygous carriers (Jasin M., Oncogene, 21(58), 8981-93 (2002); Tutt, et al., Trends Mol Med., 8(12), 571-6, (2002)). The association between BRCA1 and / or BRCA2 mutations and breast cancer is well-characterized in the art (Radice, PJ, Exp Clin Cancer Res., 21(3 Suppl), 9-12 (2002)). Amplification of the EMSY gene, which encodes a BRCA2 binding factor, is also known to be associated with breast and ovarian cancer. Carriers of mutations in BRCA1 and / or BRCA2 are also at increased risk of certain cancers, including breast, ovarian, pancreatic, prostate, hematological, gastrointestinal, and lung cancers. In some embodiments, individuals are heterozygous for one or more variations (e.g., mutations and polymorphisms) in BRCA1 and / or BRCA2, or their regulators.The detection of diversity in BRCA1 and BRCA2 is well known in the art and is described, for example, in European Patent No. 699754, No. 705903, Neuhausen, SLand Ostrander, EA, Genet. Test, 1, 75-83 (1992); Chappnis, PO and Foulkes, WO, Cancer Treat Res, 107, 29-59 (2002); Janatova M., et al., Neoplasma, 50(4), 246-505 (2003); Jancarkova, N., Ceska Gynekol., 68{1), 11-6 (2003)). The determination of amplification of the BRCA2 binding factor EMSY is described in Hughes-Davies, et al., Cell, 115, 523-535).

[0123] 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) polypeptides.

[0124] The pharmaceutical products of this disclosure can be administered containing at least one pharmaceutically suitable component. The pharmaceutically suitable component may be preferably selected and applied from formulation additives commonly used in the art, depending on the dosage or concentration of the antibody-drug conjugate and PARP1 selective inhibitor used in this disclosure. The anti-HER2 antibody-drug conjugate used in this disclosure can be administered as a pharmaceutical product containing, for example, a buffer such as histidine buffer, a vehicle such as sucrose and trehalose, and a surfactant such as polysorbate 80 and 20. The pharmaceutical product containing the antibody-drug conjugate used in this disclosure may preferably be used as an injectable, more preferably as an aqueous injectable or lyophilized injectable, and even more preferably as a lyophilized injectable. If the pharmaceutical product containing the anti-HER2 antibody-drug conjugate used in this disclosure is an aqueous injectable, the aqueous injectable may preferably be diluted with a suitable diluent and subsequently given as an intravenous injection. Examples of diluents include dextrose solution and physiological saline, preferably dextrose solution, and more preferably 5% dextrose solution. When the pharmaceutical product of this disclosure is a lyophilized injectable preparation, the required amount of the lyophilized injectable preparation, pre-dissolved in water for injection, may be diluted with a suitable diluent and subsequently given as an intravenous injectable preparation. Examples of diluents include dextrose solution and physiological saline, preferably dextrose solution, and more preferably 5% dextrose solution.

[0125] Examples of applicable routes of administration for the pharmaceutical products of this disclosure include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes, with intravenous routes being preferred.

[0126] The anti-HER2 antibody-drug conjugates used in this disclosure can be administered to humans at intervals of 1 to 180 days, preferably at intervals of 1, 2, 3, or 4 weeks, and more preferably at intervals of 3 weeks. The anti-HER2 antibody-drug conjugates used in this disclosure can be administered in doses of approximately 0.001 to 100 mg / kg per dose, preferably at doses of approximately 0.8 to 12.4 mg / kg per dose. For example, the anti-HER2 antibody-drug conjugate can be administered once every 3 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, preferably at doses of 5.4 mg / kg or 6.4 mg / kg once every 3 weeks.

[0127] PARP1 selective inhibitors can be administered in a suitable dose by any preferred route of administration. The size of the dose required for therapeutic treatment of a particular condition will inevitably vary depending on the patient being treated, the route of administration, and the severity of the disease being treated. Further information regarding routes of administration and administration regimens can be found in Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990, Volume 5, Chapter 25.3.

[0128] Compounds of formula (I), or pharmaceutically acceptable salts thereof, are usually administered orally in the form of pharmaceutical formulations containing the active ingredient or a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt, in a pharmaceutically acceptable dosage form. Depending on the disorder to be treated and the patient, the composition may be administered in various doses.

[0129] Pharmaceutical formulations of the compound of formula (I) above can be prepared for oral administration, particularly in the form of tablets or capsules, using techniques aimed at providing drug release specifically targeted to the colon (Patel, MM Expert Opin. Drug Deliv. 2011, 8(10), 1247-1258).

[0130] The pharmaceutical formulations of the compound of formula (I) described above can be conveniently administered in unit dosage form and can be prepared by any of the methods well known in the field of medicine, for example, as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985).

[0131] Pharmaceutical formulations of the compound of formula (I) suitable for oral administration may contain one or more physiologically compatible carriers and / or additives, and may be in solid or liquid form. Tablets and capsules can be prepared using binders, fillers, lubricants and / or surfactants (e.g., sodium lauryl sulfate). Liquid compositions may contain conventional additives such as suspending agents, emulsifiers and / or preservatives. Liquid compositions can be encapsulated in gelatin, for example, to form unit dosage forms. Examples of solid oral dosage forms include tablets, two-piece hard-shell capsules, and soft-elastic gelatin (SEG) capsules. Such two-piece hard-shell capsules can be prepared, for example, by filling a gelatin or hydroxypropyl methylcellulose (HPMC) shell with the compound of formula (I).

[0132] The dry shell formulations of the compound of formula (I) typically contain gelatin at a concentration of about 40% w / w to 60% w / w, a plasticizer at a concentration of about 20% to 30% (such as glycerin, sorbitol, or propylene glycol), and water at a concentration of about 30% to 40%. Other materials such as preservatives, dyes, opacifiers, and flavorings may also be present. Liquid fillers may include solid drugs dissolved, solubilized, or dispersed (with suspending agents such as beeswax, hydrogenated castor oil, or polyethylene glycol 4000), or liquid drugs in vehicles or combinations of vehicles such as mineral oils, vegetable oils, triglycerides, glycols, polyols, and surfactants.

[0133] The preferred daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof in human therapeutic treatment is approximately 0.0001 to 100 mg / kg body weight. Oral formulations are preferred, and in particular, tablets or capsules that can be formulated by methods known to those skilled in the art to provide the active compound in doses ranging from 0.1 mg to 1000 mg are preferred. [Examples]

[0134] This disclosure will be described in detail with reference to the embodiments shown below. However, this disclosure is not limited to these embodiments. Furthermore, it should not be construed as restrictive in any way.

[0135] Synthesis example of a PARP1 selective inhibitor The following synthesis examples 1 to 32 of the PARP1 selective inhibitor are as described in Examples 1 to 32 of International Publication No. 2021 / 013735.

[0136] General experimental conditions 1 ¹H NMR spectra were obtained using a Bruker 300 MHz, 400 MHz, or 500 MHz spectrometer at 27°C unless otherwise specified. Chemical shifts are expressed in parts per million (ppm, in δ units), and the solvent residue is also shown. 1The values ​​are based on H isotopologs (CHCl3: 7.24 ppm; CHDCl2: 5.32 ppm; CD3S(=O)CD2H: 2.49 ppm). Binding constants are given in Hertz (Hz). The splitting pattern represents the apparent multiplicity and is specified as s (singular), d (double), t (tripular), q (quadular), m (multiple), and br s (broad single). 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. Reported molecular ions correspond to [M+H]+ unless otherwise specified; for molecules with multiple isotopic patterns (e.g., Br, Cl), the reported values ​​are obtained at the lowest isotopic mass unless otherwise specified.

[0137] Flash chromatography was performed using a Biotage® SP1® purification system, an ISCO CombiFlash® Rf system, or a Thermo Fisher Gilson system, employing normal-phase silica FLASH+® (40M, 25M, or 12M) or SNAP® KP-Sil cartridges (340, 100, 50, or 10) and an Agela Flash Column silica-CS column in conjunction with a C18 flash column, either straight-phase or standard flash chromatography. In general, all solvents used were commercially available analytical solvents. Anhydrous solvents were those commonly used for the reaction. The phase separator used in the examples was an ISOLUTE® Phase Separator column. The intermediates and examples named below were named using ACD / Name 12.01 from Advanced Chemistry Development, Inc. (ACD / Labs). The starting materials were obtained from commercial sources or prepared through literature channels.

[0138] X-ray powder diffraction (XRPD) analysis XRPD analysis was performed using a Bruker D8 diffractometer, commercially available from Bruker AXS Inc. (Madison, Wisconsin). XRPD spectra were obtained by placing a sample of the material to be analyzed (approximately 10 mg) on ​​a silicon single-crystal wafer mount (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 revolutions per minute (to improve count statistics) and irradiated with X-rays produced by a copper long microfocus tube operated at 40 kV and 40 mA at a wavelength of 1.5406 angstroms (i.e., approximately 1.54 angstroms). The sample was exposed in theta-theta mode over a range of 5 degrees to 40 degrees 2-theta, for 1 second per 0.02 degree 2-theta increment (continuous scan mode). The operation time was approximately 15 minutes on the D8. The XRPD 2θ value can vary within a reasonable range, for example, within ±0.2°, and its XRPD intensity can vary for various reasons, including preferred orientation, when measured on essentially the same crystal morphology. The principle of XRPD is 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, H.P. & Alexander, LE (1974), X-ray Diffraction Procedures, John Wiley and Sons, New York.

[0139] DSC analysis DSC analysis was performed on samples prepared according to standard methods using a Q SERIES® Q1000 DSC calorimeter available from TA INSTRUMENTS® (New Castle, Delaware). 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. Thermal data were analyzed using standard software, e.g., Universal v.4.5A from TA INSTRUMENTS®.

[0140] The following abbreviations are used: AcOH = acetic acid; aq = aqueous solution; BAST = bis(2-methoxyethyl)aminosulfate trifluoride; Boc2O = di-tert-butyl dicarbonate; Boc = t-butyloxycarbonyl; CDCl3 = deuterated chloroform; CD3OD = deuterated methanol; CH3NO2 = nitromethane; DCE = 1,2-dichloroethane; DCM = dichloromethane; DEA = diethylamine; DEAD = diethyl azodicarboxylic acid; Des-Martin periodinane = 1,1,1-tris(acetyloxy)-1,1-di Hydro-1,2-benzoiodoxol-3-(1H)-one; DIPEA = N,N-diisopropylethylamine; DMAP = 2,6-dimethylaminopyridine; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DMSO-d6 = deuterated dimethyl sulfoxide; DPPA = diphenylphosphoradidate; dppf = 1,1'-bis(diphenylphosphinofino)ferrocene; DIAD = (E)-diazene-1,2-diisopropyl dicarboxylic acid; DSC = differential scanning calorimetry; DTAD = (E)-diazene-1 ,2-dicarboxylate di-tert-butyl;ee = enantiomer excess;eq. = equivalent;ESI = electrospray ionization;Et2O = diethyl ether SiO or EA = ethyl acetate;EtOH = ethanol;FA = formic acid;Grubbs catalyst (1,3-dimethylimidazoline-2-ylidene)(tricyclohexylphosphine)ruthenium dichloride;h = time;HATU = (dimethylamino)-N,N-dimethyl(3-oxide-1H-[1,2,3]triazolo[4,5-b]pyridinyl)methaneiminium hexafluoro Phosphate; HCl = hydrochloric acid; H2O2 = hydrogen peroxide; HP = high pressure; IPA = isopropyl alcohol; LC = liquid chromatography; LiClO4 = lithium perchlorate; mmol = millimoles; mCPBA = metachloroperbenzoic acid; MeOH = methanol; min = minute; MeCN or CH3CN = acetonitrile; MeNO2 = nitromethane; MS = mass spectrometry; NMP = N-methyl-2-pyrrolidone; NMR = nuclear magnetic resonance; Pd / C = palladium / carbon; Pd2dba3 = tris(dibenzylideneacetone)dipalladium(0);PdCl2(dppf) = 1,1'-bis(di-tert-butylphosphino)ferrocenepalladium 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-trioxatriphospholinane-2,4,6-trioxide; TBTU = 2-(1H-benzo[d][1,2,3]triazole-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate; TFA = trifluoroacetic acid; THF = tetrahydrofuran; TLC = Thin-layer chromatography; TMS = Trimethylsilyl; Xanthophos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; CBr4 = Carbon tetrabromide; HCl = Hydrochloric acid; HBr = Hydrobromic acid; Cs2CO3 = Cesium carbonate; MgSO4 = Magnesium sulfate; NaHCO3 = Sodium bicarbonate; DDQ = 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone; SOCl2 = Thionyl chloride; DIBAL-H = Diisobutylaluminum hydride; NH4HCO3 = Ammonium bicarbonate; BINAP = 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl.

[0141] Synthesis of starting materials and intermediates [ka] Intermediate 2: 7-bromo-3-ethyl-1H-1,6-naphthyridine-2-one Butyryl chloride (0.143 mL, 1.37 mmol) was added dropwise at 0°C to a stirred solution of 4-amino-6-bromopyridine-3-carbaldehyde (intermediate 1, 250 mg, 1.24 mmol), DIPEA (1.086 mL, 6.22 mmol), and DMAP (30.4 mg, 0.25 mmol) in CH2Cl2 (5 mL). The resulting solution was stirred at rt for 4 hours. More than 2 eq of butyryl chloride was added, and the reaction was continued for a further 24 hours. The reaction product was diluted with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to obtain the crude product. 1.5 mL of MeOH was added, the solid (product) was filtered, and washed with 1 mL of MeOH to obtain 7-bromo-3-ethyl-1H-1,6-naphthyridine-2-one (intermediate 2, 167 mg, 53.1%) as a white solid. ¹H NMR (DMSO-d6): 1.17 (3H, t), 2.45-2.50 (2H, m, overlaps 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.

[0142] Intermediate 3: 3-ethyl-7-vinyl-1H-1,6-naphthyridine-2-one PdCl2(dppf) (37.6 mg, 0.05 mmol) was added to a stirred mixture prepared by mixing 7-bromo-3-ethyl-1H-1,6-naphthyrizin-2-one (intermediate 2, 130 mg, 0.51 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborane (0.105 mL, 0.62 mmol), and K2CO3 (213 mg, 1.54 mmol) in 1,4-dioxane (4 mL) / water (1.333 mL) and stirring. The resulting mixture was stirred at 90°C for 1 hour. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, and concentrated to obtain the crude product. The resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 20% MeOH (in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain 3-ethyl-7-vinyl-1H-1,6-naphthyridine-2-one (intermediate 3, 93 mg, 90%) as a yellow solid. ¹H NMR (DMSO-d6): 1.18 (3H, t), 2.53 (2H, m, overlaps 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.

[0143] Intermediate 4: 3-ethyl-2-oxo-1H-1,6-naphthyridine-7-carbaldehyde To a solution of 3-ethyl-7-vinyl-1H-1,6-naphthyrizin-2-one (intermediate 3, 30 mg, 0.15 mmol), 2,6-lutidine (0.035 mL, 0.30 mmol), and sodium periodate (128 mg, 0.60 mmol) in THF (1 mL) / water (0.200 mL), osmium tetroxide (0.024 mL, 3.00 μmol) in H2O was added and the mixture was stirred overnight at rt. The reaction product was diluted with water, extracted with ethyl acetate, and the filtrate was concentrated to dryness. The resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 15% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 3-ethyl-2-oxo-1H-1,6-naphthyrizine-7-carboaldehyde (intermediate 4, 24.00 mg, 79%) as a pale yellow foam. ¹H NMR (DMSO-d6): 1.20 (3H, t), 2.55-2.62 (2H, m, overlaps 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.

