Parp1 inhibitors and their use in the treatment of cns diseases

Abstract

The present application provides PARP1 inhibitors and their use in the treatment of CNS diseases. The PARP1 inhibitors of the present application have the structure shown in Formula (I). The PARP1 inhibitors of the present application selectively inhibit PARP1 activity over PARP2 activity. The present application also provides the use of the PARP1 inhibitors in the treatment of CNS diseases. (I)

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to the field of disease treatment, specifically to PARP1 inhibitors and their application in the treatment of CNS diseases, such as the treatment of brain tumors. Background Technology

[0002] Poly(ADP-ribose) polymerase (PARP) refers to a group of proteins whose function is to synthesize NAD+. + These are substrate-catalyzed modification reactions that add ADP-ribose to receptor proteins. This is one of many post-translational protein modifications. Therefore, they can also be called ADP-ribose transferases.

[0003] PARP1 is the most abundant and characteristic member of the PARP family within cells. PARP1 is a protein composed of 1014 amino acids (NCBI Accession P09874), with a molecular weight of approximately 116 kDa. Its structure consists of two main domains: an N-terminal DNA-binding domain and a catalytic domain. PARP1 is known to play important roles in many cellular functions, including gene expression, transcription, cell division, cell differentiation, apoptosis, DNA damage response, and repair. When DNA damage occurs, PARP1 is activated and participates in base excision repair (BER), the main mechanism for repairing single-strand DNA damage. PARP1 binds to single-strand break (SSB) sites and subsequently repairs DNA via BER. In response to DNA damage, in addition to BER repair, cells have evolved two other major repair pathways: homologous recombination (HR) and non-homologous recombination end joining (NHEJ). Studies have found that HR-deficient tumors are sensitive to PARP inhibitors, indicating that homologous recombination defects and PARP1 inhibition form a pair of synthetic lethality, a point confirmed by clinical studies. Several PARP inhibitors are currently approved for the treatment of ovarian, breast, pancreatic, and prostate cancers with DNA damage repair defects (such as BRCA1 / 2 mutations). Furthermore, PARP1 inhibitors can also be used to treat diseases caused by excessive cell death, including central nervous system (CNS) diseases such as stroke and neurodegenerative diseases (Akinori Iwashita et al., 2004, J. Pharmacol. Exp. Thera. 310: 425; Marianna Mekhaeil et al., 2023, Neurootherapeutics 20: 1347-1368).

[0004] PARP2 is a protein composed of 583 amino acids (NCBI Accession NP_005475), with a molecular weight of approximately 62 kDa. Its structure includes a DNA-binding domain and a catalytic domain (Ame et al., 1999 J Biol Chem274:17860-17868). The catalytic domain of PARP2 is highly similar to that of PARP1. Studies have shown that PARP2 has similar functions to PARP1, participating in the BER mechanism for DNA damage repair (Schreiber et al., 2002 J Biol Chem277:23028-23036). Marketed PARP inhibitors, such as Olaparib, Niraparib, Talazoparib, and Rucaparib, exhibit similar inhibitory activity against PARP2, except for inhibiting PARP1. Clinical trial results show that these marketed PARP inhibitors have comparable therapeutic effects; however, their toxicities differ significantly. For example, Talazoparib has side effects similar to chemotherapy drugs, such as hair loss. A recent study showed that talazoparib, in addition to inhibiting PARP1 and PARP2, also has a high affinity for two other members of the PARP family, TNKS1 (Tankyrase 1) and TNKS2 (Tankyrase 2) (Ryan et al., 2021 J Biol Chem 296:100251 / 1-100251 / 13). TNKS1 and TNKS2 share a high degree of amino acid sequence similarity, with 83% of their overall amino acid sequences being identical, and 89% of their catalytic domain sequences being identical. They play roles in DNA repair, telomere maintenance, and Wnt / β-catenin signaling. Targeting PARPs other than PARP1 may be the reason why PARP inhibitors cause off-target toxicities (such as hair loss and diarrhea). In addition, inhibition of PARP2 activity may also lead to hematological toxicity (Farrés et al., 2013 Blood 122:44-54; Farrés et al., 2015, Cell Death and Differentiation 22:1144-1157). The toxicity of these PARP inhibitors limits their clinical application and their combination with other targeted therapies.

[0005] Central nervous system disorders, also known as central nervous system diseases, are a group of neurological disorders that affect the structure or function of the brain or spinal cord, collectively forming the central nervous system (CNS). Brain tumors are the most common and have the highest mortality rate (Daroff et al., 2014 Encyclopedia of the Neurological Sciences, eBook ISBN:9780123851581).

[0006] Brain tumors are considered one of the deadliest cancers in the world. The unique tumor microenvironment and inherent characteristics of brain tumor cells make them resistant to most traditional and advanced treatments. Given the low survival rates of current brain tumor treatment strategies, there is an urgent need for new treatment approaches. Summary of the Invention

[0007] On the one hand, the present invention provides compounds with structures as shown in the following formula (I), or their hydrates, isotope labels, or pharmaceutically acceptable salts: (I) Wherein, R1 is selected from halogens, alkyl groups and haloalkyl groups; R2 is selected from halogens and alkyl groups; R3 is selected from halogens, alkyl groups and cyano groups (i.e. –CN); R4 is selected from alkyl groups, haloalkyl groups and cycloalkyl groups.

[0008] In some embodiments of the compounds, methods, and uses of formula (I) of the present invention, (a) R1 is a halogen, C 1-3 Alkyl or halogenated C 1-3 Alkyl, or (b) R2 is a halogen or C 1-3 Alkyl group, or (c)R3 is a halogen, C 1-3 Alkyl or cyano, or (d)R4 is C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-4 Cycloalkyl, or any combination of (a) to (d), such as all of (a) to (d).

[0009] In some embodiments of the compounds, methods, and uses of formula (I) of the present invention, (e) R1 is F, methyl, or trifluoromethyl, or (f) R2 is F, Cl, or methyl, or (g) R3 is F, methyl, or cyano, or (h) R4 is C 1-3 Alkyl, C 1-3 Halogenated alkyl or C 3-4 Cycloalkyl, or any combination of (e) to (h), such as all of (e) to (h).

[0010] In some embodiments of the compounds, methods and uses of formula (I) of the present invention, (i) R1 is F, methyl and trifluoromethyl, or (j) R2 is F, Cl or methyl, or (k) R3 is F, methyl or cyano, or (l) R4 is methyl, ethyl, difluoroethyl or cyclopropyl, or any combination of (i) to (l), such as all of (i) to (l).

[0011] In some embodiments of the compound of formula (I), R1 is any one of (a), (e), and (i) above; R2 is any one of (b), (f), and (j) above; R3 is any one of (c), (g), and (k) above; and R4 is any one of (d), (h) and (l) above.

[0012] In some embodiments of the compounds, methods, and uses of formula (I) of the present invention, R1 is F, CH3, or CF3; R2 is F, Cl, or CH3; R3 is F, CH3, or cyano; and R4 is C. 1-3 Alkyl, C 1-3 Halogenated alkyl or C 3-4 Cycloalkyl. In some embodiments, R1 is F; R2 is F; R3 is a halogen or alkyl; R4 is an alkyl, haloalkyl, or cycloalkyl. In some embodiments, R1 is CH3; R2 is F; R3 is a halogen or cyano; R4 is an alkyl, haloalkyl, or cycloalkyl. In some embodiments, all R1, R2, R3, and R4 of the compound are defined by any one of the rows in rows 1-11 of the following table: .

[0013] In some embodiments of the compounds and methods of the present invention, R1 is CH3, R2 is F, R3 is F, and R4 is CH3. In some embodiments of the compounds and methods of the present invention, R1 is CH3, R2 is F, R3 is CN, and R4 is CH3. In some embodiments of the compounds and methods of the present invention, R1 is CH3, R2 is F, R3 is F, and R4 is cyclopropyl. In some embodiments of the compounds and methods of the present invention, R1 is CF3, R2 is F, R3 is F, and R4 is CH3. In some embodiments of the compounds and methods of the present invention, R1 is F, R2 is F, R3 is CH3, and R4 is CH3. In some embodiments of the compounds and methods of the present invention, R1 is F, R2 is F, R3 is F, and R4 is CH3. In some embodiments of the compounds and methods of the present invention, R1 is F, R2 is F, R3 is F, and R4 is CH2CH3. In some embodiments of the compounds and methods of the present invention, R1 is F, R2 is F, R3 is F, and R4 is CH2CHF2. In some embodiments of the compounds and methods of the present invention, R1 is F, R2 is F, R3 is F, and R4 is cyclopropyl. In some embodiments of the compounds and methods of the present invention, R1 is F, R2 is Cl, R3 is F, and R4 is CH3. In some embodiments of the compounds and methods of the present invention, R1 is F, R2 is CH3, R3 is F, and R4 is CH3.

[0014] On one hand, the present invention provides methods for preparing any of the compounds disclosed herein. In an exemplary embodiment, the present invention provides a method for preparing 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one, comprising: (a) 4-Fluoro-1H-pyrazole-5-carboxylic acid is reacted with an acylation reagent (such as SOCl2) to prepare 4-fluoro-1H-pyrazole-5-carbonyl chloride; (b) Under alkaline conditions (e.g., catalyzed by NaH or LiHMDS), 4-fluoro-1H-pyrazole-5-carbonyl chloride and 3,5-dibromo-2,6-difluoroaniline are reacted to prepare N-(3,5-dibromo-2,6-difluorophenyl)-4-fluoro-1H-pyrazole-5-carboxamide. (c) N-(3,5-dibromo-2,6-difluorophenyl)-4-fluoro-1H-pyrazole-5-carboxamide undergoes a cyclization reaction under alkaline conditions (e.g., catalyzed by K2CO3) to prepare 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one; (d) 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one was prepared by selective dehalogenation of 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one; (e) 3,6-Difluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxaline-4(5H)-one was prepared by reacting 7-bromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one with (Bu)3SnCH2OH under the catalysis of a Pd catalyst (such as Xphos Pd G2); (f) 3,6-Difluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxaline-4(5H)-one reacts with HBr to generate 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one; (g) 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one was prepared by reacting 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one with 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridine amide under alkaline conditions (e.g., catalyzed by DIEA and KI).

[0015] On one hand, the present invention provides a method for inducing regression of a central nervous system tumor in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or its hydrate, isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, tumor regression is induced after administration of the compound. In some embodiments, tumor regression is assessed 7 to 84 days after administration of the compound, for example, after any day between 7 and 84 days after administration of the compound, such as after 8 days, 9 days, etc., or at any time between any two days. In some embodiments, tumor regression is between 1% and 100%, between 5% and 100%, between 10% and 100%, between 25% and 100%, between 50% and 100%, between 75% and 100%, or any percentage value between 1% and 100%, such as 2%, 3%, 4%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, etc., or any range between any two such values. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, simultaneously with, or after administration of one or more additional anticancer therapies or drugs (such as the anticancer therapies or drugs disclosed herein). In some embodiments, the CNS tumor, such as a brain tumor, has a volume at time t that is less than the tumor volume on the first day of compound administration, and time t is day 7, day 84, or any day between day 7 and day 84. In some embodiments of the method for inducing central nervous system tumor regression in a subject, the central nervous system tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell carcinoma, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinoblastoma, schwannoma, primary central nervous system lymphoma, brainstem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and dysplastic neuroepithelial tumor. In some embodiments of the method for inducing central nervous system tumor regression in a subject, the central nervous system tumor is any central nervous system cancer disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumors disclosed herein. In some embodiments, the brain tumor is a high-grade (rapidly growing) tumor. In some implementations, brain tumors are low-grade (slow-growing) tumors.In some embodiments, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments of a method for inducing regression of a central nervous system tumor in a subject, the compound, such as a compound of formula (I) or its hydrate, an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition comprising the compound or its hydrate, an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of the compound, such as a compound of formula (I) or its hydrate, an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered.

[0016] On one hand, the present invention provides a method for inducing an increase in central nervous system tumor regression in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or its hydrate, isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, an increase in tumor regression is induced after administration of the compound. In some embodiments, the increase in tumor-induced regression is assessed 7 to 84 days after administration of the compound, for example, after any day between 7 and 84 days after administration of the compound, such as after 8 days, 9 days, etc., or at any time between any two days. In some embodiments, tumor regression is between 1% and 100%, between 5% and 100%, between 10% and 100%, between 25% and 100%, between 50% and 100%, between 75% and 100%, or any percentage value between 1% and 100%, such as 2%, 3%, 4%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, etc., or any range between any two such values. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, simultaneously with, or after the administration of one or more additional anticancer therapies or drugs (such as the anticancer therapies or drugs disclosed herein). In some embodiments of the method for inducing an increase in central nervous system tumor regression in a subject, the central nervous system tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell tumor, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinoblastoma, schwannoma, primary central nervous system lymphoma, brainstem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and dysplastic neuroepithelial tumor. In some embodiments of the method for inducing an increase in central nervous system tumor regression in a subject, the central nervous system tumor is any central nervous system cancer disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumors disclosed herein. In some embodiments, the brain tumor is a high-grade (rapidly growing) tumor. In some implementations, the brain tumor is a low-grade (slow-growing) tumor. In some implementations, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma).In some embodiments of a method for inducing an increase in the regression of central nervous system tumors in a subject, the compound, such as a compound of formula (I) or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition comprising the compound or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of the compound, such as a compound of formula (I) or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable pharmaceutical composition thereof, is administered.

