TW202525808A - Compound as parp1 inhibitor, use thereof and composition comprising the same - Google Patents

Abstract

The present invention provides PARP1 inhibitors and their applications in the treatment of CNS diseases. The PARP1 inhibitor of the present invention has a structure represented by formula (I). Compared with PARP2 activity, the PARP1 inhibitor of the present invention can selectively inhibit PARP1 activity. The present invention also provides the use of the PARP1 inhibitor in treating CNS diseases.

Description

Compounds used as PARP1 inhibitors, uses thereof, and compositions containing the same

The present invention belongs to the field of medicinal chemistry and relates to the field of disease treatment, specifically to PARP1 inhibitors and their use in treating CNS diseases, such as brain tumors.

Poly(ADP-ribose) polymerases (PARPs) refer to a group of proteins that catalyze the modification reaction of NAD ⁺ by adding ADP-ribose to receptor proteins. This is one of the various post-translational modifications of proteins. Therefore, they are also referred to as ADP-ribose transferases.

PARP1 is the most abundant and well-characterized member of the PARP family. It is a 1014-amino acid protein (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 an important role in many cellular functions, including gene expression, transcription, cell division, cell differentiation, apoptosis, and DNA damage response and repair. When DNA damage occurs, PARP1 is activated and participates in base excision repair (BER), the primary mechanism for repairing single-strand DNA damage. PARP1 binds to single-strand break (SSB) sites and subsequently repairs DNA through BER. In response to DNA damage, cells have evolved two major repair pathways, in addition to the BER repair mechanism: homologous recombination (HR) and non-homologous recombination end joining (NHEJ). Studies have found that HR-deficient tumors are sensitive to PARP inhibitors, suggesting that homologous recombination defects and PARP1 inhibition form a synthetic lethality, a finding 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). In addition, 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, Neurotherapeutics 20: 1347-1368).

PARP2 is a 583-amino acid protein (NCBI Accession NP_005475) with a molecular weight of approximately 62 kDa. Its structure comprises a DNA-binding domain and a catalytic domain (Ame et al., 1999 J Biol Chem 274:17860-17868). The catalytic domain of PARP2 shares a high degree of similarity with that of PARP1. Studies have shown that PARP2 has similar functions to PARP1, participating in the BER mechanism for repairing DNA damage (Schreiber et al., 2002 J Biol Chem 277:23028-23036). Marketed PARP inhibitors, such as olaparib, niraparib, talazoparib, and rucaparib, all exhibit similar inhibitory activity against PARP2 in addition to PARP1. Clinical trial results show that the therapeutic efficacy of these marketed PARP inhibitors is comparable, however, their toxicity profiles vary significantly. For example, talazoparib has side effects similar to chemotherapy, 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 sequences being identical and 89% of their catalytic domain sequences being identical. They play a role in DNA repair, telomere maintenance, and Wnt/β-catenin signaling. Targeting PARPs other than PARP1 may contribute to the off-target toxicities (such as hair loss and diarrhea) associated with PARP inhibitors. Furthermore, inhibition of PARP2 activity may also lead to hematologic toxicity (Farrés et al., 2013 Blood 122:44-54; Farrés et al., 2015 Cell Death and Differentiation 22:1144-1157). These toxicities limit the clinical application of PARP inhibitors and their combination with other targeted agents.

Central nervous system diseases are a group of neurological disorders that affect the structure or function of the brain or spinal cord, which together make up 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).

Brain tumors are considered one of the deadliest cancers worldwide. The unique tumor microenvironment and intrinsic characteristics of brain tumor cells render them resistant to most traditional and advanced therapies. Given the low survival rates associated with current brain tumor treatment strategies, new therapeutic strategies are urgently needed.

In one aspect, the present invention provides a compound having a structure as shown in the following formula (I) or a hydrate, isotope-labeled substance or pharmaceutically acceptable salt thereof: (I) wherein _R1 is selected from halogen, alkyl and halogenated alkyl; _R2 is selected from halogen and alkyl; _R3 is selected from halogen, alkyl and cyano (i.e., –CN); _R4 is selected from alkyl, halogenated alkyl and cycloalkyl.