[0144] Intermediate 5: 3-ethyl-7-(hydroxymethyl)-1H-1,6-naphthyridine-2-one Sodium borohydride (61.4 mg, 1.62 mmol) was slowly added at 0°C to a stirred solution of 3-ethyl-2-oxo-1H-1,6-naphthyridine-7-carbaldehyde (intermediate 4, 82 mg, 0.41 mmol) in methanol (2 mL), and the resulting mixture was stirred at room temperature for 1 hour. Methanol was removed under vacuum, and the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 and 35% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 3-ethyl-7-(hydroxymethyl)-1H-1,6-naphthyridine-2-one (intermediate 5, 68.0 mg, 82%) as a pale yellow solid. ¹H NMR (500 MHz, DMSO-d6): 1.18 (3H, t), 2.52-2.55 (2H, m, overlaps 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.

[0145] Intermediate 6: 7-(bromomethyl)-3-ethyl-1H-1,6-naphthyridine-2-one CBr4 (928 mg, 2.80 mmol) was added at 0°C to a stirred solution of 3-ethyl-7-(hydroxymethyl)-1H-1,6-naphthirizine-2-one (intermediate 5, 381 mg, 1.87 mmol) and triphenylphosphine (734 mg, 2.80 mmol) in CH2Cl2 (18.656 ml). The resulting solution was stirred at 0°C for 2 hours. The reaction product was concentrated, and the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 and 15% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 7-(bromomethyl)-3-ethyl-1H-1,6-naphthirizine-2-one (intermediate 6, 386 mg, 77%) as a white solid (containing triphenylphosphine oxide, difficult to separate). This compound was subjected to the next step without further purification. m / z(ES + )[M] + =267.

[0146] Synthesis Example 1: 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] DIPEA (0.059 mL, 0.34 mmol) was added at 20°C to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,6-naphthyllysin-2-one (intermediate 6, 30 mg, 0.11 mmol) and N-methyl-5-piperazine-1-ylpyridine-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 substance was further purified by reverse-phase chromatography (RediSep Rf Gold® C18, 0 to 90% acetonitrile (in water), with 0.1% NH4OH as an additive). The product fraction was concentrated to dryness under reduced pressure to obtain 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 1, 23.60 mg, 51.7%) as a pale yellow solid. ¹H NMR (500MHz, DMSO-d6): 1.18 (3H,br t), 2.54 (2H,m, overlaps with solvent DMSO peak), 2.67 (4H,br s), 2.79 (3H,br d), 3.38 (4H,br s), 3.75 (2H,br s), 7.34 (1H,s), 7.42 (1H,br dd), 7.77-7.88 (2H,m), 8.29 (1H,br d), 8.40 (1H,br d), 8.75 (1H,s), 11.60-12.11 (1H,m); m / z (ES + )[M+H] + =407.

[0147] Synthesis Example 2: 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide [ka] DIPEA (0.082 mL, 0.47 mmol) was added at 20°C to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,6-naphthyridine-2-one (intermediate 6, 25 mg, 0.09 mmol) and 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-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 flash silica chromatography with an eluent gradient of 0 and 20% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Synthesis Example 2, 17.00 mg, 42.8%) as a pale yellow solid. ¹H NMR (500 MHz, DMSO-d6): 1.18 (3H, t), 2.52-2.55 (2H, m, overlaps 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.

[0148] Synthesis Example 3: 6-Chloro-5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] DIPEA (0.082 mL, 0.47 mmol) was added at 20°C to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,6-naphthyridine-2-one (intermediate 6, 25 mg, 0.09 mmol) and 6-chloro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 47, 33.7 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 flash silica chromatography with an eluent gradient of 0 and 20% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 6-chloro-5-[4-[(3-ethyl-2-oxo-1H-1,6-naphthyllysine-7-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 3, 19.20 mg, 46.5%) as a white solid. ¹H NMR (500 MHz, DMSO-d6): 1.18 (3H, t), 2.53 (2H, m, overlaps 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. [ka]

[0149] Intermediate 8: 6-formyl-5-nitropyridine-3-carboxylate ethyl A mixture of ethyl 6-methyl-5-nitropyridine-3-carboxylate (intermediate 7, 10 g, 47.58 mmol) and selenium dioxide (7.92 g, 71.36 mmol) was mixed in 50 mL of 1,4-dioxane and stirred at 110°C for 20 hours. The reaction mixture was cooled to room temperature, filtered through a celite pad, and washed with ethyl acetate. The filtrates were combined and concentrated, and the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 and 70% ethyl acetate (in hexane). The product fraction was concentrated under reduced pressure to obtain ethyl 6-formyl-5-nitropyridine-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.

[0150] Intermediate 9: 6-[(E)-2-ethoxycarbonylbuta-1-enyl]-5-nitropyridine-3-carboxylate ethyl (E / Z isomer mixture) To a stirred solution of sodium hydride (9.63 g, 240.89 mmol) (60% in mineral oil) in anhydrous THF (100 mL), ethyl 2-(diethoxyphosphoryl)butanoate (60.8 g, 240.89 mmol) was added dropwise at 0°C using an addition funnel to obtain a gray mixture. The resulting mixture was stirred at 0°C for 10 minutes, then heated to room temperature over 10 minutes, and stirred at 40°C for 5 minutes. The reaction mixture was cooled to -78°C, and then a solution of ethyl 6-formyl-5-nitropyridine-3-carboxylate (intermediate 8, 22.5 g, 100.37 mmol) dissolved in 100 mL of THF was slowly added to the cooled reaction mixture. The mixture was stopped with a sat.NH4Cl solution and extracted with ethyl acetate. The combined organic layers were dried over sodium Na2SO4, filtered, and concentrated to obtain the crude product. The obtained residue was purified by flash silica chromatography with an eluent gradient of 0 and 50% ethyl acetate (in hexane). The product fraction was concentrated under reduced pressure to obtain 6-[(E)-2-ethoxycarbonylbuta-1-enyl]-5-nitropyridine-3-carboxylate ethyl (intermediate 9, 24.30 g, 75%) as a yellow oily substance (1:1 and mixture of E / Z isomers). 1H NMR(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),4.1 1-4.24(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.

[0151] Intermediate 10: 7-ethyl-6-oxo-7,8-dihydro-5H-1,5-naphthyridine-3-carboxylate ethyl A mixture of 6-[(E)-2-ethoxycarbonylbuta-1-enyl]-5-nitropyridine-3-carboxylate ethyl (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%) was mixed in ethanol (30 mL), degassed, filled with H2 (balloon), and the reaction was stirred overnight at room temperature under an H2 atmosphere. The mixture was filtered through a celite bed, and the celite 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 minutes. The mixture was diluted with ether, the solid was filtered off, washed with diethyl ether, and dried under vacuum to obtain ethyl 7-ethyl-6-oxo-7,8-dihydro-5H-1,5-naphthyridine-3-carboxylate (intermediate 10, 2.260 g, 78%) as a white solid. ¹H NMR (500 MHz, DMSO-d6) 0.94(3H,t), 1.33(3H,t), 1.41-1.51(1H,m), 1.69-1.81(1H,m), 2.41-2.48(1H,m), 2.94(1H,dd), 3.20(1H,dd), 4.35(2H,t), 7.67(1H,d), 8.61(1H,d), 10.32(1H,s); m / z (ES + )[M+H] + =249.

[0152] Intermediate 11: 7-ethyl-6-oxo-5H-1,5-naphthyridine-3-carboxylate ethyl Ethyl 7-ethyl-6-oxo-7,8-dihydro-5H-1,5-naphthiridine-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 hours. The solvent was removed under reduced pressure, and a solution of sat.NaHCO3 was added. The residue was stirred at room temperature for 1 hour. The solid was filtered off and washed with water, followed by 10 ml of diethyl ether. The obtained solid was dried under vacuum to obtain ethyl 7-ethyl-6-oxo-5H-1,5-naphthiridine-3-carboxylate (intermediate 11, 1.738 g, 78%) as a light brown solid. m / z(ES + )[M+H] + =247.

[0153] Intermediate 12: 3-ethyl-7-(hydroxymethyl)-1H-1,5-naphthyridine-2-one 2M lithium aluminum hydride in THF (29.2 mL, 58.47 mmol) was added dropwise to ethyl-6-oxo-5H-1,5-naphthyridine-3-carboxylate ethyl (intermediate 11, 7.2 g, 29.24 mmol) in tetrahydrofuran (150 mL) under nitrogen at 0°C for 45 minutes. The resulting mixture was stirred at 0°C for 1.5 hours. The reaction mixture was stopped by adding 1 M aq HCl (29 mL) dropwise. The reaction mixture was concentrated, and the solid was diluted with water (approximately 150 ml) and 29 ml of 1 M HCl solution to obtain a yellow suspension. The solid was collected by filtration, washed with water and diethyl ether, and dried to obtain the crude product as a yellow solid (contaminated with some inorganic salt). This solid was suspended in a mixture of methanol and DCM (2:1) (400 ml) and heated under reflux. The solid was filtered off. This solid was resuspended in a methanol / DCM mixture, and this procedure was repeated five times to obtain the majority of the product from this mixture. The combined filtrate was then concentrated to approximately 100 ml, the solid was collected by filtration, washed with ether, and dried under vacuum to obtain 3-ethyl-7-(hydroxymethyl)-1H-1,5-naphthyridine-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.

[0154] Synthesis Example 4: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] Thionyl chloride (6.41 mL, 88.14 mmol) was added dropwise at 0°C to a CH2Cl2 (60 mL) suspension of 3-ethyl-7-(hydroxymethyl)-1,5-naphthyridine-2(1H)-one (intermediate 12, 3 g, 14.69 mmol) and N,N-dimethylformamide (0.114 mL, 1.47 mmol). The resulting solution was stirred at room temperature for 6 hours. The mixture was concentrated to dryness to obtain crude 7-(chloromethyl)-3-ethyl-1H-1,5-naphthyridine-2-one (intermediate 17). DIPEA (12.83 mL, 73.45 mmol) was added at 20°C to a stirred solution of 7-(chloromethyl)-3-ethyl-1H-1,5-naphthyridine-2-one (intermediate 17, crude product from the above), potassium iodide (0.488 g, 2.94 mmol), and N-methyl-5-piperazine-1-ylpyridine-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 product was diluted with water, basicized with aq. NaHCO3 solution, and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to obtain the crude product. The resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 15% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 4, 3.93 g, 65.8%) as an off-white, partially crystalline solid. 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, m / z(ES + )[M] + =406. [ka]

[0155] Intermediate 14: 7-(bromomethyl)-3-ethyl-1H-1,5-naphthyridine-2-one CBr4 (219 mg, 0.66 mmol) was added at 0°C to a stirred solution of 3-ethyl-7-(hydroxymethyl)-1H-1,5-naphthirizine-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 product was concentrated under vacuum, and the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 and 15% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 7-(bromomethyl)-3-ethyl-1H-1,5-naphthirizine-2-one (intermediate 14, 84 mg, 71.4%) (contains triphenylphosphine oxide, difficult to separate). This compound was subjected to the next step without further purification. m / z(ES + )[M] + =267.

[0156] Synthesis Example 5: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide [ka] DIPEA (0.082 mL, 0.47 mmol) was added at 20°C to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,5-naphthyrizin-2-one (intermediate 14, 25 mg, 0.09 mmol) and 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-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 flash silica chromatography with an eluent gradient of 0 and 20% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Synthesis 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, overlaps 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.

[0157] Synthesis Example 6: 6-Chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] DIPEA (0.082 mL, 0.47 mmol) was added at 20°C to a stirred solution of 7-(bromomethyl)-3-ethyl-1H-1,5-naphthyridine-2-one (intermediate 14, 25 mg, 0.09 mmol) and 6-chloro-N-methyl-5-piperazine-1-ylpyridine-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 flash silica chromatography with an eluent gradient of 0 and 20% MeOH (in DCM). The product fraction was concentrated under reduced pressure to obtain 6-chloro-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 6, 19.80 mg, 48.0%) as a pale yellow solid. ¹H NMR (500MHz, DMSO-d6): 1.19 (3H,t), 2.55 (2H,m, overlaps 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. [ka]

[0158] Intermediate 16: 5-piperazine-1-ylpyridine-2-carboxylate methyl HCl in dioxane (4.67 mL, 18.67 mmol) was added to a stirred solution of 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate tert-butyl (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 obtain 5-piperazine-1-ylpyridine-2-carboxylate methyl, 2HCl (intermediate 16, 543 mg, 99%) as a pale yellow solid. 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.

[0159] Intermediate 18: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]pyridine-2-carboxylate methyl DIPEA (944 μl, 5.40 mmol) was added at 20°C to a stirred solution of 7-(chloromethyl)-3-ethyl-1H-1,5-naphthyllysine-2-one, HCl (intermediate 17, 200 mg, 0.77 mmol), sodium iodide (11.57 mg, 0.08 mmol), and 5-piperazine-1-ylpyridine-2-carboxylate methyl, 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 mixture was stirred for 10 minutes. The solid was filtered off and washed with 2 mL of water, followed by 1 mL of acetonitrile to obtain 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]pyridine-2-carboxylate methyl (intermediate 18, 158 mg, 50.2%) as an off-white solid. 1H NMR(500MHz,DMSO-d6)1.19(3H,br t),2.54-2.61(6H,m),3.40(4H,br s),3.66(2H,s),3.81(3H,s),7.35(1H,br m / z(ES + )[M+H] + =408.

[0160] Synthesis Example 7: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide [ka] Ammonia in methanol (4 mL, 28.00 mmol) was added to 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxylate methyl (intermediate 18, 60 mg, 0.15 mmol), and the resulting solution was heated at 50°C for 24 hours (in a sealed tube). The reaction mixture was cooled to room temperature, the solid was filtered off, and washed with 2 mL of methanol to obtain 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 7, 88 mg, 90%) as a light brown solid. ¹H NMR (500 MHz, DMSO-d6): 1.19 (3H, t), 2.56 (6H, m, overlaps 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. [ka]

[0161] Intermediate 20: Methyl 5-bromo-6-fluoropyridine-2-carboxylate In an oven-dried flask, methyl 5-bromopyridine-2-carboxylate (intermediate 19, 6 g, 27.77 mmol) was placed 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 obtain a light brown solid. The residue was suspended in a mixture of DCM and sat.NH4Cl solution, and the white suspension was filtered. The organic layer was 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 obtained residue was purified by flash silica chromatography with an eluent gradient of 0 and 25% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain methyl 5-bromo-6-fluoropyridine-2-carboxylate (intermediate 20, 5.98 g, yield 90%). 1 ¹H NMR (500MHz, chloroform-d): 4.01 (3H, s), 7.93 (1H, d), 8.15 (1H, t); m / z (ES + )[M] + =234.

[0162] Intermediate 21: tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate A mixture of tert-butyl piperazine-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) was mixed in 1,4-dioxane (200 mL) and stirred overnight at 80°C 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 flash silica chromatography with an eluent gradient of 0 and 100% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate tert-butyl (intermediate 21, 14.00 g, 88%) as a yellow solid; 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.

[0163] Intermediate 22: tert-butyl 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate tert-butyl (intermediate 21, 12.49 g, 36.80 mmol) was stirred at rt for 24 hours in methylamine (120 mL, 36.80 mmol, 33 wt% in ethanol). (Sealed tube). The solvent was removed under reduced pressure. The residue was dissolved in DCM, filtered through a silica gel bed, and washed with ethyl acetate. The filtrate was concentrated and dried under vacuum to obtain 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 22, 12.45 g, 100%) as a yellow solid. m / z(ES + )[M+H] + =340.