[0017] On one hand, the present invention provides a method for inhibiting the growth of a central nervous system tumor in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, tumor growth is inhibited after administration of the compound. In some embodiments, the inhibition of central nervous system tumor growth is assessed 7 to 84 days after administration of the compound, for example, after any day between 7 and 84 days after administration of the compound, for example, after 8 days, 9 days, etc., or at any time between any two days. In some embodiments, tumor growth is inhibited by 10% to 150%, for example, 30% to 150% or 50% to 120%, for example, any integer value between 10% and 150%, for example, 30%, 40%, 50%, 60%, 70%, 100%, 125%, 150%, etc., or any range between any two such values. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, concurrently with, or after the administration of one or more additional anticancer therapies or drugs (such as those disclosed herein). In some embodiments of the method for inhibiting the growth of a central nervous system tumor in a subject, the central nervous system tumor is selected from meningiomas, meningeal sarcomas, ependymomas, astrocytomas, gliomas, glioblastomas, pineal cell tumors, invasive pituitary adenomas, pituitary carcinomas, germ cell tumors, sarcomas, craniopharyngiomas, retinoblastomas, schwannomas, primary central nervous system lymphomas, brainstem gliomas, pituitary adenomas, anaplastic astrocytomas, mixed gliomas, primitive neuroectodermal tumors, hemangioblastomas, vestibular schwannomas, chordomas, spinal neurofibromas, lymphomas, optic gliomas, and dysplastic neuroepithelial tumors. In some embodiments of the method for inhibiting the growth of a central nervous system tumor in a subject, the central nervous system tumor is any central nervous system cancer disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (rapid-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma).In some embodiments of a method for inhibiting the growth of central nervous system tumors in a subject, the compound, such as a compound of formula (I) or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition comprising the compound, or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of the compound, such as a compound of formula (I) or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered.

[0018] On one hand, the present invention provides a method for inhibiting PARP1 activity, such as selectively inhibiting PARP1 activity relative to PARP2 activity in a subject, comprising administering to the subject an effective amount of a compound of formula (I), or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, after administration of the compound, the inhibition of PARP1 activity in the subject is greater than the inhibition of PARP2 activity. In some embodiments, the ratio of PARP1 activity inhibition to PARP2 activity inhibition in the subject is at least 50, at least 750, at least 1500, at least 2500, at least 5000, at least 7500, at least 10000, at least 15000, at least 20000, at least 25000, or within any two of these numbers. In some embodiments, the ratio of PARP1 activity inhibition to PARP2 activity inhibition in the subject is between 50 and 2000, between 750 and 15000, between 2500 and 15000, or between 12500 and 25000, for example, between 15000 and 25000. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, simultaneously with, or after administration of one or more additional anticancer therapies or drugs (such as the anticancer therapies or drugs disclosed herein). In some embodiments, the compound, such as a compound of formula (I), or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition comprising the compound, or its hydrate, or its isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of a compound, such as a compound of formula (I), or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt, or its pharmaceutically acceptable composition, is administered.

[0019] On one hand, the present invention provides a method for treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject suffers from a disease or condition characterized by or caused by excessive cell death. In some embodiments, the disease or condition is a stroke or a neurodegenerative disease.

[0020] On the other hand, the present invention provides a method for treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the cancer is ovarian cancer, breast cancer, pancreatic cancer, or prostate cancer. In some embodiments, the cancer has a DNA damage repair defect, such as a homologous recombination defect. In some embodiments, the subject has a central nervous system (CNS) tumor. In some embodiments, the subject has a brain tumor. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, simultaneously with, or after administration of one or more additional anticancer therapies or drugs (such as the anticancer therapies or drugs disclosed herein). In some embodiments, the subject has a central nervous system (CNS) tumor. In some embodiments, the CNS tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell carcinoma, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, glioblastoma multiforme, medulloblastoma, astrocytoma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and embryogenetic neuroepithelial tumor. In some embodiments, the central nervous system tumor is any central nervous system tumor disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumors disclosed herein. In some embodiments, the brain tumor is a high-grade (rapid-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments, the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition comprising the compound, its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

[0021] On one hand, the present invention provides the use of compounds of formula (I) disclosed herein (e.g., an effective amount of the compound), or hydrates thereof, or isotopically substituted derivatives thereof, or pharmaceutically acceptable salts thereof, in the preparation of medicaments. On the other hand, the present invention provides the use of compounds of formula (I), or hydrates thereof, or isotopically substituted derivatives thereof, or pharmaceutically acceptable salts thereof, in the preparation of medicaments for treating any condition such as cancer, such as any CNS tumor, for example, brain tumors as disclosed or described herein.

[0022] On the one hand, the present invention provides the use of the compound of formula (I) disclosed herein (e.g., an effective amount of the compound), or its hydrate, or its isotopically substituted derivative or its pharmaceutically acceptable salt, for the treatment of the conditions disclosed herein or for the purposes disclosed herein.

[0023] On the one hand, the present invention provides a method for treating the conditions disclosed herein or for the purposes disclosed herein, using a compound of formula (I) (e.g., an effective amount of the compound), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof.

[0024] On one hand, this invention provides a method for treating central nervous system (CNS) diseases or conditions (e.g., CNS tumors, such as brain tumors), comprising administering a compound of formula (I) disclosed herein, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, wherein the R group is as defined herein, and wherein the compound of formula (I) can penetrate the blood-brain barrier (BBB). In some embodiments, the compound of formula (I) penetrates the blood-brain barrier (BBB). In some embodiments, the CNS disease or condition is a brain tumor. In some embodiments, the central nervous system (CNS) disease or condition is a CNS tumor. In some embodiments, the central nervous system (CNS) disease or condition is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell carcinoma, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, glioblastoma multiforme, medulloblastoma, primary central nervous system lymphoma, brainstem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic nerve glioma, and dysplastic neuroepithelial tumor. In some embodiments, the central nervous system tumor is any central nervous system tumor disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumors disclosed herein. In some embodiments, the brain tumor is a high-grade (rapid-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments, a therapeutically effective amount of the compound, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt is administered. In some embodiments, a therapeutically effective amount of said compound, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt is administered in a pharmaceutically acceptable composition comprising said compound, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable diluents, carriers, or excipients.

[0025] On one hand, the present invention provides a pharmaceutically acceptable composition comprising a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable diluent, carrier, or excipient.

[0026] In some embodiments of the method disclosed herein, the method includes administering a therapeutically effective amount of a compound of formula I as described herein, its hydrate, its isotopically substituted derivative, or its pharmaceutically acceptable salt. In various embodiments, the pharmaceutically acceptable salt is an inorganic or organic acid salt, such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate, or oxalate. In various embodiments, the pharmaceutically acceptable salt is an inorganic or organic base salt formed with a base, such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, tromethamine), or N-methylglucosamine salt.

[0027] In any embodiment of any method and use disclosed herein, the compound of formula (I), its hydrate, its isotopically substituted derivative, or its pharmaceutically acceptable salt is administered before, simultaneously with, or in combination with one or more additional anticancer therapies or drugs, wherein the one or more additional anticancer therapies or drugs are selected from, for example, monoclonal antibodies, surgery, radiation therapy (such as ionizing radiation (IR), gamma radiation, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and whole-body radioisotopes), endocrine therapy, etc. Treatments, biological response modifiers (such as interferon, interleukin, and tumor necrosis factor), hyperthermia and cryotherapy, drugs to reduce any adverse reactions (such as antiemetics) and other approved chemotherapy drugs, spindle toxins (such as vincristine, vinorelbine, and paclitaxel), podophyllotoxins (such as etoposide, irinotecan, and vorponotecan), nitrosoureas (such as vastatin and lomustine), inorganic ions (such as cisplatin and carboplatin), enzymes (such as asparaginase), hormones (such as tamoxifen, leucine, flutamide, and medroxyprogesterone acetate), Gleevec™, doxorubicin, dexamethasone, and cyclophosphamide.

[0028] In any of the treatments disclosed herein, in some embodiments, the subject is a person in need of such treatment. In various embodiments, the subject is a human being. In some embodiments, the subject is a non-human being, such as a veterinary animal (e.g., a pig, cow, sheep, horse, dog, and cat) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

[0029] In any use of the compounds disclosed herein in treating a subject's condition, in some embodiments, the subject is a subject in need of such treatment. In various embodiments, the subject is a human being. In some embodiments, the subject is a non-human being, such as a veterinary animal (e.g., a pig, cow, sheep, horse, dog, or cat) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

[0030] In any use of the compounds disclosed herein in the preparation of a medicament for treating a subject or for treating a condition of a subject, in some embodiments, the subject is a subject in need of such treatment. In various embodiments, the subject is a human being. In some embodiments, the subject is a non-human being, such as a veterinary animal (e.g., a pig, cow, sheep, horse, dog, and cat) or a zoo or laboratory animal (e.g., any non-human primate or mammal). Detailed Implementation

[0031] As used herein, the term "alkyl" refers to an alkyl group having a straight-chain or branched group having a maximum of 10 carbon atoms. Useful alkyl groups include straight-chain or branched C4 groups. 1-10 Alkyl, such as C 1-6 Alkyl group. In some embodiments, the alkyl group is C10. 1-3 Alkyl groups. Typical alkyl groups include methyl, deuterated methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl (e.g., 3-pentyl), hexyl, and octyl, which may optionally be substituted. In some embodiments, the alkyl group is, for example, C10. 1-3 Alkyl and C 1-6 The alkyl group is unsubstituted. In some embodiments, the alkyl group, for example, is C10. 1-3 Alkyl and C 1-6 The alkyl group is substituted. In some embodiments, the substituents of the alkyl group include halogen groups (F, Cl, Br, and I), -OH, -CHO, -NH2, or combinations thereof.

[0032] As used in this article, the term "cycloalkyl" refers to a cyclic alkyl group. Useful cycloalkyl groups are C16-C ... 3-8 Cycloalkyl. In some embodiments, the cycloalkyl group is C10. 3-6 Cycloalkyl groups. Typical cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Cycloalkyl groups can be substituted with one or more substituents as described herein. In some embodiments, the cycloalkyl group is, for example, C10. 3-4 cycloalkyl and C 3-6 Cycloalkyl groups are unsubstituted.

[0033] Useful halogens or halogen groups include fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0034] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more H atoms are replaced by one or more halogen atoms. In some embodiments, the haloalkyl is a halo-C 1-3 Alkyl groups. Exemplary haloalkyl groups include trifluoromethyl, difluoroethyl, fluoromethyl, and dichloroethyl.

[0035] The term “inducement” as used in this article means “cause” or “induce”.

[0036] As used herein, the term "inhibition" includes a reduction or decrease in a parameter, such as a reduction or decrease in tumor growth. For example, inhibition of tumor growth following exposure to a compound disclosed herein indicates a reduction or decrease in tumor growth, for example, by 5%, 10%, 20%, 30%, 40%, or more, relative to similar tumors not exposed to the compound. Inhibition can be less than, equal to, or greater than 100%. If a subject's exposure to a compound results in a reduction or decrease in a parameter (e.g., tumor growth), then the compound is an "inhibitor" of said parameter (e.g., tumor growth).

[0037] The term "regression" as used in this article refers to a reduction in the size or extent of a tumor. This regression may be related to the disappearance or reduction in the number or size of tumor cells (e.g., tumor cells).

[0038] As used in this article, the term “inducing tumor regression” means causing or resulting in a reduction in the size or extent of a tumor (including causing the tumor to disappear (e.g., to become undetectable)), for example by causing the disappearance or reduction in the number or size of tumor cells (e.g., tumor cells).

[0039] As used herein, the term "increased induced regression" refers to causing an already occurring regression to proceed at a greater rate or to a greater extent, or to resulting in additional regression where previous regression has ceased. Therefore, increased induced regression of a tumor means, for example, that a regressing tumor regresses at a greater rate or to a greater extent after treatment with a compound of formula (I) of this disclosure, for example, or if the tumor has previously ceased regression, by a compound of formula (I) of this disclosure. Increased regression may occur when, for example, two different (e.g., independently acting) treatments both result in regression, and their combination has an additive or synergistic effect.