In some embodiments of the compounds of formula (I) and methods and uses of the present invention, (a) R ₁ is halogen, C _1-3 alkyl or halogenated C _1-3 alkyl, or (b) R ₂ is halogen or C _1-3 alkyl, or (c) R ₃ is halogen, C _1-3 alkyl or cyano, or (d) R ₄ is C _1-3 alkyl, halogenated C _1-3 alkyl or C _3-4 cycloalkyl, or any combination of (a) to (d), for example, all of (a) to (d).

In some embodiments of the compounds of formula (I) and methods and uses of the present invention, (e) R ₁ is F, methyl or trifluoromethyl, or (f) R ₂ is F, Cl or methyl, or (g) R ₃ is F, methyl or cyano, or (h) R ₄ is C _1-3 alkyl, C _1-3 haloalkyl or C _3-4 cycloalkyl, or any combination of (e) to (h), for example all of (e) to (h).

In some embodiments of the compounds of formula (I) and methods and uses of the present invention, (i) R ₁ is F, methyl and trifluoromethyl, or (j) R ₂ is F, Cl or methyl, or (k) R ₃ is F, methyl or cyano, or (l) R ₄ is methyl, ethyl, difluoroethyl or cyclopropyl, or any combination of (i) to (l), such as all of (i) to (l).

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.

In some embodiments of the compounds of formula (I) and methods and uses 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 _C1-3 alkyl, _C1-3 haloalkyl, or _C3-4 cycloalkyl. In some embodiments, _R1 is F; _R2 is F; _R3 is halogen or alkyl; and _R4 is alkyl, haloalkyl, or cycloalkyl. In some embodiments, _R1 is _CH3 ; _R2 is F; _R3 is halogen or cyano; and _R4 is alkyl, haloalkyl, or cycloalkyl. In some embodiments, all of _R1 , _R2 , _R3 , and _R4 in the compound are defined in any of rows 1-11 of the following table: R ₁ R ₂ R ₃ R ₄ 1 CH ₃ F F CH ₃ 2 CH ₃ F CN CH ₃ 3 CH ₃ F F Cyclopropyl 4 CF ₃ F F CH ₃ 5 F F CH ₃ CH ₃ 6 F F F CH ₃ 7 F F F CH ₂ CH ₃ 8 F F F _{CH2CHF2 ​} 9 F F F Cyclopropyl 10 F Cl F CH ₃ 11 F CH ₃ F CH ₃

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, R ₁ is F, R ₂ is F, R ₃ is F, and R ₄ is CH ₃ . In some embodiments of the compounds and methods of the present invention, R ₁ is F, R ₂ is F, R ₃ is F, and R ₄ is CH ₂ CH ₃ . In some embodiments of the compounds and methods of the present invention, R ₁ is F, R ₂ is F, R ₃ is F, and R ₄ is CH ₂ CHF ₂ . In some embodiments of the compounds and methods of the present invention, R ₁ is F, R ₂ is F, R ₃ is F, and R ₄ is cyclopropyl. In some embodiments of the compounds and methods of the present invention, R ₁ is F, R ₂ is Cl, R ₃ is F, and R ₄ is CH ₃ . In some embodiments of the compounds and methods of the invention, R ₁ is F, R ₂ is CH ₃ , R ₃ is F, and R ₄ is CH ₃ .

In one aspect, the 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-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one, comprising: (a) reacting 4-fluoro-1H-pyrazole-5-carboxylic acid with an acylation reagent (such as SOCl ₂ ) to prepare 4-fluoro-1H-pyrazole-5-carbonyl chloride; (b) reacting 4-fluoro-1H-pyrazole-5-carbonyl chloride with 3,5-dibromo-2,6-difluoroaniline under alkaline conditions (for example, under the catalysis of NaH or LiHMDS) 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 cyclization reaction under alkaline conditions (such as under the catalysis of K ₂ CO ₃ ) 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 undergoes selective dehalogenation reaction to prepare 7-bromo-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one; (e) In the presence of a Pd catalyst (such as Xphos Pd G2) catalyzed by 7-bromo-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one and (Bu) ₃ SnCH ₂ OH to prepare 3,6-difluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxalin-4(5H)-one; (f) 3,6-difluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxalin-4(5H)-one reacts with HBr to produce 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one; (g) 7-((4-(2-Fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one is prepared by reacting 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one with 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridinamide under alkaline conditions (e.g., catalyzed by DIEA and KI).