[0164] Intermediate 23: 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide HCl (4M in dioxane, 100 ml, 400.00 mmol) was added at 0°C to a solution of 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 22, 12.5 g, 36.94 mmol) in 1,4-dioxane (50 ml). The reaction mixture was stirred for 5 hours, during which time the temperature was warmed to room temperature to obtain a yellow suspension. The suspension was diluted with ether, the solid was filtered off, and washed with ether. This solid was dried under vacuum to obtain 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 23, 11.42 g, 99%) as a pale yellow solid. 1H NMR(500MHz,DMSO-d6)δ ppm 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 [ka]

[0165] Intermediate 15: tert-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate Ruphos Pd G3 (4.07 g, 4.86 mmol) was added to a degassed mixture of 1,4-dioxane (200 mL), methyl 5-bromopyridine-2-carboxylate (intermediate 19, 30 g, 138.87 mmol), piperazine-1-carboxylate tert-butyl (27.2 g, 145.81 mmol), and Cs2CO3 (90 g, 277.73 mmol). This mixture was stirred at 110°C for 6 hours under an N2 atmosphere. The mixture was then cooled to room temperature, diluted with water, and extracted with ethyl acetate (150 ml x 3). The combined organic layer was dehydrated with anhydrous Na2SO4 and filtered. 3-(diethylenetriamino)propyl-functionalized silica gel (12 g, 1.3 mmol / g loading) was added to the filtrate, and the mixture was stirred at rt for 1 hour. The mixture was filtered, and the filtrate was concentrated to approximately 100 mL. The crystalline yellow solid was filtered off, washed with ether, and dried under vacuum to obtain tert-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 15, 26.36 g, 82 mmol, 59.1%) as a yellow solid. 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.

[0166] Intermediate 24: tert-butyl 4-[6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate Methylamine (100 ml, 1155.26 mmol, 40% in water) was added to a solution of 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate tert-butyl (intermediate 15, 36 g, 112.02 mmol) in MeOH (100 ml), and the reaction mixture was stirred at room temperature for 4 hours to obtain a white suspension. The mixture was concentrated, and the residue was partitioned into a solution of sat.NH4Cl and DCM, and the layers were separated. The aqueous layer was extracted with DCM, the organic layers were combined, washed with brine, dehydrated with Na2SO4, filtered, and concentrated to obtain tert-butyl 4-[6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 24, 35.9 g, 100%) as a yellow solid. ¹H NMR (500 MHz, chloroform-d): 1.49 (9H, s), 3.02 (3H, d), 3.26-3.35 (4H, m), 3.58-3.67 (4H, m), 7.23 (1H, dd), 7.81 (1H, br d), 8.07 (1H, d), 8.16 (1H, d); m / z (ES + )[M+H] + =321.

[0167] Intermediate 13: Carboxylate N-methyl-5-piperazine-1-ylpyridine-2-carboxamide HCl (4M in dioxane, 150 ml, 600.00 mmol) was added to a suspension of 4-[6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 24, 35.9 g, 112.05 mmol) in MeOH (50 ml), and the resulting orange suspension was stirred at rt for 4 hours. Approximately 80 ml of solvent was removed under reduced pressure, and the mixture was diluted with ether and hexane (200 ml, 1 / 1). The solid was recovered by filtration, washed with hexane, dried, and dried under vacuum to obtain N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl salt (intermediate 13, 37.0 g, 100%) as a yellow solid. 1H NMR(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 [ka]

[0168] Intermediate 26: 4-(1-methoxycarbonylpropylamino)-3-nitrobenzoate methyl Sodium bicarbonate (27.0 g, 321.39 mmol) was gradually added to a mixture prepared by mixing methyl 4-fluoro-3-nitrobenzoate (intermediate 25, 16 g, 80.35 mmol), methyl 2-aminobutanoate, and HCl (14.81 g, 96.42 mmol) in THF (100 mL) and stirring. The reaction mixture was stirred overnight at room temperature. Water was added to the reaction to stop the reaction, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated aq. NaHCO3 solution, dried over MgSO4, and concentrated to dryness to obtain methyl 4-(1-methoxycarbonylpropylamino)-3-nitrobenzoate (intermediate 26, 22.86 g, 96%) as a bright yellow solid. 1H NMR(500MHz,DMSO-d6)0.91(3H,t),1.75-2.12(2H,m),3.75(3H,s),3.85(3H,s),4.63-4.82(1H,m),7.15(1H,d),8.00(1H,dd),8.52-8.76(2H,m).

[0169] Intermediate 27: 2-ethyl-3-oxo-2,4-dihydro-1H-quinoxaline-6-carboxylate methyl Pd / C (4.15 g, 3.90 mmol) was gradually added 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 an H2 atmosphere for 30 hours. Methanol was removed under vacuum, 150 mL of DMF was added, and the mixture was stirred for 10 minutes. The palladium catalyst was filtered off with ceilite and washed with 50 mL of DMF (the substance has very low solubility in organic solvents such as MeOH / DCM / siRNA). The filtrate was concentrated in Genevac to obtain methyl 2-ethyl-3-oxo-2,4-dihydro-1H-quinoxaline-6-carboxylate (intermediate 27, 15.80 g, 87%) as a gray solid. The substance was analyzed by NMR and subjected to 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.

[0170] Intermediate 28: 2-ethyl-3-oxo-4H-quinoxaline-6-carboxylate methyl DDQ (15.87 g, 69.92 mmol) was added to a suspension of 2-ethyl-3-oxo-2,4-dihydro-1H-quinoxaline-6-carboxylate methyl (intermediate 27, 15.6 g, 66.59 mmol) in 1,4-dioxane (150 mL). The reaction mixture was stirred overnight at room temperature. This mixture was slowly added to a saturated aq NaHCO3 solution (approximately 500 ml) and stirred at room temperature for 20 minutes. The precipitate was filtered, washed with water (100 ml), and dried to obtain 2-ethyl-3-oxo-4H-quinoxaline-6-carboxylate methyl as an off-white solid (intermediate 28, 11.40 g, 73.7%). 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.

[0171] Intermediate 29: 3-ethyl-7-(hydroxymethyl)-1H-quinoxaline-2-one 2M lithium aluminum hydride 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) under a nitrogen atmosphere at 0°C for 50 minutes. The resulting mixture was stirred at 0°C for 1.5 hours. The reaction mixture was slowly poured into 1M aq HCl (300 mL) at 0°C. 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 layer was concentrated to 300 ml and diluted with ether (200 ml) to obtain a suspension. The solid was collected by filtration, washed with ether, and dried under vacuum to obtain 3-ethyl-7-(hydroxymethyl)-1H-quinoxarin-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.

[0172] Intermediate 30: 7-(bromomethyl)-3-ethyl-1H-quinoxaline-2-one Hydrogen bromide (60 ml, 48 wt% in water) was added to 3-ethyl-7-(hydroxymethyl)-1H-quinoxarin-2-one (intermediate 29, 7.8 g, 38.19 mmol) (producing a clear brown solution). 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 obtain an off-white precipitate. The solid was filtered under vacuum, washed with water, then with diethyl ether, and dried to obtain 7-(bromomethyl)-3-ethyl-1H-quinoxarin-2-one as a beige solid (intermediate 30, 11.10 g, 84%) 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. [ka]

[0173] Intermediate 32: tert-butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate A mixture of piperazine-1-carboxylate tert-butyl (intermediate 31, 2.57 g, 13.80 mmol), 6-bromo-5-fluoropyridine-2-carboxylate methyl (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 hours, and LC-MS showed 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 flash silica chromatography with an eluent gradient of 0 to 50% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate tert-butyl (intermediate 32, 2.200 g, 67.7%) as a pale yellow solid. ¹H NMR (500 MHz, chloroform-d): 1.50 (9H, s), 3.05-3.20 (4H, m), 3.58-3.72 (4H, m), 3.98 (3H, s), 7.31 (1H, d), 8.06 (1H, d); m / z (ES + )[M+H] + =400.

[0174] Intermediate 33: 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl In a sealed pressure vessel, 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate tert-butyl (intermediate 32, 2.2 g, 5.50 mmol) and methylamine (22 ml, 176.72 mmol) (33 wt%) in ethanol were added, and the mixture was heated at 60°C for 2 hours. LC-MS showed complete conversion. The mixture was concentrated, and the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 and 80% siRNA (in hexane). The product fraction was concentrated under reduced pressure to dryness to obtain 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 33, 2.200 g, 100%) as a white solid. ¹H NMR (500 MHz, chloroform-d): 1.50 (9H, s), 3.02 (3H, d), 3.05-3.14 (4H, m), 3.56-3.74 (4H, m), 7.36 (1H, d), 7.68 (1H, br d), 8.11 (1H, d); m / z (ES + )[M+H] + =399.

[0175] Intermediate 34: tert-butyl 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate A mixture of 1,4-dioxane (5 ml), 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 33, 200 mg, 0.50 mmol), tributyl(vinyl) stannane (0.161 ml, 0.55 mmol), and second-generation XPhos Pd cycle (19.71 mg, 0.03 mmol) was stirred under N2 at 100°C for 2.5 hours, and LC-MS showed complete conversion. The mixture was diluted with DCM, washed with sat.NH4Cl, the organic layer was dried (anhydrous Na2SO4), filtered, and concentrated. The resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 80% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 34, 174 mg, 100%) as a white solid. m / z (ES+ ) [M+H] + = 347

[0176] Intermediate 35: 4-[2-ethyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl Pd / C (53.5 mg, 0.05 mmol) (10 wt% dry basis, wet load) was added to a solution of 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 34, 174 mg, 0.50 mmol) in MeOH (6 mL). The flask was degassed and refilled with H2 (balloon). The mixture was stirred overnight at rt. LC-MS showed that the reaction was not complete. Further Pd / C (53.5 mg, 0.05 mmol) was added, and the resulting mixture was stirred at rt under an H2 atmosphere for 5 hours. The mixture was filtered through a celite pad, washed with methanol, and the filtrate was concentrated to dryness to obtain 4-[2-ethyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (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.

[0177] Intermediate 36: 6-ethyl-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide A mixture of 4-[2-ethyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 35, 172 mg, 0.49 mmol) in HCl (4 M in dioxane, 8 ml, 32.00 mmol) was stirred at room temperature rt for 1 hour to obtain a white suspension. The mixture was diluted with ether, the solid was filtered off, and dried under vacuum to obtain 6-ethyl-N-methyl-5-piperazine-1-ylpyridine-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);m / z(ES + )[M+H] + =249.

[0178] Synthesis Example 8: 6-ethyl-5-[4-[(2-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] DIPEA (0.203 mL, 1.17 mmol) was added to a suspension of 6-ethyl-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 36, 75 mg, 0.23 mmol), and 7-(bromomethyl)-3-ethyl-1H-quinoxaline-2-one (intermediate 30, 69.3 mg, 0.23 mmol) in acetonitrile (3 mL). The resulting mixture was stirred at 60°C for 3 hours, and LC-MS showed complete conversion. The mixture was cooled to rt, concentrated, and the residue was purified by Gilson reverse-phase column (eluted from 0 to 95% ACN / water / 0.1% TFA, run for 15 minutes, and collected for 5-9 minutes). The product-containing fraction was concentrated, and the residue was then dissolved in methanol and DCM. 300 mg of tetraalkylammonium carbonite, polymer bonds (40-90 mesh, 2.5-3.5 mmol / g), and the mixture were stirred at rt for 10 minutes. The mixture was then filtered and washed with methanol. The filtrate was concentrated and redissolved in a water / CAN mixture, and this mixture was freeze-dried to obtain 6-ethyl-5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 8, 60.0 mg, 59.1%) as a pale yellow solid. 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 m / z(ES) + )[M+H] + = 435. [ka]

[0179] Intermediate 37: 4-[6-(methylcarbamoyl)-2-(trifluoromethyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl Silver(I) fluoride (176 mg, 1.39 mmol) was mixed in DMF (2 mL) and thoroughly stirred. Trimethyl(trifluoromethyl)silane (0.247 mL, 1.67 mmol) was added at room temperature. The mixture was stirred for 20 minutes, followed by the addition of copper powder (133 mg, 2.09 mmol). After stirring for 4 hours, the reaction mixture turned blue (an indicator of CuCF3 formation). 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate tert-butyl (intermediate 33, 150 mg, 0.38 mmol) was added to the mixture, and the resulting dark mixture was stirred at 90°C for 18 hours to obtain a brown suspension. LC-MS showed sufficient conversion. The mixture was diluted with ethyl acetate, and the solid was filtered off. The filtrate was washed with water, followed by washing with brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The obtained residue was purified by flash silica chromatography with an eluent gradient of 0 to 70% RINKAN (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 4-[6-(methylcarbamoyl)-2-(trifluoromethyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 37, 146 mg, 100%) as a yellow residue. 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.

[0180] Intermediate 38: N-methyl-5-piperazine-1-yl-6-(trifluoromethyl)pyridine-2-carboxamide A mixture of 4-[6-(methylcarbamoyl)-2-(trifluoromethyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 37, 146 mg, 0.38 mmol) in HCl (4 M in dioxane, 8 ml, 32.00 mmol) was stirred at rt for 2 hours. LC-MS showed complete conversion. The solvent was concentrated to 2 ml and the mixture was diluted with ether / hexane (15 ml, 5 / 1). The solid was filtered off and dried under vacuum to obtain N-methyl-5-piperazine-1-yl-6-(trifluoromethyl)pyridine-2-carboxamide, 2HCl (intermediate 38, 127 mg, 94%) as a pink solid. 1H NMR(500MHz,DMSO-d6)2.83(3H,d),3.21(8H,br s),8.09(1H,d),8.23(1H,d),8.46(1H,br d),9.08(2H,br d);m / z(ES + )[M+H] + =289.

[0181] Synthesis Example 9: 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide [ka] DIPEA (0.121 mL, 0.69 mmol) was added to a suspension of N-methyl-5-piperazin-1-yl-6-(trifluoromethyl)pyridine-2-carboxamide, 2HCl (intermediate 38 mg, 50 mg, 0.14 mmol), and 7-(bromomethyl)-3-ethylquinoxaline-2(1H)-one (intermediate 30 mg, 46.2 mg, 0.14 mmol) in acetonitrile (3 mL), and the mixture was stirred at 60°C for 3 hours. The mixture was cooled to rt, concentrated, and the residue was purified by Gilson reverse-phase column elution (eluted with 0 to 95% ACN / water / 0.1% TFA). The product-containing fraction was concentrated at room temperature. The residue was then dissolved in methanol and DCM, followed by the addition of 250 mg of tetraalkylammonium carbonite polymer conjugate (40-90 mesh, 2.5-3.5 mmol / g), and the mixture was stirred at room temperature for 10 minutes. Next, the solid was filtered off, washed with methanol, and the filtrate was concentrated to obtain a solid. This solid was then redissolved in a water / CH3CN mixture and freeze-dried to obtain 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methyl-6-(trifluoromethyl)pyridine-2-carboxamide (Synthesis Example 9, 40.0 mg, 60.9%) as a white solid. 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 m / z(ES),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+H] + = 475. [ka]

[0182] Intermediate 39: tert-butyl 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate Osmium tetroxide (0.050 mL, 6.35 μmol) in H2O was added to a solution of 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 34, 110 mg, 0.32 mmol), 2,6-lutidine (0.074 mL, 0.64 mmol), and sodium periodate (272 mg, 1.27 mmol) in THF (5 mL) / water (1 mL) / tert-butanol (0.304 mL, 3.18 mmol). The mixture was stirred overnight at rt to obtain a yellow suspension. LC-MS and TLC showed complete conversion. The reaction product was diluted with water and extracted with ethyl acetate. After concentration, the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 100% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 39, 100 mg, 90%) as a yellow solid. 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.

[0183] Intermediate 40: tert-butyl 4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 39, 99 mg, 0.28 mmol) in CH2Cl2 (2 mL) was cooled to 0°C, and DAST (0.710 mL, 0.71 mmol) (1 M in DCM) was added. The resulting mixture was stirred at room temperature for 3 hours. TLC and LCMS showed complete conversion. The reaction was stopped by adding sat.NaHCO3 solution dropwise, and the mixture was extracted with DCM. The combined organic matter was dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 100% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 40, 94 mg, 89%) as an off-white solid. 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.