[0040] As used herein, the terms "pharmaceutically acceptable" or "medicinal" mean a substance that, to a reasonable extent of medical judgment, is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and is proportionate to a reasonable benefit / risk ratio. Therefore, pharmaceutically acceptable refers to a substance that is not biologically or otherwise unacceptable. This material can be administered to an individual in conjunction with the associated active compound without causing clinically unacceptable biological effects or interacting in a harmful manner with any other component of a pharmaceutical composition containing the material.

[0041] As used herein, the terms "therapeutic effective amount" or "effective amount" for a therapeutic agent or a composition comprising a therapeutic agent mean an amount sufficient to treat, diagnose, prevent, and / or delay the onset of one or more symptoms of a disease, condition, and / or symptom when administered to a subject who has or is susceptible to the disease, condition, and / or symptom (e.g., to slow or prevent an increase in the severity of any such symptom, or to reduce the severity of any such symptom). "Therapeutic effective amount" and "effective amount" also include amounts sufficient to affect or cause a change in activity when administered to a subject, such as enzyme activity, like PARP1 activity or PARP2 activity. Those skilled in the art will understand that a therapeutic effective amount or effective amount is generally administered by a dosing regimen comprising at least one unit dose. Non-limiting examples of a "therapeutic effective amount" or "effective amount" of a compound of formula (I) of the present invention, its hydrate, its isotopically substituted derivative, its pharmaceutically acceptable salt, or a composition comprising a compound of formula (I) are amounts that can be used to inhibit, block, or reverse cell activation, migration, or proliferation, or to effectively treat cancer or improve cancer symptoms. Another non-limiting example of a "therapeutic effective amount" or "effective amount" of a compound of formula (I) of the present invention, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt or composition comprising a compound of formula (I) is an amount that can be used to inhibit PARP1 activity.

[0042] As used herein, the term "treatment" means any method used to partially or completely reduce, improve, alleviate, suppress, prevent, delay the onset, severity, and / or incidence of one or more symptoms or features of a particular disease, condition, and / or symptom. Treatment may be administered to subjects who do not exhibit signs of disease and / or only exhibit early signs of disease in order to reduce the risk of developing disease-related pathology.

[0043] Tumor cells (or cancer cells) as described herein are generally characterized by their abnormal proliferation relative to normal cells and the formation of clusters or tumors in an individual with cancer. “Cancer” encompasses a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the blood and lymphatic systems. Examples of various cancers include cancers of the central nervous system, such as brain cancer and spinal cord cancer. The terms “tumor” and “cancer” are used interchangeably herein. For example, both terms include solid and liquid, such as diffuse or circulating tumors. In some embodiments, the terms “cancer” or “tumor” include malignant cancer and tumors, as well as advanced cancer and tumors. As used herein, “tumor” and “cancer” refer to both malignant (e.g., spread from the tissue of origin to another tissue) and non-malignant (e.g., growing but still confined to the tissue of origin), such as benign growth.

[0044] Central nervous system tumors (also referred to as CNS tumors in this article) are known in this field (DN Louis et al., 2021 WHO Classification of Tumors of the Central Nervous System: a summary, Neuro Oncol. Aug 2021; 23(8): 1231–1251 doi:10.1093 / neuonc / noab106). Central nervous system tumors include brain tumors and spinal cord tumors. Central nervous system tumors include astrocytomas, oligodendrogliomas, glioblastomas, diffuse astrocytomas, angiocentric gliomas, juvenile pleomorphic low-grade neuroepithelial tumors, diffuse low-grade gliomas, diffuse midline gliomas, diffuse hemispherical gliomas, juvenile high-grade gliomas, pleomorphic xanthoastrocytomas, subependymal giant cell astrocytomas, chordoid gliomas, astroblastomas, gangliocytomas, dysembryonic neuroepithelial tumors, etc. Diffuse gliomas with oligodendroglioma-like features and nuclear clusters, papillary gliomas, multinodular and vacuolar neuronal tumors, cerebellar gangliocytomas with developmental abnormalities (Ihermitt-Duclos disease), external neurocytomas, supratentorial ependymomas, posterior fossa ependymomas, spinal ependymal carcinomas, medulloblastomas, atypical teratomas / rhabdoid tumors, embryonal tumors with multi-layered rosettes, neuroblastomas of the central nervous system, and other similar tumors. bcor Internally tandem repeating central nervous system tumors, myxoid tumors of the pineal region, meningiomas, solitary fibromas, meningeal melanomas, amelio (epithelial) craniopharyngiomas, and papillary craniopharyngiomas.

[0045] A brain tumor is a cancerous (malignant) or non-cancerous (benign) abnormal growth of cells or a mass within the brain. Brain tumors can occur within brain tissue. They can also occur near brain tissue. Nearby locations include nerves, the pituitary gland, the pineal gland, and the membranes covering the surface of the brain. Brain tumors can originate in the brain itself. These are called primary brain tumors. Sometimes, cancer spreads from other parts of the body to the brain. These tumors are secondary brain tumors, also known as metastatic brain tumors. There are many different types of primary brain tumors. Some brain tumors are not cancerous. These are called non-cancerous brain tumors or benign brain tumors. Non-cancerous brain tumors may grow over time and compress brain tissue. Other brain tumors are brain cancers, also known as malignant brain tumors. Brain cancers can grow rapidly. Cancer cells can invade and destroy brain tissue. Brain tumors are a subset of tumors of the central nervous system. Brain tumors include, but are not limited to: gliomas, embryonal brain tumors (such as medulloblastomas), ependymomas, glioblastomas, primary central nervous system lymphomas, pineal region tumors (such as germ cell tumors or pineal cell tumors), pituitary adenomas, meningiomas, and acoustic neuromas (vestibular schwannomas). Some brain tumors are high-grade (rapidly growing). Some brain tumors are lower-grade (slow-growing).

[0046] Specifically, the present invention provides compounds of formula (I), or their hydrates, or isotopically substituted derivatives, or pharmaceutically acceptable salts thereof: (I) Wherein, R1 is selected from halogens, alkyl groups and haloalkyl groups; R2 is selected from halogens and alkyl groups; R3 is selected from halogens, alkyl groups and cyano groups (i.e. –CN); R4 is selected from alkyl groups, haloalkyl groups and cycloalkyl groups.

[0047] In one or more embodiments of the compound of formula (I), R1 is a halogen, C 1-3 Alkyl or halogenated C 1-3 Alkyl group. In some embodiments, R1 is F, methyl, or trifluoromethyl.

[0048] In one or more embodiments of the compound of formula (I), R2 is a halogen or C 1-3 Alkyl group. In some embodiments, R2 is F, Cl, or methyl.

[0049] In one or more embodiments of the compound of formula (I), R3 is a halogen, C 1-3 Alkyl or cyano. In some embodiments, R3 is F, methyl, or cyano.

[0050] In one or more embodiments of the compound of formula (I), R4 is C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-4 Cycloalkyl. In some embodiments, R4 is methyl, ethyl, difluoroethyl, or cyclopropyl.

[0051] In one or more embodiments of the compound of formula (I), R1 is CH3, R2 is F, R3 is F or cyano, and R4 is CH3 or cyclopropyl.

[0052] In one or more embodiments of the compound of formula (I), R1 is F, R2 is F, Cl or CH3, R3 is F or CH3, and R4 is methyl, ethyl, difluoroethyl or cyclopropyl.

[0053] In some embodiments of the compound of formula (I), one or more of R1, R2, R3, and R4 are alkyl, haloalkyl, or cycloalkyl and are not further substituted. In some embodiments of the compound of formula (I), one or more of R1, R2, R3, and R4 are alkyl, haloalkyl, or cycloalkyl as defined herein, and only R4 is further substituted, for example, by F, Cl, Br, I, -OH, -CHO, -NH2, or combinations thereof.

[0054] Example compounds of formula (I) include, but are not limited to: 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 1); 7-((4-(2-cyano-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 2); 7-((4-(2-fluoro-6-(cyclopropylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 3); 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3-trifluoromethyl-6-flupyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 4); 7-((4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 5); 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 6); 7-((4-(2-fluoro-6-(ethylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 7); 7-((4-(2-fluoro-6-(2,2-difluoroethylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 8); 7-((4-(2-fluoro-6-(cyclopropylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 9); 3-Fluoro-7-((4-(2-Fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-chloropyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 10); 3-Fluoro-7-((4-(2-Fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one (Example 11); Or its hydrates, isotopically substituted derivatives, or pharmaceutically usable salts.

[0055] Some of the compounds of this invention may exist as stereoisomers, including optical isomers. This invention includes all stereoisomers and racemic mixtures of such stereoisomers, as well as individual enantiomers that can be isolated according to methods well known to those skilled in the art.

[0056] Examples of medicinal salts include inorganic and organic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate, and oxalate; as well as inorganic and organic base salts formed with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, tromethamine), and N-methylglucosamine.

[0057] Examples of prodrugs of the compounds of the present invention include simple esters of compounds containing carboxylic acids (e.g., by means of methods known in the art, via C...). 1-4 Esters obtained by alcohol condensation); esters of compounds containing hydroxyl groups (e.g., obtained by condensation with C according to methods known in the art). 1-4 Carboxylic acid, C 3-6 esters obtained by condensation of diacids or their anhydrides, such as succinic anhydride and fumaric anhydride; imines of compounds containing amino groups (e.g., obtained by condensation with C according to methods known in the art). 1-4 Imines obtained by condensation of aldehydes or ketones; carbamates of compounds containing amino groups, such as those by Leu et al. ( J.Med.Chem.42:3623-3628 (1999) and Greenwald et al. ( J.Med.Chem. Those esters described in 42:3657-3667 (1999); aldol acetals or keto acetals of compounds containing alcohols (e.g., those acetals obtained by condensation with chloromethyl methyl ether or chloromethyl ethyl ether according to methods known in the art).

[0058] The PARP1 inhibitors of the present invention can be prepared using methods known to those skilled in the art or the novel methods of the present invention. Specifically, the compounds of formula (I) of the present invention can be prepared by the exemplary reaction shown in Scheme 1. 4-Methyl-1H-pyrazole-5-carboxylic acid reacts with SOCl2 to give the product 4-methyl-1H-pyrazole-5-carbonyl chloride. 4-Methyl-1H-pyrazole-5-carbonyl chloride reacts with 3-bromo-2,6-difluoroaniline under alkaline conditions (e.g., catalyzed by NaH or LiHMDS) to give the product N-(3-bromo-2,6-difluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide. N-(3-bromo-2,6-difluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide undergoes a cyclization reaction under alkaline conditions (e.g., catalyzed by K2CO3) to give the product 7-bromo-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one. 7-Bromo-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one and (Bu)3SnCH2OH were reacted with a Pd catalyst (such as Xphos Pd G2) to give the product 6-fluoro-7-(hydroxymethyl)-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one. 6-Bromo-7-(hydroxymethyl)-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one was then reacted with HBr to give the product 7-(bromomethyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one. 7-(bromomethyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one undergoes a substitution reaction with 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridine amide under alkaline conditions (such as under the catalysis of DIEA and KI) to give the target compound 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one.

[0059] Reaction Scheme 1

[0060] Other related compounds can be prepared using similar methods. For example, by replacing 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide with 6-cyano-N-methyl-5-(piperazin-1-yl)pyridineamine, the target compound 7-((4-(2-cyano-6-(methylcarbamoyl)pyridin-3-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one is prepared. By replacing 4-methyl-1H-pyrazol-5-carboxylic acid with 4-fluoro-1H-pyrazin-5-carboxylic acid, the target compound 7-( (4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one. The target compound 3-fluoro-7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-chloropyrazolo[1,5-a]quinoxaline-4(5H)-one was prepared by replacing 3-bromo-2-chloro-6-fluoroaniline with 3-bromo-2-chloro-6-fluoroaniline.

[0061] The compounds of the present invention can be prepared as illustrated in the exemplary reaction of reaction scheme 2. The reaction of 4-fluoro-1H-pyrazole-5-carboxylic acid with SOCl2 yields the product 4-fluoro-1H-pyrazole-5-carbonyl chloride. The 4-fluoro-1H-pyrazole-5-carbonyl chloride reacts with 3,5-dibromo-2,6-difluoroaniline under alkaline conditions (e.g., catalyzed by NaH or LiHMDS) to give the product N-(3,5-dibromo-2,6-difluorophenyl)-4-fluoro-1H-pyrazole-5-carboxamide. Under alkaline conditions (e.g., catalyzed by K2CO3), N-(3,5-dibromo-2,6-difluorophenyl)-4-fluoro-1H-pyrazole-5-carboxamide reacts to give the product 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one. In the presence of sodium ascorbate, CuI, and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one undergoes selective dehalogenation to give the product 7-bromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one. 3,6-Difluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxaline-4(5H)-one reacts with HBr to give the product 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one. Under alkaline conditions (e.g., catalyzed by DIEA and KI), 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one reacts with 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridine amide to give the target compound 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-ylmethyl)-3,3-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one.