In one aspect, 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, an isotopically substituted derivative, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is in need of such treatment. In some embodiments, tumor regression is induced following administration of the compound. In some embodiments, induced tumor regression is assessed 7 to 84 days after administration of the compound, e.g., any day between 7 and 84 days, e.g., 8 days, 9 days, etc., or any time between any two days. In some embodiments, the 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 non-human animal, such as a veterinary animal or zoo animal, such as disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer therapies or drugs, such as those disclosed herein. In some embodiments, the volume of the CNS tumor, such as a brain tumor, at time t is less than the volume of the tumor 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 regression of a central nervous system tumor in a subject, the central nervous system tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinal In some embodiments of the method for inducing regression 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 central nervous system tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a 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 tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments of the method for inducing regression of a central nervous system tumor in a subject, the compound, such as a compound of formula (I) or a hydrate thereof, an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound or a hydrate thereof, 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 a compound, such as a compound of formula (I) or a hydrate thereof, an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

In one aspect, the present invention provides a method for inducing increased 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, an isotopically substituted derivative, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is in need of such treatment. In some embodiments, increased tumor regression is induced following administration of the compound. In some embodiments, increased induced tumor regression is assessed 7 to 84 days after administration of the compound, e.g., any day between 7 and 84 days, e.g., 8 days, 9 days, etc., or any time between any two days. In some embodiments, the 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 non-human animal, such as a veterinary animal or zoo animal, such as disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer therapies or drugs, such as those disclosed herein. In some embodiments of the method for inducing increased regression of a central nervous system tumor in a subject, the central nervous system tumor is selected from the group consisting of meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinal Meningioblastoma, neurotheliomas, primary central nervous system lymphoma, brain stem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and dysembryonic neuroepithelial tumor. In some embodiments of the method for inducing increased regression 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 central nervous system tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a 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 tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments of the method for inducing increased regression of a central nervous system tumor in a subject, the compound, such as a compound of formula (I) or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I) or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable pharmaceutical composition thereof, is administered.

In one aspect, 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 in need of such treatment. In some embodiments, after administration of the compound, tumor growth is inhibited. In some embodiments, inhibition of central nervous system tumor growth is assessed 7 to 84 days after administration of the compound, e.g., any day between 7 and 84 days, e.g., 8 days, 9 days, etc., or any time between any two days. In some embodiments, tumor growth is inhibited by 10% to 150%, such as 30% to 150% or 50% to 120%, such as any integer value between 10% and 150%, such as 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 non-human animal, such as a veterinary animal or a zoo animal, such as disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer 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 the group consisting of meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinal 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 central nervous system tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a 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 tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments of the method for inhibiting the growth of a central nervous system tumor in a subject, the compound, such as a compound of formula (I) or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I) or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

In one aspect, the present invention provides a method for inhibiting PARP1 activity, for example, 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 a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is in need of such treatment. In some embodiments, after administration of the compound, inhibition of PARP1 activity in the subject is greater than inhibition of PARP2 activity. In some embodiments, the ratio of inhibition of PARP1 activity to inhibition of PARP2 activity in the subject is at least 50, at least 750, at least 1500, at least 2500, at least 5000, at least 7500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, or a range between 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, such as between 15000 and 25000. In some embodiments, the subject is a human or non-human animal, such as a veterinary animal or a zoo animal, such as disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer therapies or drugs, such as those disclosed herein. In some embodiments, the compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

In one aspect, the present invention provides a method for treating a subject in need thereof, 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 stroke or a neurodegenerative disease.

In another aspect, the present invention provides a method for treating cancer 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 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 non-human animal, such as a veterinary animal or zoo animal, such as those disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer therapies or drugs (e.g., the anti-cancer 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, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, 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 dysembryoplastic neuroepithelial tumor. In some embodiments, the CNS tumor is any CNS tumor disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a medulloblastoma), an ependymoma, a glioblastoma, a primary CNS lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments, the compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

In one aspect, the present invention provides the use of a compound of formula (I) disclosed herein (e.g., an effective amount of the compound), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament. In one aspect, the present invention provides the use of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating any condition, such as cancer, such as any CNS tumor, for example, a brain tumor as disclosed or described herein.