[0184] Intermediate 41: 6-(difluoromethyl)-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide A mixture of 1,4-dioxane (6 ml, 24.00 mmol) and 4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 40, 92 mg, 0.25 mmol) in 4 M HCl was stirred at rt for 1.5 hours to obtain an orange suspension. This mixture was diluted with ether, filtered, and the solid was redissolved in methanol. The mixture was concentrated to dryness to obtain 6-(difluoromethyl)-N-methyl-5-piperazine-1-ylpyridine-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.

[0185] Synthesis Example 10: 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] DIPEA (0.127 mL, 0.73 mmol) was added to a suspension of 6-(difluoromethyl)-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 41 mg, 50 mg, 0.15 mmol), and 7-(bromomethyl)-3-ethylquinoxaline-2(1H)-one (intermediate 30 mg, 48.6 mg, 0.15 mmol) in acetonitrile (3 mL). The resulting mixture was stirred at 60°C for 3 hours, and LC-MS showed complete conversion. The mixture was concentrated, and the residue was purified by Gilson reverse-phase column elution (eluted with 0 to 95% ACN / water / 0.1% TFA). The product-containing fraction was concentrated at room temperature. Next, the residue was dissolved in methanol and DCM, followed by the addition of 250 mg of tetraalkylammonium carbonite polymer bonds (40-90 mesh, 2.5-3.5 mmol / g), and the mixture was stirred at room temperature for 10 minutes. The solid was then filtered off, washed with methanol, and the filtrate was concentrated to obtain a solid. This solid was then redissolved in a water / CH3CN mixture and freeze-dried to obtain 6-(difluoromethyl)-5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (Synthesis 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. [ka]

[0186] Synthesis Example 11: 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] 7-(bromomethyl)-3-ethylquinoxaline-2(1H)-one (intermediate 30, 0.147 g, 0.55 mmol) and N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 13, 0.161 g, 0.55 mmol) were added to a 20 mL vial. The vial was sealed, evacuated, and refilled with N2. Acetonitrile (3 mL) and DIPEA (0.481 mL, 2.75 mmol) were added to the vial and placed in a heating block preheated to 70°C. The reaction mixture was stirred at the same temperature for 2 hours and then cooled to room temperature. The volume of the reaction mixture was reduced to 1 / 3 of its initial volume under vacuum, and NaHCO3 aqueous solution (2 mL) was added. The reaction mixture was stirred for 30 minutes, filtered, and the solid was washed with water (50 mL). The crude product was purified by flash silica chromatography using 0 to 30% MeOH (in DCM) to obtain 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 11, 93.0 mg, 41.6%) as a pale yellow solid. 1 ¹H NMR (500MHz, DMSO-d6): 1.22 (3H,t), 2.52-2.60 (4H,m), 2.73-2.85 (5H,m), 3.30 (4H,m, overlapping with water peak), 3.62 (2H,s), 7.22-7.31 (2H,m), 7.39 (1H,dd), 7.69 (1H,d), 7.83 (1H,d), 8.23-8.31 (1H,m), 8.39 (1H,br d), 12.13-12.36 (1H,m); m / z (ES + )[M+H] + =407. [ka]

[0187] Synthesis Example 12: 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide [ka] 7-(bromomethyl)-3-ethylquinoxaline-2(1H)-one (intermediate 30, 150 mg, 0.56 mmol) was added to 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-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 × 150 mm; mobile phase A: water (10 MMOL / L NH4HCO3, 0.1% NH3·H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: from 28% B to 38% B over 8 minutes; 254; 220 nm; RT: 8.02 min). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Synthesis Example 12, 9 mg, 42.9%) as a white solid. 1 H NMR(400MHz,CD3OD)δ 1.33(3H,t),2.65-2.72(4H,m),2.87-2.95(5H,m),3.26-3.30(4H,m),3 .71(2H,s),7.33-7.41(2H,m),7.52(1H,dd),7.76(1H,d),7.90(1H,dd); 19 F NMR(376MHz,CD3OD)δ -73.40;m / z(ES + )[M+H] + = 425. [ka]

[0188] Intermediate 43: 5-bromo-N,6-dimethylpicolinamide A 2M solution of methylamine in THF (20 mL, 40.00 mmol) was added to methyl 5-bromo-6-methyl picolinate (intermediate 42, 2.0 g, 8.69 mmol), and the resulting mixture was stirred at 80°C for 18 hours. The solvent was removed by distillation under reduced pressure. The crude product was purified by reverse-phase chromatography using an eluent gradient of 5-80% MeOH in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 5-bromo-N,6-dimethylpicolinamide (intermediate 43, 1.5 g, 75%) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 2.65(3H,s),2.82(3H,d),7.75(1H,d),8.17(1H,d),8.57 - 8.76(1H,m);m / z(ES + )[M+H] += 229.

[0189] Intermediate 44: tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridine-3-yl)piperazine-1-carboxylate 5-Bromo-N,6-dimethylpicolinamide (intermediate 43, 1.0 g, 4.37 mmol) was added under nitrogen to toluene (20 mL) 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). The resulting mixture was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by reverse-phase chromatography using an eluent gradient of 5-30% MeOH in water (0.4% HCO2H). The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridine-3-yl)piperazine-1-carboxylate (intermediate 44, 1.2 g, 82%) as a brown solid. 1 H NMR(300MHz,CD3OD)δ 1.50(9H,s),2.58(3H,s),2.92 - 3.00(7H,m),3.62(4H,m),7.50(1H,d),7.88(1H,d);m / z(ES + )[M+H] + =335.

[0190] Intermediate 45: N,6-dimethyl-5-(piperazine-1-yl)picolinamide Tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridine-3-yl)piperazine-1-carboxylate (intermediate 44, 1.18 g, 3.53 mmol) was added to a 4 M solution of HCl 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 obtain N,6-dimethyl-5-(piperazine-1-yl)picolinamide (intermediate 45, 0.77 g, 81%) as a yellow solid. 1 H NMR(300MHz,CD3OD)δ 2.86(3H,s),3.02(3H,s),3.42 - 3.54(8H,m),8.29(2H,d);m / z(ES + )[M+H] + =235.

[0191] Synthesis Example 13: 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide [ka] 7-(bromomethyl)-3-ethylquinoxaline-2(1H)-one (intermediate 30, 100 mg, 0.37 mmol) was added to N,6-dimethyl-5-(piperazin-1-yl)picolinamide (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 × 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 minutes; 254; 220 nm; RT: 6.43 min). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Synthesis Example 13, 68.7 mg, 43.6%) as an off-white solid. 1 H NMR(400MHz,CD3OD)δ 1.33(3H,t),2.55(3H,s),2.71(4H,s),2.87 - 2.99(5H,m),3.05(4H,t),3.73(2H,s),7.35(1H,s),7.38(1H,d),7.49(1H,d),7.77(1H,d),7.87(1H,d);m / z(ES+)[M+H] + =421. [ka]

[0192] Intermediate 47: Methyl 6-chloro-5-(piperazin-1-yl) picolinate Piperazine (1.0 g, 11.61 mmol) was added to methyl 6-chloro-5-fluoropicolinate (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 by distillation under reduced pressure. The crude product was purified by reverse-phase chromatography using an eluent gradient of 5-60% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain methyl 6-chloro-5-(piperazin-1-yl)picolinate (intermediate 47, 1.28 g, 95%) as a red oil. 1 1H NMR (400MHz, 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.

[0193] Intermediate 48: 6-Chloro-N-methyl-5-(piperazine-1-yl)picolinamide A 2M solution of methylamine in THF (40 mL, 80.00 mmol) was added to methyl 6-chloro-5-(piperazin-1-yl) picolinate (intermediate 47, 1.26 g, 4.93 mmol). The resulting mixture was stirred at 80°C for 18 hours. The solvent was removed by distillation under reduced pressure. The crude product was purified by reverse-phase chromatography using a 5-60% MeCN eluent gradient in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 6-chloro-N-methyl-5-(piperazin-1-yl) picolinamide (intermediate 48, 1.12 g, 89%) as a pale yellow oil. 1 ¹H NMR (300MHz, 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-NH proton not shown); m / z (ES + )[M+H] + =255.

[0194] Synthesis Example 14: 6-Chloro-5-[4-[(2-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] 7-(bromomethyl)-3-ethylquinoxaline-2(1H)-one (intermediate 30, 200 mg, 0.75 mmol) was added to 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide (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 minutes; 254; 220 nm; RT: 7.3 min). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-5-[4-[(2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 14, 52.6 mg, 30.4%) as a white solid. 1 HNMR(400MHz,CD3OD)δ 1.33(3H,t),2.71(4H,s),2.87 - 2.96(5H,m),3.23(4H,s),3.73(2H,s),7.33 - 7.41(2H,m),7.62(1H,d),7.77(1H,d),8.00(1H,d);m / z(ES + )[M+H] + =441. [ka]

[0195] Intermediate 50: 7-bromo-3-(trifluoromethyl)quinoxaline-2(1H)-one 4-bromobenzene-1,2-diamine (intermediate 49, 0.9 g, 4.81 mmol) was added to methyl 3,3,3-trifluoro-2-oxopropanoate (0.9 g, 5.77 mmol) in toluene (10 mL). The resulting mixture was stirred at 100°C for 60 minutes. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography using an eluent gradient of 0-50% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain a positional isomer mixture of 7-bromo-3-(trifluoromethyl)quinoxaline-2(1H)-one and 6-bromo-3-(trifluoromethyl)quinoxaline-2(1H)-one (intermediate 50 + intermediate 51, 1.28 g, 45.4%) as an off-white solid. The mixture of positional isomers was isolated. 1 The 1H NMR spectrum was not interpreted; m / z (ES) + )[M+H] + =295.

[0196] Intermediate 52: 7-(hydroxymethyl)-3-(trifluoromethyl)quinoxaline-2(1H)-one Pd(Ph3P)4 (0.3 g, 0.26 mmol) was added to a mixture of 7-bromo-3-(trifluoromethyl)quinoxaline-2(1H)-one and 6-bromo-3-(trifluoromethyl)quinoxaline-2(1H)-one (intermediate 50 + intermediate 51, 1.2 g, 2.05 mmol) and (tributylstanyl)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 reverse-phase chromatography using eluent gradient 5 with 50% MeOH (in water (0.1% HCO2H)). The pure fraction was evaporated to dryness to obtain 7-(hydroxymethyl)-3-(trifluoromethyl)quinoxaline-2(1H)-one (intermediate 52, 0.32 g, 64.0%) as an off-white solid. 1 H NMR(300MHz,DMSO-d6,)δ 4.63(2H,d),5.52(1H,t),7.30(1H,dd),7.38(1H,d),7.83(1H,d),13.05(1H,s);m / z(ES+ )[M+H] + =245.

[0197] Synthesis Example 15: N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]pyridine-2-carboxamide [ka] A 33% HBr solution in AcOH (3 mL, 18.23 mmol) was added to 7-(hydroxymethyl)-3-(trifluoromethyl)quinoxaline-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)picolinamide (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 minutes; 254; 220 nm; RT: 5.77). The fraction containing the desired compound was evaporated to dryness to obtain N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 15, 44.0 mg, 21.71%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 2.55-2.62(m,4H),2.78(d,3H),3.34-3.38(t,4H),3.69(s,2H),7.34 - 7.44(m,3H),7.80-7.91(m,2H),8.27(d,1H),8.36-8.41(m,1H),12.97(s,1H); 19 F NMR(376MHz,DMSO-d6)δ -68.36;m / z(ES +)[M+H] + =447. [ka]

[0198] Synthesis Example 16: 6-Chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]pyridine-2-carboxamide [ka] 33% HBr in AcOH (3 mL, 18.23 mmol) 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)picolinamide (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: from 10B to 50B over 7 minutes; 254; 220 nm; RT: 6.75). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 16, 22.00 mg, 25.9%) as an off-white solid. 1H NMR(400MHz,DMSO-d6)δ 2.56-2.64(s,4H),2.79(d,3H),3.09-3.17(m,4H),3.71(s,2H),7.36-7.42(m ,2H),7.67(d,1H),7.88(d,1H),7.94(d,1H),8.39-8.44(m,1H),12.89(s,1H); 19 F NMR(376MHz,DMSO)δ -68.41;m / z(ES+)[M+H] + =481. [ka]

[0199] Synthesis Example 17: 6-Fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]pyridine-2-carboxamide [ka] 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)picolinamide (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 minutes; 254; 220 nm; RT: 7.2). The fraction containing the desired compound was evaporated to dryness to obtain 6-fluoro-N-methyl-5-[4-[[3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 17, 66.0 mg, 33.9%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 2.55-2.69(m,4H),2.77(d,3H),3.15-3.23(m,4H),3.69(s,2H),7.33 - 7.46(m,2H),7.58(dd,1H),7.78 - 7.93(m,2H),8.37-8.42(m,1H),12.99(s,1H); 19 F NMR(376MHz,DMSO-d6)δ -68.36,-72.52;m / z(ES + )[M+H] + =465. [ka]

[0200] Intermediate 54: 2-Methyl aminopentanoate hydrochloride SOCl2 (17 mL, 232.94 mmol) was added dropwise to 2-aminopentanoic acid (intermediate 53, 10.0 g, 85.36 mmol) in MeOH (200 mL) at 0°C. The resulting mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to obtain methyl 2-aminopentanoate hydrochloride (intermediate 54; 15.78 g, 110%) as a white solid. 1 H NMR(DMSO-d6,400MHz)δ 0.88(3H,t),1.19 - 1.51(2H,m),1.67 - 1.83(2H,m),3.74(3H,s),3.89-3.93(1H,m),8.64(3H,s);m / z(ES+)[M+H] + = 132.

[0201] Intermediate 55: 4-(1-methoxy-1-oxopentan-2-ylamino)-3-methyl nitrobenzoate 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-nitrobenzoate (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 by distillation under reduced pressure. The reaction mixture was diluted with dimethyl phosphate (150 mL) and washed sequentially with water (100 mL × 1), saturated NaHCO3 (100 mL × 1), and saturated brine (100 mL × 1). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain methyl 4-(1-methoxy-1-oxopentan-2-ylamino)-3-nitrobenzoate (intermediate 55, 14.09 g, 100%) as a yellow oil. 1 H NMR(400MHz,DMSO-d6)δ 0.89(3H,t),1.26 - 1.41(2H,m),1.84 - 1.94(2H,m),3.73(3H,s),3.83(3H,s),4.68-4.75(1H,m),7.12(1H,d),8.00(1H,d),8.60(1H,d),8.63(1H,d);m / z(ES+)[M+H] + =311.

[0202] Intermediate 56: 3-Oxo-2-propyl-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl Pd(OH)2 / C (20 wt%, 1.58 g, 2.25 mmol) was added to methyl 4-((1-methoxy-1-oxopentan-2-yl)amino)-3-nitrobenzoate (intermediate 55, 14.05 g, 45.28 mmol) in MeOH (300 mL). The resulting mixture was stirred under H2 at room temperature for 30 hours. The reaction mixture was filtered. The precipitate was washed with DMF (100 mL), and the filtrate was evaporated to dryness to obtain the crude product. The crude product was washed with DCM (10 mL), dried under vacuum, and methyl 3-oxo-2-propyl-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 56, 9.12 g, 81%) was obtained as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 0.87(3H,t),1.32 - 1.46(2H,m),1.57-1.64(2H,m),3.74(3H,s),3.88-3.93(1H,m),6.70(1H,d),6.83(1H,d),7.32(1H,d),7.40(1H,dd),10.38(1H,s);m / z(ES + )[M+H] + =249.

[0203] Intermediate 57: 3-Oxo-2-propyl-3,4-dihydroquinoxaline-6-carboxylate methyl 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 obtain methyl 3-oxo-2-propyl-3,4-dihydroquinoxaline-6-carboxylate (intermediate 57, 7.86 g, 87%) as an off-white solid. 1H NMR(400MHz,DMSO-d6)δ 0.98(3H,t),1.68-1.80(2H,m),2.75-2.83(2H,m),3.89(3H,s),7.73 - 7.85(2H,m),7.88(1H,d),12.45(1H,s);m / z(ES + )[M+H] + =247.