[0062] Reaction Scheme 2

[0063] Other related compounds can be prepared using similar methods. For example, by replacing 4-fluoro-1H-pyrazol-5-carboxylic acid with 4-methyl-1H-pyrazin-5-carboxylic acid, the target compound 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one can be prepared. By replacing 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridine amide with N,6-dimethyl-5-(piperazin-1-yl)pyridine amide, the target compound 7-((4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one can be prepared.

[0064] Pharmaceutically acceptable salts of the PARP1 inhibitors of the present invention are also included within the scope of this disclosure. Acid addition salts are formed by mixing a solution of the compound of the present invention with a solution of a pharmaceutically acceptable non-toxic acid (such as hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, etc.). Base addition salts are formed by mixing a solution of the compound of the present invention with a solution of a pharmaceutically acceptable non-toxic alkali, such as sodium hydroxide, potassium hydroxide, quinoline, sodium carbonate, tris(hydroxymethyl)aminomethane, N-methylglucosamine, etc.

[0065] The compounds of the present invention can be administered as unprocessed pharmaceutical products. The compounds of the present invention can also be administered as part of a suitable pharmaceutical formulation containing a pharmaceutically acceptable carrier (including excipients and adjuvants). These pharmaceutically acceptable carriers facilitate the processing of the compounds into pharmaceutically acceptable pharmaceutical formulations. In some embodiments, the pharmaceutical formulation, such as oral or other formulations, such as tablets, lozenges, and capsules, and solutions suitable for injection or oral administration, comprises about 0.01% w / w to 99% w / w, such as from about 0.25% w / w to 75% w / w, of the active compound and excipients.

[0066] The PARP1 inhibitors of the present invention can be administered in pharmaceutical compositions containing pharmaceutically acceptable carriers, wherein the pharmaceutical compositions comprise all pharmaceutical formulations containing compounds of the present invention, the amount of which can effectively achieve their intended purpose. Although individual needs vary, those skilled in the art can determine the optimal dosage of each portion of the pharmaceutical formulation. Generally, the compounds, or their hydrates or isotopically substituted derivatives or available medicinal salts thereof, are administered orally to mammals daily at a dosage of about 0.0025 to 50 mg / kg body weight. In some embodiments, the dosage is about 0.01 to 10 mg / kg orally. If a known anticancer drug is also administered, its dosage should effectively achieve its intended purpose. The optimal dosages of these known anticancer drugs are well known to those skilled in the art.

[0067] A single oral dose may comprise about 0.01 to 50 mg, preferably about 0.1 to 10 mg, of the compound of the present invention. A single dose may be administered once or multiple times daily as one or more tablets, each tablet containing about 0.1 to 50 mg, preferably about 0.25 to 10 mg, of the compound of the present invention or a solvate thereof.

[0068] In topical formulations, the concentration of the compounds of the present invention can be from about 0.01 to 100 mg per gram of carrier.

[0069] The present invention also provides compositions. In some embodiments, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. Exemplary diluents, carriers, and excipients used in the compositions disclosed herein are known to those skilled in the art and include, but are not limited to, those disclosed, for example, in the Handbook of Pharmaceutical Excipients (Raymond C. Rowe et al., 6th edition, 2009), the entire contents of which are incorporated herein by reference. Furthermore, exemplary compositions, such as the dosage forms of the compositions disclosed herein, are known to those skilled in the art and include, for example, those disclosed, for example, in Loyd V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (8th edition, 2005), the entire contents of which are incorporated herein by reference.

[0070] In one embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of at least one compound of formula (I) disclosed herein (e.g., a therapeutically effective amount of one compound disclosed herein), or any pharmaceutically acceptable form thereof (e.g., a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as salts of phosphates and organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey), the entire disclosure of which is incorporated herein by reference for all purposes.

[0071] The pharmaceutical formulations disclosed herein can be administered to any mammal, provided they can experience the therapeutic effects of the compounds disclosed herein. Among such mammals, humans and veterinarians are of paramount importance, although the disclosure is not limited thereto.

[0072] The pharmaceutical formulation disclosed herein can be administered in any manner to achieve its intended purpose. For example, administration can be via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, intranasal, or local routes. Alternatively, or concurrently, administration can be via oral route. The dosage will depend on the subject's age, health status and weight, the type of concurrent treatment, the frequency of treatment, and the nature of the desired effect.

[0073] The pharmaceutical formulations of the present invention can be manufactured using known methods. For example, they can be manufactured by conventional mixing, granulation, tableting, dissolving, or freeze-drying processes. When manufacturing oral formulations, solid excipients and active compounds can be combined, and the mixture can be selectively ground. If desired or necessary, appropriate excipients can be added, and the granular mixture can be processed to obtain tablets or tablet cores.

[0074] Suitable excipients, especially fillers, include sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and / or calcium phosphates, such as tricalcium phosphate or dicalcium phosphate; and binders, such as starch pastes including corn starch, wheat starch, rice starch, potato starch, gelatin, astragalus gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrants, such as the starches mentioned above, as well as carboxymethyl starch, croscarmellose, agar, or alginate or its salts, such as sodium alginate, may be added. Adjuvants, especially flow conditioners and lubricants, include silica, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate, and / or polyethylene glycol. If desired, a suitable coating that resists gastric juices can be provided to the tablet core. For this purpose, a concentrated sugar solution can be applied. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solution, and suitable organic solvents or solvent mixtures. To prepare a gastric juice-resistant coating, a suitable cellulose solution, such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate, can be used. Dyes or pigments may be added to the coating of the tablet or tablet core, for example, for identification or to characterize the dosage of the active ingredient.

[0075] Other orally edible pharmaceutical formulations include compressible capsules made of gelatin, and sealed soft capsules made of gelatin and plasticizers such as glycerin or sorbitol. The compressible capsule may contain an active compound in particulate form, mixed with fillers such as lactose; binders such as starch; and / or lubricants such as talc or magnesium stearate, and stabilizers. In soft capsules, the active compound is preferably dissolved or suspended in a suitable liquid such as oils or liquid paraffin, in which stabilizers may be added.

[0076] Suitable formulations for parenteral administration include aqueous solutions of the active compound, such as solutions of water-soluble salts and alkaline solutions. Additionally, oily injectable suspensions of the appropriate active compound can be administered. Suitable lipophilic solvents or carriers include oils such as sesame oils, synthetic fatty acid esters such as ethyl oleate or triglycerides or polyethylene glycol 400, or hydrogenated castor oil, or cyclodextrin. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, and / or dextran. Suspension stabilizers may also be included.

[0077] According to one aspect of the invention, the compounds of the invention are formulated for external and parenteral use and are used to treat skin cancer.

[0078] The topical formulations of the present invention can be formulated into oils, creams, emulsions, ointments, etc., using preferred suitable carriers. Suitable carriers include plant or mineral oils, white mineral oil (white paraffin), branched-chain fatty acids or oils, animal fats, and high molecular weight alcohols (greater than C12). Preferred carriers are those in which the active ingredient can be dissolved. Emulsifiers, stabilizers, moisturizers, and antioxidants may also be included, as well as agents that impart color or fragrance if desired. Furthermore, these topical formulations may contain transdermal penetration enhancers. Examples of such enhancers can be found in U.S. Patent Nos. 3,989,816 and 4,444,762.

[0079] Creams are preferably formulated with a mixture of mineral oil, self-emulsifying beeswax, and water, mixed with an active ingredient dissolved in a small amount of oil, such as almond oil. A typical example of a cream includes approximately 40 parts water, 20 parts beeswax, 40 parts mineral oil, and 1 part almond oil.

[0080] Ointments can be formulated by mixing a plant oil containing active ingredients, such as almond oil, with warm paraffin wax, and then allowing the mixture to cool. A typical example of an ointment consists of approximately 30% by weight almond oil and 70% by weight white paraffin wax.

[0081] Another embodiment of the present invention relates to a pharmaceutical composition that can effectively treat cancer, comprising a PARP1 inhibitor in combination with at least one known anticancer drug or a pharmaceutically acceptable salt of an anticancer drug. In particular, it is used in combination with other anticancer drugs involved in DNA damage and repair mechanisms, such as HDAC inhibitors vorinostat, romidesin, pabistat, and belistat, etc. It is also used in combination with other anticancer drugs involved in cell division, including Chk1 / 2 inhibitors, CDK4 / 6 inhibitors such as palbociclib, Wee1 inhibitors, ATM inhibitors, ATR inhibitors, DNA-PK inhibitors, etc. Furthermore, it is used in combination with other targeted anticancer drugs, including USP1 inhibitors, PRMT5 inhibitors, Polθ inhibitors, RAD51 inhibitors, etc.Other known anticancer drugs that can be used in combination therapy include, but are not limited to, alkylating agents such as busulfan, melphalan, chlorambucil, cyclophosphamide, ifosfamide, temozolomide, bendamustine, cisplatin, mitomycin C, bleomycin, and carboplatin; topoisomerase I inhibitors such as camptothecin, irinotecan, and topotecan; topoisomerase II inhibitors such as doxorubicin, epirubicin, aclarubicin, mitoxantrone, methyl hydroxyrosine, and mentholtoporp; RNA / DNA antimetabolites such as 5-azacytidine, gemcitabine, 5-fluorouracil, and methotrexate; DNA antimetabolites such as 5-fluoro-2′-deoxyuridine, fludarabine, nelarabine, cytarabine, pralatrexate, pemetrexed, hydroxyurea, and thioguanine; and antimitotic agents. Examples of active ingredients include colchicine, vincristine, vinorelbine, paclitaxel, ixapril, cabazitaxel, and docetaxel; examples of antibodies include monoclonal antibodies such as panitumumab, nezotuzumab, nivolumab, pembrolizumab, ramucirumab, bevacizumab, pertuzumab, trastuzumab, cetuximab, oxetuzumab, ofamumumab, rituximab, alemtuzumab, teimomab, tosimob, daratumumab, erlotuzumab, ofatumumab, dinutuximab, blinatumomab, ipilimumab, Avastin, Herceptin, and rituximab; examples of antibody-drug conjugates (ADCs) such as trastuzumab-metanexine conjugate T-DM1, and humanized anti-HER2. Antibody-drug conjugates Trastuzumab Deruxtecan, Trastuzumab Emtansine, humanized anti-TROP2 monoclonal antibody-drug conjugates Datopotamab Deruxtecan, Gemtuzumab Ozogamicin, CD30-guided antibody-drug conjugates Brentuximab Vedotin, Inotuzumab Ozogamicin, Sacituzumab govitecan, Enfortumab Vedotin and Belantamab Mafodotin; kinase inhibitors such as imatinib, gefitinib, erlotinib, ostinib, afatinib, celitinib, alectinib, crizotinib, erlotinib, lapatinib, sorafenib, regorafenib, vemurafenib, dabrafenib, aflibercept, sunitinib, nilotinib, dasatinib, bosutinib, pralitinib, ibrutinib, cabozantinib, lenvatinib, vandetanib, trametinib, carbitinib, axitinib, tesimolimus, idalalisib, pazopanib, temastrox, and everolimus.Other known anticancer drugs that can be used in combination therapy include tamoxifen, letrozole, fulvestrant, mitoxantridine, octreotide, retinoid, arsenic, zoledronic acid, bortezomib, carfilzomib, Ixazomib, vemodega, sondega, denosumab, thalidomide, lenalidomide, venetoclax, Aldesleukin (recombinant human interleukin-2), and Sipueucel-T (prostate cancer treatment vaccine).

[0082] Furthermore, in any embodiment of any method and use disclosed herein, or after administration of one or more additional anticancer therapies or drugs, or in combination with one or more additional anticancer therapies or drugs, said one or more additional anticancer therapies or drugs are selected from those disclosed herein, such as monoclonal antibodies, surgery, radiation therapy (e.g., ionizing radiation (IR), gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and whole-body radioisotopes), endocrine therapy, biological response modifiers (e.g., interferon, leukopenia), etc. Intercytokines and tumor necrosis factor (TNF), hyperthermia and cryotherapy, drugs to reduce any adverse reactions (such as antiemetics) and other approved chemotherapy drugs, spindle toxins (such as vincristine, vinorelbine, and paclitaxel), podophyllotoxins (such as etoposide, irinotecan, and vorponotecan), nitrosoureas (such as valerate and lomustine), inorganic ions (such as cisplatin and carboplatin), enzymes (such as asparaginase), hormones (such as tamoxifen, leuprorelin, flutamide, and medroxyprogesterone acetate), Gleevec™, doxorubicin, dexamethasone, and cyclophosphamide.

[0083] This disclosure also provides the use of PARP1 inhibitors in the preparation of medicaments for treating central nervous system tumors (e.g., brain tumors) in an individual. Central nervous system tumors may be selected from any of those disclosed herein. Brain tumors may be selected from any of those disclosed herein. In some embodiments, the PARP1 inhibitor is a compound of formula (I) as described herein, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. This disclosure also provides methods for treating central nervous system tumors, such as brain tumors, in subjects (e.g., humans, veterinarians, or zoo animals) of the disclosed formula (e.g., humans, veterinarians, or zoo animals). In some embodiments, the PARP1 inhibitor is a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, as described herein.

