Methods for treating brain tumors and neuroblastomas

Abstract

The present disclosure relates to a method of treating a brain tumor or neuroblastoma in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a substituted azaquinolone compound having activity as a poly(ADP-ribose) polymerase (PARP) inhibitor.

Description

[Technical Field]

[0001] This disclosure relates to a method for treating a brain tumor or neuroblastoma in a patient requiring treatment for the brain tumor or neuroblastoma, comprising administering to the patient a therapeutically effective amount of a substituted azaquinolone compound, which is a poly(ADP-ribose) polymerase (PARP) inhibitor. Brain tumors and neuroblastomas may include alpha-thalassemia / X-linked intellectual disability syndrome (ATRX) deficiency phenotype. The PARP inhibitor may be administered to the patient in combination with alkylating chemotherapeutic agents and / or ionizing radiation. [Background technology]

[0002] Current therapeutic strategies for treating brain tumors such as gliomas include surgical resection, ionizing radiation, and alkylating chemotherapy such as temozolomide (TMZ). Unfortunately, many of these brain tumors, such as glioblastoma (GBM), are associated with a very poor clinical prognosis. While surgical, instrumental, diagnostic, and radiotherapy / chemotherapy strategies have slowly evolved over time, this has not translated into a significant increase in patient survival rates. Therefore, new treatment strategies for brain tumors such as GBM are needed.

[0003] Gliomas are classified using several molecular markers. Gliomas can be classified based on whether they contain mutations in the isocitrate dehydrogenase (IDH) 1 and / or 2 genes (Non-Patent Literature 1) and / or in the alpha-thalassemia / mental retardation syndrome X-linked (ATRX) gene (Non-Patent Literature 2). Furthermore, genetic alterations in the ATRX gene are associated with a subgroup of patients with poor outcomes in neuroblastoma patients (Non-Patent Literature 3). In addition, MGMT promotermethylation has been used as a marker to predict promising outcomes in GBM patients undergoing alkylating chemotherapy (Non-Patent Literature 4).

[0004] Temozolomide (TMZ) is the standard of care (SOC) chemotherapy for GBM (Non-Patent Documents 5; 6). Mechanistically, TMZ methylates DNA at specific sites, which, if left unrepaired, leads to DNA adducts that cause single-strand and double-strand breaks, cell cycle arrest, and apoptosis (Non-Patent Document 7). Importantly, PARP (particularly PARP1) is a key enzyme involved in the early stages of the DNA damage repair (DDR) process as a response to single-strand and double-strand breaks. In fact, one of the key functions of PARP1 is during base excision repair (BER), where DNA adducts generated by TMZ are removed and downstream factors are recruited to repair the damage. Various studies have investigated the combined benefits of PARP inhibitors (PARPi) and TMZ (Non-Patent Documents 8, 9, and 10).

[0005] Ionizing radiation (IR) induces single-strand and double-strand DNA breaks, leading to cell cycle arrest and cell death (Non-Patent Literature 11). Importantly, PARP1 is involved in repairing these types of DNA damage. Various studies have investigated the combined benefits of PARPi and IR (Non-Patent Literature 12). In addition to increasing the sensitivity of gliomas to radiation, it has also been reported that PARPi increases the sensitivity of ependymomas to radiation (Non-Patent Literature 13).

[0006] Examples of PARP inhibitors and their mechanisms of action are taught, for example, in Patent Document 1.

[0007] PARP1 and PARP2 are the most widely studied PARPs for their roles in DNA damage repair. PARP1 is activated by DNA damage cleavage and functions to catalyze the addition 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 DNA damage.

[0008] After this mobilization role is complete, PARP auto-PARization triggers the release of bound PARP from the DNA, completing the repair through access to other DNA repair proteins. Therefore, PARP's binding to the damage site, its catalytic activity, and its final release from the DNA are all crucial steps in cancer cells' response to DNA damage caused by chemotherapy and radiotherapy (Non-Patent Literature 14).

[0009] PARP inhibitors with improved selectivity for PARP1 are thought to have improved efficacy and reduced toxicity compared to other clinical PARP1 / 2 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.

[0010] Therefore, the development of PARP inhibitors that can be used as monotherapy and in combination with alkylating chemotherapy and ionizing radiation for the safe and effective treatment of brain tumors and neuroblastoma is required in this technology. Such PARP inhibitors may exhibit selectivity for PARP1.

[0011] The applicant has discovered that the azaquinolone described herein, surprisingly, possesses PARP inhibitory activity and may therefore be useful in treating diseases and symptoms in which PARP function has pharmacological significance. Furthermore, the azaquinolone described herein exhibits remarkably high selectivity for PARP1 compared to other PARP family members such as PARP2, PARP3, PARP5a, and PARP6.

[0012] The applicant has further discovered that the azaquinolone described herein is, remarkably, able to penetrate the blood-brain barrier (BBB) ​​and is useful in the treatment of brain tumors such as gliomas.

[0013] The applicant demonstrated, using both in vitro and in vivo models, that PARP inhibitors possessing these properties can be used to treat gliomas. As described herein, PARP inhibitors were demonstrated to have potent activity as monotherapy in ATRX mutant cell lines, both in IDH1 mutant and IDH1 wild-type backgrounds. [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] International Publication No. 2004 / 080976 Pamphlet [Non-patent literature]

[0015] [Non-Patent Document 1] Hartmann et al. 2009 [Non-Patent Document 2] Haase et al. 2018 [Non-Patent Document 3] George et al. 2020 [Non-Patent Document 4] Weller et al. 2010 [Non-Patent Document 5] Stupp et al., 2014 [Non-Patent Document 6] Stupp et al., 2010 [Non-Patent Document 7] Singh et al., 2021 [Non-Patent Document 8] Gupta et al., 2018 [Non-Patent Document 9] Higuchi et al., 2020 [Non-Patent Document 10] Murai et al., 2014 [Non-Patent Document 11] Reisz et al., 2014 [Non-Patent Document 12] Jannetti et al., 2020 [Non-Patent Document 13] van Vuurden et al., 2011 [Non-Patent Document 14] Bai 2015 [Summary of the Invention] [Means for Solving the Problems]

[0016] In a first aspect, the applicant provides a method of treating a brain tumor or neuroblastoma in a patient in need of treatment of a brain tumor or neuroblastoma, comprising administering to the patient a therapeutically effective amount of a poly(ADP-ribose) polymerase (PARP) inhibitor, wherein the brain tumor or neuroblastoma comprises an alpha-thalassemia / mental retardation syndrome X-linked (ATRX) deficient phenotype, and the PARP inhibitor is a compound of formula (I): [Chemical Formula] (wherein R 1 is each independently selected from H, C 1~4 alkyl, C 3~6 cycloalkyl, C 1~4 fluoroalkyl, and C 1~4 alkyloxy; R 2 is each independently selected from H, halo, C 1~4 alkyl, and C 1~4 fluoroalkyl; R 3 is H or C 1~4 alkyl; R 4 is halo or C 1~4 alkyl) or a pharmaceutically acceptable salt thereof, and the method is made available.

[0017] In some embodiments, the brain tumor or neuroblastoma further includes an isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) deficiency phenotype (e.g., considered to be an IDH1 mutant and / or an IDH2 mutant). In other embodiments, the brain tumor or neuroblastoma does not include an IDH1 or IDH2 phenotype (e.g., considered to be IDH1 wild-type and / or IDH1 wild-type).

[0018] In some embodiments, brain tumors or neuroblastomas involve O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation.

[0019] In some embodiments, the method further includes the step of diagnosing the patient as having a brain tumor or neuroblastoma containing an ATRX deficiency phenotype before administering a PARP inhibitor. In other embodiments, the patient to be treated has been previously diagnosed as having a brain tumor or neuroblastoma containing an ATRX deficiency phenotype. Methods for diagnosing the patient are described herein and may include, for example, assaying cells obtained from a brain tumor or neuroblastoma, or from cerebrospinal fluid (CSF) derived from the patient.

[0020] Furthermore, the applicant has demonstrated that the PARP inhibitors disclosed herein exhibit synergistic antitumor activity when used in combination with alkylating chemotherapeutic agents, including temozolomide (TMZ) and Val-083, and / or with ionizing radiation.

[0021] Therefore, in some embodiments, PARP inhibitors are used in patients, i) Alkylating chemotherapeutic agents; and / or ii) Radiation therapy agents It is administered in combination with [another drug].

[0022] In a second embodiment, the applicant provides a method for treating a brain tumor or neuroblastoma in a patient requiring treatment for the brain tumor or neuroblastoma, comprising administering to the patient a therapeutically effective amount of a poly(ADP-ribose) polymerase (PARP) inhibitor, and i) Alkylating chemotherapeutic agents; and / or ii) Radiotherapy agents administered at doses of 10 Gy or more This includes administering, The PARP inhibitor is a compound of formula I as defined in the first embodiment, making the method available.

[0023] In some embodiments, brain tumors or neuroblastomas are: (i) Alphathalassemia / X-linked (ATRX) deficiency phenotype; (ii) isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) deficiency phenotype; and / or (iii)O 6 - Methylguanine-DNA methyltransferase (MGMT) promoter-methylation Includes.

[0024] In some embodiments, the alkylating chemotherapeutic agent is temozolomide (TMZ) or dianhydrogalactitol (Val-083).

[0025] The combination of the PARP inhibitor and TMZ described herein has been demonstrated to provide antitumor benefits without requiring high doses of TMZ. This is beneficial because it suggests that brain tumors and neuroblastomas can be treated with reduced doses of alkylating chemotherapeutic agents using combination therapies including PARP inhibitors, which can advantageously enable effective cancer treatment with reduced toxic side effects associated with chemotherapy. Similarly, the use of the PARP inhibitor of the present invention has been demonstrated to effectively treat gliomas in combination with reduced doses of clinically relevant ionizing radiation. This suggests that effective cancer treatment with reduced toxic side effects associated with radiotherapy can be achieved.

[0026] Therefore, in some embodiments, TMZ: (i) Approximately 200mg / m 2 Doses less than approximately 150 mg / m² 2Less than a certain dose, approximately 125 mg / m² 2 Doses less than 100 mg / m², or approximately 100 mg / m² 2 Less than a dose; (ii) Approximately 50~200mg / m 2 The dosage is approximately 75-150 mg / m². 2 The dosage, or approximately 75 mg / m² 2 ~125 mg / m² 2 dosage It is administered via [method / service name].

[0027] In some embodiments, VAL-083 is: (i) Approximately 50mg / m 2 Less than a certain dose, approximately 40 mg / m² 2 Less than a dose, approximately 30 mg / m² 2 Less than a dose of approximately 20 mg / m² 2 Less than a dose; (ii) Approximately 10~50mg / m 2 The dosage is approximately 10-40 mg / m². 2 The dosage, or approximately 10 mg / m² 2 ~30mg / m 2 dosage It is administered via [method / service name].

[0028] The listed dosages of TMZ and Val-083, described in more detail herein, are typically daily doses.

[0029] In some embodiments, radiotherapy agents: (i) doses less than approximately 60 Gy, less than approximately 55 Gy, less than approximately 50 Gy, less than approximately 45 Gy, or less than approximately 40 Gy; or (ii) doses of approximately 20 Gy to 60 Gy, approximately 20 Gy to 55 Gy, approximately 20 Gy to 50 Gy, approximately 20 to 45 Gy, approximately 20 to 40 Gy, approximately 30 Gy to 60 Gy, approximately 30 Gy to 55 Gy, approximately 30 Gy to 50 Gy, approximately 30 Gy to 45 Gy, or approximately 30 Gy to 40 Gy It is administered via [method / service name].

[0030] Ionizing radiation, as described in more detail herein, is typically administered as subdivided radiotherapy.

[0031] In some embodiments, the brain tumor is a glioma or ependymoma. In some embodiments, the brain tumor is a glioma. The glioma may be an adult glioma or a paediatric glioma, and may be a high-grade glioma or a low-grade glioma. In some embodiments, the glioma is a paediatric glioma. In some embodiments, the glioma is a high-grade glioma, for example, a glioma selected from the list consisting of oligodendroglioma, undifferentiated astrocytoma, glioblastoma, and diffuse median glioma. In some embodiments, the glioma is a glioblastoma.

[0032] Furthermore, as demonstrated herein, the PARP inhibitors of the present invention, combined with ionizing radiation, have been shown to treat a cell line model of H3K27M mutant diffuse median glioma. H3K27M mutant diffuse median glioma is a high-grade pediatric brain tumor with a poor prognosis that is difficult to treat with current therapeutic strategies. The data demonstrated herein support the potential of the PARP inhibitors of the present invention to be used as a method for treating this difficult glioma.

[0033] Therefore, in some embodiments, the glioma is an H3K27M mutant glioma.

[0034] Also available are PARP inhibitors used in a method for treating a brain tumor or neuroblastoma in a patient, the method comprising administering a therapeutically effective amount of a PARP inhibitor to the patient, the brain tumor or neuroblastoma comprising the alpha-thalassemia / mental retardation syndrome X-linked (ATRX) deficiency phenotype, and the PARP inhibitor being a compound of formula I as defined in the first embodiment.

[0035] Also available are PARP inhibitors used in methods to treat brain tumors or neuroblastomas in patients, the method being a therapeutically effective amount of PARP inhibitor, and i) Alkylating chemotherapeutic agents; and / or ii) Ionizing radiation administered at doses of 10 Gy or more This includes administering to the patient (for example, separately, sequentially, or simultaneously), PARP inhibitors are compounds of formula I as defined in the first embodiment.

[0036] Also available are alkylating chemotherapeutic agents used in methods for treating brain tumors or neuroblastomas in patients, the method comprising administering an alkylating chemotherapeutic agent and a therapeutically effective amount of a PARP inhibitor to the subject (e.g., separately, sequentially, or simultaneously), the PARP inhibitor being a compound of formula I as defined in the first embodiment.

[0037] In one embodiment of the above-mentioned model, the compound of formula (I) is: [ka] (In the formula, R 1 H and C are independent of each other. 1~4 Alkyl, C 1~4 Fluoroalkyl, and C 1~4 Selected from alkyloxy, R 2 These are H, Halo, and C, independently. 1~4 Alkyl and C 1~4 Selected from fluoroalkyl groups, R 3 H or C 1~4 It is alkyl, R 4 is a halo or C 1~4 (It is alkyl.) It is a compound of or a pharmaceutically acceptable salt thereof.

[0038] In one embodiment of the above-mentioned embodiment, R of formula (I) or formula (Ia) 1 R is selected from one of methyl, ethyl, isopropyl, cyclopropyl, 1,1-difluoroethyl, 1-fluoroethyl, trifluoromethyl, difluoromethyl, and methoxy. In a particular embodiment, R 1 It is either methyl or ethyl.

[0039] In one embodiment of the above-mentioned embodiment, R of formula (I) or formula (Ia) 2 R is selected from one of H, chloro, fluoro, methyl, and difluoromethyl. In one embodiment, R 2 It is either fluoro or methyl.

[0040] In one embodiment of the above-mentioned embodiment, R of formula (I) or formula (Ia) 3 It is either methyl or ethyl.

[0041] In one embodiment of the above-mentioned embodiment, R of formula (I) or formula (Ia) 4 R is selected from one of chloro, fluoro, and methyl. In a particular embodiment, R 4 It is fluoro.

[0042] In one embodiment of the above-mentioned embodiment, formula (I) or formula (Ia) (wherein R 1 is C 1~4 It is alkyl, R 2 It is a halo, R 3 is C 1~4 It is alkyl, R 4 is Halo or C 1~4 Compounds (which are alkyl) or pharmaceutically acceptable salts thereof are provided.

[0043] In a further embodiment, the compound of formula (I) or formula (Ia) is in the form of a free base.

[0044] In one embodiment, a compound of formula (I), which is 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, or a pharmaceutically acceptable salt thereof is provided.

[0045] In one embodiment, a compound of formula (I), which is 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, or a pharmaceutically acceptable salt thereof is provided.

[0046] In one embodiment, a compound of formula (I) which is crystalline form B of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, or a pharmaceutically acceptable salt thereof, is provided.

[0047] In one embodiment, a compound of formula (I) which is crystalline form D of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, or a pharmaceutically acceptable salt thereof, is provided.

[0048] In one embodiment, a compound of formula (I), which is 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide mesylate, is provided as crystalline form C by option.

[0049] Further embodiments will become apparent to those skilled in the art by reading this specification.

[0050] 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. Blockade of hERG may cause safety problems such as arrhythmias. Advantageously, the compound of formula (I) has low hERG activity. In one embodiment, the compound of formula (I) has an IC50 of >10 μM. In another embodiment, the compound of formula I has an IC50 of >20 μM.

[0051] To minimize the risk of off-target effects, it is desirable that drug molecules have selectivity for specific targets. The compound of formula I is advantageously more selective for PARP1 than other members of the PARP family, including PARP2, PARP3, PARP5a, and PARP6. Advantageously, the compound of formula (I) is more selective for PARP1 than for PARP2. In one embodiment, the compound of formula (I) is 10 times more selective for PARP1 than for PARP2. In one embodiment, the compound of formula (I) is 100 times more selective for PARP1 than for PARP2.

[0052] In one embodiment, the patient is heterozygous for one or more variations, e.g., mutations and polymorphisms, in BRCA1 and / or BRCA2, or their regulators. Detection of variations in BRCA1 and BRCA2 is well known in the art and is described, for example, in European Patent No. 699754; European Patent No. 705903; Neuhausen and Ostrander 1997; Chappnis and Foulkes 2002; Janatova et al., 2003; Jancarkova 2003. Determination of amplification of the BRCA2 binding factor EMSY is described in Hughes-Davies, et al. (Hughes-Davies, et al 2003).

[0053] Cancer-associated mutations and polymorphisms can be detected at the nucleic acid level by detecting the presence of variant nucleic acid sequences, or at the protein level by detecting the presence of variant (i.e., mutant or allele variant) polypeptides.

[0054] definition 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 5~6It is an alkyl group. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl, such as methyl or n-hexyl.

[0055] Cycloalkyl groups are saturated cyclic alkyl groups. 3~6 Cycloalkyls are saturated cyclic alkyl groups having 3 to 6 carbon atoms. 3~6 Examples of cycloalkyl compounds include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. 3~6 As a cycloalkyl, C 3~5 Cycloalkyl and C 3~4 Cycloalkyl groups are examples.

[0056] A fluoroalkyl group is an alkyl group in which one or more H atoms are replaced by 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-), 1-fluoroethyl (CH3CHF-), and 2-fluoroethyl (CH2FCH2-).

[0057] Halo means fluoro, chloro, bromo, and iodine. In one embodiment, halo is fluoro or chloro.

[0058] An alkyloxy group is an alkyl group that is bonded to the remainder of a molecule via an oxygen atom. Suitable examples of C1-4 alkyloxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, sec-butoxy, and t-butoxy.

[0059] 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.

[0060] In this specification, unless otherwise specified, the term “effective dose” means the amount of compound or composition sufficient to significantly and positively alter (e.g., produce a positive clinical response) the sign and / or symptom being treated. The effective dose of the active ingredient used in a pharmaceutical composition will vary depending on the specific symptom being treated, the severity of the symptom, the duration of treatment, the nature of the combination therapy, the specific active ingredient used, the specific pharmaceutically acceptable excipients / carriers used, and similar factors within the knowledge and expertise of the attending physician.

[0061] As used herein, the term “to treat” means, unless otherwise indicated, to restore, alleviate, inhibit the progression of, delay the worsening of, delay the onset of, or prevent the disorder or symptom to which such term applies, or one or more signs of such disorder or symptom. As used herein, the term “treatment” means, unless otherwise indicated, the act of treating as defined above. The term “to treat” also includes adjuvant and neoadjuvant treatments of the subject. To avoid ambiguity, references to “treatment” herein include curative, palliative, and prophylactic treatments, and the administration of drugs used in such treatments.

[0062] Compounds of formula (I) or formula (Ia) can form stable, pharmaceutically acceptable acidic or basic salts, in which case it may be appropriate to administer the compound as a salt. Examples of acid addition salts include acetate, adipine, ascorbate, benzoate, benzenesulfonate, bicarbonate, bisulfate, butyrate, camphorate, camphor sulfonate, choline, citrate, cyclohexylsulfamate, diethylenediamine, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxyethylsulfonate, heptanoate, hexanoate, hydrochloride, hydrochloride, hydroiodide, hydroxymaleate, and lactic acid. Examples include salts, malates, maleates, methanesulfons (mesylates), meglumines, 2-naphthalenesulfons, nitrates, oxalates, pamoates, persulfates, phenylacetates, phosphates, diphosphates, picrates, pivalates, propions, quinates, salicylates, stearates, succinates, sulfamates, sulfanilates, sulfates, tartrates, tosylates (p-toluenesulfons), trifluoroacetates, and undecanoates. Non-toxic, physiologically acceptable salts are preferred, but other salts may also be useful, for example, in the isolation or purification of the product.

[0063] Salts can be formed by conventional means, for example, by reacting a product in free base form with one or more equivalents of suitable acids in a solvent or medium in which the salt is insoluble, or in a solvent such as water (which is removed by vacuum), or by freeze-drying, or by exchanging anions of an existing salt on a suitable ion-exchange resin with other anions.

[0064] Compounds of formula (I) or formula (Ia) may have multiple chiral centers, and naturally, this application encompasses all individual stereoisomers, enantiomers, and diastereoisomers, as well as mixtures thereof. Therefore, naturally, insofar as compounds of formula I can exist as optically active or racemic compounds due to one or more chiral carbon atoms, this application includes, within its definition, all optically active or racemic compounds having the above-described activity. This application encompasses all such stereoisomers having the activity defined herein.

[0065] Therefore, throughout this specification, when referring to a compound of formula (I) or formula (Ia), the term compound naturally includes diastereoisomers, mixtures of diastereoisomers, and enantiomers that are PARP1 inhibitors.

[0066] Furthermore, naturally, a compound of a particular formula (I) or formula (Ia) and its pharmaceutically acceptable salts can exist in solvated and non-solvated forms, such as 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 (e.g., hydrated) form of the salt of the compound.

[0067] Formulas (I) or (Ia) described herein are intended to encompass all isotopes of their 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, and 14 Includes 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 and18 This includes all isotopic forms of fluorine, such as F; etc. 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 lower abundances. For example, 1 H is usually present in amounts exceeding 99.98%, but in one embodiment, any compound of any of the formulas shown herein has H at one or more positions. 2 H or 3 H may be enriched. In another embodiment, a radioactive isotope of any compound of any of the formulas shown herein, for example, 3 H and 14 When enriched with 1C, the compound may be useful in drug and / or substrate tissue distribution assays. Naturally, this application encompasses all such isotopic forms.

[0068] Compounds of formula (I) or formula (Ia), 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.

[0069] Pharmaceutical formulations of the compounds of formula (I) or formula (Ia) described 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, 2011).

[0070] Pharmaceutical formulations of the compounds of formula (I) or formula (Ia) described above can be conveniently administered in unit dosage form and can be prepared by any method well known in pharmaceutical technology, for example, as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA. (1985).

[0071] Pharmaceutical formulations suitable for oral administration may contain one or more physiologically compatible carriers and / or excipients and may be in solid or liquid form. Tablets and capsules may be prepared with binders, fillers, lubricants, and / or surfactants, such as sodium lauryl sulfate. Liquid compositions may contain conventional additives such as suspending agents, emulsifiers, and / or preservatives. Liquid compositions may be encapsulated in gelatin, for example, to provide 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 may be prepared, for example, by filling a gelatin or hydroxypropyl methylcellulose (HPMC) shell with the compound of formula (I).

[0072] Dry shell formulations typically contain gelatin at a concentration of approximately 40 w / w% to 60 w / w%, plasticizers at a concentration of approximately 20% to 30% (such as glycerin, sorbitol, or propylene glycol), and water at a concentration of approximately 30% to 40%. Other materials such as preservatives, dyes, opacifiers, and flavorings may also be present. Liquid fillers contain 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 oil, vegetable oil, triglycerides, glycols, polyols, and surfactants.

[0073] The appropriate daily dose of a compound of formula (I) or formula (Ia) or a pharmaceutically acceptable salt thereof in human therapeutic treatment is approximately 0.0001 to 100 mg / kg of body weight.

[0074] 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.

[0075] Molecular markers Brain tumors, such as gliomas and neuroblastomas, can be characterized based on their genetic and molecular classifications.

[0076] In some embodiments, the brain tumor or neuroblastoma includes the alpha-thalassemia / mental retardation syndrome X-linked (ATRX) deficiency phenotype.

[0077] The human ATRX gene occupies approximately 300 kbp of the q21.1 band on the long arm of the X chromosome. The gene, containing 36 exons, encodes a 2492-amino acid protein with a molecular weight of 282,586 kDa, although spliced ​​transcript variants have also been identified. The NCBI Entrez Gene ID for human ATRX is Gene ID: 546. The protein contains an ATPase / helicase domain and a domain called ADD(ATRX-DNMT3-DNMT3L, ADDATRX) that binds to histone H3. The amino acid sequence for the longest isoform of human ATRX is provided as NCBI reference sequence: NP_000480.3.

[0078] Inactivating mutations in the ATRX gene in gliomas have been reported to be uniformly distributed throughout the gene and to be present in 7% of adult glioblastomas (GBMs) and 14–31% of pediatric GBMs (Jiao et al. 2012). ATRX loss has been reported to promote tumor growth and impair non-homologous end-joining DNA repair in gliomas (Koschmann et al. 2017).

[0079] As used herein, a brain tumor or neuroblastoma containing an ATRX-deficient phenotype means that ATRX activity is reduced or inactivated within the cancer cells. ATRX activity is typically eliminated or inactivated by mutations in the ATRX gene. Therefore, in some embodiments, a brain tumor or neuroblastoma containing an ATRX-deficient phenotype includes cancer cells having mutations in the ATRX gene. Such mutations within the cancer cells are typically heterozygous, and the cancer cells contain one mutant copy and one wild-type copy of the ATRX gene.

[0080] In some embodiments, the brain tumor or neuroblastoma includes an isocitrate dehydrogenase (IDH) 1 and / or 2 deficiency phenotype. In some embodiments, the brain tumor or neuroblastoma includes an IDH1 deficiency phenotype. In some embodiments, the brain tumor or neuroblastoma includes an IDH2 deficiency phenotype. In some embodiments, the brain tumor or neuroblastoma includes both IDH1 and IDH2 deficiency phenotypes. In some embodiments, the brain tumor or neuroblastoma does not include an IDH1 deficiency phenotype (i.e., it is IDH1 wild-type). In some embodiments, the brain tumor or neuroblastoma does not include an IDH2 deficiency phenotype (i.e., it is IDH2 wild-type). In some embodiments, the brain tumor or neuroblastoma does not include an IDH1 or IDH2 deficiency phenotype (i.e., it is IDH1 and IDH2 wild-type).

[0081] Mutations in IDH1 and IDH2 are common in human malignancies. In gliomas, IDH1 and IDH2 mutations are recognized in >80% of World Health Organization (WHO) grade II / III cases. Cancer-associated IDH1 and IDH2 mutations tend to localize to arginine residues important for isocitrate recognition and enzyme catalytic activity (R132 for IDH1, and R140 or R172 for IDH2). By reducing or inactivating the catalytic activity of these enzymes, the ability of cells to combat reactive oxygen species is affected (Cohen et al. 2013). The NCBI Entrez Gene ID for human IDH1 is Gene ID: 3417, and the NCBI Entrez Gene ID for human IDH2 is Gene ID: 3418. The amino acid sequence for human IDH1 is provided as NCBI reference sequence: NP_001269315.1, and the amino acid sequence for human IDH2 is provided as NCBI reference sequence: NP_001276839.1.

[0082] As used herein, brain tumors or neuroblastomas comprising the IDH1 and / or IDH2 deficiency phenotype mean that IDH1 and / or IDH2 activity is reduced or inactivated in cancer cells. IDH1 or IDH2 activity is typically removed or inactivated by mutations in the IDH1 or IDH2 gene, respectively. Therefore, in some embodiments, brain tumors or neuroblastomas comprising the IDH1 and / or IDH2 deficiency phenotype comprise cancer cells having mutations in the IDH1 and / or IDH2 gene. Such mutations in cancer cells are typically heterozygous, and the cancer cells contain one mutant copy and one wild-type copy of the IDH1 gene. Mutations in the IDH1 gene may be nonsynonymous mutations in the nucleotide encoding amino acid residue R132 of IDH1, while mutations in the IDH2 gene may be nonsynonymous mutations in the nucleotide encoding amino acid residues R140 or R174 of IDH2.

[0083] In some embodiments, brain tumors or neuroblastomas are O 6 -Includes methylguanine-DNA methyltransferase (MGMT) promoter methylation.

[0084] The MGMT gene in humans is located on chromosome 10q26.3 and encodes an evolutionarily highly conserved and ubiquitously expressed enzyme involved in DNA repair. Methylation of the MGMT gene promoter induces a reduction in MGMT protein levels, which has been observed in approximately 50% of GBMs.

[0085] In some embodiments, the method includes a further step of diagnosing a patient with a brain tumor or neuroblastoma containing one or more of the above-mentioned molecular markers (ATRX mutation, IDH mutation, and MGMT methylation). Various methods can be used to perform genomic characterization of these tumors and to perform diagnosis. For example, the diagnostic step may include surgically isolating cells from the tumor and assaying cells obtained from a patient-derived brain tumor or neuroblastoma (i.e., tumor biopsy). In addition, non-invasive methods can be used. For example, there have been reports of obtaining cell-free circulating tumor DNA from cerebrospinal fluid (CSF) of brain tumor patients and assaying the CSF sample (Martinez-Ricarte et al. 2018).

[0086] Patient-derived samples (e.g., tumor-derived cells and / or CSF samples) can be evaluated for ATRX mutations, IDH1 and / or IDH2 mutations using any method known to those skilled in the art. Such methods, though not limited to them, include: magnetic resonance, chemical assays (e.g., high-performance liquid chromatography (HPLC)), sequencing, and PCR.

[0087] Various assays are known and available for evaluating MGMT gene promoter methylation. Suitable assays include those described in Mansouri et al. 2019, such as methylation-specific PCR, pyrosequencing, quantitative real-time PCR high-resolution fusion, methylation-specific multiplex ligation-dependent probe amplification, and immunohistochemistry.

[0088] Furthermore, brain tumors or neuroblastomas can be characterized based on their level of genomic instability. Studies have demonstrated that DNA damage checkpoint signaling is abnormally and constitutively active in gliomas. This can be measured using markers of ongoing DNA replication stress such as γgH2AX, as well as phosphorylation of Chk2, Chk1, and Rad17 (Bartkova et al. 2010). In some embodiments, brain tumors or neuroblastomas are characterized by a high level of genomic instability. This can be demonstrated by high levels of γgH2AX (e.g., higher than in non-tumor cells), as well as phosphorylation of Chk2, Chk1, and Rad17.

[0089] therapeutic use As used herein, the term “to treat” means, unless otherwise indicated, to restore, alleviate, inhibit the progression of, delay the worsening of, delay the onset of, or prevent the disorder or symptom to which such term applies, or one or more signs of such disorder or symptom. As used herein, the term “treatment” means the act of treating as defined above, unless otherwise indicated. The term “to treat” also includes adjuvant and neoadjuvant treatments of the subject (patient). To avoid ambiguity, references to “treatment” herein include curative, palliative, and prophylactic treatments, and the administration of drugs used in such treatments.

[0090] In this specification, unless otherwise specified, the term “effective dose” means the amount of compound or composition sufficient to significantly and positively alter (e.g., produce a positive clinical response) the sign and / or symptom being treated. The effective dose of the active ingredient used in a pharmaceutical composition will vary depending on the specific symptom being treated, the severity of the symptom, the duration of treatment, the nature of the combination therapy, the specific active ingredient used, the specific pharmaceutically acceptable excipients / carriers used, and similar factors within the knowledge and expertise of the attending physician.

[0091] The PARP inhibitor of the present invention may be formulated as a pharmaceutical composition for clinical use, and may comprise a pharmaceutically acceptable carrier, diluent, or adjuvant. The composition may be formulated for topical, parenteral, intravenous, intramuscular, intrathecal, intraocular, subcutaneous, oral, inhalation, or transdermal routes of administration, which may include injection. Injectable formulations may comprise the selected compound in a sterile or isotonic medium.

[0092] The PARP inhibitor of the present invention will generally be administered via the oral route in the form of a pharmaceutical preparation comprising 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 at various doses.

[0093] The pharmaceutical formulation of the PARP inhibitor described herein can be prepared for oral administration, particularly in the form of tablets or capsules, using techniques specifically aimed at providing colon-targeted drug release (Patel, 2011).

[0094] In therapeutic treatment for humans, a suitable daily dose of the PARP inhibitor described herein is about 0.0001 to 100 mg per kg of body weight.

[0095] Oral formulations are preferred, particularly tablets or capsules which can be formulated by methods known to those skilled in the art and provide a dose of the active compound ranging from 0.1 mg to 1000 mg. In some embodiments, the amount of the PARP inhibitor administered to a patient is 10 mg / day to 500 mg / day, 20 mg / day to 500 mg / day, 40 mg / day to 500 mg / day, 10 mg / day to 400 mg / day, 20 mg / day to 400 mg / day, or 40 mg / day to 400 mg / day.

[0096] Combination In some embodiments, the method includes administering a PARP inhibitor in combination with an alkylating chemotherapeutic agent and / or ionizing radiation. Such combination treatment may include separate, sequential, or simultaneous administration of the PARP inhibitor and the alkylating chemotherapeutic agent and / or ionizing radiation.

[0097] Also offered are pharmaceuticals comprising i) a PARP inhibitor as defined herein, and ii) an alkylating chemotherapeutic agent. PARP inhibitors and alkylating chemotherapeutic agents typically exist in separate dosage forms.

[0098] The alkylating chemotherapeutic agent may be temozolomide (TMZ).

[0099] TMZ is the standard of care (SOC) chemotherapy for GBM (Stupp et al., 2014; Stupp et al, 2010). Treatment involves administering 150-200 mg / m² for 5 days every 28 days. 2 This may include administering a daily dose. Temozolomide has the following structure: [ka] It has.

[0100] Therefore, in some embodiments, this method involves administering a therapeutically effective amount (e.g., 10 mg / day to 500 mg / day) of a PARP inhibitor described herein, and TMZ at a dose of 150 to 200 mg / m². 2 This may include administering it at a rate of / day. This procedure may occur throughout the entire 28-day cycle, where TMZ is administered daily for the first 5 days and then stopped for the remainder of the cycle. PARP inhibitors may be administered daily in the 28-day cycle, or only when TMZ is being administered (e.g., only for the first 5 days of the 28-day cycle).

[0101] Furthermore, the data provided herein demonstrates that the PARP inhibitor described herein can provide anti-tumor benefits in in vitro models and in vivo models at a relatively low dose of TMZ. While not wishing to be bound by theory, it is believed that the PARP inhibitor described herein may achieve clinical benefits (e.g., tumor reduction) at a lower dose of TMZ (e.g., lower than the standard-of-care dose of TMZ for treating the disease in question).

[0102] Therefore, in some embodiments, TMZ is administered (e.g., daily) at the following dose: less than about 200 mg / m 2 , less than about 175 mg / m 2 , less than about 150 mg / m 2 , less than about 125 mg / m 2 , less than about 100 mg / m 2 , less than about 75 mg / m 2 , or less than about 50 mg / m 2 .

[0103] In some embodiments, TMZ is administered (e.g., daily) at the following dose: about 50 to 200 mg / m 2 , about 50 to 175 mg / m 2 , about 50 to 150 mg / m 2 , about 50 to 125 mg / m 2 , about 50 to 100 mg / m 2 , about 50 to 75 mg / m 2 , about 75 to 200 mg / m 2 , about 75 to 175 mg / m 2 , about 75 to 150 mg / m 2 , about 75 to 125 mg / m 2 , about 75 to 100 mg / m 2 , about 100 to 200 mg / m 2 , about 125 to 175 mg / m 2 , or about 125 to 150 mg / m 2 .