In one aspect, the present invention provides a compound of formula (I) disclosed herein (e.g., an effective amount of the compound), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, for use in treating a disease or for a purpose disclosed herein.

In one aspect, the present invention provides a compound of formula (I) disclosed herein (e.g., an effective amount of the compound), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, for use in a method of treating a disease disclosed herein or for a purpose disclosed herein.

In one aspect, the present invention provides a method for treating a central nervous system (CNS) disease or condition (e.g., a CNS tumor, such as a brain tumor), 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 BBB. In some embodiments, the CNS disease or condition is a brain tumor. In some embodiments, the CNS disease or condition is a CNS tumor. In some embodiments, the central nervous system (CNS) disease or disorder is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, glioblastoma multiforme, medulloblastoma, primary central nervous system lymphoma, brain stem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and dysembryoplastic neuroepithelial tumor. In some embodiments, the CNS tumor is any CNS tumor disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a medulloblastoma), an ependymoma, a glioblastoma, a primary CNS lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments, a therapeutically effective amount of a compound, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered. In some embodiments, a therapeutically effective amount of the compound, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients.

In one aspect, 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.

In some embodiments of the methods disclosed herein, the method comprises administering a therapeutically effective amount of a compound of Formula I as described herein, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments of the methods disclosed herein, the method comprises administering a therapeutically effective amount of a compound of Formula I as described herein, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In various embodiments, the pharmaceutically acceptable salt is an inorganic or organic acid salt, such as a 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 hydroxylate, tris(hydroxymethyl)aminomethane (TRIS, tromethamine), or N-methyl-glucamine salt.

In any embodiment of any method and use disclosed herein, the compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered before, simultaneously with, or after, 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 systemic radioisotopes), endocrine therapy, biologic response modifiers (such as interferons, interleukins, and tumor necrosis factor), hyperthermia and cryotherapy, medications to mitigate any adverse effects (such as antiemetics) and other approved chemotherapy drugs, steroid toxins (such as vincristine, vinpoxetine, vinorelbine, and paclitaxel), podophyllotoxins (such as etofoside, irinotecan, and vopotex), nitrosoureas (such as vastatin and lomustine), inorganic ions (such as cisplatin and carboplatin), enzymes (such as asparaginase), hormones (such as tamoxifen, leucine, flutamide, and megestrol acetate), Glivir™, doxorubicin, dexamethasone, and cyclophosphamide.

In any of the treatment methods disclosed herein, in some embodiments, the subject is a subject in need of such treatment. In various embodiments, the subject is a human. In some embodiments, the subject is a non-human, such as a veterinary animal (e.g., pigs, cows, sheep, horses, dogs, and cats) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

In any use of the compounds disclosed herein for treating a condition in a subject, in some embodiments, the subject is a subject in need of such treatment. In various embodiments, the subject is a human. In some embodiments, the subject is a non-human, such as a veterinary animal (e.g., pigs, cows, sheep, horses, dogs, and cats) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

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

As used herein, the term "alkyl" refers to an alkyl group having a linear or branched chain of up to 10 carbon atoms. Useful alkyl groups include linear or branched _C1-10 alkyl groups, such as _C1-6 alkyl groups. In some embodiments, the alkyl group is a _C1-3 alkyl group. Typical alkyl groups include methyl, deuterated methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl (e.g., 3-pentyl), hexyl, and octyl groups, which may be optionally substituted. In some embodiments, alkyl groups such as _C1-3 alkyl and _C1-6 alkyl groups are unsubstituted. In some embodiments, alkyl groups such as _C1-3 alkyl and _C1-6 alkyl groups are substituted. In some embodiments, substituents on the alkyl group include halogen groups (F, Cl, Br, and I), -OH, -CHO, _-NH2 , or combinations thereof.