[0084] This invention also provides the use of the PARP1 inhibitor as described herein in the preparation of a medicament for treating a central nervous system tumor (e.g., a brain tumor) in a subject of need. This invention also provides the use of a PARP1 inhibitor or a pharmaceutical composition containing a PARP1 inhibitor in a method of treating a central nervous system tumor (e.g., a brain tumor) in a subject of need. This invention further provides a method of treating a central nervous system tumor, such as a brain tumor, in a subject of need, comprising administering to the subject an effective amount of a PARP1 inhibitor or a pharmaceutical composition containing the inhibitor. In some embodiments, the PARP1 inhibitor is a compound of formula (I) as described herein, or a hydrate thereof, an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof.

[0085] On the other hand, the present invention provides a method for inducing regression of a central nervous system tumor in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, tumor regression is induced after administration of the compound. In some embodiments, tumor regression is assessed 7 to 84 days after administration of the compound, for example, after any day between 7 and 84 days after administration of the compound, such as after 8 days, 9 days, etc., or at any time between any two days. In some embodiments, tumor regression is between 1% and 100%, between 5% and 100%, between 10% and 100%, between 25% and 100%, between 50% and 100%, between 75% and 100%, or any percentage value between 1% and 100%, such as 2%, 3%, 4%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, etc., or any range between any two such values. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, simultaneously with, or after administration of one or more additional anticancer therapies or drugs (such as the anticancer therapies or drugs disclosed herein). In some embodiments, the CNS tumor, such as a brain tumor, has a volume at time t that is less than the tumor volume on the first day of compound administration, and time t is day 7, day 84, or any day between day 7 and day 84. In some embodiments of the method for inducing central nervous system tumor regression in a subject, the central nervous system tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell carcinoma, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinoblastoma, schwannoma, primary central nervous system lymphoma, brainstem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and dysplastic neuroepithelial tumor. In some embodiments of the method for inducing central nervous system tumor regression in a subject, the central nervous system tumor is any central nervous system cancer disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumors disclosed herein. In some embodiments, the brain tumor is a high-grade (rapidly growing) tumor. In some implementations, brain tumors are low-grade (slow-growing) tumors.In some embodiments, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments of a method for inducing regression of a central nervous system tumor in a subject, the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, or its pharmaceutically acceptable salt, is administered in a pharmaceutically acceptable composition comprising the compound, its hydrate, its isotopically substituted derivative, or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, its pharmaceutically acceptable salt, or its pharmaceutically acceptable composition, is administered.

[0086] On one hand, the present invention provides a method for inducing an increase in central nervous system tumor regression in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, an increase in tumor regression is induced after administration of the compound. In some embodiments, the increase in tumor-induced regression is assessed 7 to 84 days after administration of the compound, for example, after any day between 7 and 84 days after administration of the compound, such as after 8 days, 9 days, etc., or at any time between any two days. In some embodiments, tumor regression is between 1% and 100%, between 5% and 100%, between 10% and 100%, between 25% and 100%, between 50% and 100%, between 75% and 100%, or any percentage value between 1% and 100%, such as 2%, 3%, 4%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, etc., or any range between any two such values. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, simultaneously with, or after the administration of one or more additional anticancer therapies or drugs (such as the anticancer therapies or drugs disclosed herein). In some embodiments of the method for inducing an increase in central nervous system tumor regression in a subject, the central nervous system tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell tumor, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinoblastoma, schwannoma, primary central nervous system lymphoma, brainstem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and dysplastic neuroepithelial tumor. In some embodiments of the method for inducing an increase in central nervous system tumor regression in a subject, the central nervous system tumor is any central nervous system cancer disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumors disclosed herein. In some embodiments, the brain tumor is a high-grade (rapidly growing) tumor. In some implementations, the brain tumor is a low-grade (slow-growing) tumor. In some implementations, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma).In some embodiments of a method for inducing an increase in the regression of central nervous system tumors in a subject, the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, or its pharmaceutically acceptable salt, is administered in a pharmaceutically acceptable composition comprising the compound, its hydrate, its isotopically substituted derivative, or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, its pharmaceutically acceptable salt, or its pharmaceutically acceptable pharmaceutical composition, is administered.

[0087] On one hand, the present invention provides a method for inhibiting the growth of a central nervous system tumor in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, tumor growth is inhibited after administration of the compound. In some embodiments, the inhibition of central nervous system tumor growth is assessed 7 to 84 days after administration of the compound, for example, after any one day between 7 and 84 days after administration of the compound, for example, after 8 days, 9 days, etc., or at any time interval between any two days. In some embodiments, tumor growth is inhibited by 10% to 150%, for example, 30% to 150% or 50% to 120%, for example, any integer value between 10% and 150%, for example, 30%, 40%, 50%, 60%, 70%, 100%, 125%, 150%, etc., or in any range between any two such values. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, concurrently with, or after the administration of one or more additional anticancer therapies or drugs (such as those disclosed herein). In some embodiments of the method for inhibiting the growth of a central nervous system tumor in a subject, the central nervous system tumor is selected from meningiomas, meningeal sarcomas, ependymomas, astrocytomas, gliomas, glioblastomas, pineal cell tumors, invasive pituitary adenomas, pituitary carcinomas, germ cell tumors, sarcomas, craniopharyngiomas, retinoblastomas, schwannomas, primary central nervous system lymphomas, brainstem gliomas, pituitary adenomas, anaplastic astrocytomas, mixed gliomas, primitive neuroectodermal tumors, hemangioblastomas, vestibular schwannomas, chordomas, spinal neurofibromas, lymphomas, optic gliomas, and dysplastic neuroepithelial tumors. In some embodiments of the method for inhibiting the growth of a central nervous system tumor in a subject, the central nervous system tumor is any central nervous system cancer disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (rapid-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma).In some embodiments of a method for inhibiting the growth of central nervous system tumors in a subject, the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition comprising the compound, its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

[0088] On one hand, the present invention provides a method for inhibiting PARP1 activity, such as selectively inhibiting PARP1 activity relative to PARP2 activity in a subject, comprising administering to the subject an effective amount of a compound of formula (I), or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need of such treatment. In some embodiments, after administration of the compound, the inhibition of PARP1 activity in the subject is greater than the inhibition of PARP2 activity. In some embodiments, the ratio of PARP1 activity inhibition to PARP2 activity inhibition in the subject is at least 50, at least 750, at least 1500, at least 2500, at least 5000, at least 7500, at least 10000, at least 15000, at least 20000, at least 25000, or within any two of these numbers. In some embodiments, the ratio of PARP1 activity inhibition to PARP2 activity inhibition in the subject is between 50 and 2000, between 750 and 15000, between 2500 and 15000, or between 12500 and 25000, for example, between 15000 and 25000. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, simultaneously with, or after administration of one or more additional anticancer therapies or drugs (such as the anticancer therapies or drugs disclosed herein). In some embodiments, the compound, such as a compound of formula (I), or its hydrate, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition comprising the compound, or its hydrate, or its isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of a compound, such as a compound of formula (I), or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt, or its pharmaceutically acceptable composition, is administered.

[0089] On one hand, the present invention provides a method for treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject suffers from a disease or condition characterized by or caused by excessive cell death. In some embodiments, the disease or condition is a stroke or a neurodegenerative disease.

[0090] On the other hand, the present invention provides a method for treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the cancer is ovarian cancer, breast cancer, pancreatic cancer, or prostate cancer. In some embodiments, the cancer has a DNA damage repair defect, such as a homologous recombination defect. In some embodiments, the subject has a central nervous system (CNS) tumor. In some embodiments, the subject has a brain tumor. In some embodiments, the subject is a human or a non-human animal, such as a veterinary animal or a zoo animal, as disclosed herein. In some embodiments, the compound is administered before, simultaneously with, or after administration of one or more additional anticancer therapies or drugs (such as the anticancer therapies or drugs disclosed herein). In some embodiments, the subject has a central nervous system (CNS) tumor. In some embodiments, the CNS tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell carcinoma, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, glioblastoma multiforme, medulloblastoma, astrocytoma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and embryogenetic neuroepithelial tumor. In some embodiments, the central nervous system tumor is any central nervous system tumor disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumors disclosed herein. In some embodiments, the brain tumor is a high-grade (rapid-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments, the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition comprising the compound, its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In some embodiments, a therapeutically effective amount of the compound, such as a compound of formula (I), its hydrate, its isotopically substituted derivative, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

[0091] On one hand, the present invention provides the use of compounds of formula (I) disclosed herein (e.g., an effective amount of the compound), or hydrates thereof, or isotopically substituted derivatives thereof, or pharmaceutically acceptable salts thereof, in the preparation of medicaments. On the other hand, the present invention provides the use of compounds of formula (I), or hydrates thereof, or isotopically substituted derivatives thereof, or pharmaceutically acceptable salts thereof, in the preparation of medicaments for treating any condition such as cancer, such as any CNS tumor, for example, brain tumors as disclosed or described herein.

[0092] On the one hand, the present invention provides the use of the compound of formula (I) disclosed herein (e.g., an effective amount of the compound), or its hydrate, or its isotopically substituted derivative or its pharmaceutically acceptable salt, for the treatment of the conditions disclosed herein or for the purposes disclosed herein.

[0093] On the one hand, the present invention provides a method for treating the conditions disclosed herein or for the purposes disclosed herein, using a compound of formula (I) (e.g., an effective amount of the compound), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof.

[0094] On one hand, this invention provides a method for treating central nervous system (CNS) diseases or conditions (e.g., CNS tumors, such as brain tumors), comprising administering a compound of formula (I) disclosed herein, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, wherein the R group is as defined herein, and wherein the compound of formula (I) can penetrate the blood-brain barrier (BBB). In some embodiments, the compound of formula (I) penetrates the blood-brain barrier (BBB). In some embodiments, the CNS disease or condition is a brain tumor. In some embodiments, the central nervous system (CNS) disease or condition is a CNS tumor. In some embodiments, the central nervous system (CNS) disease or condition is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell carcinoma, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, glioblastoma multiforme, medulloblastoma, primary central nervous system lymphoma, brainstem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic nerve glioma, and dysplastic neuroepithelial tumor. In some embodiments, the central nervous system tumor is any central nervous system tumor disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as the brain tumors disclosed herein. In some embodiments, the brain tumor is a high-grade (rapid-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a glioma, an embryonal brain tumor (e.g., medulloblastoma), an ependymoma, a glioblastoma, a primary central nervous system lymphoma, a pineal region tumor (e.g., a germ cell tumor or pineal cell tumor), a pituitary adenoma, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments, a therapeutically effective amount of the compound, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt is administered. In some embodiments, a therapeutically effective amount of said compound, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt is administered in a pharmaceutically acceptable composition comprising said compound, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt, and one or more pharmaceutically acceptable diluents, carriers, or excipients.

[0095] On one hand, the present invention provides a pharmaceutically acceptable composition comprising a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable diluent, carrier, or excipient.

[0096] In some embodiments of the method disclosed herein, the method includes administering a therapeutically effective amount of a compound of formula (I) as described herein, its hydrate, its isotopically substituted derivative, or its pharmaceutically acceptable salt. In various embodiments, the pharmaceutically acceptable salt is an inorganic or organic acid salt, such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate, or oxalate. In various embodiments, the pharmaceutically acceptable salt is an inorganic or organic base salt formed with a base, such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, tromethamine), or N-methylglucosamine salt.

[0097] In any embodiment of any method and use disclosed herein, the compound of formula (I), its hydrate, its isotopically substituted derivative, or its pharmaceutically acceptable salt is administered before, simultaneously with, or in combination with one or more additional anticancer therapies or drugs, wherein the one or more additional anticancer therapies or drugs are selected from, for example, monoclonal antibodies, surgery, radiation therapy (such as ionizing radiation (IR), gamma radiation, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and whole-body radioisotopes), endocrine therapy, etc. Treatments, biological response modifiers (such as interferon, interleukin, and tumor necrosis factor), hyperthermia and cryotherapy, drugs to reduce any adverse reactions (such as antiemetics) and other approved chemotherapy drugs, spindle toxins (such as vincristine, vinorelbine, and paclitaxel), podophyllotoxins (such as etoposide, irinotecan, and vorponotecan), nitrosoureas (such as vastatin and lomustine), inorganic ions (such as cisplatin and carboplatin), enzymes (such as asparaginase), hormones (such as tamoxifen, leucine, flutamide, and medroxyprogesterone acetate), Gleevec™, doxorubicin, dexamethasone, and cyclophosphamide.