[0104] The alkylating chemotherapeutic agent may be dianhydrogalactitol (Val-083). Val-083 is a blood-brain barrier-penetrating alkylating agent that induces interstrand crosslinking of DNA strands, causing double-strand DNA breaks (Jimenez-Alcazar et al., 2021).

[0105] Val-083 was previously an approved FDA and EMA orphan drug designation for the treatment of advanced GBM in relation to the TMZ treatment plan. Currently, Val-083 is in clinical trials in both MGMT-methylated GBM and MGMT-unmethylated GBM patients (NCT03050736, NCT02717962, and NCT03138629), and its structure: [ka] It has.

[0106] 30 mg / m² per day 2 VAL-083 was described as generally safe and well-tolerated when used in combination with radiotherapy. VAL-083 was administered on days 1, 2, and 3 every 21 days.

[0107] Therefore, in some embodiments, this method involves administering a therapeutically effective amount (e.g., 10 mg / day to 500 mg / day) of a PARP inhibitor described herein, and 10 to 50 mg / m² of Val-083. 2 / day (for example, 30 mg / m²) 2 This may include administering the drug at a rate of / day. This procedure may occur throughout the entire 21-day cycle, where VAL-083 is administered daily for the first three days and then stopped for the remainder of the cycle. PARP inhibitors may be administered daily in the 21-day cycle, or only when VAL-083 is being administered (e.g., only for the first three days of the 21-day cycle).

[0108] In some embodiments, VAL-083 is administered at the following dose (e.g., daily): approximately 50 mg / m² 2Less than approximately 40 mg / m² 2 Less than approximately 30 mg / m² 2 Less than or approximately 20 mg / m² 2 less than.

[0109] In some embodiments, VAL-083 is administered at the following doses (e.g., daily): approximately 10-50 mg / m² 2 , about 10~40mg / m 2 , or approximately 10 mg / m² 2 ~30mg / m 2 .

[0110] In some embodiments, the PARP inhibitors described herein may be administered in combination with ionizing radiation (for example, for the treatment of H3K27M gliomas). Ionizing radiation may be administered in addition to or instead of alkylating chemotherapeutic agents.

[0111] In certain embodiments, the dose of ionizing radiation is approximately 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, 70 Gy, 75 Gy, 80 Gy, 90 Gy, or 100 Gy. In some embodiments, the dose of ionizing radiation is less than approximately 100 Gy, less than approximately 90 Gy, less than approximately 80 Gy, less than approximately 70 Gy, less than approximately 60 Gy, less than approximately 50 Gy, less than approximately 45 Gy, less than approximately 40 Gy, less than approximately 35 Gy, less than approximately 30 Gy, less than approximately 25 Gy, or less than approximately 20 Gy. In certain embodiments, the radiation therapy dose is 10 Gy or more, over approximately 15 Gy, over approximately 20 Gy, over approximately 25 Gy, over approximately 30 Gy, over approximately 35 Gy, over approximately 40 Gy, over approximately 45 Gy, or over approximately 50 Gy. In some embodiments, the radiation therapy dose is approximately 10 Gy to approximately 100 Gy. In some embodiments, the radiation therapy dose is approximately 20 Gy to approximately 80 Gy. In some embodiments, the radiation therapy dose is approximately 20 Gy to approximately 60 Gy.

[0112] The dose of ionizing radiation may be administered as fractionated radiotherapy (i.e., a portion of the total dose is administered daily over several days / weeks). For example, the above-mentioned dose may be administered in 20 to 30 fractions over 4 to 6 weeks.

[0113] The dose of ionizing radiation to be administered may depend on the tumor to be treated and / or the age of the patient (e.g., adult or child). For example, a dose of 60 Gy may be used in the treatment of primary GBM (IDH-wildtype and ATRX-wildtype), while a dose of 35 Gy may be used in the treatment of recurrent GBM (which may be IDH-mutant / ATRX-mutant).

[0114] Furthermore, the data provided herein demonstrate that the PARP inhibitors described herein can provide antitumor benefits in in vitro models at relatively low doses of ionizing radiation. Without wishing to be bound by theory, it is contemplated that the PARP inhibitors described herein may achieve clinical benefits (e.g., tumor reduction) at lower doses of ionizing radiation (e.g., lower than the standard of care dose of ionizing radiation for treating the disease in question).

[0115] Therefore, in some embodiments, ionizing radiation is administered at a dose of less than about 60 Gy, less than about 55 Gy, less than about 50 Gy, less than about 45 Gy, or less than about 40 Gy. In some embodiments, ionizing radiation is administered at a dose of from about 20 Gy to about 60 Gy, from about 20 Gy to about 55 Gy, from about 20 Gy to about 50 Gy, from about 20 Gy to about 45 Gy, from about 20 Gy to about 40 Gy, from about 30 Gy to about 60 Gy, from about 30 Gy to about 55 Gy, from about 30 Gy to about 50 Gy, from about 30 Gy to about 45 Gy, or from about 30 Gy to about 40 Gy.

[0116] Diseases The methods described herein are for the treatment of brain tumors and neuroblastoma.

[0117] Examples of brain tumors that can be treated by this method include gliomas and ependymomas. In some embodiments, the brain tumor to be treated is a glioma.

[0118] Suitable gliomas for treatment include adult and pediatric gliomas, as well as high-grade and low-grade gliomas. Examples of high-grade gliomas include oligodendroglioma, anaplastic astrocytoma, glioblastoma, and diffuse median glioma. Examples of low-grade gliomas include pilocytic astrocytoma, melanoptic glioma, tectonic glioma, oligodendroglioma, ganglion glioma, and pleomorphic stellate astrocytoma. In some embodiments, the brain tumor to be treated is a high-grade glioma.

[0119] In some embodiments, the brain tumor to be treated is glioblastoma. In some embodiments, the brain tumor to be treated is glioblastoma, for example, adult glioblastoma. In some embodiments, the brain tumor to be treated is diffuse median glioma, for example, pediatric diffuse median glioma. In some embodiments, the brain tumor to be treated is H3K27M mutant glioma. [Brief explanation of the drawing]

[0120] [Figure 1] The X-ray powder diffractogram of form B of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide is shown. [Figure 2] The DSC trace of form B of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide is shown. [Figure 3]The X-ray powder diffractogram of morphology D of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide is shown. [Figure 4] This shows the single crystal structure of morphology D (ORTEP50) of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide. [Figure 5] The X-ray powder diffractogram of the MSA salt form C of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide is shown. [Figure 6] The DSC trace of the MSA salt form C of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide is shown. [Figure 7] This graph shows the cell confluence activity of potent PARP inhibitor monotherapy in glioma cells that have ATRX mutations (SJG2IDH1wt and SJG2IDH1mt) but lack wild-type ATRX (U87 IDH1wt and U87 IDH1mt). A representative graph is shown, and the error bars represent + / -SD. [Figure 8] This graph shows cellular confluence graphs illustrating PARP inhibitor enhancement of TMZ efficacy in U87 IDH1wt(A) and U87 IDH1mt R132H(B) isogenic pairs. Representative graphs are shown from three biological replicates, and the error bars represent + / -SD. [Figure 9] This graph shows cell viability in SJ-G2 IDH1wt (A) and SJ-G2 IDH1mt R132H (B) isogenic pairs, illustrating the enhancement of TMZ efficacy by PARP inhibitors. A representative graph is shown, and the error bars represent + / -SD. [Figure 10]The antitumor efficacy (A) and weight change (B) of U87MG xenograft models administered the following: i) vehicle; ii) compound 20 alone (3 mg / kg QD); iii) low-dose TMZ (6.25 mg / kg QD on days 1-5 and 29-33); iv) high-dose TMZ (25 mg / kg QD on days 1-5 and 29-33); and v) combination of compound 20 and low-dose TMZ. [Figure 11] This graph shows cell confluence graphs illustrating PARP inhibitor enhancement of Val-083 efficacy in U87 IDH1wt(A) and U87 IDH1mt R132H(B) isogenic pairs. Representative graphs are shown, with error bars representing + / -SD. [Figure 12] This graph shows cell confluence illustrating PARP inhibitor enhancement of Val-083 efficacy in SJ-G2 IDH1wt (A) and SJ-G2 IDH1mt R132H (B) isogenic pairs. A representative graph is shown, and the error bars represent + / -SD. [Figure 13] This graph shows cell confluence graphs illustrating the enhanced IR efficacy of PARP inhibitors in BT245 H3K27M pediatric gliomas. A representative graph is shown, with error bars representing + / -SD. [Modes for carrying out the invention]

[0121] Examples The compounds of this application will be further described with reference to the following non-limiting examples.

[0122] General experimental conditions 1 Unless otherwise specified, 1H NMR spectra were obtained using a Bruker 300 MHz, 400 MHz, or 500 MHz spectrometer at 27°C. 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). Splitting patterns represent apparent multiplicity and are 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 for the lowest isotopic mass unless otherwise specified.

[0123] 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 flash chromatography or standard flash chromatography. Generally, all solvents used were commercially available analytical grade solvents. Anhydrous solvents were those commonly used for reactions. The phase separator used in the examples was an ISOLUTE® Phase Separator column. The intermediates and examples described below were named using ACD / Name 12.01 from Advanced Chemistry Development, Inc. (ACD / Labs). Starting materials were obtained from commercial sources or prepared through literature channels.

[0124] 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 irradiated with X-rays produced by a copper long microfocus tube operating at 40 kV and 40 mA, with a wavelength of 1.5406 angstroms (i.e., approximately 1.54 angstroms), rotating at 30 revolutions per minute (to improve count statistics). 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 running time for the D8 was approximately 15 minutes.

[0125] The XRPD 2θ value can vary within a reasonable range, for example, within ±0.2°, and its XRPD intensity can vary when measured for essentially the same crystal morphology for various reasons, such as preferred orientation. 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.

[0126] 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 was collected between 22°C and 300°C using a dynamic heating rate of 10°C / min. The thermal data was analyzed using standard software, e.g., TA INSTRUMENTS® Universal v.4.5A.

[0127] The following abbreviations are used: AcOH = acetic acid, aq = aqueous solution, BAST = bis(2-methoxyethyl)aminosulfur trifluoride, Boc2O = di-tert-butyl dicarbonate, Boc = t-butyloxycarbonyl, CDCl3 = deuterated chloroform, CD3OD = deuterated methanol, CH3NO2 = nitromethane, DAST = diethylaminosulfur trifluoride, DCE = 1,2-dichloroethane, DCM = dichloromethane, DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, DEA = diethylamine, DEAD = diethyl azodicarboxylate, Des-martinperiodinane = 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benzoiodoxol-3-(1H)-one, DIPEA = N,N-diisopropylethylamine, DMAP = 2,6-dimethylaminopyridine, DMF = N,N-dimethylmethyl Lumamide, DMSO = dimethyl sulfoxide, DMSO-d6 = deuterated dimethyl sulfoxide, DPPA = diphenylphosphoradidate, dppf = 1,1'-bis(diphenylphosphin)ferrocene, DIAD = di-isopropyl(E)-diazene-1,2-dicarboxylate, DSC = differential scanning calorimetry, DTAD = di-tert-butyl(E)-diazene-1,2-dicarboxylate, ee = enantiomer Remainder, eq. = equivalent, ESI or ES = 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 hexafluorophosphate, HCl = hydrochloric acid, H2O2 = hydrogen peroxide, HP = high pressure, IPA = isopropyl alcohol, KF = potassium fluoride, LC = liquid chromatography, LiClO4 = lithium perchlorate, mmol = millimoles, mCPBA = meta-chloroperbenzoic acid, MeOH = methanol, min = minutes, MeCN or CH3CN or ACN = acetonitrile, MeNO2 = nitromethane, MS = mass spectrometry, NBS = N-bromosuccinimide, NH4Cl = ammonium chloride, NMP = N-methyl-2-pyrrolidone, NMR = nuclear magnetic resonance, Pd / C = palladium-supported 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, Pd-PEPPSI(trademark)-IPent = dichloro[1,3-bis(2,6-di-3-pentylphenyl)imidazole-2-ylidene](3-chloropyridyl)palladium(II), [1,3-bis(2,6-di-3-pentylphenyl)imidazole-2-ylidene](3-chloropyridyl)dichloropalladium(II), [1,3-bis(2,6-di-3-pentylphenyl)imidazole-2-ylidene](3-chloropyridyl)palladium(II) dichloride, Xphos Pd G2 = Chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II), X-Phosaminobiphenylpalladium chloride, CataCXium A-Pd-G2 = Chloro[(di(1-adamantyl)-N-butylphosphine)-2-(2-aminobiphenyl)]palladium(II), sat = saturated, SFC = supercritical fluid chromatography, T3P = 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinan 2,4,6-Trioxide, PPh3O = Triphenylphosphine oxide, 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, 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(diphenylphosphinon)-1,1'-binaphthyl, SM = starting material, CH2Cl2 = dichloromethane, Et3N = triethylamine, HCO2H = formic acid, LCMS = liquid chromatography-mass spectrometry, N2 = dinitrogen, Na2SO4 = sodium sulfate, NH4CO3 = ammonium carbonate, UV = ultraviolet light, XPhos Pd G2 = chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II), Pd(OAc)2 = palladium(II) acetate, ppt = precipitate.

[0128] Preparation of Examples [ka]

[0129] Intermediate 2: 1-bromo-4-fluoro-2-methyl-3-nitrobenzene To a 50 mL solution of 1-fluoro-3-methyl-2-nitrobenzene (intermediate 1) in TFA (50 mL), concentrated H2SO4 (20 mL) was slowly added at 0°C, followed by the addition of NBS (13.50 g, 75.87 mmol) in small portions. After addition, the mixture was stirred at room temperature for 4 hours. The resulting mixture was poured onto ice, and the formed precipitate was collected by filtration. After washing with water and drying under vacuum, 1-bromo-4-fluoro-2-methyl-3-nitrobenzene (intermediate 2) was obtained as a white solid (14.80 g, 92%). ¹H NMR (500 MHz, chloroform-d): 2.43 (3H, s), 7.03 (¹H, t), 7.68 (¹H, dd).

[0130] Intermediate 3: 2-(4-bromo-3-methyl-2-nitro-anilino)propanoic acid A mixture of 1-bromo-4-fluoro-2-methyl-3-nitrobenzene (13.8 g, 58.97 mmol) (intermediate 2), alanine (6.30 g, 70.76 mmol), and potassium carbonate (24.45 g, 176.90 mmol) in DMF (15 mL) was stirred at 100 °C for 5 hours, then the temperature was raised to 110 °C and stirred for 5 hours. The mixture was poured onto ice and slowly quenched at 0 °C with 1 M aqueous HCl solution (approximately 300 ml) to obtain a yellow suspension. The solid was collected by filtration, washed with water, and dried in a vacuum oven at 50 °C for 2 days to obtain 2-(4-bromo-3-methyl-2-nitro-anilino)propanoic acid (14.03 g, 78%) (intermediate 3) as a yellow solid (with some impurities). 1H NMR(500MHz,DMSO-d6)1.39(3H,d),2.28(3H,s),4.20(1H,quin),6.12(1H,br d),6.68(1H,d),7.58(1H,d),12.98(1H,br s);m / z(ES + )[M+H] + =303.

[0131] Intermediate 4: Methyl 2-(4-bromo-3-methyl-2-nitro-anilino)propanoate To a solution of 2-(4-bromo-3-methyl-2-nitro-anilino)propanoic acid (14.9 g, 49.16 mmol) (intermediate 3) in MeOH (150 mL), thionyl chloride (10.76 mL, 147.47 mmol) was added dropwise at 0°C, and the mixture was stirred overnight at room temperature. LC-MS showed complete conversion. The reaction mixture was slowly quenched at 0°C with saturated aqueous solution of NaHCO3 (approximately 300 mL) to obtain an orange suspension. The solid was collected by filtration, washed with water, and dried to obtain the crude product (14.6 g). The solid was purified by silica gel column chromatography (eluted with 0-25% ethyl acetate in hexane) to obtain methyl 2-(4-bromo-3-methyl-2-nitro-anilino)propanoate (intermediate 4) as a bright orange solid (12.74 g, 82%). ¹H NMR (500 MHz, chloroform-d): 1.52 (3H,d), 2.43 (3H,s), 3.76 (3H,s), 4.14 (¹H,quin), 5.83 (¹H,br d), 6.45 (¹H,d), 7.48 (¹H,d); m / z (ES + )[M+H] + =317.

[0132] Intermediate 5: 7-bromo-3,8-dimethyl-3,4-dihydro-1H-quinoxaline-2-one A small piece of ice was added at 0°C to a stirred mixture of methyl 2-(4-bromo-3-methyl-2-nitro-anilino)propanoate (11.6 g, 36.58 mmol) (intermediate 4), zinc (23.91 g, 365.77 mmol), and ammonium chloride (19.56 g, 365.77 mmol) in MeOH (100 mL) (exothermic reaction). The reaction mixture was then stirred at 0°C (ice bath) for 15 minutes. Water (2 mL) was added, and the resulting mixture was stirred at room temperature for 15 minutes. The bright orange color disappeared. The mixture was filtered through filter paper, washed with methanol, and the filtrate was concentrated under vacuum. The residue was diluted with ethyl acetate and washed with water, and then with brine. The organic layer was dried (anhydrous Na2SO4), filtered, and concentrated to obtain a mixture of methyl 2-(2-amino-4-bromo-3-methyl-anilino)propanoate and 7-bromo-3,8-dimethyl-3,4-dihydro-1H-quinoxarin-2-one (9.8 g).

[0133] To a 100 mL solution of the above solid in MeOH, 2 ml of 4 M HCl in dioxane was added at room temperature, and the mixture was stirred at room temperature for 10 minutes. Another 100 ml of methanol was added (to create a free suspension), and the resulting suspension was stirred at room temperature for 1 hour. This mixture was diluted with ether (approximately 200 ml), the solid was collected by filtration, and washed with ether. The filtrate was concentrated until the solid precipitated, and the solid was collected by filtration. This procedure was repeated several times to obtain the first portion of the product, 7.2 g. The filtrate was concentrated and purified by silica gel column chromatography (eluted with 0-100% ethyl acetate in hexane), the product fraction was concentrated, and the resulting substance was combined with the above substance to obtain 7-bromo-3,8-dimethyl-3,4-dihydro-1H-quinoxalin-2-one (9.10 g, 98%) (intermediate 5) as an off-white solid. 1H NMR(500MHz,DMSO-d6)1.23(3H,d),2.24(3H,s),3.68(1H,q),3.75(br,1H),(6.54(1H,d),7.00(1H,d),9.77(1H,s);m / z(ES + )[M+H] + =255.

[0134] Intermediate 6: 7-bromo-3,8-dimethyl-1H-quinoxaline-2-one DDQ (8.91 g, 39.24 mmol) was added at room temperature to a CH2Cl2 (400 mL) suspension of 7-bromo-3,8-dimethyl-3,4-dihydro-1H-quinoxalin-2-one (9.1 g, 35.67 mmol) (intermediate 5), and the mixture was stirred overnight. LC-MS showed a conversion free of impurities. The solvent was removed under reduced pressure, and a saturated NaHCO3 solution (approximately 300 ml) was added. The yellow suspension was stirred at room temperature for 4 hours. The solid was collected by filtration and washed with water. The solid was slurryed in saturated NaHCO3 (100 ml) and stirred at room temperature for 1 hour. The solid was filtered, washed with water, then with ether, and dried to obtain 7-bromo-3,8-dimethyl-1H-quinoxalin-2-one (7.29 g, 81%) (intermediate 6) as an off-white solid. ¹H NMR (500MHz, DMSO-d6): 2.40 (3H, s), 2.50 (3H, s) (overlap with DMSO-d6 peak), 7.32-7.65 (2H, m), 11.76 (1H, br s); m / z (ES + )[M+H] + =253.

[0135] Intermediate 7: 7-(hydroxymethyl)-3,8-dimethyl-1H-quinoxaline-2-one A mixture of (tributylstannyl)methanol (1142 mg, 3.56 mmol), 7-bromo-3,8-dimethyl-1H-quinoxarin-2-one (600 mg, 2.37 mmol) (intermediate 6), and Xphos Pd G2 (280 mg, 0.36 mmol) in 1,4-dioxane (40 mL) was stirred at 80°C for 18 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluted with 0-15% methanol in DCM) to obtain 7-(hydroxymethyl)-3,8-dimethyl-1H-quinoxarin-2-one (225 mg, 46%) (intermediate 7) as an off-white solid. 1H NMR(500MHz,DMSO-d6)2.31(3H,s),2.40(3H,s),4.58(2H,d),5.22(1H,t),7.33(1H,d),7.52(1H,d),11.53(1H,br s);m / z(ES + )[M+H] + =205.

[0136] Intermediate 8: 7-(bromomethyl)-3,8-dimethyl-1H-quinoxaline-2-one 7-(hydroxymethyl)-3,8-dimethyl-1H-quinoxalin-2-one (223 mg, 1.09 mmol) (intermediate 7) was stirred in HBr (15 ml, 132.59 mmol) (48 w% in water) at 80°C for 3.5 hours. The solvent was removed under reduced pressure, diethyl ether was added to the residue, and the mixture was sonicated. The solid was collected to obtain 7-(bromomethyl)-3,8-dimethyl-1H-quinoxalin-2-one (408 mg, 107%) (intermediate 8) as a yellow solid. ¹H NMR (500 MHz, DMSO-d6) 2.36-2.45 (6H, m), 4.83 (2H, s), 7.34 (1H, d), 7.53 (1H, d), 11.63 (1H, br s); m / z (ES + )[M+H] + =267,269.

[0137] Example 1: 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide To a suspension of 7-(bromomethyl)-3,8-dimethyl-1H-quinoxalin-2-one and HBr (37 mg, 0.10 mmol) (intermediate 8), ACN (5 ml), 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (31.5 mg, 0.10 mmol) (intermediate 32), and DIPEA (79 μl, 0.45 mmol) were added, and the reaction mixture was stirred at 70°C for 1 hour to obtain a pale yellow suspension. The suspension was cooled to room temperature, one drop of water was added, the solid was collected by filtration, washed three times with acetonitrile, and dried to obtain 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (0.016 g, 33%) (Example 1) as a yellow solid. 1H NMR(500MHz,DMSO-d6)2.07(3H,br s),2.42(3H,br d),2.56(4H,br s),2.76(3H,br s),3.14(4H,br s),3.61(2H,br s),7.23(1H,br d),7.42-7.67(2H,m),7.83(1H,br d),8.38(1H,br s),11.13-11.97(1H,m);m / z(ES + )[M+H] + = 425. [ka]

[0138] Example 2: 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide 7-(bromomethyl)-3,8-dimethylquinoxaline-2(1H)-one, HBr (240 mg, 0.69 mmol) (intermediate 8), and N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (202 mg, 0.69 mmol) (intermediate 31) were suspended in acetonitrile (13 mL), and DIPEA (0.723 mL, 4.14 mmol) was added. The resulting mixture was stirred at 70°C for 3 hours. The mixture was concentrated and purified by reverse-phase chromatography (C18 column, eluted with 0-100% ACN / water (0.2% ammonium hydroxide)) to obtain the product as a brown solid. The solid was suspended in a mixture of DCM and MeOH (2:1), concentrated to remove DCM, filtered, and washed with methanol. The solid was suspended in ACN (3 ml), 0.8 ml of 1 M HCl in water was added, and the mixture was diluted with water (approximately 3 ml). The mixture was then freeze-dried to obtain the HCl salt (0.033 g, 11%) of the product 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 2). 1HNMR(500MHz,DMSO-d6)2.45(3H,s),2.54(3H,s),2.81(3H,br d),3.24-3.56(6H,m),3.88-4.02(2H,m),4.54(2H,br s),7.48-7.71(3H,m),7.97(1H,br d),8.33(1H,br d),8.59(1H,br s),11.28(1H,br s),11.48-11.95(1H,m);m / z(ES + )[M+H] + =407. [ka]

[0139] Example 3: 6-Chloro-5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide HBr (2 mL, 0.18 mmol) (33 wt%) in AcOH was added to a solution of 7-(hydroxymethyl)-3,8-dimethyl-1H-quinoxalin-2-one (36 mg, 0.18 mmol) (intermediate 7) in NMP (2 mL). The resulting mixture was stirred at 100°C for 1 hour. The solvent was removed under reduced pressure. DIPEA (0.25 mL, 1.43 mmol) was added to a solution of 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide (48 mg, 0.19 mmol) (intermediate 30) in NMP (2 mL). The resulting mixture was stirred at 100°C for 18 hours. The crude product was purified by preparative HPLC (column: YMC-Actus Triart C18, 30*250, 5 μm; mobile phase A: water (0.05% NH3H2O), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 41B to 61B; 254; 220 nm in 7 min). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (33.0 mg, 42%) (Example 3) as a white solid. 1H NMR(400MHz,DMSO-d6)2.41(3H,s),2.43(3H,s),2.54-2.62(4H,m),2.78(3H,d),3.05-3.11(4H,m),3. 62(2H,s),7.24(1H,d),7.51(1H,d),7.65(1H,d),7.92(1H,d),8.41-8.45(1H,m),11.56(1H,s);m / z(ES + )[M+H] + =441. [ka] Example 4: 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide HBr (2 mL, 12.15 mmol) (33 wt%) in AcOH was added to a solution of 7-(hydroxymethyl)-3,8-dimethyl-1H-quinoxalin-2-one (43 mg, 0.21 mmol) (intermediate 7) in NMP (2 mL). The resulting mixture was stirred at 80°C for 1 hour. The solvent was removed under reduced pressure. To the resulting solid solution of NMP (3 mL), DIPEA (0.25 mL, 1.43 mmol) and N,6-dimethyl-5-(piperazin-1-yl)picolinamide (42 mg, 0.18 mmol) (intermediate 33) were added. The resulting mixture was stirred at 100°C for 18 hours. The crude product was purified by preparative HPLC (column: YMC-Actus Triart C18, 30*250, 5 μm; using water in acetonitrile (0.05% NH4OH)). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 4) (18.40 mg, 24%) as a white solid. NMR(400MHz,DMSO-d6)2.40(3H,s),2.43(3H,s),2.48(3H,s),2.55-2.63(4H,m),2.79(3H,d),2.87-2.94(4H, m),3.62(2H,s),7.24(1H,d),7.46(1H,d),7.51(1H,d),7.78(1H,d),8.39-8.44(1H,m),11.56(1H,s);m / z(ES + )[M+H] + =421. [ka]

[0140] Intermediate 10: Methyl 5-bromo-6-fluoropyridine-2-carboxylate In a dry flask, methyl 5-bromovicolinate (intermediate 9) (24 g, 111.09 mmol) and silver(II) fluoride (50 g, 342.78 mmol) were added to acetonitrile (300 ml), and the mixture was stirred at room temperature under N2 for 1 day. LC-MS showed approximately 70% conversion. Another batch of AgF2 (16 g) was added, and the resulting mixture was stirred continuously overnight at room temperature. The mixture was filtered through ceilite, washed with acetonitrile, and then with DCM, and the filtrate was concentrated to obtain a pale brown solid. The residue was partitioned into DCM and a saturated NH4Cl solution to obtain a white suspension. The solid was filtered off and discarded. The filtrate was transferred to a separatory funnel, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (150 ml x 3). The organic compounds were combined, dried (anhydrous Na2SO4), filtered, and concentrated until a solid precipitate appeared. The solid was collected by filtration, washed with ether, and dried to obtain a flaky, off-white solid. The combined filtrate was concentrated again, and the solid was collected by filtration to obtain 19.96 g of the combined product. The remainder of the filtrate was concentrated and purified by silica gel column (eluted with 0-25% ethyl acetate in hexane) to obtain 3.5 g of the second portion of the desired product as a flaky, white solid. All the substances were combined to obtain methyl 5-bromo-6-fluoropyridine-2-carboxylate (23.46 g, 90%) (intermediate 10). ¹H NMR (500 MHz, DMSO-d6) 3.89 (3H, s), 7.93 (1H, d), 8.51 (1H, t); m / z (ES + )[M+H] + =234.

[0141] Intermediate 11: tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate A mixture of tert-butylpiperazine-1-carboxylate (28.0 g, 150.37 mmol), methyl 5-bromo-6-fluoropyridine-2-carboxylate (intermediate 10) (23.46 g, 100.25 mmol), RuPhos Pd G3 (5.45 g, 6.52 mmol), and Cs2CO3 (82 g, 250.61 mmol) in 1,4-dioxane (400 mL) was stirred overnight at 80°C under N2. The reaction mixture was diluted with water (250 ml) and extracted with ethyl acetate (250 ml). The organic layer was washed with brine, the aqueous layer was extracted with ethyl acetate (100 ml x 1), the organic matter was dried (anhydrous Na2SO4), filtered, concentrated, and the residue was purified by silica gel column (eluted with 0-50% ethyl acetate in hexane) to obtain the product as a yellow solid. This solid was recrystallized from ethyl acetate / hexane, filtered, washed with hexane, and dried to obtain the product as a white crystalline solid (24.8 g). The filtrate was concentrated and re-purified by silica gel column to obtain an additional 1.9 g of the product. The total yield was tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 11) (26.7 g, 78%). ¹H NMR (500 MHz, chloroform-d): 1.51 (9H, s), 3.12-3.28 (4H, m), 3.48-3.67 (4H, m), 3.98 (3H, s), 7.27 (1H, d), 7.99 (1H, dd); m / z (ES + )[M+H] + =340.

[0142] Intermediate 12: Methyl 6-fluoro-5-piperazine-1-ylpyridine-2-carboxylate To a mixture of tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (1.9 g, 5.60 mmol) (intermediate 11) and MeOH (10 mL), 4 M HCl in dioxane (10 mL, 40.00 mmol) was added at room temperature, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was diluted with ether, the solid was collected by filtration, washed with ether, and dried under vacuum to obtain methyl 6-fluoro-5-piperazine-1-ylpyridine-2-carboxylate (1.360 g, 78%) (intermediate 12) as a white solid. 1H NMR(500MHz,DMSO-d6)3.24(4H,br s),3.46(4H,br s),3.84(3H,s),7.65(1H,br t),7.94(1H,br d),9.43(2H,br s);m / z(ES + )[M+H] + =240.

[0143] Intermediate 13: Methyl 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoropyridine-2-carboxylate 7-(hydroxymethyl)-3,8-dimethylquinoxaline-2(1H)-one (223 mg, 1.09 mmol) (intermediate 7) was stirred in HBr (15 ml, 132.59 mmol) (48 w%) in water at 80°C for 3.5 hours. The solvent was removed under reduced pressure, DCM was added to the residue, and the mixture was concentrated to obtain 7-(bromomethyl)-3,8-dimethylquinoxaline-2(1H)-one as a yellow solid.

[0144] To a 20 ml solution of the above solid in acetonitrile, methyl 6-fluoro-5-piperazine-1-ylpyridine-2-carboxylate, 2HCl (260 mg, 0.83 mmol) (intermediate 12) and DIPEA (1.907 ml, 10.92 mmol) were added at room temperature, and the reaction mixture was stirred at 70°C for 2 hours. The mixture was cooled to room temperature, 0.5 ml of water was added, and the solid was collected by filtration and washed with acetonitrile. The solid was dried to obtain an off-white solid as methyl 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoropyridine-2-carboxylate (0.371 g, 80%) (intermediate 13). 1H NMR(500MHz,DMSO-d6)2.42(6H,m),2.52-2.59(4H,m),3.20(4H,br d),3.61 (2H,s),3.82(3H,s),7.23(1H,d),7.41-7.59(2H,m),7.91(1H,d),11.55(1H,s); + )[M+H] + = 426.

[0145] Example 5: 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoropyridine-2-carboxamide In a sealed 40 ml vial, methyl 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoropyridine-2-carboxylate (360 mg, 0.85 mmol) (intermediate 13) and ammonia (15 ml, 105.00 mmol, 7 N in methanol) were added, and the mixture was stirred overnight at 50°C. LC-MS showed that some starting material still remained. The mixture was concentrated, and 10 ml of 7 N ammonia in methanol was added to the solid. The mixture was stirred at 50°C for 4 hours to obtain a white suspension. The solid was collected by filtration, washed with hexane, and dried to obtain 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-fluoropyridine-2-carboxamide (Example 5) (340 mg, 98%) as a white solid. 1H NMR(500MHz,DMSO-d6)2.40(3H,s),2.43(3H,s),2.56(4H,br s),3.14(4H,br s),3.61(2H,s),7.23(1H,d),7.46(1H,br s),7.48-7.58(2H,m),7.76(1H,br s),7.84(1H,br d),11.11-11.70(1H,m);m / z(ES + )[M+H] + =411. [ka]

[0146] Intermediate 15: tert-butyl 4-(6-methoxycarbonyl-2-methyl-3-pyridyl)piperazine-1-carboxylate A 40 mL vial fitted with a septum cap was filled with methyl 5-bromo-6-methyl picolinate (intermediate 14) (2 g, 8.69 mmol), tert-butylpiperazine-1-carboxylate (3.24 g, 17.39 mmol), Cs2CO3 (5.66 g, 17.39 mmol), and Ruphos Pd G3 (0.727 g, 0.87 mmol). The reaction vial was evacuated under vacuum and filled with nitrogen. 1,4-dioxane (20 mL) was added, and the reaction vial was placed in a heating block preheated to 80°C and stirred for 16 hours. The reaction mixture was cooled, diluted with water, extracted with ethyl acetate, the organic layer was washed with brine, dried on anhydrous Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (eluted with 0-50% ethyl acetate in hexane) to obtain tert-butyl 4-(6-methoxycarbonyl-2-methyl-3-pyridyl)piperazine-1-carboxylate (intermediate 15) (2.090 g, 72%) as a pale yellow solid. ¹H NMR (500 MHz, dichloromethane-d2): 1.49 (9H, s), 2.59 (3H, s), 2.88-3.00 (4H, m), 3.55-3.65 (4H, m), 3.92 (3H, s), 7.32 (1H, d), 7.92 (1H, d); m / z (ES + )[M+H] + =336.

[0147] Intermediate 16: Methyl 6-methyl-5-piperazine-1-ylpyridine-2-carboxylate A 4M solution of 1,4-dioxane (31.2 ml, 124.63 mmol) of hydrogen chloride was added to a 30 mL stirred solution of tert-butyl 4-(6-(methoxycarbonyl)-2-methylpyridine-3-yl)piperazine-1-carboxylate (intermediate 15) (4.18 g, 12.46 mmol) in DCM. The resulting solution was stirred at room temperature for 18 hours. The solvent was removed under vacuum, and the resulting solid was slurryed in diethyl ether. The solid was collected by filtration to obtain methyl 6-methyl-5-piperazine-1-ylpyridine-2-carboxylate (intermediate 16) (3.80 g, 99%) as a pale yellow solid. m / z(ES) + ) [M+H] +=236.

[0148] Intermediate 18: Methyl 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate Triphenylphosphine (1.584 g, 6.04 mmol) (calculated based on the addition of 4.4 g and a PPh3 loading of 1.6 mmol / g) was added at room temperature to a 40 mL DCM-stirred slurry of 8-fluoro-7-(hydroxymethyl)-3-methylquinoxaline-2(1H)-one (419 mg, 2.01 mmol) (intermediate 17) and perbromomethane (1.335 g, 4.03 mmol). The resulting mixture was stirred for 1 hour. The reaction mixture was filtered, washed with DCM and THF, and the filtrate was concentrated under vacuum to obtain 7-(bromomethyl)-8-fluoro-3-methylquinoxaline-2(1H)-one as a pale yellow solid.

[0149] To the acetonitrile (25 mL) slurry of the freshly prepared 7-(bromomethyl)-8-fluoro-3-methylquinoxaline-2(1H)-one described above, methyl 6-methyl-5-piperazine-1-ylpyridine-2-carboxylate, 2HCl (590 mg, 1.91 mmol) (intermediate 16) and N-ethyl-N-isopropylpropan-2-amine (1754 μl, 10.07 mmol) were added, and the reaction mixture was heated at 70°C for 1 hour. The reaction mixture was cooled to room temperature, concentrated, quenched with saturated aqueous solution of NaHCO3, and stirred for 1 hour. The solid was isolated by filtration and washed with water. The crude solid was purified by silica column chromatography using 0-10% MeOH in DCM to obtain methyl 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate (intermediate 18) (0.263 g, 31%). 1H NMR(500MHz,DMSO-d6)2.40-2.49(6H,m),2.62(4H,br s),2.97(4H,br s),3.72(2H,s),3.83(3H,s),7.30(1H,t),7.44(1H,d),7.52(1H,d),7.85(1H,d),12.45(1H,br s);19F NMR(471MHz,DMSO-d6)-135.54(1F,s);m / z(ES + )[M+H] + = 426.