[0204] Intermediate 58: 7-(hydroxymethyl)-3-propylquinoxaline-2(1H)-one A 1M 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) at 0°C. The resulting mixture was stirred at room temperature for 18 hours. The reaction was stopped with MeOH (5 ml) and aqueous solution of saturated potassium sodium tartrate tetrahydrate (20 ml), and the organic layer was evaporated to obtain 7-(hydroxymethyl)-3-propylquinoxaline-2(1H)-one (intermediate 58, 1.2 g, 17.34%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 0.97(3H,t),1.36-1.77(2H,m),2.71 - 2.79(2H,m),4.59(2H,s),5.39(1H,s),7.18(1H,dd),7.27(1H,d),7.65(1H,d),12.30(1H,s);m / z(ES + )[M+H] + =219.

[0205] Intermediate 59: 7-(bromomethyl)-3-propylquinoxaline-2(1H)-one 33% HBr in AcOH (74.6 μl, 1.37 mmol) was added to 7-(hydroxymethyl)-3-propylquinoxaline-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 obtain 7-(bromomethyl)-3-propylquinoxaline-2(1H)-one (intermediate 59, 600 mg, 155%) as a brown solid (the crude product was not pure and contained AcOH and other impurities). The product was used in the next step without further purification. 1 The 1H NMR spectrum was not clear and was not interpreted; m / z(ES) + )[M+H] + = 282.

[0206] Synthesis Example 18: N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide [ka] 7-(bromomethyl)-3-propylquinoxaline-2(1H)-one (intermediate 59, 200 mg, 0.71 mmol) and N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 13, 80 mg, 0.36 mmol) were mixed in NMP (3 mL) with DIPEA (200 μL, 1.15 mmol). 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 × 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: 38B to 50B over 7 minutes; 254 / 220 nm; RT: 6.20). The fraction containing the desired compound was evaporated to dryness to obtain N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 18, 71.0 mg, 46.5%) as a white solid. 1H NMR(400MHz,DMSO-d6)δ 0.97(3H,t),1.66-1.80(2H,m),2.55-2.61(4H,m),2.73 - 2.85(5H,m),3.33-3.40(4H,m),3.62(2H,s),7.19 - 7.31(2H,m),7.40(1H,dd),7.68(1H,d),7.83(1H,d),8.27(1H,d),8.35-8.45(1H,m),12.26(1H,s);m / z(ES+)[M+H] + =421. [ka]

[0207] Synthesis Example 19: 6-Chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide [ka] 7-(bromomethyl)-3-propylquinoxaline-2(1H)-one (intermediate 59 mg, 200 mg, 0.71 mmol) and 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 48 mg, 80 mg, 0.31 mmol) were added to NMP (3 mL) with DIPEA (200 μL, 1.15 mmol). 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 × 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 minutes; 254 / 220 nm; RT: 5.93). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 19, 52.0 mg, 36.4%) as a white solid. 1H NMR(400MHz,DMSO-d6)δ 0.97(3H,t),1.66-1.79(2H,m),2.55-2.65(4H,m),2.71 - 2.85(5H,m),3.06-3.12(4H,m),3.64(2H,s),7.20 - 7.32(2H,m),7.64-7.72(2H,m),7.94(1H,d),8.40-8.50(1H,m),12.27(1H,s);m / z(ES + )[M+H] + =455. [ka]

[0208] Synthesis Example 20: 6-Fluoro-5-[4-[(3-Fluoro-2-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] DIPEA (500 μl, 2.86 mmol) was added to 7-(bromomethyl)-3-propylquinoxaline-2(1H)-one (intermediate 59, 200 mg, 0.71 mmol), 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide, and 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 purified by preparative HPLC (column: SunFire C18 OBD preparative column, 100 Å, 5 μm, 19 mm × 250 mm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 10B to 20B over 13 minutes; 254 / 220 nm; RT: 12.13). The fraction containing the desired compound was evaporated to dryness to obtain 6-fluoro-N-methyl-5-[4-[(3-oxo-2-propyl-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-pyridine-2-carboxamide (Synthesis Example 20, 71.0 mg, 50.4%) as a white solid. 1H NMR(400MHz,DMSO-d6)δ 0.97(3H,t),1.66-1.78(2H,m),2.54-2.60(4H,m),2.71 - 2.83(5H,m),3.14-3.25(4H,m),3.62(2H,s),7.19 - 7.33(2H,m),7.57(1H,dd),7.68(1H,d),7.85(1H,dd),8.37-5.43(1H,m),12.27(1H,s); 19 F NMR(376MHz,DMSO-d6)δ -72.51;m / z(ES + )[M+H] + = 439. [ka]

[0209] Intermediate 61: 2-Methyl aminobutanoate hydrochloride SOCl2 (17 mL, 232.94 mmol) was added dropwise to 2-aminobutanoic acid (intermediate 60, 10.0 g, 96.97 mmol) in MeOH (100 mL) at 0°C. The resulting mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to obtain methyl 2-aminobutanoate hydrochloride (intermediate 61, 14.84 g, 100%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 0.91(3H,t),1.75 - 1.95(2H,m),3.73(3H,s),3.93(1H,t),8.72(3H,s);m / z(ES + )[M+H] + = 118.

[0210] Intermediate 62: 2-Fluoro-4-(1-methoxy-1-oxobutan-2-ylamino)-5-methyl nitrobenzoate DIPEA (4.02 mL, 23.03 mmol) was added to methyl 2,4-difluoro-5-nitrobenzoate (1.0 g, 4.61 mmol) and methyl 2-aminobutanoate hydrochloride (intermediate 61, 0.707 g, 4.61 mmol) in NMP (10 mL). The resulting mixture was stirred at rt for 5 hours. The crude product was purified by reverse-phase chromatography using an eluent gradient of 5 with 80% MeOH (in water (0.1% NH4HCO3)). The pure fraction was evaporated to dryness to obtain methyl 2-fluoro-4-(1-methoxy-1-oxobutan-2-ylamino)-5-nitrobenzoate (intermediate 62, 1.2 g, 83%) as a black solid. 1 H NMR(400MHz,DMSO-d6)δ 0.88(3H,t),1.78 - 2.03(2H,m),3.75(3H,s),3.83(3H,s),4.73 - 4.80(1H,m),7.06(1H,d),8.66 - 8.72(2H,m);m / z(ES + )[M+H] + =315.

[0211] Intermediate 63: 2-ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl Methyl 2-fluoro-4-((1-methoxy-1-oxobutan-2-yl)amino)-5-nitrobenzoate (intermediate 62, 1.15 g, 3.66 mmol) was added under hydrogen 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 not completed. The reaction mixture was filtered. The organic layer was evaporated to obtain the crude product, methyl 2-ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 63, 0.780 g, 85%), as a brown gum-like substance. This crude product was used directly in the next step without further purification. The crude product was not clean. 1 The 1H NMR spectrum was not interpreted; m / z (ES) + )[M+H] + = 253.

[0212] Intermediate 64: 2-ethyl-7-fluoro-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl 2-ethyl-7-fluoro-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl (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 completed. The resulting mixture was concentrated under reduced pressure to obtain a brown solid. A saturated solution of aq NaHCO3 (10 mL) was added to the solid and stirred at room temperature for 1 hour. The precipitate was filtered and rinsed with additional aq NaHCO3 solution (10 mL x 5). The solid was dried under vacuum to obtain 2-ethyl-7-fluoro-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl (intermediate 64, 750 mg, 99%) as a brown solid. 1 H NMR(300MHz,DMSO-d6)δ 1.20(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.

[0213] Intermediate 65: 3-ethyl-6-fluoro-7-(hydroxymethyl)quinoxaline-2(1H)-one A 1M solution of diisobutylaluminum hydride in THF (15.35 mL, 15.35 mmol) was gradually added 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 stopped at 0°C with saturated potassium sodium tartrate aqueous solution (20 mL) and MeOH (10 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered and washed with THF (50 mL x 3). The organic layer was evaporated to dryness to obtain the crude product. The crude product was purified by reverse-phase chromatography using an eluent gradient of 5-60% MeOH in water (0.4% HCO2H). The pure fraction was evaporated to dryness to obtain 3-ethyl-6-fluoro-7-(hydroxymethyl)quinoxaline-2(1H)-one (intermediate 65, 110 mg, 19.37%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ 1.21(3H,t),2.80(2H,q),4.63(2H,d),5.49(1H,t),7.41(1H,d),7.49(1H,d),12.36(1H,s);m / z(ES + )[M+H] + =223.

[0214] Synthesis Example 21: 5-[4-[(2-ethyl-7-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide [ka] 3-ethyl-6-fluoro-7-(hydroxymethyl)quinoxaline-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 obtain 7-(bromomethyl)-3-ethyl-6-fluoroquinoxaline-2(1H)-one (crude product). This product was used directly in the next step without further purification. 7-(bromomethyl)-3-ethyl-6-fluoroquinoxaline-2(1H)-one and 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 23, 70 mg, 0.29 mmol) in NMP (2 mL) were mixed with DIPEA (0.196 mL, 1.13 mmol). The resulting mixture was stirred at 80°C for 2 hours. The obtained mixture was purified by preparative HPLC (column: Sunfire preparative C18 column, 30 × 150 mm, 5 μm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 10B to 35B over 8 minutes; 254 / 220 nm; RT: 7.37). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2-ethyl-7-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Synthesis Example 21, 55.0 mg, 53.7%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ 1.21(3H,t),2.61(4H,m),2.73 - 2.85(5H,m),3.18(4H,m),3.68(2H,s),7.38(1H,d),7.51 - 7.61(2H,m),7.84(1H,dd),8.13(0.29H,s),8.38(1H,m),12.29(1H,s); 19 F NMR(376MHz,DMSO-d6)δ -72.53,-124.31;m / z(ES+)[M+H] + =443. [ka]

[0215] Intermediate 67: 4-(3-hydroxy-1-methoxy-1-oxobutan-2-ylamino)-3-methyl nitrobenzoate DIPEA (8.77 mL, 50.22 mmol) was added to DMF (20 mL) containing methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and methyl 2-amino-3-hydroxybutanoate hydrochloride (intermediate 66, 2.04 g, 12.05 mmol). The resulting mixture was stirred at rt for 16 hours. The reaction mixture was diluted with HCl (100 mL) and washed sequentially with saturated NaH4Cl aqueous solution (1 x 100 mL) and brine (4 x 100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the desired product, methyl 4-((3-hydroxy-1-methoxy-1-oxobutan-2-yl)amino)-3-nitrobenzoate (intermediate 67, 2.9 g, 92%), as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 1.15 - 1.27(3H,m),3.64 - 3.74(3H,m),3.83(3H,s),4.08 - 4.44(1H,m),4.61 - 4.72(1H,m),5.39 - 5.60(1H,m),7.03 - 7.15(1H,m),7.90 - 8.03(1H,m),8.62 - 8.69(1H,m),8.73 - 8.89(1H,m);m / z(ES+)[M+H] + =313.

[0216] Intermediate 68: 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl 20% Pd(OH)2 / C (0.648 g, 0.92 mmol) was added under hydrogen to methyl 4-((3-hydroxy-1-methoxy-1-oxobutan-2-yl)amino)-3-nitrobenzoate (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 celite. The organic layer was evaporated to obtain methyl 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 68, 2.290 g, 99%) as a gray solid. 1 H NMR(400MHz,DMSO-d6)δ 1.07(3H,m),2.81(1H,d),3.72(1H,m),3.74(3H,s),4.78(1H,d),6.70 - 6.86(2H,m),7.27(1H,d),7.37(1H,dd),10.38(1H,d);m / z(ES + )[M+H] + =251.

[0217] Intermediate 69: 2-(1-hydroxyethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl DDQ (2.265 g, 9.98 mmol) was added to 2-(1-hydroxyethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl (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 solution of aq NaHCO3 (100 mL) was added to the solid and stirred at room temperature for 1 hour. The precipitate was filtered and rinsed with additional aq NaHCO3 solution (30 mL x 3). The solid was dried under vacuum to obtain 2-(1-hydroxyethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl (intermediate 69, 2.24 g, 99%) as a gray solid. 1¹H NMR (400MHz, DMSO-d6) δ 1.40 (3H,d), 3.88 (3H,s), 4.94 (1H,q), 7.69 (1H,dd), 7.77 (1H,d), 7.90 (1H,d) (2 protons are not shown); m / z (ES + )[M+H] + =249.

[0218] Intermediate 70: 2-acetyl-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl Dess Martin periodinane (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 obtain the crude product. The crude product was purified by reverse-phase chromatography using eluent gradient 5 with 30% MeCN (in water (0.4% HCO2H)). The pure fraction was evaporated to dryness to obtain 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)δ 2.58(3H,s),3.91(3H,s),7.84(1H,dd),7.91 - 8.03(2H,m),12.86(1H,s);m / z(ES + )[M+H] + =247.

[0219] Intermediate 71: 2-(1,1-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl 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 obtain the crude product. The crude product was purified by reverse-phase chromatography using eluent gradient 5 with 30% MeCN (in water (0.4% HCO2H)). The pure fraction was evaporated to dryness to obtain methyl 2-(1,1-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 71, 174 mg, 26.6%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ 2.07(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.

[0220] Intermediate 72: 3-(1,1-difluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one A 1 M solution of diisobutylaluminum hydride in THF (2.39 mL, 2.39 mmol) was added at 0°C 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. The reaction was stopped at 0°C with saturated potassium sodium tartrate aqueous solution (3 mL) and MeOH (1 mL). The resulting mixture was stirred for 1 hour. The reaction was filtered and washed with THF (10 mL × 3). The organic layer was evaporated to obtain 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. 1H NMR(400MHz,DMSO-d6)δ 2.06(3H,t),4.63(2H,s),5.47(1H,s),7.26(1H,dd),7.35(1H,d),7.78(1H,d),12.75(1H,br s);m / z(ES+)[M+H] + =241.

[0221] Synthesis Example 22: 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] 3-(1,1-difluoroethyl)-7-(hydroxymethyl)quinoxaline-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 obtain 7-(bromomethyl)-3-(1,1-difluoroethyl)quinoxaline-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)quinoxaline-2(1H)-one (crude product) and N-methyl-5-(piperazin-1-yl)picolinamide (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, 30 × 150 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 minutes; 254; 220 nm; RT: 5.70). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[[2-(1,1-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 22, 47.8 mg, 43.2%) as a yellow solid.1 H NMR(400MHz,DMSO-d6)δ 2.06(3H,t),2.52-2.62(4H,m),2.78(3H,d),3.30-3.40(4H,m),3.67(2H,s),7.3 2-7.42(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. [ka]

[0222] Intermediate 74: 4-(4,4-difluoro-1-methoxy-1-oxobutan-2-ylamino)-3-methyl nitrobenzoate DIPEA (8.77 mL, 50.22 mmol) was added to DMF (20 mL) containing methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and methyl 2-amino-4,4-difluorobutanoate hydrochloride (intermediate 73, 2.0 g, 10.55 mmol). The resulting mixture was stirred at 40°C for 8 hours. The reaction mixture was diluted with HCl (100 mL) and washed sequentially with saturated NaH4Cl (1 x 100 mL) and brine (4 x 100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the desired product, methyl 4-((4,4-difluoro-1-methoxy-1-oxobutan-2-yl)amino)-3-nitrobenzoate (intermediate 74, 2.5 g, 74.9%), as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ 2.50 - 2.76(2H,m),3.71(3H,s),3.82(3H,s),4.95(1H,q),6.22(1H,tt),7.18(1H,d),7.99(1H,dd),8.63(1H,d),8.66(1H,d);m / z(ES+)[M+H] + =333.