[0098] In any of the treatments disclosed herein, in some embodiments, the subject is a person in need of such treatment. In various embodiments, the subject is a human being. In some embodiments, the subject is a non-human being, such as a veterinary animal (e.g., a pig, cow, sheep, horse, dog, and cat) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

[0099] In any use of the compounds disclosed herein in treating a subject's condition, in some embodiments, the subject is a subject in need of such treatment. In various embodiments, the subject is a human being. In some embodiments, the subject is a non-human being, such as a veterinary animal (e.g., a pig, cow, sheep, horse, dog, or cat) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

[0100] In any use of the compounds disclosed herein in the preparation of a medicament for treating a subject or for treating a condition of a subject, in some embodiments, the subject is a subject in need of such treatment. In various embodiments, the subject is a human being. In some embodiments, the subject is a non-human being, such as a veterinary animal (e.g., a pig, cow, sheep, horse, dog, and cat) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

[0101] The following examples are illustrative and not intended to limit the methods and formulations of the present invention. Other appropriate modifications and improvements to various conditions and parameters that will be apparent to those skilled in the art and that are commonly encountered in clinical treatment are all within the spirit and scope of the present invention.

[0102] Example

[0103] All reagents used were commercially available, and all solvents were dried and purified according to standard methods. Mass spectrometry samples were analyzed using a single quadrupole mass spectrometer with electrospray ionization. Recording was performed using an NMR spectrometer at 400 MHz. 1 1H NMR spectra, chemical shifts recorded in ppm starting from low field with TMS as internal standard (0.00 ppm), coupling constant. J The value is in Hz.

[0104] Example 1

[0105] 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one

[0106] a) Preparation of 4-methyl-1H-pyrazole-5-carbonyl chloride: 4-methyl-1H-pyrazole-5-carboxylic acid (2.0 g, 15.9 mmol) was dissolved in SOCl2 (20 mL) and stirred at 80 °C for 16 hours under nitrogen protection. The target product (2.2 g, off-white solid, yield 91.7%) was obtained by concentration under reduced pressure.

[0107] b) Preparation of N-(3-bromo-2,6-difluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide: 3-bromo-2,6-difluoroaniline (2.5 g, 16.7 mmol) was dissolved in THF (20 mL), and NaH (60% mineral oil, 2.776 g, 69.4 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 30 minutes under nitrogen protection. Then, 4-methyl-1H-pyrazole-5-carbonyl chloride (2.0 g, 13.9 mmol) was added at 0 °C. After the reaction was complete, the reaction solution was slowly poured into ice water (100 mL) and extracted with ethyl acetate (200 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 2), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (PE:EA = 10:1) to obtain the target product (2.7 g, gray solid, yield: 67.0%). MS (ESI): 315.90 [M+H) + .

[0108] c) Preparation of 7-bromo-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one: K₂CO₃ (2.4 g, 17.2 mmol) was added to a DMSO (27 mL) solution of N-(3-bromo-2,6-difluorophenyl)-4-methyl-1H-pyrazol-5-carboxamide (2.7 g, 8.6 mmol), and the mixture was stirred overnight at 120 °C under nitrogen protection. After the reaction was complete, the reaction solution was poured into ice-salt water, and the resulting solid was filtered to obtain the target product (2.5 g, white solid, yield: 86.8%). MS(ESI): 293.95 [M⁻¹] - .

[0109] d) Preparation of 6-fluoro-7-(hydroxymethyl)-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one: At room temperature, (Bu)3SnCH2OH (816.3 mg, 2.55 mmol) and Xphos Pd G2 (133.2 mg, 0.2 mmol) were added to a solution of 7-bromo-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one (500.0 mg, 1.7 mmol) in dioxane (10 mL). The resulting mixture was then heated under nitrogen protection at 90 °C. oThe mixture was stirred at C for 3 hours. After the reaction was complete, KF aqueous solution (1 M, 20 mL) was added at room temperature. The mixture was stirred at room temperature for 10 minutes, filtered, and the filtrate was extracted with EA (500 mL × 3). The collected organic phase was washed with saturated brine (100 mL × 2), dried over anhydrous Na2SO4, concentrated under reduced pressure to remove the solvent, and the crude product was slurried with a mixed solvent of PE and EA (V / V = 1:1). The solid was collected by filtration and dried to obtain the crude target product (500 mg, gray solid). MS (ESI): 248.05 [M+1] + , 246.05 [M-1] - .

[0110] e) Preparation of 7-(bromomethyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one: A solution of 6-fluoro-7-(hydroxymethyl)-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one (500.0 mg, 2.0 mmol) in HBr (48% aqueous solution, 5 mL) was heated at 80 °C. o The mixture was stirred at C for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain the crude target product (500 mg, white solid, yield: 79.9%). MS (ESI): 310.90 [M+1] + 308.85 [M-1] - .

[0111] f) Preparation of 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one: 7-(bromomethyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one (200.0 mg, 0.67 mmol), 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide (182.2 mg, 0.8 mmol), KI (9.1 mg, 0.07 mmol), and DIEA (332.1 mg, 2.0 mmol) were dissolved in acetonitrile (5 mL). The resulting mixture was stirred at 80 °C for 1 hour. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The crude product was purified by preparative thin-layer chromatography (DCM:MeOH = 10:1) to obtain the target compound (19.8 mg, white solid, yield: 6.6%).

[0112]

[0113] Examples 2-5

[0114] The compounds in Examples 2-5 were prepared using a synthetic method similar to that in Example 1 (Reaction Scheme 1). The results are shown below.

[0115]

[0116] Example 6

[0117] 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one

[0118] a) Preparation of 4-methyl-1H-pyrazole-5-carbonyl chloride: A solution of 4-fluoro-1H-pyrazole-5-carboxylic acid (30 g, 230 mmol) in SOCl2 (300 mL) was stirred at 80 °C for 2 hours. The mixture was concentrated under reduced pressure to give the target product (22.6 g, white solid, yield: 66%).

[0119] b) Preparation of N-(3,5-dibromo-2,6-difluorophenyl)-4-fluoro-1H-pyrazole-5-carboxamide: Under nitrogen atmosphere at 0 °C, sodium bis(trimethylsilyl)amino (NaHMDS, 2M tetrahydrofuran solution, 295.0 mL, 589.6 mmol) was added to a THF (100 mL) solution of 3,5-dibromo-2,6-fluoroaniline (81.2 g, 283.0 mmol). The mixture was stirred at 0 °C for 30 minutes. 4-fluoro-1H-pyrazole-5-carbonyl chloride (35.0 g, 235.9 mmol) was slowly added under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 30 minutes, heated to room temperature, and stirred for 1.5 hours. The mixture was quenched with water (500 mL) and treated with EA (500 mL). 3) Extraction. The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain a crude product, washed twice with PE (1200 mL) and twice with DCM (1200 mL) to obtain the target compound (105.0 g, yellow solid, yield: 93%). MS (ESI): 399.80 [M+H) + .

[0120] c) Preparation of 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one: Under nitrogen atmosphere, K₂CO₃ (83.2 g, 603.1 mmol) was added to a solution of N-(3,5-dibromo-2,6-difluorophenyl)-4-fluoro-1H-pyrazol-5-carboxamide (60.15 g, 150.8 mmol) in DMSO (600 mL). The mixture was stirred overnight at 100 °C. The mixture was cooled to room temperature. Water (600 mL) was added, and the resulting mixture was filtered. The solid was washed with water (100 mL). 3). The crude product was dried under reduced pressure to obtain the target product (56.0 g, yellow solid, yield: 98%). MS (ESI): 377.75 [MH] - .

[0121] d) Preparation of 7-bromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one: Sodium ascorbate (18.4 g, 92.9 mmol), CuI (8.8 g, 46.4 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 17.7 g, 116.1 mmol) were added to a solution of 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one (44.0 g, 116.1 mmol) in DMF (420 mL) and H2O (70 mL) under nitrogen atmosphere. The mixture was stirred overnight at 120 °C. The mixture was then cooled to room temperature. Add water (600 mL), filter the resulting mixture, wash the solid three times with (DCM / MeOH = 15 / 1, 200 mL), and concentrate under reduced pressure. Extract the crude product with EA (2000 mL × 3), collect the organic phase and concentrate to give the target product (17 g, yellow solid, yield: 49%). MS (ESI): 299.90 [M+H) + .

[0122] e) Preparation of 3,6-difluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxaline-4(5H)-one: Under nitrogen atmosphere, Xphos Pd G2 (5.8 g, 7.4 mmol) and (tributyltin)methanol (47.6 g, 148.1 mmol) were added to a solution of 7-bromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one (22.22 g, 74.1 mmol) in dry dioxane (230 mL). The mixture was stirred at 80 °C for 16 hours. The mixture was cooled to room temperature and KF aqueous solution (1 M, 250 mL) was added. The resulting mixture was filtered, and the filtrate was extracted with EA (200 mL × 3). The organic phase was collected and concentrated under reduced pressure. The solid was washed with DMF (100 mL × 3) and concentrated under reduced pressure. The crude product was washed with a mixed solvent of PE and EA (PE / EA = 3 / 1, 100 mL × 3) and a mixed solvent of DCM and methanol (DCM / MeOH = 15 / 1, 50 mL × 2). The crude product was dried to obtain the target product (14.7 g, white solid, yield: 79%). MS (ESI): 252.05 [M+H] + .

[0123] f) Preparation of 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one: A solution of 14.2 g (56.5 mmol) of 3,6-difluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxaline-4(5H)-one in HBr (48% aqueous solution, 200 mL) was stirred overnight at 80 °C under nitrogen. The mixture was concentrated under reduced pressure to give the target product (16.6 g, white solid, yield: 93%). MS (ESI): 313.90 [M+H) + .

[0124] Preparation of 7-((4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one: Under nitrogen protection at room temperature, KI (875.9 mg, 5.3 mmol), 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridineamide hydrochloride (15.4 g, 52.8 mmol) and DIEA (61.3 g, 474.9 mmol) were added to a solution of 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one (16.6 g, 52.8 mmol) in acetonitrile (200 mL). The mixture was stirred at 80 °C for 4 hours. After the reaction was complete, the solvent was removed under reduced pressure, DMF was added to the residue, the resulting mixture was stirred at 45°C for 15 minutes, cooled to room temperature and filtered to obtain the target compound (8.3 g, white solid).

[0125]

[0126] Examples 7-11

[0127] The compounds in Examples 7-11 were prepared using a synthetic method similar to that in Example 1 (Reaction Scheme 1). The results are shown below.

[0128]

[0129] Example 12

[0130] Chemiluminescence detection of PARP1 and PARP2

[0131] Diluent containing recombinant poly(ADP-ribotransferases) 1 and 2 (PARP1 and PARP2) (40 ng enzyme / well) and the test compound were added to 96-well plates coated with recombinant proteins and incubated at room temperature for 1 hour. Then, 50 μL of 0.3 ng / mL horseradish peroxidase streptavidin (HRP) was added to each well, and the plates were incubated at room temperature for 30 minutes. Finally, ELISA ECL substrate was added, and the plates were read using an EnviSion instrument. The chemiluminescence signal was recorded, and the inhibition rate of the test compound on PAPP1 and PARP2 enzyme activities was calculated using the following formula.

[0132]

[0133] IC was obtained by fitting the S-type dose-response curve equation using XL Fit software. 50 Value. The equation of the curve is Y = 100 / (1 + 10^(logC - logIC)). 50)), where C is the concentration of the compound.

[0134] Table 1 summarizes the inhibitory effects (IC50) of the compounds of this invention on PARP1 and PARP2 enzyme activities. 50 ).

[0135] Table 1

[0136] Compared to PARP2 enzyme, the compounds of this invention exhibit good selective inhibition of PARP1 enzyme activity.

[0137] Example 13

[0138] The inhibitory effect of the compounds of this invention on the growth of BRCA-mutated human breast cancer cells MDA-MB-436.

[0139] After cell resuscitation, cells were cultured and passaged in complete medium (DMEM + 10% FBS + insulin + glutathione). When cell confluence reached approximately 80%, cells were gently aspirated from the bottom of the culture dish using a 1 mL pipette, and the cell suspension was collected and centrifuged at 500 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in complete medium. Cells were seeded at an appropriate ratio into culture dishes and incubated statically at 37°C with 5% CO2. Cells were passaged until they reached good growth and approximately 80% confluence and were then used in experiments. Cells in the logarithmic growth phase were centrifuged, and the supernatant was removed. Cells were resuspended in fresh complete medium and counted. The resuspended cells were seeded at 3000 cells / well in 96-well plates and incubated overnight at 37°C with 5% CO2. The compound was prepared by adding 5 μL of 1000... The test compound solution (25-fold dilution) was prepared to a concentration of 40 μL. Solution of the test compound. The solution was mixed by shaking. 0.1% dimethyl sulfoxide was used as a control.