[0150] Example 6: 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide 7N ammonia in methanol (16.47 ml, 115.26 mmol) was added to methyl 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate (intermediate 18) (0.2452 g, 0.58 mmol) in a 40 mL scintillation vial, sealed, and stirred at room temperature for 18 hours. An additional 15 mL of 7N NH3 solution was added to the reaction mixture and stirred overnight at 50°C. The reaction mixture was concentrated under vacuum and slurryed in 5 mL of MeOH. The solid was filtered off, washed with methanol, and dried to obtain 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-methylpyridine-2-carboxamide (Example 6) (0.151 g, 64%) as an off-white solid. 1H NMR(500MHz,DMSO-d6)2.42(3H,s),2.45-2.49(3H,m),2.52-2.69(4H,m),2.94(4H,br m / z(ES) + )[M+H] + =411. [ka]

[0151] Intermediate 19: Methyl 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate 7-(hydroxymethyl)-3,8-dimethylquinoxaline-2(1H)-one (intermediate 7) (223 mg, 1.09 mmol) was stirred in HBr (15 ml, 132.59 mmol) (48 w% in water) at 80°C for 4 hours. The solvent was removed under reduced pressure, DCM was added to the residue, and the mixture was sonicated and concentrated to obtain 7-(bromomethyl)-3,8-dimethylquinoxaline-2(1H)-one as a yellow solid.

[0152] To a slurry of the above mixture in acetonitrile (20 ml), methyl 6-methyl-5-piperazine-1-ylpyridine-2-carboxylate, 2HCl (intermediate 16) (337 mg, 1.09 mmol) and DIPEA (1.907 ml, 10.92 mmol) were added. The reaction mixture was stirred at 70°C for 2 hours to obtain a clear solution. The resulting mixture was cooled to room temperature, half of the solvent was removed, and 0.5 ml of water was added. The solid was collected by filtration, washed with acetonitrile, and dried to obtain a yellow solid. This solid was purified using a silica gel column (eluted with 0-20% methanol in DCM) to obtain methyl 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate (intermediate 19) (213 mg, 46%) as an off-white solid. 1H NMR(500MHz,DMSO-d6)2.41(3H,s),2.43(3H,s),2.47(3H,s),2.58(4H,br s),2.95(4H,br m / z(ES + )[M+H] + = 422.

[0153] Example 7: 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide In a sealed 40 ml vial, methyl 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate (intermediate 19) (210 mg, 0.50 mmol) and ammonia (15 ml, 105.00 mmol, 7 N in methanol) were added, and the reaction mixture was stirred overnight at 50°C. The reaction was not completed. The mixture was concentrated, and 10 ml of 7 N ammonia in methanol was added to the solid. The vial was capped, and the mixture was stirred at 50°C for 4 hours to obtain a white suspension. The mixture was cooled to room temperature, the solid was collected by filtration, washed with hexane, and dried to obtain 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-methylpyridine-2-carboxamide (Example 7) (191 mg, 94%) as a white solid. 1H NMR(500MHz,DMSO-d6)2.41(3H,s),2.44(3H,s),2.49(3H,s),2.58(4H,br s),2.92(4H,br s),3.63(2H,br s),7.24(1H,br d),7.42(1H,br s),7.46(1H,br d),7.51(1H,br d),7.79(2H,br d),10.53-11.23(1H,m);m / z(ES + )[M+H] + =407. [ka]

[0154] Intermediate 21: Methyl 2-aminobutanoate A methanol (35 mL) slurry of 2-aminobutanoic acid (intermediate 20) (5 g, 48.49 mmol) was cooled in an ice bath. Thionyl chloride (11 mL, 150.72 mmol) was added dropwise to the mixture at 0°C. The reaction mixture was warmed to room temperature and stirred overnight. The clear solution was concentrated to dryness to obtain the residue. The obtained solid was suspended in ether, filtered, washed with ether, and dried to obtain methyl 2-aminobutanoate HCl (intermediate 21) (7.35 g, 99%) as a white solid HCl salt. ¹H NMR (500 MHz, DMSO-d6) 0.92 (3H, t), 1.76-1.93 (2H, m), 3.75 (3H, s), 3.95-4.05 (1H, m), 8.53 (3H, br s).

[0155] Intermediate 23: Methyl 2-(4-bromo-3-chloro-2-nitro-anilino)butanoate In a flask, 1,4-dioxane (30 mL) was mixed with methyl 2-aminobutanoate,HCl (intermediate 21) (1.811 g, 11.79 mmol) and 1-bromo-2-chloro-4-fluoro-3-nitrobenzene (intermediate 22) (2.0 g, 7.86 mmol). DIPEA (8.24 mL, 47.16 mmol) was added, and the mixture was stirred at 105°C for 24 hours. The mixture was concentrated, and the residue was purified by silica gel column chromatography (eluted with 0-50% ethyl acetate in hexane) to obtain methyl 2-(4-bromo-3-chloro-2-nitro-anilino)butanoate (intermediate 23) (2.100 g, 76%) as a bright yellow oil. After standing, it turned into a yellow solid. ¹H NMR (500 MHz, chloroform-d): 0.99 (3H, t), 1.78-1.89 (1H, m), 1.91-2.02 (1H, m), 3.77 (3H, s), 4.04 (1H, q), 5.63 (1H, br d), 6.55 (1H, d), 7.52 (1H, d); m / z (ES + )[M+H] + =351.

[0156] Intermediate 24: 7-bromo-8-chloro-3-ethyl-3,4-dihydro-1H-quinoxaline-2-one Sodium dithionite (3.05 g, 17.49 mmol) was added to a stirred mixture of methyl 2-(4-bromo-3-chloro-2-nitro-anilino)butanoate (intermediate 23) (2.05 g, 5.83 mmol) in DMSO (50 mL), and the mixture was stirred at 120 °C for 3 hours. The mixture was quenched with water and extracted with ethyl acetate (50 ml x 2). The organic layer was dried (anhydrous Na2SO4), filtered, concentrated, and the residue was purified by silica gel column chromatography (0-55% ethyl acetate in hexane). Peak 1 was identified as 7-bromo-8-chloro-3-ethyl-1H-quinoxalin-2-one (intermediate 25) (0.319 g, 19%), obtained as a pale yellow solid. ¹H NMR (500 MHz, methanol-d4): 1.31 (3H, t), 2.89 (2H, q), 7.56-7.67 (2H, m); m / z (ES + )[M+H] + =287,289. Also, peak 2 was identified as 7-bromo-8-chloro-3-ethyl-3,4-dihydro-1H-quinoxaline-2-one (intermediate 24) (0.895g, 53%), obtained as a yellow oil, which turned into a yellow solid upon standing. ¹H NMR (500MHz, chloroform-d): 1.04 (3H,t), 1.75-1.84 (1H,m), 1.85-1.93 (1H,m), 3.89 (1H,dd), 6.51 (1H,d), 7.12 (1H,d), 7.82 (1H,br s). m / z (ES + )[M+H] + =289,291.

[0157] Intermediate 25: 7-bromo-8-chloro-3-ethyl-1H-quinoxaline-2-one DDQ (772 mg, 3.40 mmol) was added at room temperature to a mixture of 7-bromo-8-chloro-3-ethyl-3,4-dihydro-1H-quinoxalin-2-one (intermediate 24) (895 mg, 3.09 mmol) in 1,4-dioxane (20 mL), and the resulting suspension was stirred at room temperature for 3 hours. LC-MS showed complete conversion. The solvent was removed under reduced pressure, and the residue was treated with a saturated NaHCO3 solution and stirred at room temperature for 2 hours. The solid was collected by filtration, washed with a saturated NaHCO3 solution and water, and dried to obtain 7-bromo-8-chloro-3-ethyl-1H-quinoxalin-2-one (intermediate 25) (780 mg, 88%) as an off-white solid. ¹H NMR (500 MHz, methanol-d4): 1.31 (3H, t), 2.89 (2H, q), 7.56-7.67 (2H, m); m / z (ES + )[M+H] + = 287, 289.

[0158] Intermediate 26: 8-Chloro-3-ethyl-7-vinyl-1H-quinoxaline-2-one A mixture of 7-bromo-8-chloro-3-ethyl-1H-quinoxalin-2-one (intermediate 25) (1.05 g, 3.65 mmol), tributyl(vinyl) stannane (1.737 g, 5.48 mmol), and Pd(PPh3)4 (0.422 g, 0.37 mmol) in toluene (50 mL) was stirred under N2 at 110 °C for 2 hours. LC-MS showed that approximately 44% of the starting material remained. The mixture was stirred at this temperature for 4.5 hours, then at 80 °C overnight. The mixture was concentrated and purified by silica gel column chromatography (eluted with 0-100% ethyl acetate in hexane) to obtain the desired product, 8-chloro-3-ethyl-7-vinyl-1H-quinoxalin-2-one (intermediate 26) (0.850 g, 99%), a poorly soluble pale yellow solid. m / z(ES) + ) [M+H] + =235 (The product contained PPh3O).

[0159] Intermediate 27: 5-Chloro-2-ethyl-3-oxo-4H-quinoxaline-6-carboaldehyde Osmium tetroxide (0.568 mL, 0.07 mmol) in H2O was added to a solution of 8-chloro-3-ethyl-7-vinyl-1H-quinoxalin-2-one (intermediate 26) (850 mg, 3.62 mmol), 2,6-lutidine (0.844 mL, 7.24 mmol), and sodium periodate (3099 mg, 14.49 mmol) in THF (50 mL) / water (10 mL) / tert-butanol (3.46 mL, 36.22 mmol). The mixture was stirred overnight at room temperature to obtain a yellow suspension. The reaction mixture was concentrated and partitioned into saturated aqueous NH4Cl solution and DCM to separate the layers. The aqueous layer was extracted with DCM, and the combined organic layers were dried (anhydrous Na2SO4), filtered, and concentrated. The residue was purified using a silica gel column (eluted with 0-50% ethyl acetate in hexane) to obtain 5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-carboaldehyde (intermediate 27) (808 mg, 94%) as a pale yellow solid. ¹H NMR (500 MHz, chloroform-d): 1.34-1.42 (3H, m), 3.03 (2H, q), 7.88 (2H, d), 8.99-9.38 (1H, m), 10.54 (1H, s); m / z (ES + )[M+H] + =237.

[0160] Intermediate 28: 8-Chloro-3-ethyl-7-(hydroxymethyl)-1H-quinoxaline-2-one 5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-carboaldehyde (intermediate 27) (808 mg, 3.41 mmol) was cooled to 0°C in MeOH (30 mL), and sodium borohydride (1292 mg, 3.41 mmol) (supported at 10 wt% on basic alumina) was added all at once. The reaction mixture was continuously stirred at 0°C for 40 minutes. LC-MS showed that some of the starting material remained. Another 213 mg of NaBH4 (10 wt%) was added to the mixture, and stirring was continued at 0°C for 10 minutes. The mixture was concentrated with 1 ml of water, and the residue was purified by silica gel column chromatography (eluted with 0-25% methanol in DCM) to obtain 8-chloro-3-ethyl-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 28) (625 mg, 77%) (containing 26% overreduced byproduct). ¹H NMR (500 MHz, methanol-d4): 1.27-1.34 (3H, m), 2.91 (2H, q), 4.79-4.82 (2H, m), 7.54 (1H, d), 7.75 (1H, d); m / z (ES + )[M+H] + =239.

[0161] Intermediate 29: 7-(bromomethyl)-8-chloro-3-ethyl-1H-quinoxaline-2-one Carbon tetrabromide (1612 mg, 4.86 mmol) was added in one go at 0°C to a solution of 8-chloro-3-ethyl-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 28) (580 mg, 2.43 mmol) and triphenylphosphine (1275 mg, 4.86 mmol) in CH2Cl2 (40 mL), and the mixture was stirred at 0°C for 1 hour. LC-MS showed complete conversion. The solvent was removed under reduced pressure, and the residue was purified by silica gel column (eluted with 0-50% ethyl acetate in hexane) to obtain pure 7-(bromomethyl)-8-chloro-3-ethyl-1H-quinoxalin-2-one (intermediate 29) (200 mg, 27%) as a white solid. 1H NMR(500MHz,DMSO-d6)1.22(3H,t),2.83(2H,q),4.85(2H,s),7.51(1H,d),7.71(1H,d),11.89(1H,br s);m / z(ES+ )[M+H] + =301,303.

[0162] Example 8: 6-Chloro-5-[4-[(5-Chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide DIPEA (0.058 mL, 0.33 mmol) was added to a stirred suspension in acetonitrile (4 mL) containing 7-(bromomethyl)-8-chloro-3-ethyl-1H-quinoxaline-2-one (intermediate 29) (25 mg, 0.08 mmol) and 6-chloro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide,2HCl (intermediate 30) (27.2 mg, 0.08 mmol). The resulting mixture was stirred at 70°C for 1.5 hours to obtain a suspension. LC-MS showed complete conversion. The solvent was removed under reduced pressure, and the solution was subjected to an analytical purification group. After purification, 6-chloro-5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 8) (24.00 mg, 61%) was obtained as a yellow solid. Purification conditions (achiral): Column (Xbridge C18 19mm×100mm 5μm, mobile phase A: H2O with 0.2% NH4OH, pH 10, mobile phase B: acetonitrile; gradient B%: 13 to 95% B over 8 minutes; flow rate: 20 mL / min; concentration: 35 mg / ml in DMSO; loading (mg / injection): 15; column temperature: room temperature. 1H NMR (500 MHz, DMSO-d6) 1.23 (3H, t), 2.66 (4H, br s), 2.76-2.92 (5H, m), 3.13 (4H, br s), 3.77 (2H, s), 7.44 (1H, d), 7.69 (2H, dd), 7.94 (1H, d), 8.43 (1H, q), 10.75-11.45 (1H, m); m / z (ES + )[M+H] + = 475. [ka]

[0163] Example 9: 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide DIPEA (0.116 mL, 0.66 mmol) was added to a stirred suspension in acetonitrile (4 mL) containing 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 32) (51.6 mg, 0.17 mmol) and 7-(bromomethyl)-8-chloro-3-ethylquinoxaline-2(1H)-one (intermediate 29) (50 mg, 0.17 mmol). The resulting mixture was stirred at 70°C for 1.5 hours. LC-MS showed complete conversion. The solvent was removed under reduced pressure, and the residue was purified by silica gel column (eluted with 0-20% methanol in DCM) to obtain a mixture of the product and PPh3O. The substance was subjected to an analytical group for purification, and after purification, 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Example 9) (47.0 mg, 62%) was obtained as a yellow solid. Purification conditions (achiral): Column (Xbridge C18 19 mm × 100 mm 5 μm, mobile phase A: H2O with 0.2% NH4OH, pH 10, mobile phase B: acetonitrile; gradient B%: 13 to 95% B over 8 minutes; flow rate: 20 mL / min; concentration: 35 mg / ml in DMSO; loading (mg / injection): 15; column temperature: room temperature. 1H NMR(500MHz,DMSO-d6)1.22(3H,t),2.63(4H,br s),2.76(3H,d),2.82(2H,q),3.19(4H,br m / z(ES) + )[M+H] + =459. [ka]

[0164] Example 10: 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide DIPEA (0.081 mL, 0.46 mmol) was added to a stirred suspension in acetonitrile (4 mL) containing N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (intermediate 31) (34.0 mg, 0.12 mmol) and 7-(bromomethyl)-8-chloro-3-ethylquinoxaline-2(1H)-one (intermediate 29) (35 mg, 0.12 mmol). The resulting mixture was stirred at 70°C for 1.5 hours. LC-MS showed complete conversion. The solvent was removed under reduced pressure, and the resulting residue was subjected to an analytical group for purification. After purification, 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (Example 10) (13.00 mg, 20%) was obtained as a white solid. Purification conditions: (Achiral) column (Xbridge C18 19 mm × 100 mm 5 μm, mobile phase A: H2O with 0.2% NH4OH, pH 10, mobile phase B: acetonitrile; gradient B% 13 to 95% B over 8 minutes; flow rate: 20 mL / min; concentration: 35 mg / ml in DMSO; loading (mg / injection): 15; column temperature: room temperature. 1H NMR(500MHz,DMSO-d6)1.24(3H,t),2.79(3H,d),2.86(2H,q),3.22-3.37(8H,m,integrated into water peak),4.39-4.65(2H,m),7.46( 1H,dd),7.57(1H,brd),7.79-7.90(2H,m),8.32(1H,d),8.43(1H,brd),11.87-12.21(1H,m).m / z(ES+)[M+H]+=441. [ka]

[0165] Example 11: 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide DIPEA (0.111 mL, 0.64 mmol) was added to a stirred suspension in acetonitrile (10 mL) containing N,6-dimethyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl (intermediate 33) (48.9 mg, 0.16 mmol) and 7-(bromomethyl)-8-chloro-3-ethyl-1H-quinoxaline-2-one (intermediate 29) (48 mg, 0.16 mmol). The resulting mixture was stirred at 70°C for 2 hours to obtain a suspension. LC-MS showed complete conversion. The mixture was cooled to room temperature, the solid was collected by filtration, washed with water, and dried to obtain 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 11) (42.0 mg, 58%) as a white solid. ¹H NMR (500MHz, DMSO-d6): 1.22 (3H,t), 2.65 (4H,br s), 2.78-2.88 (5H,m), 2.95 (4H,br s), 3.38 (3H,s, overlaps with water peak), 3.76 (2H,s), 7.47 (2H,dd), 7.71 (1H,d), 7.79 (1H,d), 8.42 (1H,br d), 11.59-11.99 (1H,m); m / z (ES + )[M+H] += 455. [ka]

[0166] Intermediate 35: 1-bromo-2,4-difluoro-3-nitrobenzene A mixture of 1,3-difluoro-2-nitrobenzene (intermediate 34) (19.5 g, 122.57 mmol) and NBS (26.2 g, 147.08 mmol) in sulfuric acid (150 mL) was stirred overnight at 80°C. LC-MS showed complete conversion. The mixture was cooled to room temperature and slowly poured onto ice. This mixture was extracted with ethyl acetate (200 ml), and the organic layer was washed with water (50 ml x 2), saturated NaHCO3 solution (50 ml x 2), and brine. The mixture was dried (anhydrous Na2SO4), filtered, and concentrated. The residue was purified by silica gel column chromatography (eluted with 0-20% ethyl acetate in hexane) to obtain 1-bromo-2,4-difluoro-3-nitrobenzene (intermediate 35) (26.8 g, 92%) as a pale yellow oil. 1H NMR(500MHz,DMSO-d6)7.42-7.73(1H,m),8.06-8.26(1H,m);m / z(ES + )[M+H] + =238.

[0167] Intermediate 36: Methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-hydroxy-butanoate DIPEA (8.56 mL, 48.99 mmol) was slowly added at room temperature to a 1,4-dioxane (50 mL) stirring solution of 1-bromo-2,4-difluoro-3-nitrobenzene (intermediate 35) (5.3 g, 22.27 mmol) and methyl 2-amino-3-hydroxybutanoate,HCl (4.53 g, 26.72 mmol). The resulting mixture was stirred at 40°C for 3 hours. LC-MS showed that some of the starting material remained. 800 mg of DL-threonine methyl ester HCl was added to this mixture, and the mixture was stirred continuously overnight at 40°C. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluted with 0-30% ethyl acetate in hexane) to obtain methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-hydroxy-butanoate (intermediate 36) (4.69 g, 60%) as a bright orange solid (H NMR showed that this was a mixture of diastereomers). ¹H NMR (500 MHz, chloroform-d) 1.30-1.44 (3H,m), 3.81 (3H,s), 4.00-4.22 (1H,m), 4.22-4.48 (1H,m), 6.32-6.68 (1H,m), 7.40-7.66 (2H,m); m / z (ES + )[M+H] + =351,353.

[0168] Intermediate 37: Methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-fluoro-butanoate DAST (0.919 mL, 6.95 mmol) was slowly added over 10 minutes at 0°C to a mixture of methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-hydroxy-butanoate (intermediate 36) (2.22 g, 6.32 mmol) in CH2Cl2 (40 mL), and the mixture was stirred at 0°C for 20 minutes. LCMS and TLC showed that the starting material remained. 0.4 mL of DAST was added to the mixture, and the reaction mixture was stirred for another 10 minutes. The mixture was quenched with a saturated NaHCO3 solution and extracted by DCM. The organic layer was dried (anhydrous Na2SO4), filtered, and concentrated to obtain a yellow oil. The obtained residue was purified using a silica gel column (eluted with 0-30% ethyl acetate in hexane). Peak 2 was obtained as methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-fluorobutanoate (1.110 g, 50%) (intermediate 37) as a yellow oil, and peak 4 was obtained as the starting material methyl 2-((4-bromo-3-fluoro-2-nitrophenyl)amino)-3-hydroxybutanoate (0.300 g, 13%) along with other by-products. 1H NMR(500MHz,chloroform-d)1.37-1.51(3H,m),2.80-2.93(0.5H,m),3.00(0.5H,br d),3.75(1.5H,s),3.85(1.5H,s),4.40-4.56(0.5H,m),5.01-5.23(0.5H ,m),6.43-6.58(0.5H,m),6.71-6.74(0.5H,m),7.38-7.69(2H,m).m / z(ES + )[M+H] + =353.

[0169] Intermediate 38: 7-bromo-8-fluoro-3-(1-fluoroethyl)-3,4-dihydro-1H-quinoxaline-2-one A mixture of methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-fluorobutanoate (intermediate 37) (1.11 g, 3.14 mmol), zinc (2.466 g, 37.72 mmol), and ammonium chloride (3.36 g, 62.87 mmol) in MeOH (20 mL) was mixed with water (2 mL), and the mixture was stirred at room temperature for 10 minutes. The orange color disappeared (exothermic), and LC-MS showed complete conversion. The mixture was filtered, the solid was washed with methanol, and the filtrate was concentrated. The resulting residue was dissolved in DCM, the organic matter was washed with water, dried (anhydrous Na2SO4), filtered, and concentrated to obtain the crude product. The residue was purified using a silica gel column (0-30% ethyl acetate in hexane) to obtain methyl 2-((2-amino-4-bromo-3-fluorophenyl)amino)-3-fluorobutanoate (0.533 g, 52%) as a pale yellow solid.

[0170] The above yellow solid was dissolved in 15 ml of methanol, and 0.5 1 M HCl was added to the methanol. The reaction mixture was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The solvent was removed, and the residue was diluted with DCM / methanol (5:1). The organic layer was washed once with 50% NaHCO3 solution, dried (anhydrous Na2SO4), and concentrated. The residue was purified by silica gel column chromatography (eluted with 0-31% ethyl acetate in hexane) to obtain 7-bromo-8-fluoro-3-(1-fluoroethyl)-3,4-dihydro-1H-quinoxarin-2-one (intermediate 38) (0.337 g, 37%) as a white solid. ¹H NMR (500 MHz, methanol-d4): 1.27-1.46 (3H, m), 4.26 (1H, dd), 4.90-5.12 (1H, m), 6.50 (1H, dd), 6.97 (1H, dd). m / z (ES + )[M+H] + =291,293.

[0171] Intermediate 39: 7-bromo-8-fluoro-3-(1-fluoroethyl)-1H-quinoxaline-2-one DDQ (289 mg, 1.27 mmol) was added to a CH2Cl2 (10 mL) slurry of 7-bromo-8-fluoro-3-(1-fluoroethyl)-3,4-dihydro-1H-quinoxalin-2-one (intermediate 38) (337 mg, 1.16 mmol), and the mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and the residue was diluted with a saturated NaHCO3 solution (approximately 20 ml). This suspension was stirred at room temperature for 3 hours. The solid was collected by filtration, washed with water, and dried to obtain 7-bromo-8-fluoro-3-(1-fluoroethyl)-1H-quinoxalin-2-one (intermediate 39) (333 mg, 100%) as a white solid. ¹H NMR (500 MHz, methanol-d4): 1.65-1.84 (3H, m), 5.87-6.18 (1H, m), 7.50-7.60 (1H, m), 7.61-7.78 (1H, m). m / z (ES + )[M+H] + =289,291.

[0172] Intermediate 40: 8-Fluoro-3-(1-fluoroethyl)-7-(hydroxymethyl)-1H-quinoxaline-2-one A mixture of Pd-PEPPSI™-IPent catalyst (26 mg, 0.03 mmol), (tributylstannyl)methanol (1638 mg, 5.10 mmol), and 7-bromo-8-fluoro-3-(1-fluoroethyl)-1H-quinoxarin-2-one (intermediate 39) (590 mg, 2.04 mmol) in 1,4-dioxane (25 mL) was degassed, refilled with N2, and the mixture was stirred at 80°C for 17 hours. The mixture was concentrated, and the residue was purified by silica gel column chromatography (eluted with 0-50% ethyl acetate in hexane (to recover the remaining SM), followed by 0-20% methanol in DCM) to obtain 8-fluoro-3-(1-fluoroethyl)-7-(hydroxymethyl)-1H-quinoxarin-2-one (intermediate 40) (282 mg, 57%). 1H NMR(500MHz,DMSO-d6)1.58-1.81(3H,m),4.67(2H,br d),5.46(1H,br t),5.87-6.24(1H,m),7.33-7.48(1H,m),7.65(1H,br d),12.65-12.83(1H,m);m / z(ES + )[M+H] + =241.

[0173] Intermediate 41: 7-(bromomethyl)-8-fluoro-3-(1-fluoroethyl)-1H-quinoxaline-2-one A suspension of triphenylphosphine (1223 mg, 4.66 mmol) and 8-fluoro-3-(1-fluoroethyl)-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 40) in CH2Cl2 (15 mL) was cooled to 0°C, and carbon tetrabromide (1546 mg, 4.66 mmol) was added. The mixture instantly became clear and purple. When this mixture was stirred continuously at this temperature for 10 minutes, the mixture became a yellow solution, and LC-MS showed complete conversion (though not perfectly clear). The mixture was concentrated and purified by silica gel column chromatography (eluted with 0-20% methanol in DCM), and the main peak was obtained as 7-(bromomethyl)-8-fluoro-3-(1-fluoroethyl)-1H-quinoxalin-2-one (intermediate 41), which contained some impurities. The mixture was concentrated to obtain 2.5 g of solid (theoretical mass is 353 mg, and assuming a 100% yield, it was carried over to the next step). m / z(ES + )[M+H] + =303,305.

[0174] Examples 12 and 13: 6-Fluoro-5-[4-[[5-Fluoro-2-[(1S and 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide DIPEA (0.265 mL, 1.52 mmol) was added to a mixture of 7-(bromomethyl)-8-fluoro-3-(1-fluoroethyl)-1H-quinoxalin-2-one (intermediate 41) (115 mg, 0.38 mmol) and 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (94 mg, 0.30 mmol) (intermediate 32) in acetonitrile (20 mL). The resulting mixture was stirred at 70°C for 1 hour. LC-MS showed complete conversion. The mixture was concentrated, and the residue was dissolved in DMSO (approximately 4 mL) and purified by C18 reverse-phase column chromatography (eluted with 0-100% ACN / water / 0.1% TFA). The products containing the fractions were combined and freeze-dried. The substance was re-purified using Gilson (elution with 0-80% ACN / water / 0.1% TFA) to obtain the product 6-fluoro-5-[4-[[5-fluoro-2-[(1S and 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide. Enantiomers were separated by chiral column. Chiral purification conditions: Column information: chiralpak OD 4.6 mm × 100 mm 5 μm, mobile phase A: CO2 (100%), mobile phase B: methanol with 0.2% NH4OH, isocratic 25% B for 6 minutes, flow rate: 4.0 mL / min, diluent: methanol, column temperature: room temperature, outlet pressure (SFC): N / A.

[0175] Peak 1: The white solid was diluted with water and 0.1 mL of ACN aqueous solution. 0.5 M HCl was added, and the mixture was freeze-dried to obtain isomer 1, 6-fluoro-5-[4-[[5-fluoro-2-[(1S or 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 12, absolute stereochemistry undetermined) (5.42 mg, 10.90 μmol, 3%), as the HCl salt, a yellow solid. 1H NMR(500MHz,DMSO-d6)1.54-1.73(3H,m),2.70-2.87(3H,m),3.53-3.90(8H,m),4.57(2H,br m / z(ES) + )[M+H] + =461,>95%ee.

[0176] Peak 2: The white solid was diluted with water and 0.1 mL of ACN aqueous solution. 0.5 M HCl was added, and the mixture was freeze-dried to obtain isomer 2, 6-fluoro-5-[4-[[5-fluoro-2-[(1S or 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide, as the HCl salt (Example 13, absolute stereochemistry undetermined). 1H NMR(500MHz,DMSO-d6)1.49-1.83(3H,m),2.77(3H,br d),3.46-3.91(8H,m),4.56(2H,br s),5.80-6.26(1H,m),7.52-7.80(3H,m),7.87(1H,br d),8.43(1H,br d),11.44-11.82(1H,m),12.77-13.24(1H,m);m / z(ES + )[M+H] + =461,>95% ee. [ka]

[0177] Examples 14 and 15: 5-[4-[[5-fluoro-2-[(1S and 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide 7-(bromomethyl)-8-fluoro-3-(1-fluoroethyl)quinoxaline-2(1H)-one (intermediate 41) (138 mg, 0.41 mmol) and N,6-dimethyl-5-(piperazin-1-yl)picolinamide, 2HCl (126 mg, 0.41 mmol) (intermediate 33) were suspended in acetonitrile (13 mL), and DIPEA (429 μl, 2.46 mmol) was added. The resulting mixture was stirred at 70°C for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (eluted with 0-20% methanol in DCM) to obtain the racemic product 5-[4-[[5-fluoro-2-[(1S / 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide as a pale yellow solid (169 mg). Enantiomers were separated by chiral separation. Chiral purification conditions: Column information: chiralpak OD 21.2mm × 250mm 5μm, mobile phase A: CO2 (100%), mobile phase B: methanol containing 0.2% NH4OH, isocratic: 25% B for 12 minutes, flow rate: 70.0 mL / min, concentration: 8.45 mg / ml in methanol, load: 4.23 mg / injection, column temperature: room temperature, outlet pressure (SFC): N / A.

[0178] After chiral separation, each isomer was re-purified using a reversed-phase column (eluted with 0-60% ACN / water / 0.2% ammonium hydroxide) to obtain the following results.

[0179] Peak 1: 5-[4-[[5-fluoro-2-[(1S or 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide, isomer 1 (Example 14, absolute stereochemistry undetermined) (30.7 mg, 0.067 mmol, 16%) as a white solid. 1H NMR(500MHz,DMSO-d6)1.40-1.70(3H,m),2.48(3H,s),2.54-2.69(4H,m),2.79(3H,d),2.94(4H,br m / z(ES) + )[M+H] + =457;>98% ee.

[0180] Peak 2: 5-[4-[[5-fluoro-2-[(1S or 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide, isomer 2 (Example 15, absolute stereochemistry undetermined) (37 mg, 0.081 mmol, 20%) as a white solid. 1H NMR(500MHz,chloroform-d)1.68-1.88(3H,m),2.51(3H,s),2.71(4H,br s),2.86-3.12(7H,m),3.81(2H,s),5.92-6.29(1H,m),7.34(1H,d),7.44(1H,t),7.76(1H,d),7.95(1H,br d),7.99(1H,d),10.24(1H,br s);m / z(ES + )[M+H] + =457;94.5% ee. [ka]

[0181] Intermediate 43: Methyl 2-(4-bromo-3-chloro-2-nitro-anilino)propanoate In a flask, methyl alaninate,HCl (intermediate 42) (1.851 g, 13.26 mmol) and 1-bromo-2-chloro-4-fluoro-3-nitrobenzene (intermediate 22) (2.25 g, 8.84 mmol) were added to 1,4-dioxane (70 mL). DIPEA (9.27 mL, 53.06 mmol) was added, and the mixture was stirred at 105°C for 24 hours to obtain a brown solution. LC-MS indicated that the reaction was complete. The mixture was concentrated, and the residue was purified by silica gel column chromatography (eluted with 0-30% ethyl acetate in hexane) to obtain methyl 2-(4-bromo-3-chloro-2-nitro-anilino)propanoate (intermediate 43) (2.120 g, 71%) as a bright yellow oil. After standing, this turned into a yellow solid. ¹H NMR (500 MHz, chloroform-d): 1.52 (3H,d), 3.77 (3H,s), 6.53 (1H,d), 7.15 (1H,t), 7.53 (1H,d), 7.78 (1H,dd); m / z (ES + )[M+H] + =337.

[0182] Intermediate 44: 7-bromo-8-chloro-3-methyl-3,4-dihydro-1H-quinoxaline-2-one Sodium dithionite (3.28 g, 18.84 mmol) was added to a 50 mL DMSO-stirred solution of methyl 2-(4-bromo-3-chloro-2-nitro-anilino)propanoate (intermediate 43) (2.12 g, 6.28 mmol), and the mixture was stirred at 120°C for 5 hours. LC-MS and TLC showed complete conversion. The mixture was quenched with water and extracted with ethyl acetate (50 ml x 2). The organic layer was dried (anhydrous Na2SO4), filtered and concentrated, and the residue was purified by silica gel column (0-55% ethyl acetate in hexane) to obtain 7-bromo-8-chloro-3-methyl-1H-quinoxalin-2-one (intermediate 45) (0.055 g, 3%), m / z(ES+)[M+H]+273,275, and 7-bromo-8-chloro-3-methyl-3,4-dihydro-1H-quinoxalin-2-one (intermediate 44) (0.190 g, 11%), m / z(ES + )[M+H] + We obtained 275 and 277.

[0183] Intermediate 45: 7-bromo-8-chloro-3-methyl-1H-quinoxaline-2-one DDQ (157 mg, 0.69 mmol) was added to a mixture of 7-bromo-8-chloro-3-methyl-3,4-dihydro-1H-quinoxalin-2-one (intermediate 44) (190 mg, 0.69 mmol) in 1,4-dioxane (10 mL), and the resulting mixture was stirred overnight at room temperature. LC-MS showed complete conversion. The mixture was concentrated, and the residue was treated with a saturated NaHCO3 solution. The mixture was stirred at room temperature for 4 hours, and the solid was isolated by filtration and washed with a saturated NaHCO3 solution and water. The solid was then purified by silica gel column chromatography (eluted with 0-20% methanol in DCM) to obtain 7-bromo-8-chloro-3-methyl-1H-quinoxalin-2-one (intermediate 45) (122 mg, 65%) as a yellow solid. m / z(ES + )[M+H] + = 273, 275.

[0184] Intermediate 46: 8-Chloro-3-methyl-7-vinyl-1H-quinoxaline-2-one A mixture of 7-bromo-8-chloro-3-methyl-1H-quinoxalin-2-one (intermediate 45) (122 mg, 0.45 mmol), tetrakis(triphenylphosphine)palladium (0) (51.5 mg, 0.04 mmol), and tributyl(vinyl)stannane (212 mg, 0.67 mmol) in toluene (15 ml) was stirred at 110°C for 16 hours under N2. LC-MS indicated completion of the reaction. The mixture was concentrated, and the residue was purified by silica gel column chromatography (eluted with 0-16% methanol in DCM) to obtain 8-chloro-3-methyl-7-vinyl-1H-quinoxalin-2-one (intermediate 46) (98 mg, 100%) as a brown solid (contaminated with PPh3O). m / z(ES) + )[M+H] + =221.