[0223] Intermediate 75: 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl 20% Pd(OH)2 / C (0.465 g, 0.66 mmol) was added under hydrogen to methyl 4-((4,4-difluoro-1-methoxy-1-oxobutan-2-yl)amino)-3-nitrobenzoate (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 celite. The filtrate was evaporated to obtain methyl 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 75, 1.64 g, 92%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 2.24-2.32(2H,m),3.76(3H,s),4.10-4.18(1H,m),6.27(1H,tt),6.73(1H,d),6.89(1H,s),7.37(1H,d),7.44(1H,dd),10.58(1H,s);m / z(ES + )[M+H] + =271.

[0224] Intermediate 76: 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl DDQ (1.478 g, 6.51 mmol) was added to 2-(2,2-difluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl (intermediate 75, 1.6 g, 5.92 mmol) in DCM (100 mL). The resulting mixture was stirred at room temperature for 3 hours. The mixture was removed under reduced pressure to obtain a brown solid. A saturated solution of aq NaHCO3 (100 mL) was added to the solid and stirred at room temperature for 1 hour. The precipitate was filtered and rinsed with additional aq NaHCO3 solution (30 mL x 3). The solid was dried under vacuum to obtain 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl (intermediate 76, 1.58 g, 99%) as an off-white solid. 1H NMR(400MHz,DMSO-d6)δ 3.46(2H,td),3.90(3H,s),6.57(1H,t),7.79 - 7.92(3H,m),12.68(1H,s);m / z(ES + )[M+H] + =269.

[0225] Intermediate 77: 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one A 1 M solution of diisobutylaluminum hydride in THF (22.37 mL, 22.37 mmol) was gradually added at 0°C 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. The reaction was stopped at 0°C with saturated potassium sodium tartrate aqueous solution (20 mL) and MeOH (10 mL). The resulting mixture was stirred for 1 hour. The reaction was filtered and washed with THF (30 mL x 3). The organic layer was evaporated to obtain 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 reverse-phase chromatography using an eluent gradient of 5-60% MeOH in water (0.4% HCO2H). The pure fraction was evaporated to dryness to obtain 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one (intermediate 77, 500 mg, 69.4%) as a red solid. 1 H NMR(300MHz,DMSO-d6)δ 3.42(2H,td),4.61(2H,s),5.42(1H,brs),6.56(1H,tt),7.23(1H,dd),7.32(1H,d),7.71(1H,d),12.55(1H,s);m / z(ES+)[M+H] + =241.

[0226] Intermediate 78: 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carbaldehyde Dess Martin periodinane (530 mg, 1.25 mmol) was added to 3-(2,2-difluoroethyl)-7-(hydroxymethyl)quinoxaline-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 reaction mixture was evaporated to obtain the crude product. The crude product was purified by reverse-phase chromatography using eluent gradient 5 with 30% MeCN (in water (0.4% HCO2H)). The pure fraction was evaporated to dryness to obtain 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carbaldehyde (intermediate 78, 160 mg, 81%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 3.47(2H,td),6.58(1H,tt),7.77-7.85(2H,m),7.90 - 7.98(1H,m),10.09(1H,s),12.79(1H,s);m / z(ES + )[M+H] + =239.

[0227] Synthesis Example 23: 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] Titanium isopropoxide (65.6 mg, 0.23 mmol) was added to 2 mL of THF containing 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carbaldehyde (intermediate 78 mg, 55 mg, 0.23 mmol) and N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 13 mg, 60 mg, 0.23 mmol). 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 reaction was stopped with MeOH (0.1 mL). The reaction mixture was evaporated to obtain the crude product, which was purified by preparative HPLC (column: XBridge Shield RP18 OBD column, 30 × 150 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 minutes; 254; 220 nm; RT: 5.70). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 23, 8.76 mg, 8.57%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 2.56(4H,m),2.78(3H,d),3.32-3.48(6H,m),3.64(2H,s),6.55(1H,tt),7.27-7.33(2 H,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. [ka]

[0228] Synthesis Example 24: 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide [ka] Titanium isopropoxide (59.7 mg, 0.21 mmol) was added to 2 mL of THF containing 2-(2,2-difluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carbaldehyde (intermediate 78 mg, 50 mg, 0.21 mmol) and 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 23 mg, 50.0 mg, 0.21 mmol). 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 stopped with MeOH (0.1 mL). The reaction mixture was evaporated to obtain the crude product. The crude product was purified by preparative HPLC (column: Sunfire preparative C18 column, 30 × 150, 5 μm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 2B to 27B over 7 minutes; 254 / 220 nm; RT: 6.78). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[[2-(2,2-difluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Synthesis Example 24, 21.72 mg, 22.13%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 2.54 - 2.61(4H,m),2.76(3H,d),3.14 - 3.22(4H,m),3.41(2H,td),3.64(2H,s),6.39 - 6.71(1H,m),7.26 - 7.33(2H,m),7.57(1H,dd),7.73(1H,d),7.82 - 7.86(1H,m),8.13(0.16H,s),8.37(1H,m),12.49(1H,s); 19F NMR(376MHz,DMSO-d6)δ -72.52,-114.29;m / z(ES + )[M+H] + =461. [ka]

[0229] Intermediate 80: 4-(4-fluoro-1-methoxy-1-oxobutan-2-ylamino)-3-methyl nitrobenzoate DIPEA (8.77 mL, 50.22 mmol) was added to DMF (20 mL) containing methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and methyl 2-amino-4-fluorobutanoate hydrochloride (intermediate 79, 1.81 g, 10.55 mmol). The resulting mixture was stirred at 40°C for 8 hours. The reaction mixture was diluted with HCl (100 mL) and washed sequentially with saturated NaH4Cl (1 x 100 mL) and brine (4 x 100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the desired product, methyl 4-((4-fluoro-1-methoxy-1-oxobutan-2-yl)amino)-3-nitrobenzoate (intermediate 80, 2.5 g, 79%), as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ 2.25 - 2.35(1H,m),2.35 - 2.45(1H,m),3.71(3H,s),3.82(3H,s),4.36 - 4.58(1H,m),4.56 - 4.74(1H,m),4.84(1H,q),7.14(1H,d),7.99(1H,dd),8.63(1H,d),8.67(1H,d);m / z(ES+)[M+H]+=315.

[0230] Intermediate 81: 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl 20% Pd(OH)2 / C (0.547 g, 0.78 mmol) was added under hydrogen to methyl 4-((4-fluoro-1-methoxy-1-oxobutan-2-yl)amino)-3-nitrobenzoate (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 celite. The filtrate was evaporated to obtain methyl 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (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.

[0231] Intermediate 82: 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl DDQ (1.83 g, 8.07 mmol) was added to 2-(2-fluoroethyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl (intermediate 81, 1.85 g, 7.33 mmol) in DCM (100 mL). The resulting mixture was stirred at room temperature for 3 hours. The mixture was removed under reduced pressure to obtain a brown solid. A saturated solution of aq. NaHCO3 (100 mL) was added to the solid and stirred at room temperature for 1 hour. The precipitate was filtered and rinsed with additional aq. NaHCO3 solution (30 mL x 3). The solid was dried under vacuum to obtain 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate methyl (intermediate 82, 1.8 g, 98%) as a gray solid. 1 H NMR(400MHz,DMSO-d6)δ 3.23(2H,dt),3.89(3H,s),4.90(2H,dt),7.76 - 7.85(2H,m),7.88(1H,d),12.55(1H,s);m / z(ES+ )[M+H] + =251.

[0232] Intermediate 83: 3-(2-fluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one A 1M solution of diisobutylaluminum hydride in THF (15.99 mL, 15.99 mmol) was gradually added at 0°C to methyl 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carboxylate (intermediate 82, 1.0 g, 4.00 mmol) in THF (100 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction was stopped at 0°C with saturated potassium sodium tartrate aqueous solution (20 mL) and MeOH (10 mL). The resulting mixture was stirred for 1 hour. The reaction was filtered and washed with THF (30 mL x 3). The organic layer was evaporated to obtain the crude product. The crude product was purified by reverse-phase chromatography using an eluent gradient of 5-60% MeOH in water (0.4% HCO2H). The pure fraction was evaporated to dryness to obtain 3-(2-fluoroethyl)-7-(hydroxymethyl)quinoxaline-2(1H)-one (intermediate 83, 0.49 g, 55.2%) as a brown solid. 1 H NMR(300MHz,DMSO-d6)δ 3.20(2H,dt),4.60(2H,d),4.90(2H,dt),5.41(1H,t),7.21(1H,dd),7.30(1H,d),7.68(1H,d),12.42(1H,s);m / z(ES+)[M+H] + =223.

[0233] Intermediate 84: 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carbaldehyde Dess Martin periodinane (229 mg, 0.54 mmol) was added to 3-(2-fluoroethyl)-7-(hydroxymethyl)quinoxaline-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 obtain the crude product. The crude product was purified by reverse-phase chromatography using eluent gradient 5 with 30% MeCN (in water (0.4% HCO2H)). The pure fraction was evaporated to dryness to obtain 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carbaldehyde (intermediate 84, 93 mg, 94%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ 3.20-3.28(2H,m),4.90(2H,dt),7.74-7.80(2H,m),7.91(1H,d),10.06(1H,s),12.66(1H,s);m / z(ES+)[M+H] + =221.

[0234] Synthesis Example 25: 5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] Titanium isopropoxide (64.5 mg, 0.23 mmol) was added to 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carbaldehyde (intermediate 84, 50 mg, 0.23 mmol) and N-methyl-5-(piperazin-1-yl)picolinamide (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 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: 20B to 35B over 7 minutes; 254 / 210 nm; RT: 6.38). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[[2-(2-fluoromethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 25, 4.83 mg, 2.54%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 2.53 - 2.59(4H,m),2.78(3H,d),3.17(1H,t),3.23(1H,t),3.32 - 3.38(4H,m),3.63(2H,s),4.83(1H,t),4.95(1H,t),7.25-7.32(2H,m),7.39 (1H,dd),7.71(1H,d),7.83(1H,d),8.26(1H,d),8.37(1H,d),12.36(1H,s); 19 F NMR(376MHz,DMSO-d6)δ -217.70;m / z(ES + )[M+H] + = 425. [ka]

[0235] Synthesis Example 26: 6-Fluoro-5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide [ka] Titanium isopropoxide (90 mg, 0.32 mmol) was added to 2-(2-fluoroethyl)-3-oxo-3,4-dihydroquinoxaline-6-carbaldehyde (intermediate 84 mg, 70 mg, 0.32 mmol) and 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 23 mg, 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 was stopped with MeOH (0.1 mL). The reaction was evaporated to obtain the crude product. 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: from 28B to 35B over 8 minutes; 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 purified by preparative HPLC (column: Xselect CSH OBD column 30×150 mm 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 minutes; 254; 220 nm; RT: 6.83). The fraction containing the desired compound was evaporated to dryness to obtain 6-fluoro-5-[4-[[2-(2-fluoroethyl)-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 26, 3.79 mg, 2.65%) as a yellow solid. 1H NMR(400MHz,DMSO-d6)δ 2.55 - 2.60(4H,m),2.76(3H,d),3.14 - 3.25(6H,m),3.63(2H,s),4.89(2H,dt),7.24 - 7.31(2H,m),7.57(1H,dd),7.70(1H,d),7.84(1H,d),8.24(0.174H,s),8.38(1H,d),12.37(1H,s); 19 F NMR(376MHz,DMSO-d6)δ -72.51,-217.71;(ES + )[M+H] + =443. [ka]

[0236] Intermediate 86: 3-Nitro-4-(4,4,4-trifluoro-1-methoxy-1-oxobutan-2-ylamino)methyl benzoate DIPEA (8.77 mL, 50.22 mmol) was added to DMF (20 mL) containing methyl 4-fluoro-3-nitrobenzoate (2.0 g, 10.04 mmol) and methyl 2-amino-4,4,4-trifluorobutanoate hydrochloride (intermediate 85, 2.2 g, 10.55 mmol). The resulting mixture was stirred at 50°C for 10 hours. The reaction mixture was diluted with HCl (100 mL) and washed sequentially with saturated NaH4Cl aqueous solution (1 x 100 mL) and brine (4 x 100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the desired product, methyl 3-nitro-4-((4,4,4-trifluoro-1-methoxy-1-oxobutan-2-yl)amino)benzoate (intermediate 86, 3.0 g, 85%), as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 2.99 - 3.28(2H,m),3.73(3H,s),3.84(3H,s),5.18(1H,td),7.28(1H,d),8.01(1H,dd),8.65(1H,d),8.71(1H,d);m / z(ES + )[M+H] + =351.

[0237] Intermediate 87: 3-Oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinoxaline-6-methyl carboxylate 20% Pd(OH)2 / C (0.601 g, 0.86 mmol) was added under hydrogen to methyl 3-nitro-4-((4,4,4-trifluoro-1-methoxy-1-oxobutan-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 celite. The filtrate was evaporated to dryness to obtain methyl 3-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (intermediate 87, 2.3 g, 93%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ 2.64 - 2.83(2H,m),3.76(3H,s),4.32-4.37(1H,m),6.78(1H,d),6.90(1H,d),7.37(1H,d),7.43(1H,dd),10.64(1H,s);m / z(ES + )[M+H] + =289.

[0238] Intermediate 88: 3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinoxaline-6-carboxylate methyl DDQ (1.975 g, 8.70 mmol) was added to 3-oxo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroquinoxaline-6-carboxylate methyl (intermediate 87, 2.28 g, 7.91 mmol) in DCM (100 mL). The resulting mixture was stirred at room temperature for 3 hours. The mixture was removed under reduced pressure to obtain a brown solid. A saturated solution of aq. NaHCO3 (100 mL) was added to the solid and stirred at room temperature for 1 hour. The precipitate was filtered and rinsed with additional aq. NaHCO3 solution (30 mL x 3). The solid was dried under vacuum to obtain 3-oxo-2-(2,2,2-trifluoroethyl)-3,4-dihydroquinoxaline-6-carboxylate methyl (intermediate 88, 2.2 g, 97%) as a brown solid. 1H NMR(400MHz,DMSO-d6)δ 3.88 - 3.98(5H,m),7.81(1H,dd),7.86 - 7.94(2H,m),12.75(1H,s);m / z(ES + )[M+H] + =287.

[0239] Intermediate 89: 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinoxaline-2(1H)-one A 1M solution of diisobutylaluminum hydride in THF (20.96 mL, 20.96 mmol) was gradually added at 0°C 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. The reaction was stopped at 0°C with saturated potassium sodium tartrate aqueous solution (20 mL) and MeOH (10 mL). The resulting mixture was stirred for 1 hour. The reaction was filtered and washed with THF (30 mL x 3). The organic layer was evaporated to obtain an off-white solid, which was purified by flash silica chromatography using an eluent gradient of 5 to 55% MeOH (in water (0.4% HCO2H)). The pure fraction was evaporated to dryness to obtain 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinoxaline-2(1H)-one (intermediate 89, 650 mg, 72.2%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ 3.88(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.

[0240] Synthesis Example 27: N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide [ka] 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 obtain 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)picolinamide (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 preparative C18 column, 30 × 150, 5 μm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 10B to 25B over 7 minutes; 254 / 220 nm; RT: 6.57). The fraction containing the desired compound was evaporated to dryness to obtain N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 27, 41.5 mg, 46.6%) as an off-white solid. 1 H NMR(400MHz,DMSO-d6)δ 2.56(4H,m),2.78(3H,d),3.35(4H,m),3.65(2H,s),3.88(2H,q),7.29 - 7.42(3H,m),7.79(2H,m),8.25 - 8.30(1H,m),8.38(1H,m),12.60(1H,br s); 19 F NMR(376MHz,DMSO-d6)δ -61.53;m / z(ES + )[M+H] + =461. [ka]

[0241] Synthesis Example 28: 6-Fluoro-N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide [ka] 7-(hydroxymethyl)-3-(2,2,2-trifluoroethyl)quinoxaline-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 obtain 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 (0.203 mL, 1.16 mmol) was added to 7-(bromomethyl)-3-(2,2,2-trifluoroethyl)quinoxaline-2(1H)-one (crude product) and 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 23 mg, 60 mg, 0.25 mmol) in NMP (2 mL). The resulting mixture was stirred at 80°C for 2 hours. The obtained mixture was purified by preparative HPLC (column: Sunfire preparative C18 column, 30 × 150, 5 μm; mobile phase A: water (0.1% HCO2H), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 12B to 30B over 7 minutes; 254 / 220 nm; RT: 6.25). The fraction containing the desired compound was evaporated to dryness to obtain 6-fluoro-N-methyl-5-[4-[[3-oxo-2-(2,2,2-trifluoroethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 28, 49.0 mg, 43.3%) as an off-white solid. 1H NMR(400MHz,DMSO-d6)δ 2.53-2.63(4H,m),2.76(3H,d),3.15-3.22(4H,m),3.65(2H,s),3.88(2H,q),7.28-7.35(2H ,m),7.57(1H,dd),7.76(1H,d),7.84(1H,dd),8.17(0.185H,s),8.38(1H,m),12.57(1H,s); 19 F NMR(376MHz,DMSO-d6)δ -61.54,-72.52;m / z(ES + )[M+H] + = 479.