[0140] The following day, the 96-well plate inoculated with cells was removed from the incubator, and the culture supernatant was discarded. Then, 195 μL of fresh culture medium was added to each well, followed by 5 μL of the aforementioned 40% culture medium to the corresponding well. The test compound solution was prepared, and the culture plate was then placed in a 37℃, 5% CO2 incubator for 7 days. On the fourth day, the medium containing the compound was replaced. After 7 days, 20 μL of CCK-8 was added to each well, and the plate was shaken and incubated for another 5 minutes after each incubation. The absorbance values ​​(OD value = absorbance) were then read at 450 nm or 650 nm wavelengths using a multi-function reader. 450nm -Absorbance 650nm ).

[0141] Data were analyzed using GraphPad Prism 6.0 software. The inhibitory activity of the compound on cell proliferation was plotted on a coordinate system of cell viability and compound concentration. Cell viability % = (OD) / (Cell viability % = 0.05%) ... 化合物 -OD 背景 ) / (OD DMSO -OD 背景 ) × 100. IC 50 The value was fitted with an S-shaped dose-response curve equation, which is: Y = 100 / (1 + 10^(LogC - LogIC)) 50 C is the concentration of the compound.

[0142] Table 2 summarizes the data on the inhibitory effects of the compounds on the growth of human breast cancer cells MDA-MB-436 (IC50, 100%). 50 ).

[0143] Table 2

[0144] The compound of this invention has a good inhibitory effect on the proliferation of BRCA-mutated human breast cancer cells MDA-MB-436.

[0145] Example 14

[0146] Pharmacokinetic studies of compounds in mouse plasma and brain after a single oral administration

[0147] The compounds of the present invention were formulated into a homogeneous suspension of 0.5% methylcellulose / water and administered to CD-1 (ICR) mice by gavage at a dose of 10 mg / kg. Mice were euthanized by CO2 inhalation at 0.5, 4, and 8 hours post-administration, and blood and brain samples were collected from each mouse. Approximately 0.1 mL of blood was collected from the saphenous vein or other suitable site from each mouse and placed in pre-chilled EDTA-K2 tubes. After collection, the brain was washed with cold saline, dried, and weighed. The brain was homogenized using homogenization buffer (15 mM PBS (pH 7.4): MeOH = 2:1) at a ratio of 1:4 (1 g tissue to 4 mL buffer, dilution ratio 5). The tissue homogenate was maintained at -60°C or lower prior to LC-MS / MS analysis. The concentrations of the compounds in plasma and brain were determined by LC-MS / MS.

[0148] Compound concentration, calculated AUC 0-8h The B / P ratios are summarized in Table 3.

[0149] Table 3

[0150] Note: The data on the relationship between compound concentration and time were analyzed using WinNonlin software via a non-compartmental method, and the AUC was calculated using the linear logarithmic trapezoidal method. 0-8h .

[0151] The compounds of this invention have good exposure levels in the brains of mice.

[0152] Example 15

[0153] K of compounds in mice p,uu

[0154] The ratio of unbound drugs in the brain to unbound drugs in plasma (K) was determined using the following method. p,uu ) to be measured.

[0155] Determination of free components in mouse plasma

[0156] On the day of the experiment, mouse plasma was thawed under running cold tap water and centrifuged at 3220×g for 5 minutes to remove any clots. The pH value was checked and recorded. Only plasma within the pH range of 7.0 to 8.0 was used.

[0157] The test compound or positive control warfarin was incorporated into frozen mouse plasma at a final concentration of 2 μM. 150 µL of the compound-spikeped plasma sample was aliquoted into one side of a 96-well balanced dialysis plate (HTD dialysis), and an equal volume of dialysis buffer was added to the other side. Aliquots of the plasma sample were collected before incubation and used as T0 samples for recovery calculation. Three incubations were performed. The plate was then incubated at 37°C in a humidified incubator containing 5% CO2 for 4 hours. After incubation, 50 µL of sample was collected from both the plasma and buffer sides. The plasma sample was mixed with an equal volume of blank buffer; the buffer sample was mixed with an equal volume of blank plasma. The matrix-matched samples were quenched with a stop solution containing an internal standard (IS). The samples were analyzed by LC-MS / MS. In the absence of a standard curve, the concentration of the test compound in the plasma and buffer samples was determined based on the peak area ratio of the analyte to the IS.

[0158] Determination of free components in mouse brain homogenate

[0159] On the day of the experiment, CD-1 mouse brain homogenates were thawed in a water bath at room temperature and incubated at 37°C for 10 minutes before use. The experimental compound or the control compound propranolol was added to the blank brain homogenate to a final concentration of 2 µM.

[0160] Aliquots of 100 µL of the compound-infused brain homogenate were added to one side of a 96-well equilibrium dialysis plate (HTD dialysis), and an equal volume of dialysis buffer (100 mM sodium phosphate and 150 mM NaCl, 7.4 ± 0.1) was added to the other side of the plate. The aliquots of the brain homogenate were harvested before incubation and used as T0 samples for recovery calculation. Three incubations were performed.

[0161] The brain homogenate samples were then rotated at approximately 100 rpm at 37°C and 5% CO2 for 4 hours in a humidified incubator. After incubation, 50 µL of sample was taken from both the brain homogenate side and the buffer side. The brain homogenate sample was mixed with an equal volume of buffer; the buffer sample was then mixed with an equal volume of blank brain homogenate. The matrix-matched samples were quenched with a stop solution containing an internal standard.

[0162] The samples were analyzed using LC-MS / MS. The concentrations of compounds in the brain homogenate and buffer samples were determined based on the peak area ratio of the analyte to the standard index (IS), and no standard curve was provided.

[0163] K p,uu The value is calculated using the following formula: AUC 脑 and AUC 血浆 AUC in plasma and brain obtained in Example 14, respectively 0-8h ;f u,血浆 and f u,脑 These figures represent the percentage of unbound compounds in plasma and brain, respectively. The K values ​​of these compounds in mice... p,uu The values ​​are summarized in Table 4.

[0164] Table 4

[0165] The tested compounds have good K p,uu .

[0166] Example 16

[0167] Pharmacokinetic studies of compounds administered orally in mice after a single oral dose

[0168] The example compounds of the present invention were formulated into a homogeneous suspension of 0.5% methylcellulose / water and administered to CD-1 (ICR) mice by gavage at a dose of 10 mg / kg. Plasma samples were collected at eight time points: 0.250, 0.500, 1.00, 2.00, 4.00, 6.00, 8.00, and 24.0 hours post-administration. Approximately 0.1 mL of blood was collected from the saphenous vein or other suitable site of each mouse at each time point and placed in pre-chilled EDTA-K2 tubes. The concentrations of the compounds were determined by LC-MS / MS.

[0169] The obtained plasma concentration and time data were analyzed using a non-compartmental model with WinNonlin software, and pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. The mouse pharmacokinetic parameters of the compounds are shown in Table 5, where the half-life tm is... 1 / 2 Indicates the time required for half of the drug concentration to be eliminated; C max AUC represents the highest drug concentration achieved after administration. 0-t AUC represents the area under the plasma concentration-time curve from time 0 to the last time. 0-inf This represents the area under the plasma concentration-time curve from time 0 to infinity.

[0170] Table 5

[0171] The compound was found to have good exposure levels in mouse plasma.

[0172] Example 17

[0173] Antitumor efficacy study in a subcutaneous breast cancer model of MDA-MB-436 individuals

[0174] Human breast cancer MDA-MB-436 cells were purchased from ATCC (HTB-130). Cells were cultured in RPMI-1640 medium containing 10 µg / mL insulin, 16 µg / mL glutathione, and 10% fetal bovine serum at 37°C in a 5% CO2 incubator, and passaged two to three times per week. Cells in the exponential growth phase were collected, counted, and then seeded.

[0175] 0.2 mL (1×10 7 A suspension of tumor cells containing 50% artificial basement membrane was subcutaneously inoculated into the right posterior back of each mouse. When the average tumor volume reached ~150 mm... 3 Mice were randomly grouped and administered drugs at that time.

[0176] After administration to the groups, the animals were monitored daily. Body weight and tumor size were measured three times a week.

[0177] Regularly monitor animal weight as an indirect measure of toxicity.

[0178] Measure the tumor diameter using calipers, three times a week. Tumor volume (mm) 3 The formula for calculating ) is: , where a and b represent the long and short diameters of the tumor, respectively.

[0179] The tumor growth inhibition rate (TGI) was calculated using tumor size, and the antitumor efficacy of the compound was evaluated using the TGI (%). V给药组-1 and V 溶媒组-1 The mean tumor volume refers to the average tumor volume at the time of administration in the compound-treated group and the solvent-controlled group (i.e., on the first day of administration); V 给药组-t and V 溶媒组-t These refer to the average tumor volume after administration in the compound-treated group and the solvent-controlled group, respectively. When the TGI (%) value is greater than 100%, the tumor elimination rate (%) is calculated using the formula... The calculation yielded the result.

[0180] Research-1

[0181] The antitumor effects of compounds 1 and 6 (i.e., compounds of Examples 1 and 6) were evaluated in the MDA-MB-436 human breast cancer subcutaneous xenograft model.

[0182] Grouping and dosing regimens are shown in Table 6.

[0183] Table 6

[0184] Note: a. N is the number of animals per group, 8 tumor-bearing nude mice per group; b. The solvent is 0.5% MC aqueous solution; c. Adjust the dosage volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the dosing regimen; po refers to oral administration; e. QD refers to once a day; continue administration for 28 days. NA indicates not applicable.

[0185] result: 1) Mortality rate, morbidity rate, and weight gain or loss Throughout the study, no significant weight loss was observed, nor were any deaths or signs of illness detected.

[0186] 2) Tumor growth inhibition and tumor regression

[0187] Based on data from day 28, the tumor growth inhibition effects, such as tumor volume and TGI value, compared with the control group are shown in Table 7.

[0188] Table 7

[0189] Notes: a. Mean ± standard error; NA indicates that it is not applicable.

[0190] In summary, all treatment groups showed significant tumor growth inhibition compared to the solvent control group. Compound 1 exhibited a favorable dose-dependent relationship at both 0.2 mg / kg and 1 mg / kg. Furthermore, compound 6 demonstrated tumor elimination at both 0.2 mg / kg and 1 mg / kg doses.

[0191] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity, indicating that the compound was well tolerated in mice at the tested dose level.

[0192] Research-2

[0193] The antitumor effects of compounds 5 and 6 (i.e., compounds of Examples 5 and 6) were evaluated in the MDA-MB-436 human breast cancer subcutaneous xenograft model.

[0194] Grouping and dosing regimens are shown in Table 8.

[0195] Table 8

[0196] Note: a. N is the number of animals per group, 8 tumor-bearing nude mice per group; b. The solvent is 0.5% MC aqueous solution; c. Adjust the dosage volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the dosing regimen; po means oral administration; e. QD means once a day; continue administration for 28 days. NA means not applicable.

[0197] result: 1) Mortality rate, morbidity rate, and weight gain or loss Throughout the study, no significant weight loss was observed, nor were any deaths or signs of illness detected.

[0198] 2) Tumor growth inhibition and tumor regression

[0199] Based on data from day 28, the tumor growth inhibition effects, such as tumor volume and TGI value, compared with the control group are shown in Table 9.

[0200] Table 9

[0201] Notes: a. Mean ± standard error; NA indicates that it is not applicable.

[0202] In summary, all treatment groups showed significant inhibition of tumor growth compared to the solvent control group. Compounds 5 and 6 showed similar effects at a dose of 0.1 mg / kg. Compound 6 resulted in tumor regression at both 1 mg / kg and 10 mg / kg doses.

[0203] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity, indicating that compounds 5 and 6 were well tolerated in mice at the tested dose levels.

[0204] Example 18

[0205] DLD-1 BRCA2 - / - Antitumor efficacy study in human colorectal cancer epithelial-subcutaneous model

[0206] Evaluation of compound 6 in DLD-1 BRCA2 - / - Antitumor effects in a human colorectal cancer epithelial subcutaneous xenograft model.

[0207] The laboratory of Beijing Aisiyipu Biotechnology Co., Ltd. constructed DLD-1 BRCA2 by knocking out BRCA2 in DLD-1 wild-type cells (purchased from ATCC) using CRISPR / Cas9 technology. - / - cell.

[0208] DLD-1 BRCA2 - / - Cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 100 μg / mL Hygromycin B; cells were cultured in a 37°C, 5% CO2 incubator. Cells in the exponential growth phase were collected, counted, and then seeded.

[0209] 0.1 mL (1×10 5 A suspension of tumor cells containing 50% artificial basement membrane was subcutaneously inoculated into the right anterior shoulder and back of each mouse. When the average tumor volume reached ~175 mm... 3 Mice were randomly assigned to groups and administered drugs. Grouping and administration protocols are shown in Table 10.