[0185] Intermediate 47: 5-Chloro-2-methyl-3-oxo-4H-quinoxaline-6-carboaldehyde Osmium tetroxide (0.1 mL, 0.01 mmol) in H2O was added to a solution of 8-chloro-3-methyl-7-vinyl-1H-quinoxalin-2-one (140 mg, 0.63 mmol) (intermediate 46), 2,6-lutidine (0.148 ml, 1.27 mmol), and sodium periodate (543 mg, 2.54 mmol) in THF (10 mL) / water (2 mL) / tert-butanol (0.607 mL, 6.34 mmol). The mixture was stirred overnight at room temperature to obtain a yellow suspension. LC-MS and TLC showed that some of the starting material remained. To the mixture, THF (10 ml) / water (2.000 ml), 200 mg of sodium periodate, and 0.3 ml of osmium tetroxide were added, and the mixture was continuously stirred at room temperature for 5 hours. LC-MS showed complete conversion. The reaction mixture was diluted with water, saturated with NH4Cl solution, and extracted by DCM. The combined organic layers were dried (anhydrous Na2SO4), filtered, and concentrated. The residue was purified by silica gel column (eluted with 0-20% methanol in DCM) to obtain 5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-carboaldehyde (intermediate 47) (141 mg, 100%) as a yellow solid (not very pure, and carried over to the next step). m / z(ES) + )[M+H] + =223.

[0186] Intermediate 48: 8-Chloro-7-(hydroxymethyl)-3-methyl-1H-quinoxaline-2-one Sodium borohydride (23.96 mg, 0.63 mmol) was added to a cooled solution of 5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-carboaldehyde (intermediate 47) (141 mg, 0.63 mmol) in a mixture of MeOH (16 mL) and DCM (8.00 mL) cooled to 0°C. The mixture was continuously stirred at 0°C for 1 hour. LC-MS showed complete conversion. 1 ml of water was added to the mixture, and it was concentrated. The residue was purified by silica gel column chromatography (eluted with 40-100% ethyl acetate in hexane, followed by 0-20% methanol in DCM) to obtain 8-chloro-7-(hydroxymethyl)-3-methyl-1H-quinoxaline-2-one (intermediate 48) (142 mg, 100%) as a yellow solid. 1H NMR(500MHz,DMSO-d6)2.42(3H,s),4.65(2H,br d),5.53(1H,br t),7.46(1H,br d),7.69(1H,br d),11.77(1H,br s);m / z(ES + )[M+H] + =225.

[0187] Intermediate 49: 7-(bromomethyl)-8-chloro-3-methyl-1H-quinoxaline-2-one 8-chloro-7-(hydroxymethyl)-3-methyl-1H-quinoxalin-2-one (intermediate 48) (142 mg, 0.63 mmol) and triphenylphosphine (332 mg, 1.26 mmol) were cooled to 0°C in CH2Cl2 (20 ml). Perbromomethane (419 mg, 1.26 mmol) was added all at once, and the mixture was stirred at 0°C for 1 hour and then at room temperature for 2 hours. LC-MS showed no reaction progress. The second portion of triphenylphosphine (332 mg, 1.26 mmol) and perbromomethane (419 mg, 1.26 mmol) were added to the mixture at room temperature, and the mixture was stirred for 1 hour. LC-MS showed complete conversion. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluted with 0-100% ethyl acetate in hexane) to obtain the product 7-(bromomethyl)-8-chloro-3-methyl-1H-quinoxalin-2-one as a yellow solid (intermediate 49) (32 mg, 18%). Further elution with 20% methanol in DCM yielded the second part, 100 mg (55%) of the product, as a brown solid (purity 47%). m / z(ES + )[M+H] + = 287, 289.

[0188] Example 16: 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide DIPEA (0.053 mL, 0.31 mmol) was added to a mixture of N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (22.43 mg, 0.08 mmol) (intermediate 31) and 7-(bromomethyl)-8-chloro-3-methyl-1H-quinoxaline-2-one (intermediate 49) (22 mg, 0.08 mmol) in acetonitrile (4 mL). The resulting suspension was stirred at 70°C for 1 hour. LC-MS showed complete conversion. The mixture was concentrated, the residue dissolved in DMSO, and purified by reverse-phase C18 column elution (0-100% ACN / water / 0.1% TFA). The fraction containing the pure product was freeze-dried to obtain 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (20 mg, 56%). 1 mL of 2 M HCl was added to ether, and the solvent was removed under vacuum to obtain the corresponding HCl salt as a yellow solid (Example 16). ¹H NMR (500 MHz, methanol-d4): 2.96-3.03 (3H, m), 3.48-3.86 (6H, m), 4.04-4.41 (2H, m), 4.78 (2H, s), 4.86 (3H, d, integrated into water peak), 7.74 (1H, d), 7.84 (1H, d), 8.14 (1H, dd), 8.31 (1H, d), 8.47 (1H, d); m / z (ES + )[M+H] + = 427. [ka]

[0189] Example 17: 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide DIPEA (0.061 mL, 0.35 mmol) was added to a mixture of 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (intermediate 32) (27.1 mg, 0.09 mmol) and 7-(bromomethyl)-8-chloro-3-methyl-1H-quinoxalin-2-one (intermediate 49) (50 mg, 0.09 mmol) (purity approximately 50%) in acetonitrile (5 mL), and the resulting solution was stirred at 70°C for 1 hour. LC-MS showed complete conversion. The mixture was concentrated, the residue was dissolved in DMSO, and purified by reverse-phase C18 column (elution with 0-100% ACN / water / 0.1% TFA), followed by a second purification by reverse-phase C18 column (elution with 0-100% ACN / water / 0.1% TFA). Finally, the substance was purified for the third time using a reverse-phase column (eluted by 0-100% ACN / water / ammonium hydroxide, pH approximately 10) to obtain 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Example 17) (16.50 mg, 43%) as a white solid. 1H NMR(500MHz,DMSO-d6)2.37-2.47(3H,m),2.63(4H,br s),2.76(3H,d),3.13-3.23(4H,m),3.74(2H,s),7.45(1H,d),7.57(1H,dd),7.68(1H,d),7.84(1H,d),8.39(1H,br d),10.71-12.11(1H,m);m / z(ES + )[M+H] + =445. [ka]

[0190] Example 18: 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide DIPEA (0.061 mL, 0.35 mmol) was added to a mixture of N,6-dimethyl-5-(piperazin-1-yl)picolinamide, 2HCl (intermediate 33) (26.7 mg, 0.09 mmol) and 7-(bromomethyl)-8-chloro-3-methyl-1H-quinoxalin-2-one (50 mg, 0.09 mmol) (intermediate 49, purity approximately 50%) in acetonitrile (5 mL), and the resulting solution was stirred at 70°C for 1 hour. LC-MS showed complete conversion. The mixture was concentrated, the residue was dissolved in DMSO, and the residue was purified by reverse-phase C18 column chromatography (eluted with 0-100% ACN / water / 0.1% TFA). After concentration of the fraction, the residue was re-purified by reverse-phase C18 column chromatography (eluted with 0-100% ACN / water / ammonium hydroxide, pH approximately 10). The pure fraction was freeze-dried to obtain 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 18) (18.4 mg, 48%) as the free base. ¹H NMR (500 MHz, methanol-d4): 2.53 (6H, d), 2.76 (4H, br s), 2.94 (3H, s), 3.04 (4H, br t), 3.86 (2H, s), 7.48 (1H, d), 7.51-7.60 (1H, m), 7.69 (1H, d), 7.86 (1H, d); m / z (ES + )[M+H] + =441. [ka]

[0191] Intermediate 50: 7-bromo-8-fluoro-3-(1-hydroxyethyl)-3,4-dihydro-1H-quinoxaline-2-one Ammonium chloride (6.70 g, 125.31 mmol) was added at 0°C to a suspension of methyl 2-((4-bromo-3-fluoro-2-nitrophenyl)amino)-3-hydroxybutanoate (intermediate 36) (4.4 g, 12.53 mmol) and zinc (8.19 g, 125.31 mmol) in MeOH (65 mL). Water (2 mL) was added, and the mixture was stirred at 0°C for 60 minutes. The orange color disappeared, indicating complete conversion, and LC-MS showed that the reaction was complete. The mixture was filtered, washed with methanol, and the filtrate was concentrated. The residue was diluted with ethyl acetate / methanol (10 / 1), the organic matter was washed with water (approximately 20 ml) and brine, dried (anhydrous Na2SO4), and concentrated to obtain intermediate methyl 2-((2-amino-4-bromo-3-fluorophenyl)amino)-3-hydroxybutanoate.

[0192] The above solid was slurryed in methanol (approximately 30 ml), and 4 M HCl (approximately 1 ml) was added to dioxane. The mixture was stirred at room temperature for 2 hours. LC-MS showed complete conversion. The mixture was concentrated, and the residue was purified by silica gel column chromatography (eluted with 0-100% ethyl acetate in hexane) to obtain 7-bromo-8-fluoro-3-(1-hydroxyethyl)-3,4-dihydro-1H-quinoxarin-2-one (intermediate 50) (3.20 g, 88%) as a yellow solid (a mixture of diastereomers). m / z(ES) + )[M+H] + =289,291.

[0193] Intermediate 51: 7-bromo-8-fluoro-3-(1-hydroxyethyl)-1H-quinoxaline-2-one DDQ (432 mg, 1.90 mmol) was added to a CH2Cl2 (30 mL) suspension of 7-bromo-8-fluoro-3-(1-hydroxyethyl)-3,4-dihydro-1H-quinoxalin-2-one (intermediate 50) (500 mg, 1.73 mmol), and the mixture was stirred overnight at room temperature. LCMS showed a conversion free of impurities. The solvent was removed under reduced pressure, and a saturated NaHCO3 solution (approximately 100 ml) was added. The mixture was stirred at room temperature for 3 hours. The solid was collected by filtration, washed with water, and dried to obtain 7-bromo-8-fluoro-3-(1-hydroxyethyl)-1H-quinoxalin-2-one (intermediate 51) (439 mg, 88%). 1H NMR(500MHz,DMSO-d6)1.39(3H,d),4.78-5.31(2H,m),7.39-7.71(2H,m),12.72(1H,br s);m / z(ES + )[M+H] + = 287, 289.

[0194] Intermediate 52: 3-acetyl-7-bromo-8-fluoro-1H-quinoxaline-2-one A DCM solution of DMSO (0.651 mL, 9.17 mmol) was added dropwise to a stirred solution of oxalyl chloride (3.06 mL, 6.12 mmol) (2 M in DCM) in dichloromethane (20 ml) at -78°C. A solution of 7-bromo-8-fluoro-3-(1-hydroxyethyl)-1H-quinoxalin-2-one (intermediate 51) (439 mg, 1.53 mmol) was slowly added to the reaction mixture, and the resulting slurry was stirred at -78°C for 15 minutes. Triethylamine (1.279 mL, 9.17 mmol) was added dropwise, and the resulting slurry was stirred at 0°C for another 30 minutes. LC-MS showed the formation of the desired product. Water (30 ml) was added, and the mixture was extracted with dichloromethane / MeOH (5:1) (2 × 50 ml). The organic phases were combined and dried on magnesium sulfate. The solvent was removed under vacuum, and the residue was purified by reverse-phase C18 column chromatography (elution with 0-100% ACN / water / 0.1% TFA) to obtain 3-acetyl-7-bromo-8-fluoro-1H-quinoxaline-2-one (intermediate 52) (85 mg, 19%) as a yellow solid. ¹H NMR (500 MHz, DMSO-d6) 2.52-2.66 (3H, m), 7.42-7.76 (2H, m), 13.03 (1H, br s). m / z (ES + )[M+H] + = 285, 287.

[0195] Intermediate 53: 7-bromo-3-(1,1-difluoroethyl)-8-fluoro-1H-quinoxaline-2-one DAST (0.148 mL, 1.12 mmol) was added at room temperature to a CH2Cl2 (20 mL) suspension of 3-acetyl-7-bromo-8-fluoro-1H-quinoxalin-2-one (intermediate 52) (80 mg, 0.28 mmol), and the resulting suspension was stirred at room temperature for 24 hours. LC-MS showed 42% product formation. The mixture was continuously stirred over the weekend. Water was added to the mixture and extracted by DCM. The organic layer was dried (anhydrous Na2SO4), filtered, and concentrated. The residue was purified by silica gel column (eluted with 0-20% methanol in DCM) to obtain 7-bromo-3-(1,1-difluoroethyl)-8-fluoro-1H-quinoxalin-2-one (intermediate 53) (65.0 mg, 75%) as a pale yellow solid. m / z(ES + )[M+H] + =307,309. (The substance was not very pure and was carried over to the next step).

[0196] Intermediate 54: 3-(1,1-difluoroethyl)-8-fluoro-7-(hydroxymethyl)-1H-quinoxaline-2-one A mixture of (tributylstannyl)methanol (102 mg, 0.32 mmol), Xphos Pd G2 (24.98 mg, 0.03 mmol), and 7-bromo-3-(1,1-difluoroethyl)-8-fluoro-1H-quinoxarin-2-one (intermediate 53) (65 mg, 0.21 mmol) in 1,4-dioxane (10 mL) was stirred at 80°C for 6 hours under an N2 atmosphere. LC-MS showed complete conversion. The solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (eluted with 0-20% methanol in DCM) to obtain 3-(1,1-difluoroethyl)-8-fluoro-7-(hydroxymethyl)-1H-quinoxarin-2-one (intermediate 54) (55.0 mg, 100%) as a brown solid. m / z(ES + )[M+H] + =259.

[0197] Intermediate 55: 7-(bromomethyl)-3-(1,1-difluoroethyl)-8-fluoro-1H-quinoxaline-2-one CBr4 (129 mg, 0.39 mmol) was added at 0°C to a mixture of 3-(1,1-difluoroethyl)-8-fluoro-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 54) (67 mg, 0.26 mmol) and triphenylphosphine (102 mg, 0.39 mmol) in CH2Cl2 (6 mL). The resulting mixture was stirred overnight at room temperature. LC-MS showed complete conversion. The solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (eluted with 0-100% ethyl acetate in hexane) to obtain 7-(bromomethyl)-3-(1,1-difluoroethyl)-8-fluoro-1H-quinoxalin-2-one (intermediate 55) (56.0 mg, 67%) as a white solid. m / z(ES + )[M+H] + =321,323.

[0198] Example 19: 5-[4-[[2-(1,1-difluoroethyl)-5-fluoro-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide 7-(bromomethyl)-3-(1,1-difluoroethyl)-8-fluoro-1H-quinoxalin-2-one (intermediate 55) (56 mg, 0.16 mmol) and N,6-dimethyl-5-(piperazin-1-yl)picolinamide,2HCl (intermediate 33) (48.2 mg, 0.16 mmol) were suspended in acetonitrile (4 mL), to which DIPEA (0.164 mL, 0.94 mmol) was added, and the resulting mixture was stirred at 70 °C for 1.5 hours. LC-MS showed complete conversion. The mixture was concentrated, and the residue was purified by reverse-phase Gilson column elution (0-70% ACN / water / 0.1% TFA). The pure fractions were combined, 0.5 ml of 1 M HCl aqueous solution was added to the combined fraction, and the mixture was freeze-dried to obtain 5-[4-[[2-(1,1-difluoroethyl)-5-fluoro-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide as the HCl salt (Example 19) (35.0 mg, 44%) as a yellow solid. 1H NMR(500MHz,DMSO-d6)2.08(3H,br t),2.52(3H,s),2.80(3H,br d),3.02-3.54(8H,m),4.61(2H,br s),7.57(1H,br d),7.67-8.04(3H,m),8.52(1H,br d),11.74(1H,br s),12.77-13.55(1H,m);m / z(ES + )[M+H] + = 475. [ka]

[0199] Intermediate 56: Methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)propanoate DIPEA (151 ml, 867.27 mmol) was slowly added to a stirred solution of 1-bromo-2,4-difluoro-3-nitrobenzene (intermediate 35) (68.8 g, 289.09 mmol) and methyl alaninate,HCl (40.4 g, 289.09 mmol) in DMF (300 mL). The resulting solution was stirred at room temperature for 18 hours (complete conversion to the desired product by LC-MS). The reaction mixture was concentrated using a rocket evaporation system, diluted with water, and extracted with ethyl acetate. The organic layer was thoroughly washed with water, dried over sodium sulfate, filtered, and concentrated under vacuum. 100 mL of DCM was added to the above orange solid, and the suspension was stirred at room temperature for 30 minutes. The solid was filtered to obtain methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)propanoate (24.00 g, 26%) (intermediate 56) as a bright orange solid. ¹H NMR (500 MHz, dichloromethane-d²): 1.52-1.62 (3H, m), 3.80 (3H, s), 4.28 (1H, quin), 6.49 (1H, dd), 7.19-7.39 (1H, m), 7.54 (1H, dd); ¹⁹F NMR (471 MHz, dichloromethane-d²): -109.49 (1F, s); m / z (ES + )[M+H] + =321,323.

[0200] Intermediate 57: 7-bromo-8-fluoro-3-methyl-3,4-dihydro-1H-quinoxaline-2-one Zinc (78 g, 1195.88 mmol) was gradually added at 0°C to a mixture of methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)propanoate (intermediate 56) (48 g, 149.49 mmol) and ammonium chloride (64.0 g, 1195.88 mmol) in MeOH (720 ml) and water (16 ml) (exothermic reaction). This mixture was stirred at room temperature for 2 hours (complete disappearance of the orange color indicates completion of the reaction). The solid was filtered off, and the solid cake was washed with 20% MeOH in DCM. The filtrate was concentrated, water was added to the crude product, and the product was extracted with ethyl acetate. The organic layer was dried and concentrated under vacuum to obtain oil. m / z(ES + )[M+H]+ =291,293.

[0201] This substance was slurryed in ethyl acetate (50 mL) and methanol (50 mL), and 2 mL of 4N HCl in dioxane was added. The mixture was stirred for 1 hour. The reaction mixture was concentrated to obtain the crude product 7-bromo-8-fluoro-3-methyl-3,4-dihydro-1H-quinoxalin-2-one (intermediate 57) (38.7 g) as a gray solid. The crude product (38.7 g) was subjected to the next step without further purification, assuming a 100% yield of this reaction. m / z(ES) + )[M+H] + =259.

[0202] Intermediate 58: 7-bromo-8-fluoro-3-methyl-1H-quinoxaline-2-one DDQ (21.55 g, 94.95 mmol) was added all at once to a stirred solution of 7-bromo-8-fluoro-3-methyl-3,4-dihydro-1H-quinoxarin-2-one (intermediate 57) (20.5 g, 79.13 mmol) in DCM (200 mL) to obtain a very concentrated off-white slurry, to which an additional 800 mL of dichloromethane was added. The resulting slurry was stirred at room temperature for 2 hours (complete conversion to the desired product by LC-MS). The reaction mixture was concentrated under vacuum and quenched with a saturated aqueous solution of sodium bicarbonate (approximately 500 mL; vigorous foaming occurred during quenching). The slurry was stirred overnight at room temperature, the solid was filtered off, thoroughly washed with water, and the solid was dried overnight on the filter. This solid was washed with diethyl ether and dried for 30 minutes to obtain 7-bromo-8-fluoro-3-methyl-1H-quinoxalin-2-one (intermediate 58) (16.28 g, 80%) as an off-white solid. 19F NMR (471 MHz, DMSO-d6) -124.18 (1F, s); 1H NMR (500 MHz, DMSO-d6) 2.41 (3H, s), 7.45-7.54 (2H, m), 12.60 (1H, br s); m / z (ES + )[M+H] + =257.

[0203] Intermediate 17: 8-Fluoro-7-(hydroxymethyl)-3-methyl-1H-quinoxaline-2-one A mixture of (tributylstannyl)methanol (15.39 g, 47.93 mmol), 7-bromo-8-fluoro-3-methyl-1H-quinoxalin-2-one (intermediate 58) (11.2 g, 43.57 mmol), and Xphos Pd G2 (1.714 g, 2.18 mmol) in 1,4-dioxane (200 mL) was stirred at 80°C for 7 hours. LC-MS showed complete conversion. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluted with 0-15% methanol in DCM). The fraction was concentrated into a slurry, diluted with ether, and the solid was collected by filtration and dried to obtain 8-fluoro-7-(hydroxymethyl)-3-methyl-1H-quinoxalin-2-one (intermediate 17) (8.10 g, 89%) as a white solid. 1H NMR(500MHz,DMSO-d6)2.41(3H,s),4.63(2H,br d),5.39(1H,t),7.31(1H,br t),7.51(1H,d),12.41(1H,br s).m / z(ES + )[M+H] + =209.

[0204] Example 20: 6-Fluoro-5-[4-[(5-Fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide Triethylphosphan (20.90 ml, 145.06 mmol) was added dropwise to a stirred suspension of 8-fluoro-7-(hydroxymethyl)-3-methyl-1H-quinoxalin-2-one (intermediate 17) (15.1 g, 72.53 mmol) and 1,2-dibromo-1,1,2,2-tetrachloroethane (52.0 g, 159.56 mmol) in DCM (400 mL) using an addition funnel under nitrogen at 0°C. The mixture was stirred at room temperature for 3 hours to obtain a pale yellow suspension. Crude LCMS showed complete conversion. DCM was removed by distillation under vacuum. The residue was slurryed in 300 mL of diethyl ether at room temperature, and the pale yellow ppt was filtered and washed with 200 mL of ether. The solid was added to 300 mL of water and stirred at room temperature for 10 minutes. The solid was collected by filtration and thoroughly washed with water (200 mL) to remove the salts. The solid was dried overnight under vacuum (without heating). The solid was washed with hexane and vacuum-dried in a bushel funnel to obtain 7-(bromomethyl)-8-fluoro-3-methylquinoxaline-2(1H)-one (intermediate 59) (22.76 g, 116%, possibly containing some inorganic salts) as an off-white solid. This was used directly in the next reaction. ¹H NMR (500 MHz, DMSO-d6): 2.42 (3H, s), 4.65-4.93 (2H, m), 7.28-7.42 (1H, m), 7.51 (1H, d), 12.53 (1H, br s); m / z (ES + )[M+H] + =271,273.

[0205] In a flask, 7-(bromomethyl)-8-fluoro-3-methylquinoxaline-2(1H)-one (intermediate 59) (22.76 g) and 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide,2HCl (intermediate 32) (24.24 g, 77.9 mmol) were added to acetonitrile (350 ml), and DIPEA (38.0 ml, 217.59 mmol) was added at room temperature. The resulting mixture was stirred at 70°C for 4 hours. The reaction was not completed. 5 g of KI and 2 g of NaI were added to the mixture, and the mixture was stirred at 50°C for 20 hours. Further, 540 mg (approximately 0.03 eq) of 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide,2HCl (intermediate 32) was added to the mixture, and stirring was continued at 50°C for 2 hours. The solid was collected from the reaction suspension by filtration, washed with acetonitrile, and dried. The resulting substance was then suspended in water (approximately 400 ml), slurryed at room temperature for 20 minutes, filtered, and dried (purity 97% according to LC-MS). The solid was then dissolved under reflux in a mixture of DCM / MeOH(3 / 1) (approximately 1.5 L), filtered through a silica gel pad to remove most of the DCM until a solid precipitate appeared, and the mixture was held at room temperature for 20 minutes. The solid was collected by filtration, and this procedure was repeated for the filtrate to combine the solids and obtain the product 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (Example 20) (26 g, 84%) as a pale yellow solid. 1H NMR(500MHz,DMSO-d6)2.41(3H,s),2.57-2.69(4H,m),2.76(3H,d),3.16(4H,br s),3.70(2H,s),7.29(1H,br t),7.40-7.60(2H,m),7.83(1H,d),8.38(1H,br d),12.44(1H,br s);m / z(ES + )[M+H] + = 429. [ka]

[0206] Example 21: 6-(difluoromethyl)-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide DIPEA (0.052 mL, 0.30 mmol) was added to a stirred mixture of 7-(bromomethyl)-8-fluoro-3-methylquinoxaline-2(1H)-one (intermediate 59) (40 mg, 0.15 mmol) and 6-(difluoromethyl)-N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (intermediate 60) (50.6 mg, 0.15 mmol) in acetonitrile (mL), and the resulting mixture was stirred at 70°C for 2 hours. The reaction mixture was concentrated and subjected to an analytical group for purification (purification conditions: residue was purified by reverse-phase C18 column (eluted with 0-100% ACN / water / 0.1% NH4OH) to obtain 6-(difluoromethyl)-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 21) (15 mg, 22%) as a white solid. ¹H NMR (500 MHz, DMSO-d6) 2.37 (3H, s), 2.64 (4H, br s), 2.84 (3H, d), 3.01 (4H, br d), 3.70 (2H, s), 7.00-7.28 (2H, m), 7.42 (1H, br d),7.86(1H,d),8.09(1H,d),8.39(1H,q),12.24-12.63(1H,m);m / z(ES + )[M+H] + =461. [ka]

[0207] Intermediate 61: Methyl 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxylate Triphenylphosphine (1.512 g, 5.76 mmol) supported on a polymer (3.4 g added, calculated based on a loading of PPh3 at 1.6 mmol / g) was added at room temperature to a 40 mL DCM-stirred slurry of 8-fluoro-7-(hydroxymethyl)-3-methylquinoxaline-2(1H)-one (intermediate 17) (400 mg, 1.92 mmol) and perbromomethane (1.274 g, 3.84 mmol). The resulting mixture was stirred at 23°C for 1 hour. The reaction was not completed. Additional polymer-bound PPh3 (1 g) was added to complete the reaction. The reaction mixture was filtered, washed with DCM and THF, and the filtrate was concentrated under vacuum to obtain 7-(bromomethyl)-8-fluoro-3-methylquinoxaline-2(1H)-one as a pale yellow solid.

[0208] To the freshly prepared 7-(bromomethyl)-8-fluoro-3-methylquinoxaline-2(1H)-one described above, methyl 6-fluoro-5-(piperazin-1-yl)picolinate, 2HCl (intermediate 12) (600 mg, 1.92 mmol), acetonitrile (25 mL), and N-ethyl-N-isopropylpropan-2-amine (1674 μl, 9.61 mmol) were added, and the reaction mixture was heated at 70°C for 1 hour. The reaction mixture was cooled to room temperature, concentrated, and the crude solid was purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain methyl 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxylate (intermediate 61) (0.484 g, 59%) as an off-white solid. 1H NMR(500MHz,DMSO-d6)2.34-2.49(3H,m),2.52-2.62(4H,m),3.08-3.28(4H,m),3.70 (2H,s),3.83(3H,s),7.29(1H,t),7.44-7.54(2H,m),7.91(1H,dd),12.45(1H,s);19F NMR(471MHz,DMSO-d6)-135.50(1F,s),-70.49(1F,s).m / z(ES + )[M+H] + =430.

[0209] Example 22: 6-Fluoro-5-[4-[(5-Fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide Ammonia (7N ammonia in MeOH) (31.3 ml, 218.90 mmol) was added to methyl 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxylate (intermediate 61) (0.470 g, 1.09 mmol) in a 40 mL scintillation vial, sealed, and stirred at room temperature for 18 hours. LC-MS confirmed complete conversion to the desired product. The white solid was filtered to obtain 103 mg of the pure product. The filtrate was concentrated under vacuum, and the resulting off-white solid was slurryed in approximately 5 mL of methanol and filtered to obtain an additional 298 mg of the pure product. The two batches were combined to obtain 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide (Example 22) (0.401 g, 88%). 1H NMR(500MHz,DMSO-d6)2.42(3H,s),2.59(4H,br s),3.09-3.27(4H,m),3.70(2H,s),7.29(1H,br t),7.46(1H,br s),7.49-7.58(2H,m),7.76(1H,br s),7.85(1H,br d),12.35(1H,br s);19F NMR(471MHz,DMSO-d6)-135.49(1F,s),-72.40(1F,s);m / z(ES + )[M+H] + =415. [ka]

[0210] Intermediate 62: Methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)butanoate DIPEA (165 ml, 942.91 mmol) was slowly added to a stirred solution of 1-bromo-2,4-difluoro-3-nitro-benzene (intermediate 35) (74.8 g, 314.30 mmol) and methyl 2-aminobutanoate,HCl (48.3 g, 314.30 mmol) in DMF (733 mL), and the resulting solution was stirred at room temperature for 18 hours. The DMF was removed by distillation using a rocket evaporator, diluted with water, and extracted with ethyl acetate. After concentration, the crude substance was purified by flash silica chromatography using an elution gradient of 0-70% HCl in hexane. The product fraction was concentrated under reduced pressure to obtain methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)butanoate (intermediate 62) (49.4 g, 47%) as a red solid. 1H NMR(500MHz,DMSO-d6)0.82-0.98(3H,m),1.77-1.93(2H,m),3.70(3H,d),4.38-4.54(1H,m),6.77(1H,br d),7.28(1H,br d),7.64-7.80(1H,t);m / z(ES + )[M+H] + =335.

[0211] Intermediate 63: 7-bromo-3-ethyl-8-fluoro-3,4-dihydro-1H-quinoxaline-2-one Zinc (44.1 g, 674.07 mmol) was gradually added to a mixture of methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)butanoate (intermediate 62) (50.2 g, 149.79 mmol) and ammonium chloride (64.1 g, 1198.34 mmol) in MeOH (468 mL) (exothermic reaction). This mixture was stirred at room temperature for 1 hour. The solid was filtered off and washed with 20% MeOH in DCM. This substance was dissolved in methanol (120 mL), 4N HCl (10 mL) in dioxane was added, and the reaction mixture was stirred for 30 minutes. The solvent was removed under vacuum, diluted with ethyl acetate, and basicized with a saturated NaHCO3 solution. The organic layer was separated, washed with water, dried over sodium sulfate, and concentrated to obtain the crude product. The solid was tritulated with 100 mL of methanol and stirred for 10 minutes. The pale brown solid was filtered off to obtain 7-bromo-3-ethyl-8-fluoro-3,4-dihydro-1H-quinoxarin-2-one (intermediate 63) (39.8 g, 97%). ¹H NMR (500 MHz, DMSO-d6): 0.92 (3H, t), 1.49-1.77 (2H, m), 3.57-3.87 (1H, m), 6.24-6.62 (2H, m), 6.99 (1H, dd), 10.44 (1H, s); m / z (ES + )[M+H] + =273.

[0212] Intermediate 64: 7-bromo-3-ethyl-8-fluoro-1H-quinoxaline-2-one DDQ (29.7 g, 130.94 mmol) was added all at once to a stirred solution of 7-bromo-3-ethyl-8-fluoro-3,4-dihydro-1H-quinoxalin-2-one (intermediate 63) (29.8 g, 109.12 mmol) in DCM (546 mL), and the resulting solution was stirred at room temperature for 2 hours. The solvent was removed under vacuum, and the solid was slurryed with 150 mL of methanol and stirred for 30 minutes. The solid was filtered and washed with 30 mL of methanol. The solid was transferred to a 2 L round-bottom flask, 200 mL of water was added, and then 300 mL of sodium bicarbonate was slowly added. After the addition was complete, this mixture was stirred at room temperature overnight to obtain a pale yellow slurry. Stirring was stopped, and the aqueous layer was decanted. The solid was collected by filtration and thoroughly washed with water to obtain 7-bromo-3-ethyl-8-fluoro-1H-quinoxalin-2-one (intermediate 64) (24.45 g, 83%) as a yellow solid. ¹H NMR (500 MHz, DMSO-d6) 1.22 (3H, t), 2.81 (2H, q), 7.27-7.69 (2H, m), 12.59 (1H, br s); m / z (ES + )[M+H] + =271.

[0213] Intermediate 65: 3-ethyl-8-fluoro-7-(hydroxymethyl)-1H-quinoxaline-2-one Xphos Pd G2 (1.121 g, 1.43 mmol) was added to a 1,4-dioxane (143 mL) stirred degassed solution of 7-bromo-3-ethyl-8-fluoro-1H-quinoxarin-2-one (intermediate 64) (7.727 g, 28.50 mmol) and (tributylstannyl)methanol (10.98 g, 34.20 mmol). The resulting solution was stirred at 80°C for 6 hours. The solvent was removed under vacuum, and 100 mL of diethyl ether was added. The slurry was stirred for 30 minutes. The solid was filtered off and washed with 50 mL of diethyl ether to obtain 3-ethyl-8-fluoro-7-(hydroxymethyl)-1H-quinoxarin-2-one (5.88 g, 93%) (intermediate 65) as an off-white solid. 1H NMR(500MHz,DMSO-d6)1.22(3H,t),2.82(2H,q),4.64(2H,br d),5.40(1H,t),7.32(1H,br t),7.55(1H,d),12.40(1H,br s);m / z(ES + )[M+H] + =223.

[0214] Intermediate 66: 7-(bromomethyl)-3-ethyl-8-fluoro-1H-quinoxaline-2-one Triethylphosphan (19.94 ml, 135.00 mmol) was added dropwise over 5 minutes at 0°C under nitrogen to a stirred solution of 3-ethyl-8-fluoro-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 65) (10 g, 45.00 mmol) and CBr4 (49.2 g, 148.50 mmol) in DCM (355 mL). The reaction mixture was stirred at room temperature for 1 hour. The DCM was removed under vacuum, and the residue was slurryed in 150 mL of diethyl ether. The white ppt was filtered off and washed with 50 mL of diethyl ether. This solid was slurryed with water (200 mL) and stirred for 30 minutes. The solid was filtered off and thoroughly washed with water. The solid was dried overnight under vacuum to obtain 7-(bromomethyl)-3-ethyl-8-fluoroquinoxaline-2(1H)-one (intermediate 66) (11.38 g, 89%) as a light brown solid. ¹H NMR (500 MHz z, DMSO-d6): 1.22 (3H, t), 2.83 (2H, q), 4.81 (2H, s), 7.37 (1H, br t), 7.55 (1H, d), 12.53 (1H, br s); m / z (ES + )[M+H] + =285. [ka]

[0215] Example 23: 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide DIPEA (20.90 ml, 119.64 mmol) was added to a stirred slurry of 7-(bromomethyl)-3-ethyl-8-fluoroquinoxaline-2(1H)-one (intermediate 66) (11.37 g, 39.88 mmol) and N,6-dimethyl-5-piperazine-1-ylpyridine-2-carboxamide,2HCl (intermediate 33) (14.09 g, 45.86 mmol) in acetonitrile (178 mL). The resulting solution was stirred at 50°C for 2 hours. The reaction was complete. Half of the solvent was removed by evaporation, and 10 mL of saturated sodium bicarbonate was added. The mixture was stirred for 15 minutes. The solid was filtered off and washed with water, then with 50 mL of acetonitrile. The solid was dissolved in DCM / methanol (approximately 9 / 1) and filtered through a silica bed. The filtrate was concentrated to obtain a pale yellow solid. This substance was triturated with approximately 120 mL of methanol, the solid was filtered off and dried. LCMS still showed approximately 2.1% impurities (possibly from reagents). The substance was again triturated with acetonitrile, and then with 3% methanol in acetonitrile to obtain approximately 14 g of a white solid. Methanol (40 mL) was added and the mixture was stirred for 3 hours. The solid was filtered off to obtain the pure product 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 23) (12.26 g, 70%). 1HNMR(500MHz,DMSO-d6)1.23(3H,t),2.48(3H,s),2.62(4H,br s),2.76-2.88(5H,m),2.95(4H,br m / z(ES + )[M+H] + = 439. [ka]

[0216] Example 24: 6-(difluoromethyl)-5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide DIPEA (0.049 mL, 0.28 mmol) was added to a stirred mixture of 7-(bromomethyl)-3-ethyl-8-fluoroquinoxaline-2(1H)-one (intermediate 66) (40 mg, 0.14 mmol) and 6-(difluoromethyl)-N-methyl-5-(piperazin-1-yl)picolinamide,2HCl (intermediate 60) (48.1 mg, 0.14 mmol) in acetonitrile (2 mL), and the resulting mixture was stirred at 70°C for 2 hours. The reaction mixture was concentrated and subjected to an analytical group for purification (purification conditions: residue was purified by reverse-phase C18 column (eluted with 0-100% ACN / water / 0.1% NH4OH) to obtain 6-(difluoromethyl)-5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 24) (56.0 mg, 84%). ¹H NMR (500 MHz, DMSO-d6) 1.23 (3H, t), 2.65 (4H, br d), 2.77-2.86 (5H, m), 2.97-3.06 (4H, m), 3.73 (2H, s), 7.00-7.26 (1H, t), 7.30 (1H, br d),7.55(1H,br d),7.86(1H,d),8.09(1H,d),8.39(1H,q),12.45(1H,br d);m / z + )[M+H] + = 475. [ka]

[0217] Intermediate 67: Methyl 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxylate DIPEA (246 μl, 1.41 mmol) was added to a 2 mL acetonitrile-stirred slurry containing 7-(bromomethyl)-3-ethyl-8-fluoro-1H-quinoxaline-2-one (intermediate 66) (134 mg, 0.47 mmol) and methyl 5-(piperazin-1-yl) picolinate, 2HCl (intermediate 119) (138 mg, 0.47 mmol). The resulting solution was stirred at 50°C for 2 hours. The solvent was removed under vacuum, and the resulting residue was purified by flash silica chromatography using an elution gradient of 0-20% MeOH in DCM. The product fraction was concentrated under reduced pressure to obtain methyl 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxylate (intermediate 67) (0.142 g, 71.0%) as a white solid. m / z(ES + )[M+H] + = 426.