As used herein, the term "cycloalkyl" refers to a cyclic alkyl group. Useful cycloalkyl groups are _C3-8 cycloalkyl groups. In some embodiments, the cycloalkyl group is _C3-6 cycloalkyl. Typical cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Cycloalkyl groups may be substituted with one or more substituents as described herein. In some embodiments, cycloalkyl groups, such as _C3-4 cycloalkyl and _C3-6 cycloalkyl, are unsubstituted.

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

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 group is a halo C _1-3 alkyl group. Exemplary haloalkyl groups include trifluoromethyl, difluoroethyl, fluoromethyl, and dichloroethyl.

The term "induce" as used in this article means "to cause."

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

As used herein, the term "regression" refers to a reduction in the size or extent of a tumor. Such regression may be associated with the disappearance or decrease in the number or size of tumor cells (e.g., tumor cells).

As used herein, the term "inducing tumor regression" means causing or resulting in a decrease in the size or extent of a tumor, including causing the disappearance of a tumor (e.g., to an undetectable extent), such as by causing the disappearance or decrease in the number or size of tumor cells (e.g., tumor cells).

As used herein, the term "inducing increased regression" means causing regression that is already ongoing to proceed at a greater rate or to a greater extent, or causing additional regression where regression has previously ceased. Thus, inducing increased regression of a tumor means, for example, causing a tumor that is already regressing to regress at a greater rate or to a greater extent following treatment with, for example, a compound of Formula (I) disclosed herein, or causing a tumor that has previously ceased regression to regress after treatment with, for example, a compound of Formula (I) disclosed herein. Increased regression may occur when, for example, two different (e.g., independently acting) therapies are administered that both induce regression and, when combined, have an additive or synergistic effect.

As used herein, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" refers to a substance that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications, and is commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment. Thus, pharmaceutically acceptable refers to a substance that is not biologically or otherwise unacceptable. The material can be administered to a subject in conjunction with the relevant active compound without causing clinically unacceptable biological effects or interacting in a deleterious manner with any other component of a pharmaceutical composition containing the material.

As used herein, the terms "therapeutically effective amount" or "effective amount" of a therapeutic agent or a composition comprising a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and/or delay the onset of one or more symptoms of the disease, disorder, and/or condition (e.g., to slow or prevent an increase in the severity of, or reduce the severity of, any such symptom) when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition. "Therapeutically effective amount" and "effective amount" also include amounts sufficient to affect an activity or cause a change in an activity, such as an enzyme activity, such as PARP1 activity or PARP2 activity, when administered to a subject. It is understood by those skilled in the art that a therapeutically effective amount or effective amount is typically administered via a dosing regimen that includes at least one unit dose. A non-limiting example of a "therapeutically effective amount" or "effective amount" of the compound of formula (I) of the present invention, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt, or a composition comprising the compound of formula (I) is an amount that can be used to inhibit, block, or reverse cell activation, migration, or proliferation, or effectively treat cancer or improve cancer symptoms. Another non-limiting example of a "therapeutically effective amount" or "effective amount" of the compound of formula (I) of the present invention, or its hydrate, or its isotopically substituted derivative, or its pharmaceutically acceptable salt, or a composition comprising the compound of formula (I) is an amount that can be used to inhibit PARP1 activity.

As used herein, the term "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment can be administered to subjects who do not exhibit signs of disease and/or who exhibit only early signs of disease to reduce the risk of developing pathology associated with the disease.

Tumor cells (or cancer cells) as described herein are generally characterized by abnormal proliferation relative to normal cells and the formation of clusters or tumors in individuals with cancer. "Cancer" includes a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the blood and lymphatic systems. Examples of various types of cancer include central nervous system cancers, 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 bodies, such as diffuse or circulatory tumors. In some embodiments, the terms "cancer" or "tumor" include malignant cancers and tumors, as well as advanced cancers and tumors. As used herein, the terms "tumor" and "cancer" refer to both malignant (e.g., spreading from the tissue of origin to another tissue) and non-malignant (e.g., growing but remaining confined to the tissue of origin), such as benign growths.