[0242] Synthesis Example 29: 6-(difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] DIPEA (330 μl, 1.89 mmol) was added at 20°C to a stirred solution of 7-(chloromethyl)-3-ethyl-1,5-naphthyllysine-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-piperazine-1-ylpyridine-2-carboxamide,2HCl (intermediate 41,102 mg, 0.30 mmol) in acetonitrile (2.4 mL). 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 sat NaHCO3. The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 30% MeOH (in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain 6-(difluoromethyl)-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Synthesis Example 29, 52.0 mg, 42%) as a pale yellow solid. 1H 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 m / z(ES + )[M+H] + =457.

[0243] Synthesis Example 30: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methyl-6(trifluoromethyl)pyridine-2-carboxamide [ka] DIPEA (330 μl, 1.89 mmol) was added at 20°C to a stirred solution of 7-(chloromethyl)-3-ethyl-1,5-naphthyllysine-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). 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 sat NaHCO3. The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 30% MeOH (in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methyl-6(trifluoromethyl)pyridine-2-carboxamide (Synthesis Example 30, 58.0 mg, 45%) as a pale yellow solid. 1H NMR(500MHz,DMSO-d6)1.19(3H,t),2.54-2.62(6H,m),2.83(3H,d),3.04(4H,br m / z(ES) + )[M+H] + = 475.

[0244] Synthesis Example 31: 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide [ka] DIPEA (0.366 mL, 2.10 mmol) was added at 20°C to a stirred solution of 7-(bromomethyl)-3-ethyl-1,5-naphthyridine-2(1H)-one (intermediate 14, 80 mg, 0.30 mmol) and N,6-dimethyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 45, 101 mg, 0.33 mmol) in acetonitrile (2 mL). 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 sat NaHCO3. The mixture was extracted with ethyl acetate. After concentration, the resulting residue was purified by flash silica chromatography with an eluent gradient of 0 to 30% MeOH (in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Synthesis Example 31, 36.0 mg, 29%) as a pale yellow solid. 1H 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 m / z(ES) + )[M+H] + =421. [ka]

[0245] Intermediate 90: 4-[6-(ethylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl Ethanolamine in methanol (7M, 7.78 mL, 15.56 mmol) was added to a solution of 4-(6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate tert-butyl (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 obtain 4-[6-(ethylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl (intermediate 90, 0.495 g, 95%). 1 H 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).

[0246] Intermediate 91: N-ethyl-5-piperazine-1-ylpyridine-2-carboxamide HCl (0.473 mL, 15.58 mmol) in dioxane was slowly added to a stirred solution of 4-(6-(ethylcarbamoyl)pyridine-3-yl)piperazine-1-carboxylate tert-butyl (intermediate 90, 521 mg, 1.56 mmol) in methanol (10 mL). The resulting solution was stirred at rt for 17 hours. The reaction product was concentrated, and the solid was dried to obtain N-ethyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 91, 421 mg, 88%); m / z (ES + )[M+H] + = 235.

[0247] Synthesis Example 32: N-ethyl-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide [ka] DIPEA (0.320 mL, 1.83 mmol) was added at 20°C to a stirred solution of 7-(bromomethyl)-3-ethyl-1,5-naphthyridine-2(1H)-one (intermediate 14, 70 mg, 0.26 mmol) and N-ethyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 91, 89 mg, 0.29 mmol) in acetonitrile (2 mL). 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 sat NaHCO3. The mixture was extracted with ethyl acetate. After concentration, the crude product was purified by reverse-phase chromatography (column: XbridC18) with an eluent gradient of 20 using 50% MeCN (in water (containing 0.2% NH4OH)). The pure fraction was evaporated to dryness to obtain N-ethyl-5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridine-3-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide (Synthesis Example 32, 28.0 mg, 25%) as a white solid. 1H NMR(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 m / z(ES + )[M] + =420.

[0248] Synthesis Example 4 - Form A In Synthesis Example 4, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide was obtained as a partially crystalline solid by evaporating a methanol / dichloromethane solution under reduced pressure. This crystalline material was characterized as crystalline form A.

[0249] If the degree of crystallinity was low, crystalline form A was obtained by suspending 20 mg of the crude sample in 0.20 ml of water, methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, ethyl acetate, or another solvent at ambient temperature or 50°C for 1 day.

[0250] Form A was analyzed using XRPD, and the results are shown in Figure 16A and in the table below.

[0251] [Table 1]

[0252] Form A is characterized by exhibiting at least one of the following 2θ values ​​measured using CuKα radiation: 8.3, 12.4, and 19.4°.

[0253] Form A was analyzed using thermal techniques. DSC analysis showed that Form A has a melting point that starts at 254°C and has a peak at 255°C. A representative DSC trace of Form A is shown in Figure 16B.

[0254] Biological assay (PARP1 selective inhibitor) The inhibitory properties of the PARP1 selective inhibitors described herein can be determined using the following test procedure.

[0255] PARP Fluorescence Anisotropic Binding Assay Recombinant full-length 6HIS-tagged 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 4 hours with an equal volume of a 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 at less than 1% (v / v).

[0256] 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 4 hours with an equal volume of a 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 at less than 1% (v / v).

[0257] 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 4 hours with an equal volume of a 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 at less than 1% (v / v).

[0258] 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 4 hours with an equal volume of a 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 at less than 1% (v / v).

[0259] Recombinant full-length GST-tagged 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 4 hours with an equal volume of a 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 at less than 1% (v / v).

[0260] The fluorescence anisotropy of the probe when bound to a protein was measured using BMG Pherastar FS (copyright) in the presence of the test compound or solvent control, and the effect on anisotropy was assessed. 50 To determine the values, the inhibition percentage values ​​were calculated for different test compound concentrations and fitted to a 4-parameter logistic plot. If necessary, compound K i IC is calculated using the Munson-Rodbard formula defined in Anal. Biochem. 1980 Sep 1;107(1):220-39. 50 The known K of the probe that binds to the relevant PARP protein can be determined from the value. D It is based on.

[0261] hERG electrophysiological assay Electrophysiological recordings (all performed RT) from stably transfected CHO hKv11.1 cells were obtained using a Nanion Syncropatch 768PE. The test compound, vehicle, or positive control were added at different concentrations using six compound plates to allow for cumulative administration to cells (10 mM, 3.167 mM, 1 mM, 0.3167 mM, 0.1 mM, 0.03167 mM). 600 L of the compound was resuspended in 90 μL of reference buffer (mM: NaCl 80, KCl 4, CaCl 5, MgCl 1, NMDG Cl 60, 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 Nanion Syncropatch 768PE run, the extracellular solution (mM: NaCl 80, KCl 4, CaCl 5, MgCl 1, NMDG Cl 60, D-glucose monohydrate 5, HEPES) was used. The current amplitude in each cell in the presence of 10 (pH 7.4 HCl, 298 mOsm) is measured for all liquid additions performed using the Syncropatch liquid handling system. 40 μL of external solution (mM, HBPS, CaCl2 2, MgCl2 1 (pH 7.4, NaOH)) is added to a 384-well multi-hole medium-resistant recording chip, and the bottom of the plate is perfused with internal buffer (mM, KF 130, KCl 20, MgCl2 1, EGTA 10, HEPES 10, Escin 25 (all Sigma-Aldrich; pH 7.2~7.30, 10M KOH, 320 mOsm)). 20 μL of cells maintained at approximately 9°C are dispensed into each well of the chip at a density of 1e6 cells / ml, followed by 20 μL of seal enhancer (mM, NaCl 80, KCl 3, CaCl Dispense HEPES 10 and MgCl 1 (pH 7.4 NaOH). Perform a washing step, leaving a residual volume of 40 μL. To establish a stable baseline before adding the test compound, dispense 40 μL of reference buffer, and repeat this step with a 40 μL removal step after 3 minutes. Dispense 40 μL of compound concentration 1 (0.16 μM), perform "real-time" recording, expose for 3 minutes, and then remove 40 μL.This process is repeated for five further subsequent compound plates to produce a cumulative curve analysis. All data are leak-free and consist of two pulses from -80mV to 100ms with a 100ms delay. The outward K+ current is then induced by a voltage step from a holding potential of -90mV to +60mV, with each pulse delivered at a frequency of 2Hz with a pulse interval of 15s.

[0262] PARP proliferation assay (compound administered for 4 days) DLD1 and BRCA2(- / -)DLD1 cells were harvested into complete medium at densities of 1.875E4 cells / ml and 6.25E4 cells / ml, respectively, seeded at 40 μL / well in 384-well plates (Greiner, Kremsmunster, Austria; 781090) using Multidrop Combi, and incubated overnight at 37°C and 5% CO2. On the following day (Day 1), sytox green (5 μl, 2 μM) and saponin (10 μl, 0.25% stock) were added to the Day 0 plates using Multidrop Combi, the plates were sealed with a black adhesive lid, and incubated at RT for over 3 hours. Cells were imaged using Cell Insight (Thermo Fisher) with a 4× objective lens. Test compounds were added using Echo 555 and incubated for 4 days in an incubator maintained at 37°C and 5% CO2. On day 5, sytox green (5 μl, 2 μM), followed by saponin (10 μl, 0.25% stock), is added to the plate, the plate is sealed with a black adhesive lid, and incubated at RT for over 3 hours. All cells are read with Cell Insight equipped with a 4× material-resistant lens. Growth rate is determined in Geneda by evaluating the total cell count output from Cell Insight for plates on day 0 and day 5.

[0263] [Table 2]

[0264] Example 1: Production of antibody-drug conjugates According to the production method described in International Publication No. 2015 / 115091, and using an anti-HER2 antibody (an antibody containing a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 11 (amino acid residues 1-449 of SEQ ID NO: 1) and a light chain consisting of the amino acid sequence of all amino acid residues 1-214 of SEQ ID NO: 2), a drug linker represented by the following formula is produced: [ka] (In the formula, A represents the attachment site to the antibody.) An anti-HER2 antibody-drug conjugate was produced, conjugated to an anti-HER2 antibody via thioether linkage (DS-8201: trastuzumab deruxtecan). The DAR of the antibody-drug conjugate was 7.7 or 7.8.

[0265] Example 2: Production of a PARP1 selective inhibitor A PARP1 selective inhibitor of formula (I) is prepared according to the production method described herein. Specifically, 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyllysine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide: [ka] However, it can be prepared according to Synthesis Example 4 of this specification (Example 4 of International Publication No. 2021 / 013735).

[0266] Example 3: Antitumor Test Combination of antibody-drug conjugate DS-8201 (trastuzumab deruxtecan (Enhertu®))) and PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyrizine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide) method: A high-throughput combination screen was performed, in which 27 breast cancer cell lines with varying HER2 expression and one gastric cell line with high HER2 expression (Table 1) were treated with a combination of DS-8201 and AZD5305 (a PARP1 selective inhibitor).

[0267] [Table 3]

[0268] The screened values ​​were from a 7-day CellTiter-Glo cell viability assay performed as a 6×6 dose-response matrix (5-point logarithmic serial dilutions for DS-8201 and semi-logarithmic serial dilutions for AZD5305). The maximum concentration was 3 μM for AZD5305 and 10 μg / ml for DS-8201. In addition, trastuzumab and exatecan (DNA topoisomerase I inhibitors) were also screened in parallel with AZD5305 to support the deconvolution of effective combination mechanisms of action. Combination activity was evaluated based on combinations of ΔEmax and Loewe synergy scores.

[0269] result: The results for HER2-high cell lines (KPL4, NCI-N87, SKBR3, HCC1954, HCC1569, AU565) are shown in Figures 12A and 12B and Table 2, while the results for HER2-low cell lines (MDA-MB-468, MDA-MB-157, HCC1187, T47D, HCC38) are shown in Figures 13A and 13B and Table 3.

[0270] Figures 12A and 13A show the matrix of measured cell viability signals. 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 compared to DMSO control on day 7. All values ​​are normalized to the cell viability value on day 0. Values ​​between 0 and 100 represent the percentage of growth inhibition, and values ​​above 100 represent cell death.

[0271] Figures 12B and 13B show the Loewe excess matrix. The values ​​in the boxes represent the excess values ​​calculated by the Loewe additivity model.

[0272] Tables 2 and 3 show the HSA synergy score and Loewe additivity score.

[0273] [Table 4]

[0274] [Table 5]

[0275] Note: Loewe dose-additiveness predicts the expected response when two compounds act on the same molecular target through the same mechanism. It calculates additivity based on an estimate of zero interaction between compounds and is independent of the nature of dose-response correlation.

[0276] HSA (Highest Single Agent) [Berenbaum 1989] quantifies the higher effect of two single compounds at their corresponding concentrations. The combined effect is compared to the effect of each single agent at the concentrations used in that combination. An excess exceeding the highest single agent effect indicates a synergistic effect. HSA does not require the compounds to act on the same target.

[0277] 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. This predicted value is determined by the selected model. The difference between the predicted and measured values ​​may indicate synergistic or antagonistic effects, which are shown in the excess matrix. The excess matrix values ​​are summarized as a combined score of excess amount and synergistic effect score.

[0278] Figure 14 shows the combined Emax and Loewe synergy scores for various cell lines treated with DS-8201 in combination with AZD5305.

[0279] As can be seen from Figures 12A and 12B and Table 2, AZD5305 interacted synergistically with DS-8201 and increased cell death in HER2+ breast and gastric cancer cell lines. As can be seen from Figures 13A and 13B and Table 3, AZD5305 interacted synergistically with DS-8201 and increased cell death in HER2-low breast cancer cell lines at Emax (3 μM AZD5305 and 10 μg / ml DS-8201). As can be seen from Figure 14, in 11 cell lines, including HER2-low breast cancer cell lines, treatment with DS-8201 combined with AZD5305 resulted in a high combined Emax (>100) and a high Loewe synergy score (>5).

[0280] Example 4: Antitumor Test Combination of antibody-drug conjugate DS-8201 (trastuzumab deruxtecan (Enhertu®))) and PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyrizine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide) method: Cells grown under their respective conditions at densities optimal for linear growth during the assay period (4–8 days) were seeded into 96-well plates. Immediately after seeding, the specified compounds were administered to the cells in a total volume of 200 μL / well and placed in an incubator. Combinations were performed as a 6 × 8 concentration response matrix for each combination. At the endpoint, cells were fixed in 2% PFA at room temperature for 20 minutes. To obtain the cell count at the start of treatment, one additional plate was used in each experiment to fix the cells behind the conjugated cells. Subsequently, the cells were permeabilized in 0.5% Triton-X100 in PBS for 10 minutes. After washing in PBS, the cells were blocked in 5% FBS in PBS at RT for 1 hour and incubated overnight with the primary antibody in 5% FBS + 0.05% Triton at 4°C. After washing three times in PBS, the cells were incubated with the secondary antibody in 5% FBS + 0.05% Triton containing Hoechst33258 at room temperature for 1 hour. After washing three times in PBS, cells were scanned with a Cellinsight instrument equipped with a 10x objective lens and 9 fields / well. Images were analyzed using Columbus for cell count based on nuclear Hoechst staining. Relative growth in each well compared to the solvent control was calculated using total cell count / well. Growth inhibition data were analyzed using Combenefit software to calculate synergistic effect scores (Di Veroli, GY, et al., Combenefit: an interactive platform for the analysis and visualization of drug combinations. Bioinformatics, 2016, 32(18): p.2866-8).