[0218] Example 25: 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide Ammonia (7N) in methanol (3 mL, 6.00 mmol) was added to methylmethyl 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxylate (intermediate 67) (130 mg, 0.31 mmol). The resulting suspension was stirred at 50°C for 24 hours (sealed tube). After removing the solvent, the resulting residue was purified by flash silica chromatography using an elution gradient of 0-35% MeOH in DCM. The product fraction was concentrated under reduced pressure to obtain 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide (Example 25) (0.079 g, 63%) as a pale yellow solid. 1H NMR(500MHz,DMSO-d6)1.23(3H,t),2.54-2.61(4H,m),2.83(2H,q),3.32-3.40 (4H,m),3.70(2H,s),7.24-7.34(2H,m),7.38(1H,dd),7.56(1H,d),7.76(1H,br d),7.84(1H,d),8.27(1H,d),12.44(1H,br s);m / z(ES + )[M+H] + =411. [ka]

[0219] Intermediate 68: Methyl 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate Triethylphosphine (0.399 ml, 2.70 mmol) was added dropwise over 5 minutes at 0°C under nitrogen to a stirred solution of 3-ethyl-8-fluoro-7-(hydroxymethyl)quinoxaline-2(1H)-one (intermediate 65) (0.2 g, 0.90 mmol) and CBr4 (0.985 g, 2.97 mmol) in DCM (7.10 mL). The reaction mixture was stirred at room temperature for 1 hour. The DCM was removed under vacuum, and the resulting solid was slurryed in diethyl ether. The white ppt was filtered under vacuum and washed with water, then with ether. The solid was dried overnight under vacuum (without heating) to obtain 7-(bromomethyl)-3-ethyl-8-fluoroquinoxaline-2(1H)-one as a light brown solid.

[0220] To the crude product described above, methyl 6-methyl-5-(piperazin-1-yl)picolinate, 2HCl (intermediate 16) (278 mg, 0.90 mmol), acetonitrile (10 mL), and N-ethyl-N-isopropylpropan-2-amine (785 μl, 4.51 mmol) were added, and the mixture was heated at 70°C for 1 hour. The reaction mixture was cooled, concentrated, quenched with aqueous NaHCO3 solution (1 mL), and stirred at room temperature for 1 hour. Water (3 mL) was added to the mixture, and the mixture was stirred for 10 minutes. The precipitate was filtered and washed with water (50 mL). The solid was purified by normal-phase chromatography using 0-10% MeOH in DCM. The isolated product had a purity of 89% according to LC-MS. The above solid was further purified using mass-directed preparative HPLC with 20-40% acetonitrile in water containing an NH4OH modifier to obtain methyl 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate (intermediate 68) (115 mg, 0.262 mmol, 29%) as a white solid with LCMS purity of 93%. 1H NMR(500MHz,DMSO-d6)1.23(3H,t),2.45-2.49(3H,m),2.53-2.69(4H,m),2.83(2H,q),2.98(4H,br 19F NMR(471MHz,DMSO-d6)-135.54(1F,s).m / z(ES + )[M+H] + =440.

[0221] Example 26: 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide 7N ammonia in methanol (6.40 ml, 44.78 mmol) was added to methyl 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxylate (intermediate 68) (0.0984 g, 0.22 mmol) in a 40 mL scintillation vial, sealed, and stirred at room temperature for 18 hours. The reaction mixture was concentrated under vacuum, and an additional ammonia (6.40 ml, 44.78 mmol) solution was added, and the mixture was stirred at 50°C for 16 hours. The reaction mixture was concentrated under vacuum, and an additional NH3 in methanol was added, and the mixture was stirred at room temperature overnight. The reaction was completed by LC-MS. The reaction mixture was concentrated under vacuum, and the resulting solid was slurryed in diethyl ether. The solid was filtered and washed with additional ether and methanol to obtain 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-methylpyridine-2-carboxamide (Example 26) (0.095 g, 100%) as a white solid. 1H NMR(500MHz,DMSO-d6)1.22(3H,br t),2.45-2.49(3H,m),2.52-2.68(4H,m),2.82(2H,q),2.94(4H,br s),3.72(2H,br s),7.30(1H,br t),7.38-7.51(2H,m),7.55(1H,br d),7.80(2H,br d),12.41(1H,br s);19F NMR(471MHz,DMSO-d6)-135.53(1F,s);m / z(ES + )[M+H] + = 425. [ka]

[0222] Intermediate 69: 2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-carboaldehyde Des-Martin periodinane (458 mg, 1.08 mmol) was added to 3-ethyl-8-fluoro-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 65) (contaminated with its positional isomer 3-ethyl-8-fluoro-5-(hydroxymethyl)-1H-quinoxalin-2-one) (160 mg, 0.72 mmol) in DCM (5 mL). The resulting mixture was stirred at room temperature for 4 hours. The solvent was evaporated to obtain the crude product, which was purified by flash C18-flash chromatography using an elution gradient of 5-30% MeCN in water (0.4% FA). The pure fraction was evaporated to dryness to obtain 2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-carboaldehyde (containing its positional isomer 3-ethyl-8-fluoro-2-oxo-1H-quinoxaline-5-carboaldehyde) (intermediate 69) (110 mg, 69%) as a yellow solid. m / z(ES + )[M+H] + =221.

[0223] Example 27: 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide Titanium isopropoxide (64.5 mg, 0.23 mmol) was added to 2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-carboaldehyde (intermediate 69) (containing its regioisomer 3-ethyl-8-fluoro-2-oxo-1H-quinoxaline-5-carboaldehyde) (50 mg, 0.23 mmol) and 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide (54.1 mg, 0.23 mmol) in THF (3 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (intermediate 32) (192 mg, 0.91 mmol) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with MeOH (0.1 mL). The solvent was evaporated to obtain the crude product. Crude residues were purified by preparative HPLC (columns: Xselect CSH OBD column, 30*150mm 5um; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 10%B to 20%B at 10 min; 254; 220 nm; and XBridge Shield RP18 OBD column, 19*250mm, 10um; mobile phase A: water (10 MMOL / L NH4HCO3 + 0.1% NH3.H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 21B to 95B at 7 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Example 27) (6 mg, 6%) as a white solid. 1H NMR(400MHz,DMSO-d6)1.22(3H,t),2.56-2.64(4H,m),2.76(3H,d),2.82(2H,q),3.14-3.20(4H,m),3.7 1(2H,s),7.27-7.33(1H,m),7.53-7.59(2H,m),7.82-7.86(1H,m),8.38-8.45(1H,m),12.46(1H,s);19F NMR(376MHz,DMSO-d6)-72.58,-135.51;m / z(ES + )[M+H] + =443. [ka]

[0224] Example 28: 6-Chloro-5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide Titanium isopropoxide (51.6 mg, 0.18 mmol) was added to 2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-carboaldehyde (intermediate 69) (containing its positional isomer 3-ethyl-8-fluoro-2-oxo-1H-quinoxaline-5-carboaldehyde) (40 mg, 0.18 mmol) and 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 30) (50 mg, 0.20 mmol) in THF (3 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (154 mg, 0.73 mmol) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with MeOH (0.1 mL) and evaporated to obtain the crude product. The crude product 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: 21B to 41B at 7 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 28) (37.5 mg, 45%) as a white solid. 1H NMR(400MHz,DMSO-d6)1.22(3H,t),2.57-2.65(4H,m),2.76-2.86(5H,m),3.05-3.15(4H,m),3.72 (2H,s),7.29(1H,t),7.55(1H,d),7.65(1H,d),7.93(1H,d),8.40-8.45(1H,m),12.45(1H,s);19F NMR(376MHz,DMSO-d6)-135.46;m / z(ES +)[M+H] + =459. [ka]

[0225] Example 29: 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide Titanium isopropoxide (51.6 mg, 0.18 mmol) was added to 2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-carboaldehyde (intermediate 69) (containing its positional isomer 3-ethyl-8-fluoro-2-oxo-1H-quinoxaline-5-carboaldehyde) (40 mg, 0.18 mmol) and N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 31) (50 mg, 0.23 mmol) in THF (3 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (154 mg, 0.73 mmol) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with MeOH (0.1 mL) and concentrated to obtain the crude product. The crude product was purified by preparative HPLC (column: Xselect CSH OBD column, 30*150 mm, 5 μm, n; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 6B to 17B at 7 min; 254; 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 29) (17.29 mg, 22%) as a white solid. 1H NMR(400MHz,DMSO-d6)1.22(3H,t),2.53-2.63(4H,m),2.74-2.87(5H,m),3.05-3.15(4H,m,integrated into water peak),3.69(2 19F NMR(376MHz,DMSO-d6)-135.49;m / z(ES + )[M+H] + = 425. [ka]

[0226] Intermediate 70: 5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-carboaldehyde Des-Martin periodinane (1.34 g, 3.16 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-methyl-1H-quinoxalin-2-one (intermediate 17) (contaminated with 8-fluoro-5-(hydroxymethyl)-3-methyl-1H-quinoxalin-2-one) (0.33 g, 0.79 mmol) in DCM (20 ml). The resulting mixture was stirred at room temperature for 6 hours. The reaction mixture was evaporated to obtain the crude product. The crude product was purified by flash C18-flash chromatography using an elution gradient of 5-30% MeCN in water (0.4% FA). The pure fraction was evaporated to dryness to obtain 5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-carboaldehyde (contaminated with 8-fluoro-3-methyl-2-oxo-1H-quinoxaline-5-carboaldehyde) (intermediate 70) (0.300 g, 92%) as an off-white solid. m / z(ES + )[M+H] + =207.

[0227] Example 30: 6-Chloro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide Titanium isopropoxide (89 mg, 0.31 mmol) was added to 5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-carboaldehyde (contaminated with 8-fluoro-3-methyl-2-oxo-1H-quinoxaline-5-carboaldehyde) (intermediate 70) (150 mg, 0.36 mmol) and 6-chloro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 30) (80 mg, 0.31 mmol) in THF (3 mL). The resulting mixture was stirred at room temperature for 20 minutes. Sodium triacetoxyborohydride (266 mg, 1.26 mmol) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with MeOH (0.1 mL) and concentrated to obtain the crude product. 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: MeOH-for preparative separation; flow rate: 20 mL / min; gradient: 57B to 80B at 7 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 30) (35.6 mg, 25%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.42(3H,s),2.58-2.66(4H,m),2.79(3H,d),3.06-3.16(4H,m),3.72(2 19F NMR(376MHz,DMSO-d6)-135.45;m / z(ES + )[M+H] + =445. [ka]

[0228] Example 31: 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide Titanium isopropoxide (105 mg, 0.37 mmol) was added to 5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-carboaldehyde (contaminated with 8-fluoro-3-methyl-2-oxo-1H-quinoxaline-5-carboaldehyde) (intermediate 70) (150 mg, 0.36 mmol) and N,6-dimethyl-5-piperazine-1-ylpyridine-2-carboxamide,HCl (intermediate 33) (100 mg, 0.37 mmol) in THF (3 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (313 mg, 1.48 mmol) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with MeOH (0.1 mL) and evaporated to obtain the crude product. The crude product 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: 19B to 39B at 7 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 31) (12.39 mg, 8%) as an off-white solid. 1H NMR(400MHz,DMSO-d6)2.42(3H,s),2.48(3H,s),2.57-2.67(4H,m),2.80(3H,d),2.90-2.98(4H,m),3 .72(2H,s),7.30(1H,t),7.47(1H,d),7.52(1H,d),7.79(1H,d),8.39-8.46(1H,m),12.46(1H,s);19F NMR(376MHz,DMSO-d6)-135.52;m / z(ES + )[M+H] + = 425. [ka]

[0229] Example 32: 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide Titanium isopropoxide (103 mg, 0.36 mmol) was added to 5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-carboaldehyde (contaminated with 8-fluoro-3-methyl-2-oxo-1H-quinoxaline-5-carboaldehyde) (150 mg, 0.36 mmol) and N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 31) (80 mg, 0.36 mmol) in THF (3 mL). The resulting mixture was stirred at room temperature for 2 minutes. Sodium triacetoxyborohydride (308 mg, 1.45 mmol) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with MeOH (0.1 mL). The reaction mixture was evaporated to obtain the crude product. The crude product was purified by preparative HPLC (column: Xbridge Phenyl OBD column, 5 μm, 19*150 mm; mobile phase A: water (0.05% TFA), mobile phase B: MeOH-preparative; flow rate: 20 mL / min; gradient: 24B to 32B at 12 min; 254 / 220 nm, and 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: 15B to 30B at 10 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (12.4 mg, 8%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.42(3H,s),2.55-2.60(4H,m),2.78(3H,d),2.90-2.98(4H,m,integrated into water peak),3.70(2H, 19F NMR(376MHz,DMSO-d6)-135.48;m / z(ES + )[M+H] + =411. [ka]

[0230] Intermediate 72: 4-bromo-3-fluorobenzene-1,2-diamine Iron powder (5.2 g, 93.11 mmol) was added to 4-bromo-3-fluoro-2-nitroaniline (intermediate 71) (7.3 g, 31.06 mmol) and HCl (10 mL, 100.00 mmol) (10 M) in MeOH (30 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 basicized with a saturated Na2CO3 solution (100 mL). The aqueous layer was extracted with ELISA (2 × 100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain 4-bromo-3-fluorobenzene-1,2-diamine (intermediate 72) (6.05 g, 95%) as a dark solid. 1H NMR(400MHz,DMSO-d6)4.66(2H,s),4.94(2H,s),6.30(1H,dd),6.56(1H,dd);m / z(ES + )[M+H] + =205,207.

[0231] Intermediate 73: 7-bromo-8-fluoro-1H-quinoxaline-2-one 6.41 g, 31.39 mmol of 2-oxoethyl acetate in toluene was added to 4-bromo-3-fluorobenzene-1,2-diamine (intermediate 72) (4.46 g, 21.75 mmol) in toluene (30 mL). The resulting mixture was stirred at 100°C for 30 minutes. The solvent was removed under reduced pressure. The reaction mixture was diluted with (PE: 10 mL and EA: 2 mL). The precipitate was collected by filtration, washed with ELISA (5 mL), and dried under vacuum to obtain 7-bromo-8-fluoro-1H-quinoxalin-2-one (intermediate 73) (contaminated with 6-bromo-5-fluoro-1H-quinoxalin-2-one) (2.75 g, 52%) as an off-white solid. m / z(ES + )[M+H] + =243.

[0232] Intermediate 74: 8-Fluoro-7-(hydroxymethyl)-1H-quinoxaline-2-one CataCXium A-Pd-G2 (0.12 g, 0.18 mmol) was added to (tributylstannyl)methanol (1.25 g, 3.89 mmol) and 7-bromo-8-fluoro-1H-quinoxalin-2-one (intermediate 73) (1 g, 2.06 mmol) (contaminated with 6-bromo-5-fluoro-1H-quinoxalin-2-one) in 1,4-dioxane (30 mL). The resulting mixture was stirred at 100 °C under nitrogen for 18 hours. The reaction mixture was quenched with saturated KF (10 mL), filtered, and evaporated to obtain the crude product. This crude product was purified by flash C18-flash chromatography using an elution gradient of 3-30% MeCN in water (0.1% formic acid). The pure fraction was evaporated to dryness to obtain 8-fluoro-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 74) (260 mg, 69%) (contaminated with 5-fluoro-6-(hydroxymethyl)-1H-quinoxalin-2-one) as an off-white solid. m / z(ES + )[M+H] + =195.

[0233] Example 33: 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide SOCl2 (0.3 mL, 4.11 mmol) was added to 8-fluoro-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 74) (158 mg, 0.41 mmol) (contaminated with 5-fluoro-6-(hydroxymethyl)-1H-quinoxalin-2-one) in DCM (3 mL). The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. DIPEA (0.25 mL, 1.43 mmol) and N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 31) (141 mg, 0.64 mmol) were added to the mixture in NMP (3.00 mL). The resulting mixture was stirred at 80°C for 1 hour. The crude product was purified by preparative HPLC (column: XBridge preparative OBD C18 column, 19*250 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3 + 0.1% NH3.H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 18B to 24B; 254; 220 nm at 9 min). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 33) (13.00 mg, 8%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.55-2.60(4H,m),2.77(3H,d),3.28-3.33(4H,m),3.71(2H,s),7.3 0-7.42(2H,m),7.61(1H,d),7.82(1H,d),8.20(1H,s),8.25(1H,d),8.37-8.42(1H,m);19F NMR(376MHz,DMSO-d6)-129.26;m / z(ES + )[M+H] + =397. [ka]

[0234] Example 34: 6-Chloro-5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide SOCl2 (0.3 mL, 4.11 mmol) was added to 8-fluoro-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 74) (contaminated with 5-fluoro-6-(hydroxymethyl)-1H-quinoxalin-2-one) (143 mg, 0.37 mmol) in DCM (3 mL). The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. DIPEA (0.25 mL, 1.43 mmol) and 6-chloro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 30) (101 mg, 0.40 mmol) were added to the mixture in NMP (3.00 mL). The resulting mixture was stirred at 80°C for 1 hour. The crude product was purified by preparative HPLC (column: XBridge preparative OBD C18 column, 19*250 mm, 5 μm; mobile phase A: water (10 MMOL / L NH4HCO3 + 0.1% NH3.H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 25B to 28B at 9 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 34) (23.0 mg, 14%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.58-2.65(4H,m),2.78(3H,d),3.07-3.14(4H,m),3.73(2H,s),7.35(1H ,dd),7.61(1H,d),7.65(1H,d),7.93(1H,d),8.20(1H,s),8.41-8.45(1H,m),12.58(1H,s);19F NMR(376MHz,DMSO-d6)-135.18;m / z(ES + )[M+H] + =431. [ka]

[0235] Example 35: 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide SOCl2 (0.3 mL, 4.11 mmol) was added to 8-fluoro-7-(hydroxymethyl)-1H-quinoxalin-2-one (intermediate 74) (contaminated with 5-fluoro-6-(hydroxymethyl)-1H-quinoxalin-2-one) (143 mg, 0.37 mmol) (153 mg, 0.39 mmol) in DCM (3 mL). The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. DIPEA (0.25 mL, 1.43 mmol) and N,6-dimethyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 33) (137 mg, 0.58 mmol) were added to the mixture in NMP (3 mL). The resulting mixture was stirred at 80°C for 1 hour. The crude product was purified by preparative HPLC (column: XBridge preparative OBD C18 column, 19*250 mm, 5 μm; mobile phase A: water (10 MMOL / L NH4HCO3 + 0.1% NH3.H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 24B to 28B at 9 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 35) (13.0 mg, 8%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.48(3H,s),2.56-2.65(4H,s),2.79(3H,d),2.92-2.97(4H,m),3.73(2H,s),7.3 1-7.39(1H,m),7.47(1H,d),7.61(1H,d),7.78(1H,d),8.19(1H,s),8.39-8.44(1H,m),12.55(1H,s);19F NMR(376MHz,DMSO-d6)-135.25;m / z(ES + )[M+H] + =411. [ka]

[0236] Example 36: 6-Fluoro-5-[4-[(5-Fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide SOCl2 (0.3 mL, 4.11 mmol) was added to 8-fluoro-7-(hydroxymethyl)-1H-quinoxalin-2-one (contaminated with 5-fluoro-6-(hydroxymethyl)-1H-quinoxalin-2-one) (144 mg, 0.37 mmol) in DCM (3 mL). The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. DIPEA (0.25 mL, 1.43 mmol) and 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 32) (94 mg, 0.39 mmol) were added to the mixture in NMP (3.00 mL). The resulting mixture was stirred at 80°C for 1 hour. The crude product was purified by preparative HPLC (column: XBridge preparative OBD C18 column, 19*250 mm, 5 μm; mobile phase A: water (10 MMOL / L NH4HCO3 + 0.1% NH3.H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 23B to 25B at 9 min; 254 / 220 nm; RT1: 6.9, 8.46). The fraction containing the desired compound was evaporated to dryness to obtain 6-fluoro-5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 36) (16.0 mg, 10%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.56-2.62(4H,m),2.76(3H,d),3.13-3.20(4H,m),3.71(2H,d),7.33(1H, dd),7.55(1H,t),7.61(1H,d),7.83(1H,dd),8.19(1H,s),8.37-8.43(1H,m),12.56(1H,brs);19F NMR(376MHz,DMSO-d6)-72.57,-135.21;m / z(ES + )[M+H] + =415. [ka]

[0237] Example 37: 5-[4-[[2-(difluoromethyl)-5-fluoro-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide A solution of iron(II) chloride (6.18 mg, 0.05 mmol) and zinc(II) difluoromethanesulfinate (86 mg, 0.29 mmol) in water (0.5 mL) was gradually added at room temperature to a stirred solution of 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 35) (40.0 mg, 0.10 mmol) and TFA (7.51 μl, 0.10 mmol) in DMSO (3 mL). Then, tert-butyl hydroperoxide (9.44 μl, 0.10 mmol) was added, and the resulting mixture was stirred at room temperature for 2 hours. The crude product was purified by preparative HPLC (column: XBridge preparative OBD C18 column, 30 × 150 mm 5 μm; mobile phase A: water (0.05% NH3H2O), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 12% B to 32% B at 7 min; 254 / 220 nm; Rt: 6.07 min). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[[2-(difluoromethyl)-5-fluoro-3-oxo-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 37) (2.2 mg, 5%) as a pale yellow solid. 1H NMR(400MHz,DMSO-d6)2.49(3H,s),2.60-2.65(4H,m),2.80(3H,d),2.92-2.99(4H,m),3.76(2H,s ),7.08(1H,t),7.39(1H,t),7.48(1H,d),7.70(1H,d),7.79(1H,d),8.42(1H,q),13.01(1H,s);19F NMR(376MHz,DMSO-d6)-124.324,-134.183;m / z(ES + )[M+H] + =461. [ka]

[0238] Intermediate 72: 4-bromo-3-fluorobenzene-1,2-diamine Iron powder (3.56 g, 63.83 mmol) was added at room temperature to 4-bromo-3-fluoro-2-nitroaniline (intermediate 71) (3.00 g, 12.77 mmol) and concentrated hydrogen chloride (10.64 ml, 127.65 mmol) in MeOH (30 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (100 mL x 3). The organic layer was dried over MgSO4, filtered, and evaporated to obtain 4-bromo-3-fluorobenzene-1,2-diamine (intermediate 72) (2.5 g, 96%). ¹H NMR (400 MHz, DMSO-d6) 4.66 (2H, s), 4.94 (2H, s), 6.30 (dd, 1H), 6.56 (dd, 1H); m / z (ES + )[M+H] + =205.

[0239] Intermediate 75: 7-bromo-8-fluoro-3-methoxy-1H-quinoxaline-2-one 2,2,2-Methyl trimethoxyacetate (2.402 g, 14.63 mmol) was added at room temperature to 4-bromo-3-fluorobenzene-1,2-diamine (intermediate 72) (1.500 g, 7.32 mmol) and tris(((trifluoromethyl)sulfonyl)oxy)ytterbium (0.454 g, 0.73 mmol) in toluene (20 mL). The resulting mixture was stirred at 100 °C for 5 hours. The solvent was removed under reduced pressure. The crude product was purified by reverse-phase chromatography using a C18 column with an elution gradient of 5-70% MeCN in water. The pure fraction was evaporated to dryness to obtain 7-bromo-8-fluoro-3-methoxy-1H-quinoxalin-2-one (intermediate 75) (0.650 g, 32%) as a white solid. 1H NMR(300MHz,DMSO-d6)3.97(3H,s),7.31(1H,dd),7.45(1H,dd);m / z(ES + )[M+H] + =273.

[0240] Intermediate 76: 8-Fluoro-7-(hydroxymethyl)-3-methoxy-1H-quinoxaline-2-one (Tributylstannyl)methanol (882 mg, 2.75 mmol) was added to 1,4-dioxane (20 mL) with 7-bromo-8-fluoro-3-methoxyquinoxaline-2(1H)-one (intermediate 75) (300.0 mg, 1.1 mmol) and cataCXium A-Pd-G2 (73 mg, 0.11 mmol) under nitrogen at room temperature. The resulting mixture was stirred at 100 °C for 16 hours. The reaction mixture was quenched with saturated KF (10 mL) and filtered. The solvent was removed under reduced pressure. The crude product was purified by reverse-phase chromatography on a C18 column with an elution gradient of 5-100% MeOH in water. The pure fraction was evaporated to dryness to obtain 8-fluoro-7-(hydroxymethyl)-3-methoxy-1H-quinoxalin-2-one (intermediate 76) (130 mg, 53%) as a white solid. ¹H NMR (300 MHz, DMSO-d6): 3.97 (3H, s), 4.88 (2H, d), 5.36 (1H, s), 7.27-7.32 (1H, m), 7.36 (1H, d), 12.45 (1H, s); m / z (ES + )[M+H] + =225.

[0241] Intermediate 77: 7-(chloromethyl)-8-fluoro-3-methoxy-1H-quinoxaline-2-one SOCl2 (8 ml, 109.62 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-methoxy-1H-quinoxalin-2-one (intermediate 76) (50.0 mg, 0.22 mmol) in diethyl ether (50 mL) at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to obtain 7-(chloromethyl)-8-fluoro-3-methoxy-1H-quinoxalin-2-one (intermediate 77) (66.7 mg, 122%, crude product) as a yellow oil. This product was used directly in the next step without further purification. 1H NMR (300 MHz, DMSO-d6) 3.97 (3H, s), 4.88 (2H, s), 7.27-7.42 (2H, m), 12.60 (1H, s) m / z (ES +)[M+H] + =243.

[0242] Example 38: 5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 31) (150 mg, 0.68 mmol) was added at room temperature to 7-(chloromethyl)-8-fluoro-3-methoxy-1H-quinoxalin-2-one (intermediate 77) (198 mg, 0.82 mmol) and DIPEA (0.595 mL, 3.40 mmol) in MeCN (10 mL). The resulting mixture was stirred at 60 °C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography with an elution gradient of 5-70% MeCN in water. The pure fraction was evaporated to dryness to obtain the product (103.0 mg) as a yellow solid (purity 80% according to UV). The product was re-purified by flash C18-flash chromatography with an elution gradient of 5-70% MeCN in water. The pure fraction was evaporated to dryness to obtain 5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (Example 38) (36.0 mg, 12%) as a yellow solid. 1H NMR(300MHz,DMSO-d6)2.51-2.60(4H,m),2.77(3H,d),3.18-3.45(4H,m),3.66(2H,s),3.95(3H,s ),7.10-7.27(1H,m),7.27-7.42(2H,m),7.81(1H,d),8.25(1H,d),8.39(1H,q),12.30(1H,s);19F NMR(282MHz,DMSO-d6)-134.783;m / z(ES + )[M+H] + = 427. [ka]

[0243] Example 39: 6-Fluoro-5-[4-[(5-Fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide SOCl2 (0.065 mL, 0.89 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-methoxy-1H-quinoxalin-2-one (intermediate 76) (0.040 g, 0.18 mmol) in diethyl ether (10 mL) at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure. 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 32) (0.043 g, 0.18 mmol) and DIPEA (0.156 mL, 0.89 mmol) were added to the above solid in MeCN (10.00 mL) at room temperature. The resulting mixture was stirred at 60°C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC column (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: 34B to 48B at 7 min; 254 / 220 nm; RT1: 5.9). The fraction containing the desired compound was evaporated to dryness to obtain 6-fluoro-5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 39) (0.020 g, 25%) as a white solid. 1H NMR(300MHz,DMSO-d6)2.55-2.61(4H,m),2.75(3H,d),3.11-3.19(4H,m),3.67(2H,s),3.96(3H,s ),7.18-7.29(1H,m),7.35(1H,d),7.55(1H,dd),7.79-7.88(1H,m),8.41(1H,d),12.50(1H,s);19F NMR(282MHz,DMSO-d6)-72.581,-134.799;m / z(ES + )[M+H] + =445. [ka]

[0244] Example 40: 5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide N,6-dimethyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 33) (0.097 g, 0.41 mmol) was added at room temperature to 7-(chloromethyl)-8-fluoro-3-methoxyquinoxaline-2(1H)-one (intermediate 77) (0.100 g, 0.41 mmol) and DIPEA (0.360 mL, 2.06 mmol) in MeCN (10 mL). The resulting mixture was stirred at 60 °C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (column: XBridge preparative OBD C18 column, 30 × 150 mm 5 μm; mobile phase A: water (0.05% NH3H2)). O ), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 10B to 30B at 7 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 40) (0.063 g, 35%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.48(3H,s),2.58-2.63(4H,m),2.80(3H,d),2.92-2.96(4H,m),3.69(2H,s ),3.97(3H,s),7.25(1H,t),7.36(1H,d),7.47(1H,d),7.79(1H,d),8.43(1H,q),12.51(1H,s);19F NMR(376MHz,DMSO-d6)-134.815;m / z(ES + )[M+H] + =441. [ka]

[0245] Example 41: 6-Chloro-5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide SOCl2 (0.065 mL, 0.89 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-methoxy-1H-quinoxalin-2-one (intermediate 76) (0.040 g, 0.18 mmol) in diethyl ether (10 mL) at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to obtain crude 7-(chloromethyl)-8-fluoro-3-methoxy-1H-quinoxalin-2-one (0.045 g, 0.18 mmol). MeCN (10.00 mL) was added to the above solid, and then 6-chloro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 30) (0.045 g, 0.18 mmol) and DIPEA (0.156 mL, 0.89 mmol) were added. The resulting mixture was stirred at 80°C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC column (column: YMC-Actus Triart C18, 30*250, 5um; mobile phase A: water (0.05% NH3H2O), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 21B to 41B at 7 min; 254, 220 nm; RT1: 6.18). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 41) (0.041 g, 50%) as a white solid. 1H NMR(300MHz,DMSO-d6)2.56-2.66(4H,m),2.79(3H,d),3.06 19F NMR(282MHz,DMSO-d6)-134.746;m / z(ES + )[M+H] + =461. [ka]

[0246] Intermediate 78: 2-(4-bromo-3-methyl-2-nitro-anilino)butanoic acid 2-aminobutanoic acid (0.793 g, 7.69 mmol) was added at room temperature to 1-bromo-4-fluoro-2-methyl-3-nitrobenzene (intermediate 2) (1.500 g, 6.41 mmol) and K2CO3 (2.66 g, 19.23 mmol) in DMF (20 mL). The resulting mixture was stirred at 100 °C for 6 hours. The reaction mixture was poured into ice water and slowly quenched at 0 °C with 1 M HCl (20 mL) to obtain a yellow suspension. The solid was collected by filtration, washed with water, and dried to obtain 2-(4-bromo-3-methyl-2-nitro-anilino)butanoic acid (intermediate 78) (1.4 g, 74%) as a yellow solid (it was not very pure and was carried over to the next step without further purification). m / z(ES) + )[M+H] + =317.

[0247] Intermediate 79: 7-bromo-3-ethyl-8-methyl-3,4-dihydro-1H-quinoxaline-2-one Iron powder (1.585 g, 28.38 mmol) was slowly added at room temperature to 2-(4-bromo-3-methyl-2-nitro-anilino)butanoic acid (intermediate 78) (1.800 g, 5.68 mmol) and concentrated hydrogen chloride (4.73 ml, 56.76 mmol) in MeOH (100 mL). The resulting mixture was stirred at room temperature for 7 hours. The reaction mixture was filtered. The solvent was removed by distillation under reduced pressure. The reaction mixture was quenched with saturated Na2CO3 (40 mL) and extracted with siRNA (3 × 50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain a brown solid. The crude product was purified by flash C18-flash chromatography using an elution gradient of 5–50% MeCN in water. The pure fraction was evaporated to dryness to obtain 7-bromo-3-ethyl-8-methyl-3,4-dihydro-1H-quinoxarin-2-one (intermediate 79) (650 mg, 43%) as a white solid. ¹H NMR (300 MHz, DMSO-d6): 0.90 (3H,t), 1.43-1.72 (2H,m), 2.22 (3H,s), 3.56 (1H,ddd), 6.16 (1H,d), 6.56 (1H,d), 6.97 (1H,d), 9.76 (1H,s); m / z (ES + )[M+H] + =269.

[0248] Intermediate 80: 7-bromo-3-ethyl-8-methyl-1H-quinoxaline-2-one DDQ (1.316 g, 5.80 mmol) was added at room temperature to 7-bromo-3-ethyl-8-methyl-3,4-dihydro-1H-quinoxalin-2-one (intermediate 79) (1.300 g, 4.83 mmol) in 1,4-dioxane (150 mL). The resulting mixture was stirred at room temperature for 3 hours. The solvent was removed by distillation under reduced pressure. The reaction mixture was quenched with saturated NaHCO3 (150 mL). The precipitate was collected by filtration. The solid was washed with water (10 mL x 3) and dried under vacuum to obtain the desired product 7-bromo-3-ethyl-8-methyl-1H-quinoxalin-2-one (intermediate 80) (1.2 g, 93%) as a yellow solid. 1H NMR(300MHz,DMSO-d6)1.20(3H,t),2.44-2.53(3H,m),2.78(2H,q),7.48(2H,s),11.74(1H,s);m / z(ES + )[M+H] + =267.

[0249] Intermediate 81: 3-ethyl-7-(hydroxymethyl)-8-methyl-1H-quinoxaline-2-one (Tributylstannyl)methanol (1202 mg, 3.74 mmol) was added to 1,4-dioxane (40 mL) containing 7-bromo-3-ethyl-8-methylquinoxaline-2(1H)-one (intermediate 80) (400 mg, 1.50 mmol) and Pd(PPh3)4 (173 mg, 0.15 mmol) under nitrogen at room temperature. The resulting mixture was stirred at 60°C for 16 hours. The reaction mixture was quenched with KF (10 mL), and the solid was filtered off. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography using an elution gradient of 5-100% MeOH in water. The pure fraction was evaporated to dryness to obtain 3-ethyl-7-(hydroxymethyl)-8-methyl-1H-quinoxalin-2-one (intermediate 81) (100 mg, 31%) as a white solid. ¹H NMR (400 MHz, DMSO-d6): 1.22 (3H,t), 2.32 (3H,s), 2.81 (2H,q), 4.59 (2H,d), 5.25 (1H,s), 7.33 (1H,d), 7.55 (1H,d); m / z (ES + )[M+H] + =219.