[0210] Table 10

[0211] Note: a. N is the number of animals per group, 8 tumor-bearing nude mice per group; b. The solvent is 0.5% MC aqueous solution; c. Adjust the dosage volume according to the mouse's body weight at 10 μL / g; if the body weight decreases by more than 15%, adjust the dosing regimen; po refers to oral administration; e. QD refers to once a day; continue administration for 28 days. NA indicates not applicable.

[0212] After administration to the groups, the animals were monitored daily. Body weight and tumor size were measured twice a week.

[0213] Regularly monitor animal weight as an indirect measure of toxicity.

[0214] Measure the tumor diameter using calipers, three times a week. Tumor volume (mm) 3 The formula for calculating ) is: , where a and b represent the long and short diameters of the tumor, respectively.

[0215] The tumor growth inhibition rate (TGI) (%) was calculated using tumor size, and the antitumor efficacy of the compound was evaluated using the TGI (%). V 给药组-1 and V 溶媒组-1 The mean tumor volume refers to the average tumor volume at the time of administration in the compound-treated group and the solvent-controlled group (i.e., on the first day of administration); V 给药组-t and V 溶媒组-t These refer to the average tumor volume after administration in the compound-treated group and the solvent-controlled group, respectively. When the TGI (%) value is greater than 100%, the tumor elimination rate (%) is calculated using the formula... The calculation yielded the result.

[0216] result: 1) Mortality rate, morbidity rate, and weight gain or loss Throughout the study, no significant weight loss was observed, nor were any deaths or signs of illness detected.

[0217] 2) Tumor growth inhibition and tumor regression

[0218] Based on data from day 28, the tumor growth inhibition effects, such as tumor volume and TGI value, compared with the control group are shown in Table 11.

[0219] Table 11

[0220] Notes: a. Mean ± standard error; NA indicates that it is not applicable.

[0221] In summary, compared with the solvent control group, the compound 6 treatment group showed significant tumor growth inhibition, and exhibited good dose dependence at doses of 0.2 mg / kg and 1 mg / kg.

[0222] During the study, no significant weight loss was observed in any group, animal behavior was normal, and there were no signs of death or morbidity, indicating that these compounds were well tolerated in mice at the current dose levels.

[0223] Example 19

[0224] Antitumor efficacy study of MDA-MB-436-luc in an intracranial model of human breast cancer

[0225] MDA-MB-436-luc tumor cells were cultured in vitro in DMEM medium containing 10% fetal bovine serum and 1200 ng / mL puromycin at 37°C and 5% CO2. The medium was changed every 1 to 2 days. After the cells reached 80%-90% confluence, they were passaged using 0.25% trypsin-EDTA digestion, with a passage count not exceeding 4-5 times. Tumor cells in the logarithmic growth phase were then used for in vivo tumor seeding.

[0226] After anesthetizing the animal with a salbutamol injection, it was fixed prone on the operating table. The skin on the top of its head was disinfected with iodine and 75% alcohol. An incision of approximately 0.5 cm was made along the midline of the head to expose the coronal and sagittal lines. Using a brain locator, a position was established approximately 0.5-1.0 mm above the coronal line and approximately 2 mm to the right of the sagittal line. A hole was drilled using a 25-gauge syringe needle. A microsyringe needle was vertically inserted to a depth of 3 mm at this location, and 2 × 10⁶ MDA-MB-436-luc tumor cells were slowly injected (over approximately 1 minute). 5 / 2 μL suspension, leave the needle in place for 1 minute after injection, remove the needle and quickly seal the needle hole with medical bone wax and suture the wound.

[0227] After surgery, the analgesic meloxicam was administered subcutaneously at a dose of 5 mg / kg per animal. Levofloxacin (0.1 mg / mL) was added to the animals' drinking water and administered for 3-7 consecutive days.

[0228] Tumor growth is monitored through image analysis. When the average optical signal intensity (BLI, bioluminescent imaging) at the tumor site reaches 20–30 photons / s... 10 6 Mice were randomly grouped and drug administration was initiated at that time.

[0229] Imaging analysis of tumor development

[0230] Mice were intraperitoneally injected with D-luciferin (15 mg / mL, or 5 μL / g based on animal body weight). Once weekly, the animals were anesthetized by inhalation with 1%-2% isoflurane. Approximately 10 minutes after D-luciferin injection, the animals were imaged using IVISLumina III. The imaging results were analyzed using Living Image software (Perkin Elmer), and the optical signal intensity within each animal's whole-body region of interest (ROI) was calculated. The bioluminescence imaging signal (photons / s) from the ROI was quantified and used as an indicator of tumor growth and antitumor activity.

[0231] The reduction in bioluminescence imaging (BLI) signal was calculated using the following formula, expressed as a percentage: BLI 治疗组 With BLI 对照组 The values ​​are the average BLI values ​​of the treatment group and the control group on the day after tumor cell inoculation.

[0232] Research-1

[0233] The antitumor activity of compound 6 was evaluated in a human breast cancer intracranial xenograft model of MDA-MB-436-luc. Grouping and dosing regimens are shown in Table 12.

[0234] Table 12

[0235] Note: a. N is the number of animals per group, 8 tumor-bearing nude mice per group; b. The solvent is a 0.5% MC aqueous solution; c. The dosage volume is adjusted according to the mouse's body weight at 10 μL / g; if 1) the animal's body weight decreases significantly (emaciation); the body weight decreases significantly by >20% and does not recover after drug withdrawal; 2) the animal cannot obtain sufficient food and water, then the sick animal shall be euthanized. dpo refers to oral administration; e. QD refers to once a day; the solvent control group and the treatment group were administered the drugs continuously for 39 days and 42 days, respectively. NA indicates not applicable.

[0236] result: 1) Mortality rate, morbidity rate, and weight gain or loss Mice in the solvent control group showed significant weight loss and death on day 34 after tumor cell inoculation, which is likely due to intracranial tumor burden.

[0237] During the 42-day treatment with compound 6, no significant weight loss, death, or any signs of illness were observed.

[0238] 2) Tumor growth inhibition

[0239] Based on data from day 42, compared with the control group, antitumor activity data such as tumor BLI value and BLI reduction percentage are shown in Table 13.

[0240] Table 13

[0241] Notes: a. Mean ± standard error; b. Compared to the control group (i.e., the solvent group).

[0242] NA indicates that it is not applicable.

[0243] In summary, all treatment groups showed significant tumor growth inhibition compared to the solvent group. Compound 6 was well tolerated in tumor-bearing mice at the experimental dose level.

[0244] Research-2

[0245] The antitumor effects of compounds 5 and 6 were evaluated in an MDA-MB-436-luc human breast cancer intracranial xenograft model. Grouping and dosing regimens are shown in Table 14.

[0246] Table 14

[0247] Note: a. N is the number of animals per group, 8 tumor-bearing nude mice per group; b. The solvent is a 0.5% MC aqueous solution; c. Adjust the dosage volume according to the mouse's body weight at 10 μL / g; if 1) the animal's body weight decreases significantly (emaciation); the body weight decreases significantly by >20% and does not recover after drug withdrawal; 2) the animal cannot obtain sufficient food and water, then the sick animal should be euthanized. dpo refers to oral administration; e. QD refers to once a day; continuous administration for 28 days. NA indicates not applicable.

[0248] result: 1) Mortality rate, morbidity rate, and weight gain or loss One mouse in the solvent group experienced significant weight loss (>20%), and two mice on days 24 and 28 also experienced significant weight loss. These three mice were euthanized.

[0249] One mouse in group 3 (treatment group with compound 6 at a dose of 0.2 mg / kg) died. Autopsy revealed severe food accumulation in the intestines and no brain tumors.

[0250] No significant weight loss was observed in groups 2, 4, and 5, nor were any deaths or signs of illness detected.

[0251] 2) Tumor growth inhibition

[0252] Based on data from day 28, compared with the control group, antitumor activity data such as tumor BLI value and BLI reduction (%) are shown in Table 15.

[0253] Table 15

[0254] Notes: a. Mean ± standard error; b. Compared to the control group (i.e., the solvent group).

[0255] NA indicates that it is not applicable.

[0256] In summary, all treatment groups showed significant inhibition of tumor growth compared to the solvent group.

[0257] Example 20

[0258] Brain penetration in SD rats after oral administration of radiolabeled compound 6

[0259] Male and female Sprague Dawley rats were administered the medication by gavage at a dose of 5 mg / 100 µCi / kg. 14 The distribution of radiolabeled compound 6 in the blood, plasma, and tissues of Sprague Dawley rats was measured at 1, 6, 12, and 24 hours after administration. Total radioactivity in plasma and whole brain is shown in the table below.

[0260] Table 16

[0261] The results showed that compound 6 had good brain penetration.

[0262] Example 21

[0263] Pharmacokinetic studies of compound 6 in rat plasma and brain after a single oral administration

[0264] Compound 6 was formulated into a homogeneous suspension of 0.5% methylcellulose / water and administered to SD rats by gavage at a dose of 5 mg / kg. Blood samples (approximately 0.2 mL at each time point) were collected from the jugular vein or other suitable site of each rat and placed in pre-chilled EDTA-K2 tubes. Rats were euthanized by CO2 inhalation at 1, 4, and 8 hours post-administration, followed by perfusion with physiological saline, and brains were collected from each rat. After collection, the brains were washed with cold saline, dried, and weighed. The brains were homogenized using homogenization buffer (15 mM PBS (pH 7.4): MeOH = 2:1) at a ratio of 1:4 (1 g tissue to 4 mL buffer, dilution ratio 5). The tissue homogenate was maintained at -60°C. oTemperature C or lower until LC-MS / MS analysis. The concentration of compound 6 in plasma and brain was determined by LC-MS / MS.

[0265] Concentration of compound 6, calculated AUC 0-8h The B / P ratios are summarized in the table below.

[0266] Table 17

[0267] Note: The data on the relationship between compound concentration and time were analyzed using WinNonlin software via a non-compartmental method, and the AUC was calculated using the linear logarithmic trapezoidal method. 0-8h .

[0268] The results showed that compound 6 had good exposure levels in the rat brain.

[0269] While the invention has been fully described, those skilled in the art will understand that the same practices can be carried out under broad and equivalent conditions, formulations, and other parameters without affecting the scope of the invention or any embodiments thereof. All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

Claims

1. A compound having the structure of formula (I) or its hydrate or isotopically substituted derivative or its pharmaceutically acceptable salt: （I） in, R1 is selected from halogens, alkyl groups, and haloalkyl groups; R2 is selected from halogens and alkyl groups; R3 is selected from halogens, alkyl groups, and cyano groups; R4 is selected from alkyl groups, haloalkyl groups, and cycloalkyl groups.

2. The compound of claim 1, wherein: (a) R1 is a halogen, C 1-3 Alkyl or halogenated C 1-3 Alkyl, or (b) R2 is a halogen or C 1-3 Alkyl group, or (c)R3 is a halogen, C 1-3 Alkyl or cyano, or (d)R4 is C 1-3 Alkyl, Halogenated C 1-3 Alkyl or C 3-4 Cycloalkyl, or any combination of (a) to (d), such as all of (a) to (d).

3. The compound of claim 1, wherein: (e) R1 is F, methyl, or trifluoromethyl; or (f) R2 is F, Cl, or methyl; or (g) R3 is F, methyl, or cyano; or (h) R4 is C. 1-3 Alkyl, C 1-3 Halogenated alkyl or C 3-4 Cycloalkyl, or any combination of (e) to (h), such as all of (e) to (h).

4. The compound of claim 1, wherein all R1, R2, R3, and R4 of the compound are defined by any one of the rows in rows 1-11 of the following table: 。 5. The compound of claim 2, wherein: (a) R1 is CH3, R2 is F, R3 is F, and R4 is CH3; or (b) R1 is CH3, R2 is F, R3 is F, and R4 is cyclopropyl, or (c) R1 is F, R2 is F, R3 is CH3, and R4 is CH3; or (d) R1 is F, R2 is F, R3 is F, and R4 is CH3; or (e) R1 is F, R2 is F, R3 is F, and R4 is CH2CHF2, or (f) R1 is F, R2 is Cl, R3 is F, and R4 is CH3.

6. A method for treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-5, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt, optionally wherein the cancer is a central nervous system (CNS) tumor.

7. The method of claim 6, wherein the subject has a central nervous system (CNS) tumor, the CNS tumor being selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, glioma, glioblastoma, pineal cell tumor, invasive pituitary adenoma, pituitary carcinoma, germ cell tumor, sarcoma, primary central nervous system lymphoma, brainstem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic nerve glioma, and dysplastic neuroepithelial tumor.

8. A method for selectively inhibiting PARP1 activity in a subject, relative to PARP2 activity, comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1-5 or an isotopically substituted derivative thereof or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the ratio of PARP1 activity inhibition to PARP2 activity inhibition in the subject is between 750 and 15000.

10. A pharmaceutically acceptable composition comprising a compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable diluent, carrier or excipient.

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