[0281] result: The results for the HER2-high cell line (KPL4) and two HER2-low cell lines (JIMT1, MDA-MB-468) are shown in Figures 15A and 15B.

[0282] Figure 15A shows a cell number matrix, where the Y-axis represents drug A (DS-8201) and the X-axis represents drug B (AZD5305). The values ​​in the boxes represent the relative total cell (nucleus) count as a percentage of the DMSO vehicle control.

[0283] Figure 15B shows a matrix in which 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 synergy score.

[0284] The results of Examples 3 and 4 demonstrate that selective PAR1 inhibition using AZD5305 enhances the antitumor efficacy of DS-8201 in both high and low HER2-expressing cell lines in vitro. In Example 3, AZD5305 combined with DS-8201 demonstrated beneficial combinations in five HER2+ breast cancer cell lines, one HER2+ gastric cancer cell line (Figures 12A, 12B, 14 and Table 2), and five low HER2 breast cancer cell lines (Figures 13A, 13B, and 14 and Table 3). In Example 4, AZD5305 combined with DS-8201 showed synergistic activity in HER2-high (KPL4) and HER2-low (JIMT-1, MDA-MB-468) cell lines (Figures 15A and 15B).

[0285] Example 5: Antitumor test - In vivo Combination of antibody-drug conjugate DS-8201 (trastuzumab deruxtecan (Enhertu®))) and PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyrizine-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide) method: Female nude mice (Charles River) aged 5-8 weeks were used and participated in this experiment after a 7-day acclimatization period. 1 × 10⁶ of these female nude mice were placed in the flank. 7 NCI-N87 tumor cells (1:1 in Matrigel) were subcutaneously transplanted. The tumor was approximately 150 mm. 3When the tumor size reached a certain level, tumors of similar size were randomly assigned to one of the treatment groups shown in Table 4.

[0286] [Table 6]

[0287] The dosage of each compound for each animal was calculated based on the individual body weight on the day of administration. DS-8201 and AZD5305 were administered on the same day, with DS-8201 administered approximately one hour after the PO administration of AZD5305. DS-8201 was administered as a single dose of 1 mg / kg or 3 mg / kg on day 1, and AZD5305 was administered as a 1 mg / kg QD for 28 days. The total treatment period was 28 days.

[0288] DS-8201 is formulated at 3 mg / kg and 1 mg / kg. The DS-8201 dosage solution was prepared on the day of administration by diluting the stock solution (20.1 mg / ml) of DS-8201 in 25 mM histidine buffer and 9% sucrose (pH 5.5) to a concentration of 0.6 mg / ml. For the 3 mg / kg and 1 mg / kg dosage solutions, the concentration was reduced to 0.2 mg / ml. Each dosage solution was thoroughly mixed using a pipette and then administered by IV injection at a dose of 5 ml / kg.

[0289] Formulated with AZD5305 at 1 mg / kg To prepare a 1 mg / kg dosage solution, AZD5305 at a concentration of 0.1 mg / ml was prepared, yielding a dosage volume of 10 ml / kg for PO administration. A total of 49 ml of vehicle was required. 15 μl of 1 M 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 bottle. The Eppendorf tube was rinsed with 1 ml of sterile water, and then any remaining compound was also transferred to the glass bottle. The remaining sterile water (37.2 ml; 80% of the total vehicle volume) was added to the glass bottle and thoroughly mixed using an electromagnetic stirrer. The pH of the administration solution was adjusted to pH 3.74, and then the remaining vehicle (9.772 ml of sterile water) was added to the glass bottle and thoroughly mixed using an electromagnetic stirrer. The administration solution was protected from light, and small amounts were taken out daily for administration. All remaining administration solution was stored in the refrigerator for a maximum of 7 days. The final administration matrix of 1 mg / kg AZD5305 was a clear solution.

[0290] measurement Tumor growth inhibition (TGI) was calculated as follows: TGI% = {1 - (MTV treatment / MTV control)} * 100 In the formula, MTV = mean tumor volume.

[0291] Statistical significance was assessed using a one-sided t-test of (log(relative tumor volume) = log(final volume / initial volume)) at the last measurement day, compared to the vehicle control.

[0292] result Figure 17 shows tumor volume for treatment with DS-8201 or AZD5305 alone, or DS-8201 in combination with AZD5305. The data represent the change in tumor volume over time for each treatment group. The dotted line in Figure 17 indicates the end of the treatment period. For information on total dose and schedule, please refer to Table 4 above. The values ​​shown are mean ± SEM; initially, n=10 mice were treated with the vehicle, and n=8 for all other treatment groups.

[0293] Table 5 shows the TGI response (TGI%) at day 41 in NCI-N87 xenografts after treatment with DS-8201 or AZD5305 alone, or with DS-8201 in combination with AZD5305.

[0294] [Table 7]

[0295] Monotherapy with DS-8201 at 3 mg / kg showed a TGI of 62% at 41 days post-treatment. At 1 mg / kg, DS-8201 showed a TGI of 25% at 41 days post-treatment. Monotherapy with AZD5305 reached a TGI of 40% at 41 days post-treatment. Combination therapy with AZD5305 and DS-8201 at 1 mg / kg resulted in a TGI of 55% at 41 days post-treatment. Combination therapy with higher doses of DS-8201 and AZD5305 at 3 mg / kg achieved a significant TGI of 90% at 41 days post-treatment, showing a better response than any of the corresponding monotherapies.

[0296] The treatment groups were generally well-tolerated (the study was discontinued for two abnormal animals due to >15% weight loss), and the mean body weight of all treatment groups remained stable throughout the study.

[0297] Example 6: In vitro administration of the antibody-drug conjugate DS-8201 (trastuzumab deruxtecan (Enhertu®)) and the PARP1 selective inhibitor AZD5305 (5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyrizin-3-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide) in cell lines expressing high HER2, low HER2, and HER2 mutants. method: A high-throughput combination screen was performed, and four lung cancer cell lines with various HER2 expression levels (Table 6) and HER2 mutant cancer cell lines (Table 7) were screened using combinations of DS-8201 and AZD5305.

[0298] [Table 8]

[0299] [Table 9]

[0300] The values ​​displayed on the screen were from a 7-day CellTiter-Glo cell viability assay performed as a 6×6 dose-response matrix (DS-8201 and AZD5305 were semi-logarithmic serial dilutions for each combination). The maximum concentration was 3.33 or 10 μM for AZD5305 and 100 μg / ml for DS-8201. Combination activity was evaluated based on the combination of ΔEmax and Loewe synergy scores.

[0301] result: The results for HER2+, HER2-low, and HER2-low / null NSCLC cell lines (HCC1171, NCIH1573, NCIH2170, Calu6) are shown in Figures 18A, 18B, and 18C, and in Table 8. The results for the HER2 mutant cell line (5637) are shown in Figures 19A, 19B, and 19C, and in Table 9.

[0302] Figures 18A and 19A show the matrix of measured cell viability signals. 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 compared to DMSO control on day 7. All values ​​are normalized to the cell viability value on day 0. Values ​​between 0 and 100 represent the percentage of growth inhibition, and values ​​above 100 represent cell death.

[0303] Figures 18B and 19B show the Loewe excess matrix. The values ​​in the boxes represent the excess values ​​calculated by the Loewe additivity model.

[0304] Figures 18C and 19C show the HSA excess matrix. The values ​​in the boxes represent the excess values ​​calculated by the HSA (best single agent) model.

[0305] Tables 8 and 9 show the HSA synergy score and Loewe additivity score.

[0306] [Table 10]

[0307] [Table 11]

[0308] As can be seen from Figures 18A, 18B, and 18C, and Table 8, AZD5305 interacts synergistically with DS-8201 and increased 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 / null cell line Calu6 at Emax (1.25 μM AZD5305 and 100 μg / ml DS-8201). Combination activity was observed even when monotherapy activity was absent or low. A synergistic effect was observed in the cell line NCIH1573, but no cell death occurred.

[0309] As can be seen from Figures 19A, 19B, and 19C, and Table 9, AZD5305 interacts synergistically with DS-8201 and increased cell death at Emax (1.25 μM AZD5305 and 100 μg / ml DS-8201) in the HER2 mutant cell line 5637. Combination activity was also observed when AZD5305 was not active as a monotherapy.

[0310] The above description is considered sufficient to enable those skilled in the art to carry out the embodiments. The above description and examples describe in detail specific embodiments and represent the best form intended by the inventors. However, it should be understood that no matter how detailed the above description may be in writing, the embodiments can be carried out in many ways, and the claims include any equivalent thereof.

[0311] Free text for sequence listings Sequence ID 1 - Amino chain sequence of the heavy chain of the anti-HER2 antibody Sequence ID No. 2 - Amino chain sequence of the light chain of the anti-HER2 antibody SEQ ID NO: 3 - Amino acid sequence of heavy chain CDRH1 [= Amino acid residues 26-33 of SEQ ID NO: 1] SEQ ID NO: 4 - Amino acid sequence of heavy chain CDRH2 [= Amino acid residues 51-58 of SEQ ID NO: 1] SEQ ID NO: 5 - Amino acid sequence of heavy chain CDRH3 [= Amino acid residues 97-109 of SEQ ID NO: 1] SEQ ID NO: 6 - Amino acid sequence of light chain CDRL1 [= Amino acid residues 27-32 of SEQ ID NO: 2] SEQ ID NO: 7 - Amino acid sequence (SAS) containing the amino acid sequence of light chain CDRL2 [= amino acid residues 50-56 of SEQ ID NO: 2] SEQ ID NO: 8 - Amino acid sequence of light chain CDRL3 [= Amino acid residues 89-97 of SEQ ID NO: 2] SEQ ID NO: 9 - Amino acid sequence of the heavy chain variable region [= Amino acid residues 1-120 of SEQ ID NO: 1] SEQ ID NO: 10 - Amino acid sequence of the light chain variable region [= Amino acid residues 1-107 of SEQ ID NO: 2] SEQ ID NO: 11 - Amino acid sequence of the heavy chain [= Amino acid residues 1-449 of SEQ ID NO: 1]

Claims

1. A pharmaceutical product comprising an anti-HER2 antibody-drug conjugate and a PARP1 selective inhibitor for combined administration, The aforementioned anti-HER2 antibody-drug conjugate has the following formula: 【Chemistry 1】 (In the formula, A represents the attachment site to the antibody.) This is an antibody-drug conjugate in which a drug-linker represented by is conjugated to an anti-HER2 antibody via a thioether linkage. The anti-HER2 antibody is an antibody comprising a heavy chain containing CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 3, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 4, and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain containing CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 6, CDRL2 consisting of the amino acid sequence of amino acid residues 1 to 3 of SEQ ID NO: 7, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:

8. The PARP1 selective inhibitor is given by the following formula (I): 【Chemistry 2】 (In the formula, X1 and X2 are independently selected from N and C(H), X3 is independently selected from N and C(R4) (where R4 is H or fluoro), R1 is a C1-4 alkyl or C1-4 fluoroalkyl, R2 is independently selected from H, halo, C1-4 alkyl, and C1-4 fluoroalkyl. A compound represented by R3 (where R3 is H or C1-4 alkyl), or a pharmaceutically acceptable salt thereof. (however, When X1 is N, then X2 is C(H) and X3 is C(R4), When X2 is N, then X1 is C(H) and X3 is C(R4); When X3 is N, then both X1 and X2 are C(H). Pharmaceutical product.

2. In equation (I), R ３ C １～４ The pharmaceutical product according to claim 1, wherein it is alkyl.

3. In equation (I), R ３ The pharmaceutical product according to claim 1, wherein is methyl.

4. In equation (I), R １ The pharmaceutical product according to any one of claims 1 to 3, wherein the ethyl acetate is present.

5. The PARP1 selective inhibitor is given by the following formula (Ia): 【Transformation 3】 (In the formula, R １ However, C １～４ It is alkyl, R ２ is selected from H, halo, C １～４ alkyl, and C １～４ fluoroalkyl, R ３ However, H or C １～４ It is alkyl, R ４ Compounds represented by (where H is) The pharmaceutical product according to claim 1, or a pharmaceutically acceptable salt thereof.

6. In equation (Ia), R ２ The pharmaceutical product according to claim 5, wherein is H or halo.

7. In equation (Ia), R １ is ethyl, and R ２ R is selected from H, chloro and fluoro, ３ The pharmaceutical product according to claim 5, wherein is methyl.

8. The PARP1 selective inhibitor is given by the following formula: 【Chemistry 4】 AZD5305, represented by The pharmaceutical product according to claim 1, or a pharmaceutically acceptable salt thereof.

9. The pharmaceutical product according to any one of claims 1 to 8, wherein the anti-HER2 antibody is an antibody comprising a heavy chain including a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 9, and a light chain including a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO:

10.

10. The pharmaceutical product according to any one of claims 1 to 8, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of an amino acid sequence represented by SEQ ID NO:

2.

11. The pharmaceutical product according to any one of claims 1 to 8, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 11 and a light chain consisting of an amino acid sequence represented by SEQ ID NO:

2.

12. The aforementioned anti-HER2 antibody-drug conjugate has the following formula: 【Transformation 5】 A pharmaceutical product according to any one of claims 1 to 11, represented by the formula (wherein “antibody” refers to the anti-HER2 antibody conjugated to the drug-linker via a thioether bond, and n refers to the average number of units of the drug-linker conjugated per antibody molecule in the antibody-drug conjugate, with n being in the range of 7 to 8).

13. The pharmaceutical product according to any one of claims 1 to 12, wherein the anti-HER2 antibody-drug conjugate is trastuzumab deruxtecan (DS-8201).

14. The pharmaceutical product according to any one of claims 1 to 13, wherein the product is a composition comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for co-administration.

15. The pharmaceutical product according to any one of claims 1 to 13, wherein the product is a combination preparation comprising the anti-HER2 antibody-drug conjugate and the PARP1 selective inhibitor for continuous or simultaneous administration.

16. A pharmaceutical product according to any one of claims 1 to 15, for the treatment of cancer.

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

18. The pharmaceutical product according to claim 17, wherein the cancer is breast cancer.

19. The pharmaceutical product according to claim 18, wherein the breast cancer has an IHC status score of 3+.

20. The pharmaceutical product according to claim 18, wherein the breast cancer is HER2-low expressing breast cancer.

21. The pharmaceutical product according to claim 18, wherein the breast cancer has an IHC 2+ HER2 status score.

22. The pharmaceutical product according to claim 18, wherein the breast cancer has an IHC status score of 1+.

23. The pharmaceutical product according to claim 18, wherein the breast cancer has an IHC status score of IHC > 0 and < 1+.

24. The pharmaceutical product according to claim 18, wherein the breast cancer is triple-negative breast cancer.

25. The pharmaceutical product according to claim 16, wherein the cancer is gastric cancer.

26. The pharmaceutical product according to claim 16, wherein the cancer is colorectal cancer.

27. The pharmaceutical product according to claim 16, wherein the cancer is lung cancer.

28. The pharmaceutical product according to claim 27, wherein the lung cancer is non-small cell lung cancer.

29. The pharmaceutical product according to claim 16, wherein the cancer is pancreatic cancer.

30. The pharmaceutical product according to claim 16, wherein the cancer is ovarian cancer.

31. The pharmaceutical product according to claim 16, wherein the cancer is prostate cancer.

32. The pharmaceutical product according to claim 16, wherein the cancer is kidney cancer.

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