[0250] Example 42: 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide HBr (1 ml, 6.08 mmol) (33 w%) in AcOH was added to 3-ethyl-7-(hydroxymethyl)-8-methylquinoxaline-2(1H)-one (intermediate 81) (65.0 mg, 0.30 mmol) at room temperature. The resulting mixture was stirred at 60°C for 2 hours. The solvent was removed under reduced pressure. N,6-dimethyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 33) (69.8 mg, 0.30 mmol) and DIPEA (0.156 ml, 0.89 mmol) in NMP (3 mL) were added to the above solid at room temperature. The resulting mixture was stirred at 60°C for 2 hours. The crude product was purified by preparative HPLC (column: Sunfire preparative C18 column, 30*150, 5um; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 9B to 20B at 7 min; 254 / 220 nm; RT1: 5.15). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 42) (0.049 g, 38%) as a pale yellow solid. 1H NMR(300MHz,DMSO-d6)1.20(3H,t),2.42(3H,s),2.50(3H,s),2.53-2.59(4H,m),2.73-2.86(5H,m),2.87-2.9 3(4H,m),3.61(2H,s),7.23(1H,d),7.45(1H,d),7.52(1H,d),7.76(1H,d),8.39(1H,d),11.52(1H,s);m / z(ES + )[M+H] + = 435. [ka]

[0251] Example 43: 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide HBr (1 ml, 6.08 mmol) (33 w%) in AcOH was added to 3-ethyl-7-(hydroxymethyl)-8-methyl-1H-quinoxalin-2-one (intermediate 81) (65.0 mg, 0.30 mmol) at room temperature. The resulting mixture was stirred at 60°C for 2 hours. The solvent was removed under reduced pressure. 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 32) (71.0 mg, 0.30 mmol) was added to the above solid, and then DIPEA (0.156 ml, 0.89 mmol) in NMP (3 mL) was added at room temperature. The resulting mixture was stirred at 60°C for 2 hours. The crude product was purified by preparative HPLC (column: XBridge preparative OBD C18 column, 30 × 150 mm 5 μm; mobile phase A: water (0.05% NH3H2O), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 31B to 51B at 7 min; 254 / 220 nm; RT1: 6.27). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (Example 43) (0.043 g, 33%) as a pale yellow solid. 1H NMR(300MHz,DMSO-d6)1.20(3H,t),2.41(3H,s),2.49-2.59(4H,m),2.70-2.81(5H,m),3.08-3.16 (4H,m),3.59(2H,s),7.22(1H,d),7.47-7.60(2H,m),7.82(1H,dd),8.37(1H,d),11.52(1H,s);19F NMR(282MHz,DMSO-d6)-72.539;m / z(ES + )[M+H] + = 439. [ka]

[0252] Example 44: 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide HBr (1 ml, 18.42 mmol) (33 wt%) in AcOH was added to 3-ethyl-7-(hydroxymethyl)-8-methyl-1H-quinoxalin-2-one (intermediate 81) (65.0 mg, 0.30 mmol) at room temperature. The resulting mixture was stirred at 60°C for 2 hours. The solvent was removed under reduced pressure. N-methyl-5-(piperazin-1-yl)picolinamide (intermediate 31) (65.6 mg, 0.30 mmol) and DIPEA (0.156 ml, 0.89 mmol) were added to the above solid in NMP (3 mL) at room temperature. The resulting mixture was stirred at 60°C for 2 hours. The crude product was purified by preparative HPLC (column: Sunfire preparative C18 column, 30*150, 5um; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 9B to 20B at 7 min; 254 / 220 nm; RT1: 5.15). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (Example 44) (0.014 g, 10%) as a pale yellow solid. 1H NMR(400MHz,DMSO-d6)1.16-1.26(3H,m),2.41(3H,s),2.49-2.59(4H,m),2.70-2.81(5H,m),3.30-3.35(4H,m,at water peak) m / z(ES + )[M+H] + =421. [ka]

[0253] Intermediate 82: tert-butyl 4-[6-(ethylcarbamoyl)-2-fluoro-3-pyridyl]piperazine-1-carboxylate 500 mg, 1.47 mmol of tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 11) was added to 10 mL, 1.47 mmol (65 wt%) of ethylamine in water. The resulting mixture was stirred at room temperature for 2 hours. The reaction was completed. The precipitate was collected by filtration, washed with water (2 mL x 3), and dried under vacuum to obtain tert-butyl 4-[6-(ethylcarbamoyl)-2-fluoro-3-pyridyl]piperazine-1-carboxylate (intermediate 82) (0.515 g, 99%) as an off-white solid. m / z(ES + )[M+H] + =353.

[0254] Intermediate 83: N-ethyl-6-fluoro-5-piperazine-1-ylpyridine-2-carboxamide tert-butyl 4-[6-(ethylcarbamoyl)-2-fluoro-3-pyridyl]piperazine-1-carboxylate (intermediate 82) (536 mg, 1.52 mmol) was added to HCl in 1,4-dioxane (5 mL, 20.00 mmol). The resulting mixture was stirred at room temperature for 1 hour. DIPEA (5 mL) was added, and the resulting mixture was stirred at room temperature for 15 minutes. The reaction mixture was evaporated to obtain the crude product. The crude product was purified by flash C18-flash chromatography (elution gradient: 5-50% MeCN in water (0.1% NH4HCO3)). The pure fraction was evaporated to dryness to obtain N-ethyl-6-fluoro-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 83) (0.368 g, 96%) as a yellow solid. The sample was not pure and was carried over to the next step without further purification. m / z(ES + )[M+H]+ =253.

[0255] Example 45: N-ethyl-6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide Ph3P (94 mg, 0.36 mmol) was added to CBr4 (119 mg, 0.36 mmol) and 8-fluoro-7-(hydroxymethyl)-3-methylquinoxaline-2(1H)-one (intermediate 17) (50 mg, 0.24 mmol) in CH2Cl2 (3 mL). The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. N-ethyl-6-fluoro-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 83) (60 mg, 0.24 mmol) and DIPEA (1.5 mL, 8.59 mmol) were added to the mixture in NMP (3 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 flash C18-flash chromatography using an elution gradient of 0-25% MeCN in water (NH4HCO3). The pure fraction was evaporated to dryness to obtain N-ethyl-6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]pyridine-2-carboxamide (Example 45) (2.60 mg, 3%) as a white solid. 1H NMR(300MHz,DMSO-d6)1.09(3H,t),2.40(3H,s),2.52-2.62(4H,m),3.17-3.27(4H,m),3.25(2 19F NMR(282MHz,DMSO-d6)-72.58,-135.52;m / z(ES + )[M+H] + =443. [ka]

[0256] Intermediate 84: 5-bromo-N-ethyl-6-methylpyridine-2-carboxamide Ethaneamine (3 mL, 2.20 mmol) (65 wt%) in H2O was added to methyl 5-bromo-6-methylpyridine-2-carboxylate (intermediate 14) (505 mg, 2.20 mmol). The resulting mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to obtain 5-bromo-N-ethyl-6-methylpyridine-2-carboxamide (intermediate 84) (0.500 g, 94%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d6) 1.13 (3H, t), 2.66 (3H, s), 3.26-3.39 (2H, m), 7.76 (1H, d), 8.18 (1H, d), 8.67-8.72 (1H, m); m / z (ES + )[M+H] + =243.

[0257] Intermediate 85: tert-butyl 4-[6-(ethylcarbamoyl)-2-methyl-3-pyridyl]piperazine-1-carboxylate Cs2CO3 (1.340 g, 4.11 mmol) was added to 1,4-dioxane (5 mL) containing 5-bromo-N-ethyl-6-methylpyridine-2-carboxamide (intermediate 84) (0.5 g, 2.06 mmol), tert-butylpiperazine-1-carboxylate (0.575 g, 3.09 mmol), BINAP (0.128 g, 0.21 mmol), and Pd(OAc)2 (0.046 g, 0.21 mmol). The resulting mixture was stirred at 100°C under nitrogen for 18 hours. The reaction mixture was diluted with ELISA (10 mL) and washed sequentially with water (10 mL x 2) and then brine (10 mL x 1). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by flash silica chromatography (elution gradient: 0-40% ethyl ether in petroleum ether). The pure fraction was evaporated to dryness to obtain tert-butyl 4-[6-(ethylcarbamoyl)-2-methyl-3-pyridyl]piperazine-1-carboxylate (intermediate 85) (0.481 g, 67%) as a yellow solid. ¹H NMR (300 MHz, chloroform-d): 1.26 (3H,t), 1.49 (9H,s), 2.54 (3H,s), 2.85-2.98 (4H,m), 3.49 (2H,qd), 3.56-3.65 (4H,m), 7.32 (1H,d), 7.91-8.01 (2H,m); m / z (ES + )[M+H] + =349.

[0258] Intermediate 86: N-ethyl-6-methyl-5-piperazine-1-ylpyridine-2-carboxamide HCl (4 ml, 16.00 mmol, 4 M) in 1,4-dioxane was added to tert-butyl 4-[6-(ethylcarbamoyl)-2-methyl-3-pyridyl]piperazine-1-carboxylate (intermediate 85) (0.481 g, 1.38 mmol) in MeOH (10 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. The reaction mixture was basicized with DIPEA (1 mL) in MeOH (3 mL). The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography using an elution gradient of 0-20% MeCN in water (NH4HCO3). The pure fraction was evaporated to dryness to obtain N-ethyl-6-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 86) (0.189 g, 55%) as a yellow oil. ¹H NMR (400 MHz, DMSO-d6): 1.12 (3H, t), 2.81-2.92 (8H, m), 3.27-3.36 (5H, m), 7.46 (1H, d), 7.81 (1H, d), 8.43 (1H, t); m / z (ES + )[M+H] + =249.

[0259] Example 46: N-ethyl-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide Ph3P (299 mg, 1.14 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-methylquinoxaline-2(1H)-one (158 mg, 0.76 mmol) (intermediate 17) and CBr4 (378 mg, 1.14 mmol) in CH2Cl2 (3.00 mL). The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. N-ethyl-6-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 86) (188 mg, 0.76 mmol) and DIPEA (1.5 mL, 8.59 mmol) were added to the mixture in NMP (3 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 flash C18-flash chromatography using an elution gradient of 0-25% MeCN in water (NH4HCO3). The pure fraction was evaporated to dryness to obtain N-ethyl-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-6-methylpyridine-2-carboxamide (Example 46) (7.40 mg, 2%) as a white solid. 1H NMR(300MHz,DMSO-d6)1.10(3H,t),2.40(3H,s),2.50(3H,s),2.54-2.64(4H,m),2.87-2.97(4H,m),3.30 (2H,q),3.70(2H,s),7.28(1H,t),7.47(1H,d),7.52(1H,d),7.77(1H,d),8.42(1H,t),12.44(1H,s);19F NMR(282MHz,DMSO-d6)-135.54;m / z(ES + )[M+H] + = 439. [ka]

[0260] Intermediate 87: 7-bromo-8-fluoro-3-(trifluoromethyl)-1H-quinoxaline-2-one Ethyl 3,3,3-trifluoro-2-oxopropanoate (2.30 g, 13.52 mmol) was added to 4-bromo-3-fluorobenzene-1,2-diamine (intermediate 72) (2.20 g, 10.73 mmol) in toluene (10 mL). The resulting mixture was stirred at 100 °C for 18 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography (elution gradient: 3-70% MeCN in water (0.1% NH4HCO3)). The pure fraction was evaporated to dryness to obtain 7-bromo-8-fluoro-3-(trifluoromethyl)-1H-quinoxalin-2-one (intermediate 87) (contaminated with 6-bromo-5-fluoro-3-(trifluoromethyl)-1H-quinoxalin-2-one) (3.40 g, 501%) as an off-white solid. m / z(ES + )[M+H] + =311.

[0261] Intermediate 88: 8-Fluoro-7-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxaline-2-one CataCxium A Pd G2 (53 mg, 0.08 mmol) was added to 1,4-dioxane (15 mL) containing 7-bromo-8-fluoro-3-(trifluoromethyl)-1H-quinoxalin-2-one (intermediate 87) (contaminated with 6-bromo-5-fluoro-3-(trifluoromethyl)-1H-quinoxalin-2-one) (0.5 g, 0.80 mmol) and (tributylstannyl)methanol (0.5 mL, 0.80 mmol). The resulting mixture was stirred under nitrogen at 80°C for 18 hours. The reaction mixture was quenched with saturated KF (1.25 mL). The reaction solution was collected by filtration and washed with dioxane (2.5 mL). The solvent of the combined organic layers was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography using an elution gradient of 3-40% MeCN in water (0.1%, TFA). The pure fraction was evaporated to dryness to obtain 8-fluoro-7-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one (intermediate 88) (contaminated with 5-fluoro-6-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one) (0.217 g, 51%) as an off-white solid. m / z(ES + )[M+H] + =263.

[0262] Example 47: 5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide SOCl2 (0.5 mL, 6.85 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one (intermediate 88) (contaminated with 5-fluoro-6-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one) (160 mg, 0.31 mmol) in Et2O (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. DIPEA (4 mL, 22.90 mmol) and N,6-dimethyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 33) (134 mg, 0.57 mmol) were added to the mixture in MeCN (10 mL). The resulting mixture was stirred at room temperature for 24 hours. The crude product was purified by preparative HPLC (column: XBridge BEH C18 OBD preparative column, 5 μm, 19 mm x 250 mm; mobile phase A: water (10 mmol / L NH4HCO3 + 0.1% NH3.H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 24B to 33B at 10 min; 254 / 220 nm; RT1: 8.2 / 9.5). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (Example 47) (8.8 mg, 6%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.50(3H,s),2.58-2.66(4H,m),2.79(3H,d),2.90-2.99(4H,m),3.77(2H ,s),7.41(1H,t),7.47(1H,d),7.72(1H,d),7.78(1H,d),8.39-8.44(1H,m),13.21(1H,brs);19F NMR(376MHz,DMSO-d6)-68.50,-133.81;m / z(ES + )[M+H] + = 479. [ka]

[0263] Example 48: 6-Fluoro-5-[4-[[5-Fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide SOCl2 (0.4 mL, 5.48 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one (intermediate 88) (contaminated with 5-fluoro-6-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one) (120 mg, 0.23 mmol) in Et2O (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. DIPEA (2 mL, 11.45 mmol) and 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 32) (156 mg, 0.65 mmol) were added to the mixture in MeCN (10 mL). The resulting mixture was stirred at room temperature for 24 hours. The crude product was purified by preparative HPLC (column: XBridge BEH C18 OBD preparative column, 5 μm, 19 mm x 250 mm; mobile phase A: water (10 mmol / L NH4HCO3 + 0.1% NH3.H2O), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 25B to 37B at 10 min; 254 / 220 nm; RT1: 7.58 / 8.97). The fraction containing the desired compound was evaporated to dryness to obtain 6-fluoro-5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 48) (8.9 mg, 8%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.58-2.65(4H,m),2.76(3H,d),3.14-3.21(4H,m),3.75(2H,s),7.3 9(1H,t),7.56(1H,dd),7.71(1H,d),7.84(1H,dd),8.37-8.43(1H,m),13.39(1H,brs);19F NMR(376MHz,DMSO-d6)-68.48,-72.59,-133.78;m / z(ES + )[M+H] + =483. [ka]

[0264] Example 49: 6-Chloro-5-[4-[[5-Fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide SOCl2 (0.4 mL, 5.48 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one (intermediate 88) (contaminated with 5-fluoro-6-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one) (120 mg, 0.23 mmol) in Et2O (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. DIPEA (2 mL, 11.45 mmol) and 6-chloro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 30) (157 mg, 0.62 mmol) were added to the mixture in MeCN (10 mL). The resulting mixture was stirred at room temperature for 24 hours. 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: 45B to 57B at 10 min; 254 / 220 nm). The fraction containing the desired compound was evaporated to dryness to obtain 6-chloro-5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 49) (18 mg, 16%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.60-2.68(4H,m),2.78(3H,d),3.07-3.16(4H,m),3.77(2H,s),7. 40(1H,t),7.66(1H,d),7.72(1H,d),7.93(1H,d),8.40-8.43(1H,m),13.25(1H,brs);19F NMR(376MHz,DMSO-d6)-68.51,-133.73;m / z(ES + )[M+H] + = 499. [ka]

[0265] Example 50: 5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide SOCl2 (0.4 mL, 5.48 mmol) was added to 8-fluoro-7-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one (intermediate 88) (contaminated with 5-fluoro-6-(hydroxymethyl)-3-(trifluoromethyl)-1H-quinoxalin-2-one) (120 mg, 0.23 mmol) in Et2O (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. DIPEA (2 mL, 11.45 mmol) and N-methyl-5-piperazine-1-ylpyridine-2-carboxamide (intermediate 31) (259 mg, 1.18 mmol) were added to the mixture in MeCN (10 mL). The resulting mixture was stirred at room temperature for 24 hours. 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: 15B to 35B at 10 min; 254 / 220 nm; RT1: 10.18 / 11.2). The fraction containing the desired compound was evaporated to dryness to obtain 5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 50) (6 mg, 6%) as a white solid. 1H NMR(400MHz,DMSO-d6)2.51-2.57(4H,m),2.76(3H,d),3.25-3.34(4H,m),3.72(2H,s),7 .30(1H,t),7.39(1H,dd),7.65(1H,d),7.83(1H,d),8.27(1H,d),8..36-8.41(1H,m);19F NMR(376MHz,DMSO-d6)-68.34,-133.80;m / z(ES + )[M+H] + =465. [ka]

[0266] Intermediate 90: Methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-methyl-butanoate DIPEA (2.202 mL, 12.61 mmol) was slowly added to a stirred solution of 1-bromo-2,4-difluoro-3-nitrobenzene (intermediate 35) (1 g, 4.20 mmol) and methylvalinate, HCl (intermediate 89) (0.704 g, 4.20 mmol) in DMF (6 mL). The resulting solution was stirred at room temperature for 18 hours (complete conversion to the desired product by LC-MS). The reaction mixture was concentrated, diluted with water, extracted with ethyl acetate, the organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by normal-phase chromatography with hexane:ethyl acetate to obtain methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-methyl-butanoate (0.763 g, 52.0%) (intermediate 90) as a bright orange solid. ¹H NMR (500 MHz, dichloromethane-d²): 1.00-1.14 (6H, m), 2.20-2.35 (1H, m), 3.78 (3H, s), 4.06 (1H, dd), 6.52 (1H, br d), 7.39 (1H, br d), 7.52 (1H, dd); ¹⁹F NMR (471 MHz, dichloromethane-d²): -109.33 (1F, s); m / z (ES + )[M+H] + =349.

[0267] Intermediate 91: 7-bromo-8-fluoro-3-isopropyl-3,4-dihydro-1H-quinoxaline-2-one Zinc powder (1.143 g, 17.48 mmol) was gradually added at 0°C to a mixture of methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-3-methyl butanoate (0.763 g, 2.19 mmol) (intermediate 90) and ammonium chloride (0.935 g, 17.48 mmol) in MeOH (12 mL) and water (0.3 mL) (exothermic reaction). This mixture was stirred at room temperature for 2 hours (no SM remained, and the complete disappearance of the orange color indicates completion of the reaction). Zn was filtered off, and the solid cake was washed with 20% MeOH in DCM. The filtrate was concentrated under vacuum. Water was added to the crude product, and the product was extracted into an ethyl acetate layer. The organic layer was dried and concentrated under vacuum to obtain a colorless oil. The crude product was slurried in 1:1 ethyl acetate:methanol, and 0.5 mL of 4N HCl in dioxane was added. The reaction mixture was stirred for 1 hour (no non-cyclized product remained). The reaction mixture was concentrated to obtain 7-bromo-8-fluoro-3-isopropyl-3,4-dihydro-1H-quinoxarin-2-one (intermediate 91). The crude product was used as a reagent for the next step without further purification, assuming a 100% yield of this reaction. m / z(ES) + )[M+H + =287.

[0268] Intermediate 92: 7-bromo-8-fluoro-3-isopropyl-1H-quinoxaline-2-one 4,5-Dichloro-3,6-Dioxocyclohexa-1,4-diene-1,2-dicarbononitrile (595 mg, 2.62 mmol) was added all at once to a 20 mL stirred solution of 7-bromo-8-fluoro-3-isopropyl-3,4-dihydroquinoxaline-2(1H)-one (627 mg, 2.18 mmol) (intermediate 91) in DCM (20 mL). The resulting slurry was stirred at room temperature for 2 hours (complete conversion to the desired product was confirmed by LC-MS). The reaction mixture was concentrated under vacuum and quenched with a saturated aqueous solution of sodium bicarbonate. The slurry was stirred overnight at room temperature, and the solid was filtered off. The filtered solid was thoroughly washed with water, then with diethyl ether, and dried to obtain 7-bromo-8-fluoro-3-isopropyl-1H-quinoxaline-2-one (0.425 g, 68.3%) (intermediate 92) as an off-white solid. ¹H NMR (500 MHz, DMSO-d6) 1.22 (6H, d), 3.36-3.52 (1H, m), 7.45-7.58 (2H, m), 12.62 (1H, br s); ¹⁹F NMR (471 MHz, DMSO-d6) - 124.16 (1F, s); m / z (ES + )[M+H] + =285.

[0269] Intermediate 93: 8-Fluoro-7-(hydroxymethyl)-3-isopropyl-1H-quinoxaline-2-one Xphos Pd G2 (103 mg, 0.13 mmol) was added to a stirred, degassed solution of 7-bromo-8-fluoro-3-isopropylquinoxaline-2(1H)-one (375 mg, 1.32 mmol) (intermediate 92) and (tributylstannyl)methanol (507 mg, 1.58 mmol) in 1,4-dioxane (6.58 mL). The resulting solution was stirred at 80°C for 16 hours. The reaction mixture was concentrated under vacuum and purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain 8-fluoro-7-(hydroxymethyl)-3-isopropyl-1H-quinoxaline-2-one (0.255 g, 82%) (intermediate 93) as a white solid. 1H NMR(500MHz,DMSO-d6)1.22(6H,d),3.39-3.52(1H,m),4.64(2H,d),5.41(1H,t),7.33(1H,s),7.55(1H,d),12.42(1H,br s).;19F NMR(471MHz,DMSO-d6)-137.71(1F,s).;m / z(ES + )[M+H] + =237.

[0270] Intermediate 94: 7-(bromomethyl)-8-fluoro-3-isopropyl-1H-quinoxaline-2-one Triethylphosphan (0.477 ml, 3.23 mmol) was added dropwise over 5 minutes at 0°C under nitrogen to a stirred solution of 8-fluoro-7-(hydroxymethyl)-3-isopropylquinoxaline-2(1H)-one (0.2541 g, 1.08 mmol) (intermediate 93) and CBr4 (1.177 g, 3.55 mmol) in DCM (8.49 mL). The reaction mixture was stirred at room temperature for 1 hour, and the DCM was removed under vacuum. The resulting solid was slurryed in diethyl ether. The white ppt was filtered under vacuum and washed with water, then with ether. The solid was dried overnight under vacuum (without heating) to obtain 7-(bromomethyl)-8-fluoro-3-isopropyl-1H-quinoxaline-2-one (0.313 g, 97%) (intermediate 94) as a light brown solid. m / z(ES + )[M+H] + =299.

[0271] Example 51: 6-Fluoro-5-[4-[(5-Fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide 7-(bromomethyl)-8-fluoro-3-isopropylquinoxaline-2(1H)-one (100 mg, 0.33 mmol) (intermediate 94) was mixed with 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide,2HCl (104 mg, 0.33 mmol) (intermediate 32), acetonitrile (5 mL), and N-ethyl-N-isopropylpropan-2-amine (291 μL, 1.67 mmol), and the mixture was heated to 70°C. LC-MS showed complete disappearance of SM and formation of the desired product after 1 hour. The reaction mixture was cooled, concentrated, quenched with aqueous NaHCO3 solution (1 mL), and stirred at room temperature for 1 hour. Water (3 mL) was added to the mixture and stirred for 10 minutes. The precipitate was filtered and washed with a large amount of water (50 mL). The solid was purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain 6-fluoro-5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (0.050 g, 32.8%) (Example 51) as a white solid. 1H NMR(500MHz,DMSO-d6)1.22(6H,d),2.53-2.65(4H,m),2.77(3H,d),3.12-3.24(4H,m),3.36-3.52 (1H,m),3.71(2H,s),7.30(1H,t),7.52-7.59(2H,m),7.84(1H,d),8.36-8.41(1H,m),12.46(1H,br s).;19F NMR(471MHz,DMSO-d6)-135.53(1F,s),-72.59(1F,s).;m / z(ES + )[M+H] + =457. [ka]

[0272] Example 52: 5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide 7-(bromomethyl)-8-fluoro-3-isopropylquinoxaline-2(1H)-one (109 mg, 0.36 mmol) (intermediate 94) was mixed with N,6-dimethyl-5-(piperazin-1-yl)picolinamide,2HCl (112 mg, 0.36 mmol) (intermediate 33), acetonitrile (5 mL), and N-ethyl-N-isopropylpropan-2-amine (317 μl, 1.82 mmol), and the mixture was heated to 70°C. LC-MS showed complete disappearance of SM and formation of the desired product after 1 hour. The reaction mixture was cooled, concentrated, quenched with aqueous NaHCO3 solution (1 mL), and stirred at room temperature for 1 hour. Water (3 mL) was added to the mixture and stirred for 10 minutes. The precipitate was filtered and washed with a large amount of water (50 mL). The solid was purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain 5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (0.057 g, 34.6%) as a white solid (Example 52). 1H NMR(500MHz,DMSO-d6)1.22(6H,d),2.46-2.49(3H,m),2.52-2.68(4H,m),2.80(3H,d),2.94(4H,br s),3.36-3.52(1H,m),3.73(2H,s),7.30(1H,t),7.47(1H,d),7.56(1H,d),7.79(1H,d),8.37-8.44(1H,m),12.46(1H,s).;19F NMR(471MHz,DMSO-d6)-135.55(1F,s).;m / z(ES + )[M+H] + =453. [ka]

[0273] Example 53: 5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide 7-(bromomethyl)-8-fluoro-3-isopropylquinoxaline-2(1H)-one (100 mg, 0.33 mmol) (intermediate 94) was mixed with N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (98 mg, 0.33 mmol) (intermediate 31), acetonitrile (5 mL), and N-ethyl-N-isopropylpropan-2-amine (291 μl, 1.67 mmol), and the mixture was heated to 70°C. LC-MS showed complete disappearance of SM and formation of the desired product after 1 hour. The reaction mixture was cooled, concentrated, quenched with aqueous NaHCO3 solution (1 mL), and stirred at room temperature for 1 hour. Water (3 mL) was added to the mixture and stirred for 10 minutes. The precipitate was filtered and washed with a large amount of water (50 mL). The solid was purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain 5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (0.052 g, 35.5%) (Example 53) as a white solid. 1H NMR(500MHz,DMSO-d6)1.22(6H,d),2.52-2.61(4H,m),2.78(3H,d),3.26-3.30(4H,m),3.36-3.52( 1H,m),3.70(2H,s),7.31(1H,t),7.38(1H,dd),7.56(1H,d),7.82(1H,d),8.26(1H,d),8.38(1H,br d),12.45(1H,br s).;19F NMR(471MHz,DMSO-d6)-135.54(1F,s).;m / z(ES + )[M+H] + = 439. [ka]

[0274] Intermediate 96: Methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-2-cyclopropyl acetate DIPEA (2.202 mL, 12.61 mmol) was slowly added to a stirred solution of 1-bromo-2,4-difluoro-3-nitrobenzene (intermediate 35) (1 g, 4.20 mmol) and methyl 2-amino-2-cyclopropyl acetate,HCl (intermediate 95) (0.696 g, 4.20 mmol) in DMF (6 mL). The resulting solution was stirred at room temperature for 18 hours (complete conversion to the desired product by LC-MS). The reaction mixture was concentrated, diluted with water, extracted with ethyl acetate, the organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by normal-phase chromatography using hexane and ethyl acetate to obtain methyl 2-(4-bromo-3-fluoro-2-nitro-anilino)-2-cyclopropyl acetate (0.635 g, 43.5%) (intermediate 96) as a bright orange solid. ¹H NMR (500MHz, dichloromethane-d2): 0.39-0.49 (¹H, m), 0.54 (¹H, td), 0.64-0.75 (²H, m), 1.25-1.39 (¹H, m), 3.74-3.83 (⁴H, m), 6.45 (¹H, dd), 7.34 (¹H, br d), 7.52 (¹H, dd). ¹⁹F NMR (471MHz, dichloromethane-d2): -109.53 (¹F, s). m / z (ES) + )[M+H] + =347.

[0275] Intermediate 97: 7-bromo-3-cyclopropyl-8-fluoro-3,4-dihydro-1H-quinoxaline-2-one Zinc powder (957 mg, 14.63 mmol) was gradually added at 0°C to a mixture of methyl 2-((4-bromo-3-fluoro-2-nitrophenyl)amino)-2-cyclopropyl acetate (635 mg, 1.83 mmol) (intermediate 96) and ammonium chloride (783 mg, 14.63 mmol) in MeOH (12 mL) and water (0.3 mL) (exothermic reaction). This mixture was stirred at room temperature for 2 hours (no SM remained, and the complete disappearance of the orange color indicates completion of the reaction). Zn was filtered off, and the solid cake was washed with 20% MeOH in DCM. The filtrate was concentrated. The crude product showed mostly non-cyclized products. Water was added to the crude product, and the product was extracted into an ethyl acetate layer. The organic layer was dried and concentrated under vacuum to obtain oil. This substance was slurryed in 1:1 ethyl acetate:methanol, and 0.5 mL of 4N HCl in dioxane was added. The reaction mixture was stirred for 1 hour (no non-cyclized product remained). The reaction mixture was concentrated to obtain 7-bromo-3-cyclopropyl-8-fluoro-3,4-dihydro-1H-quinoxarin-2-one (intermediate 97) as a gray solid. The crude product was used as a reagent for the next step without further purification, assuming a 100% yield of this reaction. m / z(ES) + )[M+H]] + =285.

[0276] Intermediate 98: 7-bromo-3-cyclopropyl-8-fluoro-1H-quinoxaline-2-one 4,5-Dichloro-3,6-Dioxocyclohexa-1,4-diene-1,2-dicarbonitride (499 mg, 2.20 mmol) was added all at once to a 20 mL stirred solution of 7-bromo-3-cyclopropyl-8-fluoro-3,4-dihydroquinoxaline-2(1H)-one (522 mg, 1.83 mmol) (intermediate 97) in DCM. The resulting slurry was stirred at room temperature for 2 hours (complete conversion to the desired product was confirmed by LC-MS). The reaction mixture was concentrated under vacuum and quenched with a saturated aqueous solution of sodium bicarbonate. The slurry was stirred overnight at room temperature, and the solid was filtered off. The solid was thoroughly washed with water, then with diethyl ether, and dried to obtain 7-bromo-3-cyclopropyl-8-fluoro-1H-quinoxaline-2-one (0.382 g, 73.7%) (intermediate 98) as an off-white solid. m / z(ES + )[M+H] + =283.

[0277] Intermediate 99: 3-Cyclopropyl-8-fluoro-7-(hydroxymethyl)-1H-Quinoxaline-2-one Xphos Pd G2 (92 mg, 0.12 mmol) was added to a 1,4-dioxane (5.86 mL) stirred degassed solution of 7-bromo-3-cyclopropyl-8-fluoroquinoxaline-2(1H)-one (332 mg, 1.17 mmol) (intermediate 98) and (tributylstannyl)methanol (452 ​​mg, 1.41 mmol), and the resulting solution was stirred at 80°C for 16 hours. The reaction mixture was concentrated under vacuum and purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain 3-cyclopropyl-8-fluoro-7-(hydroxymethyl)-1H-quinoxaline-2-one (0.224 g, 82%) (intermediate 99) as a white solid. 1H NMR(500MHz,DMSO-d6)1.02-1.14(4H,m),2.52-2.73(1H,m),4.62(2H,d),5.38(1H,t),7.29(1H,t),7.43(1H,d),12.43(1H,br s).;19F NMR(471MHz,DMSO-d6)-137.67(1F,s).;m / z(ES + )[M+H] +=235.

[0278] Intermediate 100: 7-(bromomethyl)-3-cyclopropyl-8-fluoro-1H-quinoxaline-2-one Triethylphosphan (0.422 ml, 2.86 mmol) was added dropwise over 5 minutes at 0°C under nitrogen to a mixture of 3-cyclopropyl-8-fluoro-7-(hydroxymethyl)quinoxaline-2(1H)-one (0.223 g, 0.95 mmol) (intermediate 99) and CBr4 (1.043 g, 3.14 mmol) in diethyl ether (7.52 mL). The reaction mixture was stirred at room temperature for 1 hour. The diethyl ether was removed under vacuum, and the resulting solid was slurryed in diethyl ether. The pale greenish-white ppt was filtered under vacuum and washed with water, then with ether. The solid was dried overnight under vacuum (without heating) to obtain 7-(bromomethyl)-3-cyclopropyl-8-fluoro-1H-quinoxaline-2-one (0.193 g, 68.2%) (intermediate 100) as a pale green solid. 1H NMR(500MHz,DMSO-d6)1.03-1.17(4H,m),2.63-2.76(1H,m),4.79(2H,s),7.33(1H,t),7.43(1H,d),12.55(1H,br s).;19F NMR(471MHz,DMSO-d6)-133.65(1F,s).;m / z(ES + )[M+H] + =297.

[0279] Example 54: 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide 7-(bromomethyl)-3-cyclopropyl-8-fluoroquinoxaline-2(1H)-one (75 mg, 0.25 mmol) (intermediate 100) was mixed with 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (79 mg, 0.25 mmol) (intermediate 32), acetonitrile (5 mL), and N-ethyl-N-isopropylpropan-2-amine (220 μl, 1.26 mmol), and the mixture was heated to 70°C. LC-MS showed complete disappearance of SM and formation of the desired product after 1 hour. The reaction mixture was cooled, concentrated, quenched with aqueous NaHCO3 solution (1 mL), and stirred at room temperature for 1 hour. Water (3 mL) was added to the mixture and stirred for 10 minutes. The precipitate was filtered and washed with a large amount of water (50 mL). The solid was purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide (0.048 g, 41.8%) (Example 54) as a white solid. 1H NMR(500MHz,DMSO-d6)1.04-1.13(4H,m),2.52-2.63(4H,m),2.71(1H,s),2.77(3H,d),3.12 -3.21(4H,m),3.69(2H,s),7.26(1H,t),7.43(1H,d),7.55(1H,dd),7.84(1H,d),8.39(1H,br d),12.46(1H,br s).;19F NMR(471MHz,DMSO-d6)-135.52(1F,s),-72.58(1F,s).;m / z(ES + )[M+H] + =455. [ka]

[0280] Example 55: 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide 7-(bromomethyl)-3-cyclopropyl-8-fluoroquinoxaline-2(1H)-one (75 mg, 0.25 mmol) (intermediate 100) was mixed with N,6-dimethyl-5-(piperazin-1-yl)picolinamide,2HCl (78 mg, 0.25 mmol) (intermediate 33), acetonitrile (5 mL), and N-ethyl-N-isopropylpropan-2-amine (220 μL, 1.26 mmol), and the mixture was heated to 70°C. LC-MS showed complete conversion to the desired product after 1 hour. The reaction mixture was cooled, concentrated, quenched with aqueous NaHCO3 solution (1 mL), and stirred at room temperature for 1 hour. Water (3 mL) was added to the mixture and stirred for 10 minutes. The precipitate was filtered and washed with a large amount of water (50 mL). The solid was purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide (0.049 g, 43.1%) (Example 55) as a white solid. 1H NMR(500MHz,DMSO-d6)1.03-1.15(4H,m),2.46-2.49(3H,m),2.52-2.65(4H,m),2.65-2.75(1H,m),2.80(3H,d),2.94(4H,br s),3.71(2H,s),7.26(1H,t),7.40-7.50(2H,m),7.79(1H,d),8.37-8.44(1H,m),12.46(1H,s).;19F NMR(471MHz,DMSO-d6)-135.54(1F,s).;m / z(ES + )[M+H] + =451. [ka]

[0281] Example 56: 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide 7-(bromomethyl)-3-cyclopropyl-8-fluoroquinoxaline-2(1H)-one (43 mg, 0.14 mmol) (intermediate 100) was mixed with N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (42.4 mg, 0.14 mmol) (intermediate 31), acetonitrile (5 mL), and N-ethyl-N-isopropylpropan-2-amine (126 μl, 0.72 mmol) and heated to 70°C. LC-MS showed complete disappearance of SM and formation of the desired product after 1 hour. The reaction mixture was cooled, concentrated, quenched with aqueous NaHCO3 (1 mL), and stirred at room temperature for 1 hour. Water (3 mL) was added to the mixture and stirred for 10 minutes. The precipitate was filtered and washed with a large amount of water (25 mL). The solid was purified by normal-phase chromatography using 0-10% MeOH in DCM to obtain 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (0.020 g, 31.7%) (Example 56) as a white solid. 1H NMR (500MHz, DMSO-d6): 1.03-1.16 (4H,m), 2.53-2.60 (4H,m), 2.65-2.80 (5H,m), 3.68 (2H,s), 7.25 (1H,br t), 7.38 (1H,dd), 7.42 (1H,d), 7.82 (1H,d), 8.25 (1H,d), 8.35-8.40 (1H,m), 12.38-12.51 (1H,m) (3H missing, possible overlap with DMSO peak); 19F NMR (471MHz, DMSO-d6): -135.52 (1F,s).; m / z (ES + )[M+H] + =437 [ka]

[0282] Intermediate 102: 7-bromo-3-methoxy-8-methyl-1H-quinoxaline-2-one A mixture of 4-bromo-3-methylbenzene-1,2-diamine (1.75 g, 8.70 mmol) (intermediate 101), 2,2,2-trimethoxymethyl acetate (2.86 g, 17.41 mmol), and ytterbium(III) trifluoromethanesulfonate (0.540 g, 0.87 mmol) in toluene (10 mL) was degassed in a sealed tube, refilled with N2, and stirred overnight at 100°C to obtain a brown suspension. LC-MS showed the formation of the desired product. The mixture was cooled to room temperature, the solid was collected by filtration, washed with methanol, and dried to obtain 7-bromo-3-methoxy-8-methyl-1H-quinoxaline-2-one (1.2 g, 51.2%) (intermediate 102) as a yellow solid (containing approximately 8% of its positional isomer 6-bromo-3-methoxy-5-methylquinoxaline-2(1H)-one). ¹H NMR (500 MHz, DMSO-d6) 2.50 (3H, br s), 3.97 (3H, s), 7.32 (1H, d), 7.45 (1H, d), 11.79 (1H, br s); (m / z) (ES + )[M+H] + =269.

[0283] Intermediate 103: 7-(hydroxymethyl)-3-methoxy-8-methyl-1H-quinoxaline-2-one A mixture of (tributylstannyl)methanol (1.844 g, 5.74 mmol), 7-bromo-3-methoxy-8-methyl-1H-quinoxalin-2-one (1.03 g, 3.83 mmol) (intermediate 102), and Xphos Pd G2 (0.452 g, 0.57 mmol) in 1,4-dioxane (40 mL) was stirred overnight at 80°C under N2 to obtain a dark-colored mixture. LC-MS showed almost complete conversion. The solvent was removed under reduced pressure, and the residue was purified by silica gel column (eluted with 0-20% methanol in DCM). The fraction was concentrated to obtain a yellow solid, which was checked by LC-MS and shown to be not very pure. The product was then slurryed in 20 mL of methanol, the solid was collected by filtration, and dried to obtain a 55% pure product as a yellow solid (containing 30% starting material and 9.5% debromination by-products).

[0284] The previously obtained solid was placed in a dry flask with 1,4-dioxane (40 mL), and 900 mg of (tributylstanny)methanol and 300 mg of xphos Pd G2 were added to the flask. The mixture was degassed and stirred overnight at 80°C under N2. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (elution with 0-20% methanol in DCM) to obtain 7-(hydroxymethyl)-3-methoxy-8-methyl-1H-quinoxalin-2-one (800 mg, 95%) (intermediate 103) as a yellow solid (purity 80% according to LCMS). (m / z) (ES + )[M+H] + =221.

[0285] Intermediate 104: 7-(bromomethyl)-3-methoxy-8-methyl-1H-quinoxaline-2-one Triethylphosphan (294 μl, 2.04 mmol) was added dropwise to a CH2Cl2 (20 mL) suspension of 7-(hydroxymethyl)-3-methoxy-8-methyl-1H-quinoxalin-2-one (300 mg, 1.36 mmol) (intermediate 103) and 1,1,2,2-tetrabromo-1,2-dichloroethane (875 mg, 2.11 mmol), and the resulting mixture was incubated at room temperature for 3.5°C. After stirring for a certain time, the solvent was removed under reduced pressure, the residue was suspended in ether (10 mL), filtered, the solid was washed with ether (10 mL x 2), the solid was suspended in water (20 mL), filtered, washed with water (5 mL x 3), dried, and 7-(bromomethyl)-3-methoxy-8-methyl-1H-quinoxalin-2-one (0.250 g, 64.8%) (intermediate 104) was obtained as a pale yellow solid. (m / z)(ES + )[M+H] + =285.

[0286] Example 57: 5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide To a suspension of N,6-dimethyl-5-(piperazin-1-yl)picolinamide, 2HCl (83 mg, 0.27 mmol) (intermediate 33) and 7-(bromomethyl)-3-methoxy-8-methyl-1H-quinoxalin-2-one (85 mg, 0.27 mmol) (intermediate 104) in acetonitrile (6 mL), DIPEA (236 μL, 1.35 mmol) was added, and the resulting mixture was heated at 70°C for 2°C. The mixture was stirred for a period of time to obtain a clear solution, the mixture was cooled to room temperature to obtain a suspension, the solid was collected by filtration, washed with water and acetonitrile, and dried to obtain 5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N,6-dimethylpyridine-2-carboxamide (0.064 g, 54.3%) (Example 57) as a white solid. 1H NMR(500MHz,DMSO-d6)2.43(3H,s),2.49(3H,s),2.57(4H,br s),2.80(3H,d),2.91(4H,br (m / z)(ES + )[M+H] + =437 [ka]

[0287] Example 58: 6-Fluoro-5-[4-[(2-Methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide To a suspension of 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide, 2HCl (84 mg, 0.27 mmol) (intermediate 32) and 7-(bromomethyl)-3-methoxy-8-methyl-1H-quinoxalin-2-one (85 mg, 0.27 mmol) (intermediate 104) in acetonitrile (6 mL), DIPEA (236 μL, 1.35 mmol) was added, and the resulting mixture was stirred at 70°C for 2 hours to obtain a suspension. The mixture was cooled to room temperature, the solid was collected by filtration, washed with water and acetonitrile, dried, the solid was suspended in acetonitrile, filtered, and dried to obtain 6-fluoro-5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (0.073 g, 61.3%) (Example 58) as a beige solid. 1H NMR(500MHz,DMSO-d6)2.42(3H,s),2.55(4H,br s),2.76(3H,d),3.14(4H,br s),3.58(2H,s),3.95(3H,s),7.17(1H,br d),7.35(1H,br d),7.50-7.63(1H,m),7.83(1H,br d),8.38(1H,br d),11.58(1H,s);(m / z)(ES + )[M+H] + = 442. [ka]

[0288] Intermediate 106: Methyl 6-bromo-5-fluoropyridine-2-carboxylate Sulfuric acid (1.5 mL, 28.14 mmol) was slowly added to a mixture of 6-bromo-5-fluoropicolinic acid (500 mg, 2.27 mmol) (intermediate 105) and MeOH (8 mL). This mixture was continuously stirred at room temperature for 3 hours to obtain a white suspension. LC-MS showed complete conversion. The mixture was poured into a saturated aqueous solution of NaHCO3 and extracted with DCM (40 mL x 2). The organic layer was dried (anhydrous Na2SO4), filtered, and concentrated to obtain methyl 6-bromo-5-fluoropyridine-2-carboxylate (532 mg, 100%) (intermediate 106) as a white solid, which was used in the next step without further purification. ¹H NMR (500 MHz, chloroform-d) 4.01 (3H, s), 7.55 (1H, t), 8.15 (1H, dd); (m / z) (ES + )[M+H] + =236.

[0289] Intermediate 107: tert-butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate A mixture of tert-butylpiperazine-1-carboxylate (8.21 g, 44.06 mmol), methyl 6-bromo-5-fluoropyridine-2-carboxylate (6.065 g, 25.92 mmol) (intermediate 106), and potassium carbonate (4.66 g, 33.69 mmol) in DMF (60 mL) was stirred at 110 °C for 5 hours. LC-MS showed complete conversion. The mixture was cooled to room temperature, diluted with DCM and water, the layers were separated, the aqueous layer was extracted twice with DCM, the organic layers were combined, dried (anhydrous Na2SO4), filtered and concentrated, and the residue was purified by silica gel column chromatography (elution with 0-50% ethyl acetate in hexane, UV at 221 and 310 nm) to obtain the desired product tert-butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (7.68 g, 74.1%) (intermediate 107) as a white solid. ¹H NMR (500 MHz, chloroform-d): 1.51 (9H, s), 3.14 (4H, br t), 3.60-3.71 (4H, m), 3.99 (3H, s), 7.32 (1H, d), 8.08 (1H, d); (m / z) (ES+ )[M+H] + =402.

[0290] Intermediate 108: tert-butyl 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate tert-butyl 4-(2-bromo-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (7.67 g, 19.16 mmol) (intermediate 107) was stirred in a sealed container at 60°C for 4.5 hours in methanamine (100 mL, 19.16 mmol) (33% in ethanol). LC-MS showed complete conversion. The mixture was cooled to room temperature, concentrated, the residue dissolved in DCM, washed with saturated NH4Cl solution, dried (anhydrous Na2SO4), filtered and concentrated to obtain tert-butyl 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (7.48 g, 98%) (intermediate 108) as a white solid. ¹H NMR (500 MHz, chloroform-d): 1.50 (9H, s), 3.02 (3H, d), 3.08 (4H, br t), 3.60-3.71 (4H, m), 7.36 (1H, d), 7.68 (1H, br d), 8.11 (1H, d); (m / z) (ES + )[M+H] + =401.

[0291] Intermediate 109: tert-butyl 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate A mixture of tert-butyl 4-[2-bromo-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (1.344 g, 3.37 mmol) (intermediate 108), tributyl(vinyl) stannane (1.174 g, 3.70 mmol), and Xphos Pd G2 (0.132 g, 0.17 mmol) in 1,4-dioxane (25 ml) was stirred at 100°C for 2.5 hours under N2. LC-MS showed complete conversion. The mixture was diluted with DCM, washed with saturated NH4Cl, the organic layer was dried (anhydrous Na2SO4), filtered and concentrated, and the residue was purified by silica gel column (eluted with 0-80% ethyl acetate in hexane, UV at 226 and 293 nm) to obtain tert-butyl 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate (0.961 g, 82%) (intermediate 109) as a white solid. ¹H NMR (500 MHz, chloroform-d): 1.50 (9H, s), 2.90-3.01 (4H, m), 3.05 (3H, d), 3.55-3.68 (4H, m), 5.54 (1H, dd), 6.42 (1H, dd), 7.10 (1H, dd), 7.39 (1H, d), 7.98 (1H, br d), 8.07 (1H, d); m / z (ES + )[M+H] + =346.6,348.5.

[0292] Intermediate 110: tert-butyl 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate Osmium tetroxide (0.0435 mL, 6.00 μmol) in H2O was added to a solution of tert-butyl 4-[6-(methylcarbamoyl)-2-vinyl-3-pyridyl]piperazine-1-carboxylate (960 mg, 2.77 mmol) (intermediate 109), 2,6-lutidine (646 μl, 5.54 mmol), and sodium periodate (2371 mg, 11.08 mmol) in THF (25 mL) / water (5 mL) / tert-butanol (2650 μL, 27.71 mmol). The mixture was stirred overnight at room temperature to obtain a yellow suspension. LC-MS and TLC showed complete conversion. The reaction mixture was diluted with water and extracted with ethyl acetate. After concentration, the crude product was purified by silica column chromatography (elution with 0-100% ethyl acetate in hexane, UV at 226 and 310 nm) to obtain tert-butyl 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (0.732 g, 76%) (intermediate 110) as a yellow solid. ¹H NMR (500 MHz, chloroform-d): 1.50 (9H, s), 3.07 (3H, d), 3.15-3.30 (4H, m), 3.63-3.79 (4H, m), 7.48 (1H, d), 7.85 (1H, br d), 8.28 (1H, d), 10.10 (1H, s); (m / z) (ES + )[M+H] + =349.

[0293] Intermediate 111: tert-butyl 4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate Tert-butyl 4-[2-formyl-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (730 mg, 2.10 mmol) (intermediate 110) was cooled to 0°C in CH2Cl2 (10 mL), and DAST (692 μL, 5.24 mmol) in DCM (5 mL) was added to this mixture. The resulting mixture was then stirred at room temperature for 4 hours, and TLC and LCMS showed complete conversion. The reaction mixture was quenched by adding a saturated aqueous solution of NaHCO3 dropwise, extracted by DCM, the organic matter was dried (anhydrous Na2SO4), filtered and concentrated, and the residue was purified by silica gel column (elution with 0-100% ethyl acetate in hexane, UV at 254 and 293 nm) to obtain tert-butyl 4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (0.666 g, 86%) (intermediate 111) as a white solid. ¹H NMR (500 MHz, chloroform-d): 1.50 (9H, s), 2.93-3.02 (4H, m), 3.05 (3H, d), 3.57-3.72 (4H, m), 6.99 (1H, t), 7.62 (1H, d), 7.92 (1H, br d), 8.27 (1H, d); (m / z) (ES + )[M+H] + =371.

[0294] Intermediate 60: 6-(difluoromethyl)-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide,2HCl 4M HCl (7 mL, 28.00 mmol) in dioxane was added to a flask containing tert-butyl 4-[2-(difluoromethyl)-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (665 mg, 1.80 mmol) (intermediate 111) and a stirring bar. The mixture was stirred at room temperature for 1 hour to obtain a yellow suspension. The solvent was removed, the residue was diluted with ether, and the solid was collected by filtration and dried to obtain 6-(difluoromethyl)-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide,2HCl (0.617 g, 100%) (intermediate 60) as an orange solid. (m / z)(ES) + )[M+H] +=272.

[0295] Example 59: 6-(difluoromethyl)-5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide To a suspension of 6-(difluoromethyl)-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide,2HCl (87 mg, 0.25 mmol) (intermediate 60) and 7-(bromomethyl)-3-methoxy-8-methyl-1H-quinoxaline-2-one (80 mg, 0.25 mmol) (intermediate 104) in acetonitrile (6 mL), DIPEA (222 μL, 1.27 mmol) was added, and the resulting mixture was heated to 70°C. The mixture was stirred for 2 hours to obtain a clear solution, the mixture was cooled to room temperature to obtain a suspension, the solid was collected by filtration, washed with acetonitrile and water, and dried to obtain 6-(difluoromethyl)-5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (0.070 g, 58.3%) (Example 59) as a white solid. 1H NMR(500MHz,DMSO-d6)2.43(3H,s),2.59(4H,br s),2.83(3H,br d),2.98(4H,br s),3.60(2H,s),3.95(3H,s),6.92-7.29(2H,m),7.35(1H,d),7.85(1H,br d),8.08(1H,d),8.38(1H,br d),11.58(1H,br s);((m / z)(ES + )[M+H] + = 473. [ka]

[0296] Example 60: 6-(difluoromethyl)-5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide A mixture of 7-(bromomethyl)-3,8-dimethyl-1H-quinoxalin-2-one (196 mg, 0.73 mmol) (intermediate 8), 6-(difluoromethyl)-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide,2HCl (252 mg, 0.73 mmol) (intermediate 60), and Et3N (0.614 mL, 4.41 mmol) in acetonitrile (25 mL) was stirred at 70°C for 2 hours to obtain a clear solution. LC-MS showed complete conversion. The mixture was cooled to room temperature overnight. The solid was crystallized from the mixture, collected by filtration, washed with acetonitrile and water, and dried to obtain 141 mg of product portion 1. The filtrate was concentrated and purified using reverse-phase Gilson (elution with 5-80% ACN / water / 0.1% TFA) to obtain 92 mg of product portion 2 as the TFA salt. Overall, 6-(difluoromethyl)-5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (0.233 g, 64.0%) (Example 60) was obtained as an off-white solid. 1H NMR(500MHz,DMSO-d6)2.40(3H,s),2.43(3H,s),2.60(4H,br s),2.83(3H,d),2.98(4H,br (m / z)(ES + )[M+H] + =457. [ka]

[0297] Intermediate 113: Methyl 6-chloro-5-(piperazin-1-yl) picolinate Piperazine (1.0 g, 11.61 mmol) was added to methyl 6-chloro-5-fluoropicolinate (intermediate 112, 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 elution 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 113, 1.28 g, 95%) as a red oil. 1 H 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 proton not shown);m / z(ES + )[M+H] + =256.

[0298] Intermediate 30: 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 113, 1.26 g, 4.93 mmol). The resulting mixture was stirred at 80°C for 18 hours. The solvent was removed under reduced pressure. The crude product was purified by reverse-phase chromatography using an elution gradient of 5-60% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 6-chloro-N-methyl-5-(piperazin-1-yl) picolinamide (intermediate 30, 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. [ka]

[0299] Intermediate 114: tert-butyl 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate Tert-butyl 4-(2-fluoro-6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 11, 12.49 g, 36.80 mmol) was stirred in methylamine (120 mL, 36.80 mmol, 33 wt% in ethanol) at room temperature for 24 hours (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 tert-butyl 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 114, 12.45 g, 100%) as a yellow solid. m / z(ES + )[M+H] + =340.

[0300] Intermediate 32: 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 tert-butyl 4-[2-fluoro-6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 114, 12.5 g, 36.94 mmol) in 1,4-dioxane (50 mL). The reaction mixture was stirred for 5 hours. During this 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. The solid was dried under vacuum to obtain 6-fluoro-N-methyl-5-piperazine-1-ylpyridine-2-carboxamide,2HCl (intermediate 32, 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]

[0301] Intermediate 115: 5-bromo-N,6-dimethylpicorinamide A methylamine THF2M solution (20 mL, 40.00 mmol) was added to methyl 5-bromo-6-methyl picolinate (intermediate 14, 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 elution 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 115, 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

[0302] Intermediate 116: tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridine-3-yl)piperazine-1-carboxylate 5-Bromo-N,6-dimethylpicolinamide (intermediate 115, 1.0 g, 4.37 mmol) was added under nitrogen to toluene (20 mL) with 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 elution 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 116, 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.

[0303] Intermediate 33: N,6-dimethyl-5-(piperazine-1-yl)picolinamide Tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridine-3-yl)piperazine-1-carboxylate (intermediate 115, 1.18 g, 3.53 mmol) was added to a 4 M solution of 1,4-dioxane in HCl (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 33, 0.77 g, 81%) as a yellow solid. 1H 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. [ka]

[0304] Intermediate 117: 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 5-bromopyridine-2-carboxylate methyl (intermediate 9, 30 g, 138.87 mmol), piperazine-1-carboxylate tert-butyl (27.2 g, 145.81 mmol), and Cs2CO3 (90 g, 277.73 mmol) in 1,4-dioxane (200 mL). The 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 layers were dried over anhydrous Na2SO4 and filtered. To this filtrate, silica gel functionalized with 3-(diethylenetriamino)propyl (12 g, 1.3 mmol / load g) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was filtered, and the filtrate was concentrated to approximately 100 mL. The yellow crystalline solid was filtered off, washed with ether, and dried under vacuum to obtain tert-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 117, 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.

[0305] Intermediate 118: 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 tert-butyl 4-(6-methoxycarbonyl-3-pyridyl)piperazine-1-carboxylate (intermediate 117, 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 saturated NH4Cl solution and DCM to separate the layers. The aqueous layer was extracted with DCM, the organic layers were combined, washed with brine, dried over Na2SO4, filtered, and concentrated to obtain tert-butyl 4-[6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 118, 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.

[0306] Intermediate 119: Methyl 5-(piperazin-1-yl)picolinate 4M HCl (20 ml, 576.01 mmol) in dioxane was added at 0°C to a mixture of tert-butyl 4-(6-(methoxycarbonyl)pyridine-3-yl)piperazine-1-carboxylate (intermediate 117, 1.55 g, 4.82 mmol) in MeOH (2 mL). The reaction mixture was stirred at room temperature for 2 hours to obtain a suspension. LC-MS showed complete conversion. The mixture was diluted with ether (approximately 80 ml), the solid was collected by filtration, washed with ether, and dried to obtain methyl 5-(piperazine-1-yl)picolinate (intermediate 119) (1.384 g, 98%) as a yellow solid. 1H NMR(500MHz,DMSO-d6)3.21(4H,br s),3.66(4H,br d),3.83(3H,s),7.43-7.55(1H,m),7.95(1H,br d),8.43(1H,br s),9.49(2H,br s);(m / z)(ES+)[M+H]+=223.0.

[0307] Intermediate 31: Carboxylate N-methyl-5-piperazine-1-ylpyridine-2-carboxamide HCl (4M in dioxane, 150 mL, 600.00 mmol) was added to a MeOH (50 mL) suspension of tert-butyl 4-[6-(methylcarbamoyl)-3-pyridyl]piperazine-1-carboxylate (intermediate 118, 35.9 g, 112.05 mmol), and the resulting orange suspension was stirred at room temperature 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 collected by filtration, washed with hexane, dried, and dried under vacuum to obtain N-methyl-5-piperazine-1-ylpyridine-2-carboxamide, 2HCl salt (intermediate 31, 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.

[0308] Example 61: Preparation of crystalline form B (anhydrous form) of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide Method 1 43 mg (0.10 mmol) of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (e.g., derived from Example 20) was suspended in 1.0 ml of MeOH, and 0.11 ml of 1 M aqueous methanesulfonic acid (MSA) was added to obtain a clear solution. To this solution, 0.11 ml of 1 N aqueous NaOH was added. After the addition of the NaOH solution was complete, a white solid began to precipitate. The slurry was stirred at room temperature for 1 day. 36 mg of the white solid was filtered and dried in the air. XRPD indicates that the solid is form B of pure 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide.

[0309] Method 2 Pyridine (93.5 g) was added at 75±5°C to a solution of pure 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide mesylate (4.67 kg, prepared via Method 2 of Example 63) in water (47.9 kg) and ethanol (38.0 kg). Then, a seed of form B of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide prepared according to Method 1 (4.7 g) was added. The slurry was stirred at 75±5°C for 40 minutes, and then a 50:50v:v water:ethanol (4.2kg) solution of pyridine (651g) was gradually added over 3 hours and 40 minutes. The slurry was stirred at 75±5°C for 50 minutes, and then a 50:50v:v water:ethanol (4.1kg) solution of 4-methylmorpholine (900g) was gradually added over 3 hours and 50 minutes. The slurry was stirred at 75±5°C for 1 hour and 10 minutes, cooled to 25±5°C for 4 hours and 50 minutes, stirred at 25±5°C for 15 hours, and then filtered. The obtained solid was washed twice with 50:50v:v water:ethanol (12.5 kg x 2), and then dried under vacuum at 25°C to 50°C for 1 day to obtain form B (3.54 kg) of pure 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide in 93% yield.

[0310] Morphology B derived from Method 1 was analyzed by XRPD, and the results are summarized in the table below (Table 1), and shown in Figure 1.

[0311] [Table 1]

[0312] Morphology B is characterized by exhibiting at least one of the following 2θ values ​​measured using CuKα radiation: 6.2°, 14.3°, and 15.6°.

[0313] Morphology B (by Method 1) was analyzed using thermal methods. DSC analysis showed that morphology B has a melting point that starts at 275°C and peaks at 276°C. TGA showed that morphology B exhibits a mass loss of approximately 0.2% when heated from approximately 25°C to approximately 100°C. A representative DSC / TGA thermogram of morphology B is shown in Figure 2.

[0314] Example 62: Preparation of crystalline form D (anhydrous form) of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide. 5-6 mg of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide (Example 20) was dissolved in a mixed solvent of MeOH / DCM / H2O (0.50 ml / 0.50 ml / 0.20 ml), and the clear solution was slowly evaporated under ambient conditions to obtain a white solid. XRPD indicates that the obtained white solid is morph D of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide.

[0315] Morphology D was analyzed using XRPD, and the results are summarized in the table below (Table 2), and shown in Figure 3.

[0316] [Table 2]

[0317] Morphology D is characterized by exhibiting at least one of the following 2θ values ​​measured using CuKα radiation: 7.9°, 13.1°, and 16.3°.

[0318] A single crystal of form D was obtained by evaporation of a DMF solution (or DMF / H2O) of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide. Single crystal structure analysis confirmed that form D is the anhydrous form. The molecular structure of form D of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxalin-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide is shown in Figure 4. Crystallographic data: space group monoclinic P21 / c, lattice constants: a=17.4559(8)Å, b=5.0647(2)Å, c=22.564(1)Å, β=92.609(1)°, V=1992.8(2)Å 3 .

[0319] Example 63: Preparation of the MSA crystalline salt form C (anhydrous form) of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide. Method 1 427 mg of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (Example 20) was suspended in 8.0 ml of MeOH. 1.1 ml of 1.0 M aqueous solution of MSA (1.1 mmol) was added to this suspension to obtain a clear solution. The obtained solution was filtered to remove the solvent from the clear solution. The obtained solid was suspended in 1.0 ml of EtOH and 2.0 ml of THF to obtain a slurry. The slurry was stirred at room temperature for 1 day. The solid was collected by filtration and air-dried. 452 mg of an off-white solid was obtained. XRD shows that form C of the MSA salt of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide was obtained.

[0320] Method 2 16.8 g of methanesulfonic acid was added at 25°C to a stirred suspension of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide (80.8 g, 92.8 w / w%) in 4:1 v:v THF:ethanol (750 mL). The resulting suspension was stirred at 25°C for 16 hours and then filtered. The solid was washed with 4:1v:v THF:ethanol (300 mL) and then dried under vacuum at 35°C to obtain form C (90.3 g) of 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide mesylate in 98% yield.

[0321] MSA-morphology C derived from Method 1 was analyzed by XRPD, and the results are summarized in the table below (Table 3) and shown in Figure 5.

[0322] [Table 3]

[0323] MSA-morphology C obtained from Method 1 was analyzed by thermal methods. DSC analysis showed that MSA-morphology C began to melt and decompose at a temperature of 254°C, peaking at 258°C. TGA showed that MSA-morphology C exhibited a mass loss of approximately 0.3% when heated from approximately 25°C to approximately 100°C. A representative DSC / TGA thermogram of MSA-morphology C is shown in Figure 6.

[0324] Biological assays The inhibitory properties of the compounds described herein can be determined using the following test procedure.

[0325] 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).

[0326] 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).

[0327] 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).

[0328] 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).

[0329] 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).

[0330] The fluorescence anisotropy of the probe when bound to a protein was measured using BMG Pherastar FSX (copyright) in the presence of the test compound or solvent control, and the effect on anisotropy was determined. 50 To determine the values, the inhibition percentage values ​​were calculated for different test compound concentrations and fitted to a 4-parameter logistic plot. Compound K was used where necessary. i This can be determined from the IC50 value using the Munson-Rodbard formula defined in Anal Biochem. 1980 Sep 1;107(1):220-39, and the known K of the probe that binds to the relevant PARP protein. D Based on.

[0331] PARP proliferation assay (7-day compound administration) DLD1 and BRCA2(- / -)DLD1 cells were collected in complete medium to densities of 5000 cells / ml and 2.5E4 cells / ml, respectively. 40 μL / well was seeded into 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 room temperature 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 the plates were incubated for 7 days in an incubator maintained at 37°C and 5% CO2. On day 8, synox 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 room temperature for over 3 hours. All cells are read using Cell Insight with a 4 × objective lens. Growth rate is determined in Geneda by evaluating the total cell count output from Cell Insight for plates on day 0 and day 8.

[0332] In vitro human transporter emissions MDCKII cells expressing MDR1 and BCRP were seeded on a polyethylene membrane in a 96-well Transwell insert system at a density that formed a confluent cell monolayer. The test compound and reference compound were diluted to a concentration of 1 or 0.1 μM in transport buffer (HBSS HEPES pH 7.4). The final volume percentage of organic solvent was less than 1%. The permeability of the test compound from A to B and from B to A was determined by incubation at 37°C, 5% CO2, and 95% relative humidity for 90 minutes. At the end of incubation, samples were taken from the apical and basal outer sides and precipitated with cold acetonitrile containing an internal standard. After centrifugation at 4000 rpm, the supernatant was diluted with 0.1% formic acid aqueous solution and quantified by LC-MS / MS. The integrity of the cell monolayer was confirmed using the marker Lucifer Yellow.

[0333] The transmission coefficient (1 × 10⁻⁶ cm / s) was calculated using the following formula. Papp = (dCr / dt) × Vr / (A × C0)

[0334] (1) The emission ratio was calculated using the following formula. Emission ratio = Papp(from B to A) / Papp(from A to B)

[0335] (2) In equation (2), dCr / dt is the cumulative concentration of the compound in the receiver chamber as a function of time (unit: μM / s), Vr is the volume of solution in the receiver chamber (0.1 ml at the apical end and 0.3 ml at the basal end), A is the surface area for transport, i.e., 0.11 cm2 for the area of ​​the single layer, and C0 is the initial concentration in the donor chamber (unit: μM).

[0336] Determination of unbound fractions in plasma The unbound fraction was identified using a RED device.

[0337] The compounds were prepared as a 10 mM solution in DMSO. A 1 mM working stock was prepared by mixing up to nine test compounds (4 μL each) and one control (μL). If fewer than nine test compounds were present, an additional volume of blank DMSO was added to bring the total volume to 40 μL.

[0338] Frozen plasma was thawed in a 37°C water bath. The plasma was then centrifuged at 4,000 rpm for 2 minutes to remove the blood clot, and the supernatant was collected in a new tube. The pH of the plasma was checked, and it was used only if it was within the range of pH 7 to pH 8. 3 μL of the working solution from each cassette was added to 597 μL of blank plasma and vortexed at 1,000 rpm for 5 minutes. The final volume percentage of the organic solvent was 0.5%, and the final concentration of the test compound was 5 μM. 50 μL of the added plasma suspension was immediately transferred to a 96-well plate to serve as the T=0 control sample. The sample was processed in the same manner as the sample after incubation. The remaining plasma was kept at 37°C before initiating dialysis.

[0339] Insert the insert into the wells of the base plate with the open end facing upwards. Add 300 μL of the added plasma sample to the sample chamber indicated by the red ring. Add 500 μL of phosphate buffer (pH 7.4) to the buffer chamber. Cover the unit with a gas-permeable lid and incubate it in an orbital shaker in a CO2 incubator at 37°C for 18 hours at 300 rpm with 5% CO2. At the end of incubation, remove the lid and pipette 50 μL of post-dialysis sample from both the buffer chamber and the plasma chamber into separate 96-well plates for analysis.

[0340] The samples were matched to the matrix by adding 50 μL of blank rat plasma to the buffer sample and an equal volume of PBS to the collected plasma sample, and then vortexed and mixed. 400 μL of acetonitrile containing a suitable internal standard (IS) was added to the precipitated protein to release the compound, and the plate was mixed by vortexing for 10 minutes, followed by centrifugation at 4,000 rpm for 30 minutes. 250 μL of supernatant was transferred to a new 96-well plate and centrifuged again (4,000 rpm, 30 minutes). Then, 100 μL of supernatant was transferred to a new 96-well plate and mixed with 100 μL of distilled water for each sample by vortexing at 1,000 rpm for 5 minutes. The samples were analyzed by LC-MS / MS, and the drug concentration was determined relative to a calibration curve generated from the added blank plasma, within the typical range of 1–7500 nM.

[0341] The unbound percentage was calculated using the formula: Unbound Percentage = (Buffer Chamber Concentration / Plasma Chamber Concentration) × 100%. Unbound Percentage = Unbound Percentage / 100.

[0342] Determination of the unbound fraction in brain sections The principle of the method for determining the unbound volume in brain sections has been previously published (Development of a High-Throughput Brain Slice Method for Studying Drug Distribution in the Central Nervous System; Friden et al,; Drug Metabolism and Disposition, 2009, 37(6)1226-1233). In short: A stock solution of the compounds was prepared in DMSO at a concentration of 10 mM. A 1 mM working stock was prepared by mixing up to nine test compounds (4 μL each) and one control (4 μL). If fewer than nine test compounds were present, blank DMSO was added to bring the volume to 40 μL. On the experimental day, 4 μL was diluted in 40 mL of ECF buffer to obtain a 100 nM solution of each test compound, which was then preheated to 37°C before the start of incubation.

[0343] To prepare brain sections, rats weighing approximately 300 g were terminally anesthetized by isoflurane inhalation, their brains were carefully removed, and immersed in ice-cold oxygenated ECF buffer. The rat brains were transferred to a dish containing O2-supplemented ice-cold ECF buffer, trimmed with a razor, and then placed in the center of a microslicer tray with the posterior cut surface facing downwards, adhering to the tray. Ice-cold ECF buffer was added to cure the adhesion and moisten the brain. The tray was placed in the microslicer, and using an appropriate cutting speed, sections were cut 100-400 μm thick until the striatal region was exposed. Four to six 300 μm thick coronal sections of the striatal region were cut from each brain and placed in O2-supplemented ice-cold buffer until incubation. The six sections were transferred to an incubation tray containing 40 mL of preheated (37°C) cassette mixture. The time from brain removal to immersion of the sections in the cocktail mixture was a maximum of 20 minutes. The incubation tray was covered with a gas-permeable lid and placed in a water bath with O2 supplied by a pump at 37°C, and incubated for 5 hours at a shaking speed of 45 rpm.

[0344] Before incubation, 200 μL of non-incubated cassette solution was stored as the T=0 sample. Then, 200 μL was mixed with 200 μL of blank brain homogenate in ECF buffer (4 vol (w / v)). After incubation, the pH of the cassette solution was measured and recorded. The pH value must be greater than 7.3. 200 μL of the surface-derived cassette solution was transferred into a tube containing 200 μL of blank brain homogenate in ECF buffer (4 vol (w / v)). Each brain section was dried on filter paper and weighed in a 2 mL Eppendorf tube. After adding 9 vol (w / v) of ECF buffer, the sections were homogenized using an ultrasonic device. The samples were precipitated and diluted as follows.

[0345] 50 μL aliquots from each sample and 3 × 50 μL from each cassette solution (mixed with the blank homogenate) were transferred to a 0.6 mL centrifuge tube. The samples were precipitated with 200 μL of ice-cold acetonitrile containing an internal standard, vortexed at 2,000 rpm for 3 minutes, and then centrifuged at 14,000 rpm for 15 minutes at 4°C. 100 μL of the supernatant was transferred to a new 96-well plate for analysis, and 100 μL of distilled water was added to each sample. The plate was then shaken at 1,000 rpm for 2 minutes for LC-MS / MS analysis.

[0346] Next, the mixed section samples were further diluted in two steps. Tenfold and hundredfold double blank samples prepared with 150 μL of blank brain homogenate in ECF buffer (4 volumes (w / v)) were transferred to a 1.5 mL centrifuge tube containing 150 μL of ECF buffer and vortexed at 2,000 rpm for 2 minutes. The samples were precipitated with 1200 μL of ice-cold acetonitrile, vortexed at 2,000 rpm for 3 minutes, and then centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was then transferred to a new 96-well plate for analysis. 100 μL of distilled water was added to each sample to obtain double blank samples.

[0347] Unconnected volume of the brain (V u,brain ) to, V u =(C slice -V0*C ECF ) / (1-V0)*C ECF It was calculated as follows.

[0348] In the formula, C slice , C ECF V0, V0, and V0 represent the amount of drug in the section, the drug concentration in the ECF (the drug concentration in the brain's ECF, i.e., the free concentration), and the moisture content of the brain section (0.0931), respectively.

[0349] unconnected fraction of brain f u,brain = 1 / V u,brain

[0350] Determination of Kpuu in rats The ratio of total drug / unbound drug in plasma to total drug / unbound drug in the brain (Kp / Kpuu) was calculated as follows.

[0351] The compound was formulated as a 1:1:1 mixture of tetraethylene glycol, dimethylacetamide, and water, each at a concentration of 0.5 mM, and administered intravenously to Han Wistar rats at a rate of 2 μmol / kg / hour and a volume of 4 mL / kg. After 4 hours, the animals were sacrificed, and brain and blood samples were collected. Plasma was prepared from the blood, and all samples were frozen and stored at -20°C until analysis. After collection, the brain samples were homogenized in purified water at a ratio of 1:3 (w / v) and frozen and stored at -20°C until analysis.

[0352] Plasma and brain samples were analyzed by protein precipitation, followed by LC-MS / MS, and concentrations were determined against calibration curves generated by adding drug-containing blank rat plasma or brain homogenate over an appropriate concentration range. Brain concentrations were corrected for residual blood by subtracting 0.8% of the plasma concentration from the total brain concentration.

[0353] Next, Kp was calculated using the formula Kp = ((4 * [brain homogenate]) - (0.008 * [plasma])) / [plasma].

[0354] Next, Kpuu was calculated using the formula Kpuu = Kp * (unbound fraction in brain section / unbound fraction in plasma).

[0355] [Table 4]

[0356] [Table 5]

[0357] Further materials and methods cell line U87, SJG2, and BT245 glioblastoma cells were assayed to evaluate the activity and efficacy of the PARP inhibitors described herein in combination with the standard therapeutic chemotherapy temozolomide (TMZ), ionizing radiation (IR), and the emerging chemotherapy Val-083. The parental U87 cell line was obtained from the AstraZeneca Global Cell Bank in Alderley Park, UK. Permission was granted from ATCC to use the isocitrate dehydrogenase 1 mutant cell line (IDH1mt R132H). SJ-G2 (ATRX mutant) and BT245 (H3K27M mutant) cell lines were obtained from the Research Institute of the McGill University Health Centre, Montreal, QC H4A 3J1, Canada. The IDH1R132 mutation was induced in the SJG2 cell line to generate isogenic pairs (SJG2 IDH1mt and SJG2 IDH1wt).

[0358] Combination assay - PARP inhibitors with temozolomide (TMZ) or Val-083 To evaluate the combined activity of the PARP inhibitor (6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide; hereafter, compound 20) produced in Example 20 with TMZ or VAL-083, a nuclear count endpoint assay was used for U87 isogenic pairs, and the CellTiter-Glo (CTG) endpoint was used for SJ-G2 isogenic pairs. Cells were seeded at a low density (500 cells per well) in 96-well plates and exposed to compound 20 for a time corresponding to more than 4-5 replication cell cycle days (7-8). After cell adhesion, the plates were pre-treated with the PARP inhibitor for 1 hour using an automated digital D300 HP dispenser (Tecan), and then TMZ or VAL-083 was automatically dispensed. The drugs were added from compound stocks (PARP inhibitor 10 mM stock, TMZ 100 mM stock, and VAL-083 100 mM stock) dissolved in DMSO at the indicated titration dilutions. Each concentration was tested in two replicates on each plate. DMSO served as a vehicle control. The tested concentration range was selected to evaluate multiple dose combinations and obtain optimal PARP inhibitor, TMZ, or VAL-083 activity in the isogenic pair. Plates were incubated at 37°C and 5% CO2 for the indicated time. Cell proliferation and the absence of contamination were checked periodically under a microscope.

[0359] For the nuclear count endpoint assay, cells were fixed for approximately 15 minutes by adding 50 μl of formaldehyde (Sigma-Aldrich F8775) per well. Subsequently, the fixative was removed, and the plate was washed once with PBS at room temperature (RT) for 30 minutes with a 1:10000 dilution of stock in PBS before adding hoechst (Thermo Scientific 33342). The plate was washed again, and PBS was added. Images were obtained using CellInsight CX5 (Thermo Fisher), and the total number of hoechst-stained nuclei was analyzed by measuring. Growth inhibition was calculated using combinefit software (Di Veroli et al., 2016) by normalizing the number of nuclei in the drug-treated wells (confluence %) to DMSO control. Similarly, for the CTG endpoint assay, combination plates for SJ-G2 isogenic pairs were stopped by adding reagents according to the manufacturer's instructions (Promega, Madison, WI, USA; G7570). Cell viability (viability %) of the drug-treated wells was normalized to DMSO control, and proliferation inhibition was calculated using combinefit software (Di Veroli et al., 2016).

[0360] The synergistic effect scores and hyperactivity of each monotherapy were calculated by comparing them with an additive HSA model (Borisy et al., 2003; Tan et al., 2012). In the plots shown, the error bars represent the mean ± SD.

[0361] Combination assay - PARP inhibitors with ionizing radiation (IR) To evaluate the combined activity of compound 20 with IR, a colony formation assay was used to measure the proliferation of cells derived from a single colony over 10–14 days, allowing for at least five replication cell cycles. Cell adhesion was promoted by seeding cells at a low density (500 cells per well) in 24-well plates coated with poly-L-lysine. After cell adhesion, the plates were pre-treated with a PARP inhibitor for 1 hour using an automated digital D300 HP dispenser (Tecan), and then the cells were irradiated at 0.9 Gy via a high-pressure X-ray generator tube (Faxitron X-Ray Corporation). The PARP inhibitor was added from a compound stock (10 mM stock) dissolved in DMSO at the indicated titration dilutions. Each concentration was tested in three replicates on each plate. DMSO served as a vehicle control. The plates were incubated at 37°C in 5% CO2 for the indicated time. Cell proliferation and the absence of contamination were periodically checked under a microscope. Next, the cells were fixed and stained immediately after fixation with sulforhodamin B acid form (SRB) [Sigma, 341738-5g] reconstituted in 1% acetic acid, without any washing steps (to limit cell detachment). Plates with stained colonies were scanned at a resolution of 600 dpi using a GelCount colony counter (Oxford OPTRONIX). Colony formation was scored by quantifying the total optical density measured in ImageJ software using a 24-well plate region of interest (ROI) mask (CFU, colony formation unit). Data analysis was performed by normalizing each plate to the processing vehicle set to =100. The data were plotted normalized to the vehicle control (=100) without IR and IC was performed. 50 This was calculated using Prism GraphPad software. In the plot shown, the error bars represent the mean ± SD.

[0362] U87MG xenograft study U87MG cells (2.5 million cells in serum-free MEM medium) were subcutaneously transplanted (SC) into the dorsal flank of female nude mice (body weight over 18g). Tumors were measured twice weekly using bilateral calipers (length × width), and tumor volume was calculated using the ellipse formula (π / 6 × width × width × length). Animal body weight and tumor status were monitored throughout the study. The average tumor volume was approximately 0.2 cm³. 3 When the condition was reached, the mice were randomly assigned to the treatment group.

[0363] Animals were treated orally (PO) starting the day after randomization. Control animals were treated once daily for 10 days (QD x 10) with a vehicle (water / methanesulfonic acid (MSA) pH 3-3.2). PARP inhibitors in water / MSA pH 3-3.2 were administered at 3 mg / kg QD x 10 or QD x 33 in monotherapy and combination therapy groups, respectively. Temozolomide (TMZ) in OraPlus Perrigo was administered at 25 mg / kg QD monotherapy groups on days 1-5 and 29-33, and at 6.25 mg / kg QD monotherapy and combination therapy groups on days 1-5 and 29-33.

[0364] Tumor growth inhibition from the start of treatment was evaluated by comparing the mean change in tumor volume (TV) between the control group and the treatment group, and expressed as a percentage of tumor growth inhibition (TGI, when tumor volume (TV) ≥ start TV) or a percentage of tumor regression (reg, when TV < start TV). Statistical significance was evaluated using a one-sided t-test. Statistical significance is shown as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

[0365] Treatment with PARP inhibitors in ATRX mutation GBM models The applicants studied compound 20 for monotherapy activity in IDHmt and ATRXmt cell lines (SJ-G2 and U87 GBM models). ATRXmt GBM SJ-G2 cells showed sensitivity to compound 20 monotherapy within a clinically achievable dose range, and the addition of IDH1 mutations further reduced sensitivity in this model. The SJ-G2 ATRXmt model showed approximately 40-fold higher sensitivity to compound 20 compared to the ATRXwt U87 model. The results are shown below in Table 5 and Figure 7.

[0366] [Table 6]

[0367] These results demonstrate that compound 20 has potent monotherapy activity in ATRX mutant cell lines, and that the presence of an additional IDH1 mutation did not result in further sensitivity in this model.

[0368] Combination therapy using PARP inhibitors and TMZ The combination of compound 20 and TMZ was evaluated in IDH1 wild-type (IDH1wt) and IDH1 mutant (IDH1mt) glioblastoma (GBM) cells. Compound 20 showed increased synergistic effects with TMZ at multiple concentrations. This was well above IC95 for PARization inhibition by compound 20 (0.008 μM; data not shown) in the U87 isogenic pair (Figure 8). Importantly, compound 20 exhibited strong trapping of PARP1 at concentrations as low as 10 nM while suppressing PARP2 (data not shown). This suggests that the observed combined benefits of compound 20 and TMZ are driven by catalytic inhibition of PARP1 and increased DNA damage resulting from trapped PARP1 on DNA. Furthermore, compound 20 produces a synergistic effect with TMZ in the IDH1mt GBM strain at a concentration less than 10 times higher than that of the wild type (10 μM in IDH1wt compared to 1 μM in IDH1mt). This suggests that IDH1mt GBM is more sensitive to TMZ treatment.

[0369] Furthermore, the combination of compound 20 and TMZ was investigated in the ATRX mutant and the p53 mutant SJ-G2 GBM isogenic IDH1mt and IDH1mt models. These IDH1wt and IDH1mt SJG2 models more accurately represent the GBM patient population (Ohba, Kuwahara, Yamada, Abe, & Hirose, 2020). Importantly, compound 20 significantly enhanced the TMZ effect in the SJ-G2 isogenic pair at multiple low PARP inhibitor concentrations as low as 10 nM (Figure 9). In addition, TMZ was significantly synergistic with compound 20 at the same concentrations in the IDH1wt and IDH1mt models.

[0370] Furthermore, the antitumor efficacy of compound 20 in combination with TMZ was investigated in a U87MG xenograft model using the method described above. TMZ administered at 6.25 mg / kg QD on days 1-5 and 29-33 resulted in 28% tumor regression (2 / 8 tumors disappeared), while the combination with compound 20 (3 mg / kg QD) yielded a further antitumor benefit of 95% tumor regression (8 / 8 tumors disappeared) (Figure 10A). The difference was statistically significant. The effect of high-dose TMZ (25 mg / kg QD on days 1-5 and 29-33), which resulted in 92% tumor regression (7 / 8 tumors disappeared), was comparable to the effect of the combination treatment. Weight loss and animal welfare were monitored in place of toxicity in relation to the treatment. High-dose temozolomide (25 mg / kg) resulted in greater weight loss than the combination of low-dose temozolomide (6.25 mg / kg) with compound 20 at 3 mg / kg (Figure 10B).

[0371] Combination therapy using PARP inhibitors and Val-083 The combination of compound 20 and the alkylating agent Val-083 was evaluated in U87 IDH1wt and IDH1mt isogenic pairs. The results shown in Figure 11 demonstrate that compound 20 produces a synergistic effect with Val-083 in these cells. In particular, the benefit of the combination of compound 20 and Val-083 is independent of the IDH1 mutation status in the GBM model, because there was no dose reduction of the alkylating agent required in the IDH1mt model to produce a synergistic effect with the PARP inhibitor.

[0372] Similarly, ATRX mutant SJ-G2 IDH1wt and IDH1mt isogenic pairs show enhanced combination benefits of compound 20 and Val-083, independent of the IDH1mt status (Figure 12). These results also demonstrate that 1 μM of compound 20 is active in ATRX mutant cell lines in the absence of Val-083. This is consistent with previously reported results, shown in Figure 7, and demonstrates that compound 20 has monotherapy efficacy across multiple concentrations. Thus, as with demonstrating the potential suitability of this PARP inhibitor in monotherapy (i.e., without chemotherapy or radiotherapy), this suggests that the PARP inhibitor has a tumor-reducing effect independent of its ability to enhance the efficacy of alkylating chemotherapeutic agents such as Val-083 and TMZ.

[0373] Combination therapy using PARP inhibitors and ionizing radiation therapy H3K27M mutant diffuse median glioma is a pediatric glioma for which radiotherapy is the standard treatment. However, the prediction of this type of tumor is relatively poor. In this study, the combination of ionizing radiation and compound 20 was evaluated in a model of H3K27M mutant glioma.

[0374] The results shown in Figure 13 demonstrate that the studied PARP inhibitors show enhanced combined benefits with ionizing radiation at multiple doses of the inhibitor, including a clinically relevant reduced dose of 0.9 Gy (the clinically relevant dose is 1.8 Gy). Importantly, the IC95 for PARization inhibition induced by compound 20 in a pediatric model is 0.004 μM, indicating that the compound is within its maximum activity range. These results suggest that the disclosed PARP inhibitors can be used in combination with ionizing radiation to treat difficult gliomas, such as H3K27M mutant gliomas. Furthermore, the results suggest that it may be possible to treat tumors with PARP inhibitors along with reduced levels of ionizing radiation, which could mitigate the adverse effects associated with exposure to high doses of radiation. The present invention includes the following embodiments. [Section 1] A method for treating a brain tumor or neuroblastoma in a patient requiring treatment for the brain tumor or neuroblastoma, comprising administering to the patient a therapeutically effective amount of a poly(ADP-ribose) polymerase (PARP) inhibitor, The brain tumor or neuroblastoma includes the alpha-thalassemia / mental retardation syndrome X-linked (ATRX) deficiency phenotype, The PARP inhibitor is given by formula (I) [C1] TIFF0007927071000074.tif32160 (In the formula, R 1 H and C are independent of each other. 1~4 Alkyl, C 3~6 Cycloalkyl, C 1~4 Fluoroalkyl, and C 1~4 Selected from alkyloxy; R 2 These are H, Halo, and C, independently. 1~4 Alkyl and C 1~4 Selected from fluoroalkyl; R 3 H or C 1~4 It is alkyl; R 4 is a halo or C 1~4 (It is alkyl.) A method comprising a compound of the same, or a pharmaceutically acceptable salt thereof. [Section 2] The method according to claim 1, wherein the brain tumor or neuroblastoma further comprises an isocitrate dehydrogenase 1 and / or 2 (IDH1 and / or IDH2) deficiency phenotype. [Section 3] The brain tumor or neuroblastoma according to claim 1, wherein the brain tumor or neuroblastoma does not have an IDH1 deficiency phenotype. [Section 4] The aforementioned brain tumor or neuroblastoma is O 6 The method according to any one of claims 1 to 3, comprising methylguanine-DNA methyltransferase (MGMT) promoter methylation. [Section 5] The method according to any one of claims 1 to 4, further comprising the step of diagnosing the patient as having the brain tumor or neuroblastoma including the ATRX deficiency phenotype before administering the PARP inhibitor. [Section 6] The method according to claim 5, wherein the step of diagnosing the patient further comprises determining whether the brain tumor or neuroblastoma further includes an IDH1 or IDH2 deficiency phenotype and / or MGMT promotermethylation. [Section 7] The method according to item 5 or 6, wherein diagnosing the patient comprises assaying cells obtained from the brain tumor or neuroblastoma originating from the patient. [Section 8] The PARP inhibitor mentioned above is i) Alkylating chemotherapeutic agents; and / or ii) Ionizing radiation The method according to any one of claims 1 to 7, administered to the patient in combination with the method described above. [Section 9] A method for treating a brain tumor or neuroblastoma in a patient requiring treatment for the brain tumor or neuroblastoma, comprising administering to the patient a therapeutically effective amount of a poly(ADP-ribose) polymerase (PARP) inhibitor, and i) Alkylating chemotherapeutic agents; and / or ii) Ionizing radiation administered at doses of 10 Gy or more This includes applying, The PARP inhibitor is, Formula I [C2] TIFF0007927071000075.tif32160 (In the formula, R 1 H and C are independent of each other. 1~4 Alkyl, C 3~6 Cycloalkyl, C 1~4 Fluoroalkyl, and C 1~4 Selected from alkyloxy, R 2 These are H, Halo, and C, independently. 1~4 Alkyl and C 1~4 Selected from fluoroalkyl groups, R3 H or C 1~4 It is alkyl, R 4 is a halo or C 1~4 (It is alkyl.) A method comprising a compound of the same, or a pharmaceutically acceptable salt thereof. [Section 10] The aforementioned brain tumor or neuroblastoma is: (i) Alphathalassemia / X-linked (ATRX) deficiency phenotype; (ii) isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) deficiency phenotype; and / or (iii)O 6 - Methylguanine-DNA methyltransferase (MGMT) promoter-methylation The method described in paragraph 9, including the method described in paragraph 9. [Section 11] The method according to any one of claims 8 to 10, wherein the alkylating chemotherapeutic agent is temozolomide (TMZ) or dianhydrogalactitol (Val-083). [Section 12] TMZ reports: (i) Approximately 200mg / m 2 Doses less than approximately 150 mg / m² 2 Less than a certain dose, approximately 125 mg / m² 2 Doses less than 100 mg / m², or approximately 100 mg / m² 2 Less than a dose; (ii) Approximately 50~200mg / m 2 The dosage is approximately 75-150 mg / m². 2 The dosage, or approximately 75 mg / m² 2 ~125 mg / m² 2 dosage The method described in item 11, administered as follows: [Section 13] VAL-083 is: (i) Approximately 50mg / m 2 Less than a certain dose, approximately 40 mg / m² 2 Less than a dose, approximately 30 mg / m² 2 Less than a dose of approximately 20 mg / m² 2 Less than a dose; (ii) Approximately 10~50mg / m 2 The dosage is approximately 10-40 mg / m². 2 The dosage, or approximately 10 mg / m² 2 ~30mg / m 2 dosage The method described in item 11, administered as follows: [Section 14] The aforementioned ionizing radiation is: (i) doses less than approximately 60 Gy, less than approximately 55 Gy, less than approximately 50 Gy, less than approximately 45 Gy, or less than approximately 40 Gy; or (ii) doses of approximately 20 Gy to 60 Gy, approximately 20 Gy to 55 Gy, approximately 20 Gy to 50 Gy, approximately 20 to 45 Gy, approximately 20 to 40 Gy, approximately 30 Gy to 60 Gy, approximately 30 Gy to 55 Gy, approximately 30 to 50 Gy, approximately 30 Gy to 45 Gy, or approximately 30 Gy to 40 Gy It was applied in this manner. The method according to any one of claims 8 to 13, wherein the dose of ionizing radiation is administered as a subdivided radiotherapy, by choice. [Section 15] The method according to any one of claims 8 to 14, wherein the PARP inhibitor synergistically enhances the activity of the alkylating chemotherapeutic agent and / or radiotherapeutic agent. [Section 16] The method according to any one of claims 8 to 15, wherein the PARP inhibitor treats the brain tumor or neuroblastoma independently of any effects on the glioma induced by the administration of the chemotherapeutic agent or radiotherapeutic agent. [Section 17] R 1 The method according to any one of claims 1 to 16, wherein the present agent is selected from one of methyl, ethyl, isopropyl, cyclopropyl, 1,1-difluoroethyl, 1-fluoroethyl, trifluoromethyl, difluoromethyl, and methoxy. [Section 18] R 1 The method according to item 17, wherein is methyl or ethyl. [Section 19] R 2 The method according to any one of claims 1 to 18, wherein the material is selected from one of H, chloro, fluoro, methyl, and difluoromethyl. [Section 20] R 2 The method according to item 19, wherein is fluoro or methyl. [Section 21] R 3 The method according to any one of claims 1 to 20, wherein is methyl or ethyl. [Section 22] R 4 The method according to any one of claims 1 to 21, wherein one is selected from chloro, fluoro, and methyl. [Section 23] R 4 The method described in item 22, wherein the fluorocarbon is fluorocarbon. [Section 24] R 1 is C 1~4 It is alkyl, R 2 It is a halo, R3 is C 1~4 It is alkyl, R 4 is Halo or C 1~4 The method according to any one of claims 1 to 16, wherein the material is alkyl or a pharmaceutically acceptable salt thereof. [Section 25] The aforementioned PARP inhibitors are: 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoropyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide, 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(5-Chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[[5-Fluoro-2-[(1S and 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[[5-fluoro-2-[(1S and 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[[2-(1,1-difluoroethyl)-5-fluoro-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-(difluoromethyl)-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-(difluoromethyl)-5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[[2-(difluoromethyl)-5-fluoro-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Chloro-5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, N-ethyl-6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, N-ethyl-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide, 5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[[5-Fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[[5-Fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(2-Methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-(difluoromethyl)-5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, and 6-(difluoromethyl)-5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, Or the method according to any one of claims 1 to 16, selected from pharmaceutically acceptable salts thereof. [Section 26] The aforementioned PARP inhibitors are: The method according to any one of claims 1 to 16, wherein the salt is 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide or a pharmaceutically acceptable salt thereof. [Section 27] The aforementioned PARP inhibitors are: The method according to any one of claims 1 to 16, wherein the product is 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide. [Section 28] The method according to any one of items 1 to 27, wherein the brain tumor is a glioma or an ependymoma. [Section 29] The brain tumor is a glioma, as described in paragraph 28. [Section 30] The glioma is a pediatric glioma, as described in paragraph 29. [Section 31] The method according to paragraph 29 or 30, wherein the glioma is a high-grade glioma, optionally selected from the list consisting of oligodendroglioma, undifferentiated astrocytoma, glioblastoma, and diffuse median glioma. [Section 32] The glioma is an H3K27M mutant glioma, as described in any one of items 29 to 31. [Section 33] The method according to any one of claims 1 to 27, wherein the brain tumor or neuroblastoma is characterized by a high level of genomic instability.

[0375] References To more fully explain and disclose the present invention and the latest technologies relating thereto, several publications have been cited prior to this. A complete citation of these references is given below. Each of these references in its entirety is incorporated herein by reference. International Publication No. 2004 / 080976 Pamphlet European Patent No. 699754 European Patent No. 705903 Bai P.Biology of poly(ADP-ribose)polymerases:the factotums of cell maintenance.Mol Cell 2015;58:947-958,doi:10.1016 / j.molcel.2015.01.034 Bartkova J,Hamerlik P,Stockhausen MT,Ehrmann J,Hlobilkova A,LaursenH,Kalita O,Kolar Z,PoulsenHS,BroholmH,Lukas J,Bartek J.Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signaling inHuman gliomas.Oncogene.2010 Sep 9;29(36):5095-5102.doi:10.1038 / onc.2010.249.Epub 2010 Jun 28.PMID:20581868. Borisy,A.A.,Elliott,P.J.,Hurst,N.W.,Lee,M.S.,Lehar,J.,Price,E.R.,...Keith,C.T.(2003).Systematic discovery of multicomponent therapeutics.Proc Natl Acad Sci U S A,100(13),7977-7982.doi:10.1073 / pnas.1337088100 Chappnis,P.O.and Foulkes,W.O.,Cancer Treat Res,107,29-59(2002),doi:10.1007 / 978-1-4757-3587-1_2 Di Veroli,G.Y.,Fornari,C.,Wang,D.,Mollard,S.,Bramhall,J.L.,Richards,F.M.,& Jodrell,D.I.(2016).Combenefit:an interactive platform for the analysis and visualization of drug combinations.Bioinformatics,32(18),2866-2868.doi:10.1093 / bioinformatics / btw230 George SL,Lorenzi F,King D,Hartlieb S,Campbell J,PembertonH,Toprak UH,Barker K,Tall J,da Costa BM,van den Boogaard ML,Dolman MEM,Molenaar JJ,BryantHE,Westermann F,Lord CJ,Chesler L.Therapeutic vulnerabilities in the DNA damage response for the treatment of ATRX mutant neuroblastoma.EBioMedicine.2020 Sep;59:102971.doi:10.1016 / j.ebiom.2020.102971.Epub 2020 Aug 23.PMID:32846370;PMCID:PMC7452577 Gupta,S.K.,Smith,E.J.,Mladek,A.C.,Tian,S.,Decker,P.A.,Kizilbash,S.H.,...Sarkaria,J.N.(2018).PARP Inhibitors for Sensitization of Alkylation Chemotherapy in Glioblastoma:Impact of Blood-Brain Barrier and MolecularHeterogeneity.Front Oncol,8,670.doi:10.3389 / fonc.2018.00670 Haase,S.,Garcia-Fabiani,M.B.,Carney,S.,Altshuler,D.,Nunez,F.J.,Mendez,F.M.,Nunez,F.,Lowenstein,P.R.,& Castro,M.G.(2018).Mutant ATRX:uncovering a new therapeutic target for glioma.Expert opinion on therapeutic targets,22(7),599-613.doi:10.1080 / 14728222.2018.1487953 Hartmann C,Meyer J,Balss J,Capper D,Mueller W,Christians A,Felsberg J,Wolter M,Mawrin C,Wick W,Weller M,Herold-Mende C,Unterberg A,Jeuken JW,Wesseling P,Reifenberger G,von Deimling A.Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age:a study of 1,010 diffuse gliomas.Acta Neuropathol.2009 Oct;118(4):469-74.doi:10.1007 / s00401-009-0561-9.Epub 2009 Jun 25.PMID:19554337. Higuchi,F.,Nagashima,H.,Ning,J.,Koerner,M.V.A.,Wakimoto,H.,& Cahill,D.P.(2020).Restoration of Temozolomide Sensitivity by PARP Inhibitors in Mismatch Repair Deficient Glioblastoma is Independent of Base Excision Repair.Clin Cancer Res,26(7),1690-1699.doi:10.1158 / 1078-0432.CCR-19-2000 Himes,B.T.,Zhang,L.,& Daniels,D.J.(2019).Treatment Strategies in Diffuse Midline Gliomas With theH3K27M Mutation:The Role of Convection-Enhanced Delivery in Overcoming Anatomic Challenges.Front Oncol,9,31.doi:10.3389 / fonc.2019.00031 Hughes-Davies,et al.,Cell,115,523-535(2003)doi:10.1016 / s0092-8674(03)00930-9 Janatova M.,et al.,Neoplasma,50(4),246-250(2003) Jancarkova,N.,Ceska Gynekol.,68(1),11-6(2003),PMID:12708108 Jannetti,S.A.,Zeglis,B.M.,Zalutsky,M.R.,& Reiner,T.(2020).Poly(ADP-Ribose)Polymerase(PARP)Inhibitors and Radiation Therapy.Front.Pharmacol.,11:170.doi:10.3389 / fphar.2020.00170 Jimenez-Alcazar,M.,Curiel-Garcia,A.,Walnut,P.,Pearles-Paton,J.,Schuhmacher,AJ,Galan-Ganga,M.,...Squatrito,M.(2021).Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma.Mol Cancer Ther,20(6),1029-1038.doi:10.1158 / 1535-7163.MCT-20-0319 Koschmann , C. , Calinescu , AA , Nunez , FJ , Mackay , A. , Fazal-Salom , J. , Thomas , D. , Mendez , F. , Kamran , N. , Dzaman , M. , Mulpuri , L. , Kr asinkiewicz,J.,Doherty,R.,Lemons,R.,Brosnan-Cashman,JA,Li,Y.,Roh,S.,Zhao,L.,Appelman,H.,Ferguson,D.,Gorbunova,V. Castro,MG(2016).ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma.Science Translational Medicine,8(328),328ra28.doi:10.1126 / scitranslmed.aac8228 Mansouri,A.,Hachem,LD,Mansouri,S.,Nassiri,F.,Laperriere,NJ,Xia,D.,Lindeman,NI,Wen,PY,Chakravarti,A.,Mehta,MP,Hegi,ME,Stupp,R.,Aldape,KD,& Zadeh,G.(2019).MGMT promoter methylation status testing to guide therapy for glioblastoma: refining the approach based on emerging...

Claims

1. A pharmaceutical product for treating brain tumors or neuroblastomas in patients requiring treatment for brain tumors or neuroblastomas, comprising a poly(ADP-ribose) polymerase (PARP) inhibitor, The brain tumor or neuroblastoma includes the alpha-thalassemia / X-linked intellectual disability syndrome (ATRX) deficiency phenotype, The PARP inhibitor is given by formula (I) 【Chemistry 1】 (In the formula, R １ These are H and C, independently. １～４ Alkyl, C ３～６ Cycloalkyl, C １～４ Fluoroalkyl and C １～４ Selected from alkyloxy; R ２ These are H, Halo, and C, which are independent of each other. １～４ Alkyl and C １～４ Selected from fluoroalkyl; R ３ is H or C １～４ alkyl; R ４ is Halo or C １～４ (It is alkyl.) A pharmaceutical product, which is a compound of or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical product according to claim 1, wherein the brain tumor or neuroblastoma further comprises an isocitrate dehydrogenase 1 and / or 2 (IDH1 and / or IDH2) deficiency phenotype.

3. The pharmaceutical product according to claim 1, wherein the brain tumor or neuroblastoma does not contain the IDH1 deficiency phenotype.

4. The aforementioned brain tumor or neuroblastoma is O ６ The pharmaceutical product according to claim 1, comprising methylguanine-DNA methyltransferase (MGMT) promotermethylation.

5. The PARP inhibitor is i) Alkylating chemotherapeutic agents; and / or ii) Ionizing radiation The pharmaceutical product according to claim 1, which is administered to the patient in combination with the above.

6. A pharmaceutical product for treating brain tumors or neuroblastomas in patients requiring treatment for brain tumors or neuroblastomas, comprising a poly(ADP-ribose) polymerase (PARP) inhibitor, and i) Alkylating chemotherapeutic agents; and / or ii) Ionizing radiation administered at doses of 10 Gy or more Includes, The PARP inhibitor is, Formula I 【Chemistry 2】 (In the formula, R １ These are H and C, independently. １～４ Alkyl, C ３～６ Cycloalkyl, C １～４ Fluoroalkyl and C １～４ Selected from alkyloxy, R ２ These are H, Halo, and C, which are independent of each other. １～４ Alkyl and C １～４ Selected from fluoroalkyl groups, R ３ is H or C １～４ It is alkyl, R ４ is Halo or C １～４ (It is alkyl.) A pharmaceutical product, which is a compound of or a pharmaceutically acceptable salt thereof.

7. The aforementioned brain tumor or neuroblastoma is: (i) Alpha-thalassemia / X-linked (ATRX) deficiency phenotype; (ii) isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) deficiency phenotype; and / or (iii)O ６ - Methylguanine-DNA methyltransferase (MGMT) promoter-methylation The pharmaceutical product according to claim 6, including the above.

8. The pharmaceutically acceptable drug according to claim 5, wherein the alkylating chemotherapeutic agent is temozolomide (TMZ) or dianhydrogalactitol (Val-083).

9. TMZ is: (i) Approximately 200mg / m ２ Doses less than approximately 150 mg / m² ２ Doses less than approximately 125 mg / m² ２ Doses less than 100 mg / m², or approximately 100 mg / m². ２ Less than a dose; (ii) Approximately 50 to 200 mg / m ２ The dosage is approximately 75-150 mg / m². ２ The dosage, or approximately 75 mg / m² ２ ~125 mg / m² ２ dosage The pharmaceutical product according to claim 8, administered by [method].

10. VAL-083 is: (i) Approximately 50 mg / m ２ Less than a certain dose, approximately 40 mg / m² ２ Doses less than approximately 30 mg / m² ２ Less than a dose of approximately 20 mg / m². ２ Less than a dose; (ii) Approximately 10 to 50 mg / m ２ Dosage: approximately 10-40 mg / m² ２ The dosage, or approximately 10 mg / m² ２ ~30 mg / m² ２ dosage The pharmaceutical product according to claim 8, administered by [method].

11. The aforementioned ionizing radiation is: (i) doses of less than approximately 60 Gy, less than approximately 55 Gy, less than approximately 50 Gy, less than approximately 45 Gy, or less than approximately 40 Gy; or (ii) doses of approximately 20 Gy to 60 Gy, approximately 20 Gy to 55 Gy, approximately 20 Gy to 50 Gy, approximately 20 to 45 Gy, approximately 20 to 40 Gy, approximately 30 Gy to 60 Gy, approximately 30 Gy to 55 Gy, approximately 30 to 50 Gy, approximately 30 Gy to 45 Gy, or approximately 30 Gy to 40 Gy It was applied in this manner. The pharmaceutical product according to claim 6, wherein the dose of ionizing radiation is administered as a subdivided radiotherapy by optional choice.

12. The pharmaceutical product according to claim 5, wherein the PARP inhibitor synergistically enhances the activity of the alkylating chemotherapeutic agent and / or radiotherapy agent.

13. The pharmacopoeia according to claim 5, wherein the PARP inhibitor treats the brain tumor or neuroblastoma independently of any effects on the glioma induced by the administration of the chemotherapeutic agent or radiotherapeutic agent.

14. R １ The pharmaceutical product according to claim 1, wherein is selected from one of methyl, ethyl, isopropyl, cyclopropyl, 1,1-difluoroethyl, 1-fluoroethyl, trifluoromethyl, difluoromethyl, and methoxy.

15. R １ The pharmaceutical product according to claim 14, wherein is methyl or ethyl.

16. R ２ The pharmaceutical product according to claim 1, wherein is selected from one of H, chloro, fluoro, methyl, and difluoromethyl.

17. R ２ The pharmaceutical product according to claim 16, wherein is fluoro or methyl.

18. R ３ The pharmaceutical product according to claim 1, wherein is methyl or ethyl.

19. R ４ The pharmaceutical product according to claim 1, wherein the compound is selected from any one of chloro, fluoro, and methyl.

20. R ４ The pharmaceutical product according to claim 19, wherein is fluoro.

21. R １ is C １～４ It is alkyl, R ２ It is a halo, R ３ is C １～４ It is alkyl, R ４ is Halo or C １～４ The pharmaceutical product according to claim 1, wherein it is an alkyl group or a pharmaceutically acceptable salt thereof.

22. The aforementioned PARP inhibitors are: 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoropyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide, 5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(5-Chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-ethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[[5-Fluoro-2-[(1S and 1R)-1-Fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[[5-fluoro-2-[(1S and 1R)-1-fluoroethyl]-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(5-chloro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[[2-(1,1-difluoroethyl)-5-fluoro-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-(difluoromethyl)-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-(difluoromethyl)-5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[[2-(difluoromethyl)-5-fluoro-3-oxo-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Chloro-5-[4-[(5-fluoro-2-methoxy-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(2-ethyl-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, N-ethyl-6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]pyridine-2-carboxamide, N-ethyl-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-methylpyridine-2-carboxamide, 5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[[5-Fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Chloro-5-[4-[[5-Fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[[5-fluoro-3-oxo-2-(trifluoromethyl)-4H-quinoxaline-6-yl]methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(5-Fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(5-fluoro-2-isopropyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-6-fluoro-N-methylpyridine-2-carboxamide, 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 5-[4-[(2-cyclopropyl-5-fluoro-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N,6-dimethylpyridine-2-carboxamide, 6-Fluoro-5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, 6-(difluoromethyl)-5-[4-[(2-methoxy-5-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, and 6-(difluoromethyl)-5-[4-[(2,5-dimethyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide, The pharmaceutical product according to claim 1, or selected from pharmaceutically acceptable salts thereof.

23. The aforementioned PARP inhibitors are: The pharmaceutical product according to claim 1, wherein the product is 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide or a pharmaceutically acceptable salt thereof.

24. The aforementioned PARP inhibitors are: The pharmaceutical product according to claim 1, wherein the product is 6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide.

25. The pharmaceutical product according to claim 1, wherein the brain tumor is a glioma or an ependymoma.

26. The pharmaceutical product according to claim 25, wherein the brain tumor is a glioma.

27. The pharmaceutical product according to claim 26, wherein the glioma is a pediatric glioma.

28. The pharmacopoeci of claim 26, wherein the glioma is a high-grade glioma, optionally selected from a list consisting of oligodendroglioma, undifferentiated astrocytoma, glioblastoma, and diffuse median glioma.

29. The pharmaceutical product according to claim 26, wherein the glioma is an H3K27M mutant glioma.

30. The pharmaceutical product according to claim 1, characterized in that the brain tumor or neuroblastoma has a high level of genomic instability.

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