Central nervous system tumors (also referred to herein as CNS tumors) are known in the art (DN Louis et al., 2021 WHO Classification of Tumors of the Central Nervous System: a summary, Neuro Oncol. 2021 Aug;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 astrocytoma, oligodendroglioma, glioblastoma, diffuse astrocytoma, angiocentric glioma, juvenile pleomorphic low-grade neuroepithelioma, diffuse low-grade glioma, diffuse midline glioma, diffuse hemispheric glioma, pediatric high-grade glioma, and many astrocytoma, subependymal giant cell astrocytoma, chordoid neuroglioma, astroblastoma, ganglioneuroma, dysembryoplastic neuroepithelioma, diffuse colloid neuronoma with oligodendroglioma-like features and nuclear clustering, papillary colloid neuronoma, multinodular and vacuolar Alveolar neuron tumor, dysplastic cerebellar ganglioneuroma (Lhermitt-Duclos disease), extraventricular neuroma, supratentorial ependymoma, posterior fossa ependymoma, spinal cord ependymoma, medulloblastoma, atypical teratoma/rhabdoid tumor, embryonal tumor with multilayered rosettes, central nervous system neuroblastoma, central nervous system tumor with BCOR intraepithelial duplication, pineal region desmoplastic myxoid tumor, meningioma, solitary fibroma, meningeal melanocytoma, enamel (epithelial) pancraniocarcinoma, and papillary craniocarcinoma.

A brain tumor is an abnormal growth of cells or masses in the brain, either cancerous (malignant) or noncancerous (benign). Brain tumors can develop in the brain tissue. They can also develop near brain tissue. Nearby locations include the nerves, pituitary gland, pineal gland, and the membranes covering the brain. Brain tumors can start in the brain. These are called primary brain tumors. Sometimes, cancer spreads to the brain from other parts of the body. These tumors are secondary brain tumors, also called metastatic brain tumors. There are many different types of primary brain tumors. Some brain tumors are not cancerous. These are called noncancerous or benign brain tumors. Noncancerous brain tumors may grow over time and press on brain tissue. Other brain tumors are brain cancers, also called malignant brain tumors. Brain cancers can grow rapidly. The cancer cells can invade and destroy brain tissue. Brain tumors are a subset of central nervous system tumors. Brain tumors include, but are not limited to, neurogliomas, 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 tumors, meningiomas, and acoustic nerve tumors (vestibular schwannomas). Some brain tumors are high-grade (fast-growing). Others are low-grade (slow-growing).

Specifically, the present invention provides a compound represented by the following formula (I), or a hydrate thereof, or an isotope-substituted derivative thereof, or a pharmaceutically acceptable salt thereof: (I) wherein _R1 is selected from halogen, alkyl and halogenated alkyl; _R2 is selected from halogen and alkyl; _R3 is selected from halogen, alkyl and cyano (i.e., –CN); _R4 is selected from alkyl, halogenated alkyl and cycloalkyl.

In one or more embodiments of the compound of formula (I), R ₁ is halogen, C _1-3 alkyl, or halogenated C _1-3 alkyl. In some embodiments, R ₁ is F, methyl, or trifluoromethyl.

In one or more embodiments of the compound of formula (I), R ₂ is halogen or C _1-3 alkyl. In some embodiments, R ₂ is F, Cl or methyl.

In one or more embodiments of the compound of formula (I), R ₃ is halogen, C _1-3 alkyl, or cyano. In some embodiments, R ₃ is F, methyl, or cyano.

In one or more embodiments of the compound of formula (I), R ₄ is C _1-3 alkyl, halogenated C _1-3 alkyl, or C _3-4 cycloalkyl. In some embodiments, R ₄ is methyl, ethyl, difluoroethyl, or cyclopropyl.

In one or more embodiments of the compound of formula (I), R ₁ is CH ₃ , R ₂ is F, R ₃ is F or cyano, and R ₄ is CH ₃ or cyclopropyl.

In one or more embodiments of the compound of formula (I), R ₁ is F, R ₂ is F, Cl or CH ₃ , R ₃ is F or CH ₃ , and R ₄ is methyl, ethyl, difluoroethyl or cyclopropyl.

In some embodiments of the compound of formula (I), one or more of R ₁ , R ₂ , R ₃ , and R ₄ is alkyl, haloalkyl, or cycloalkyl, and is not further substituted. In some embodiments of the compound of formula (I), one or more of R ₁ , R ₂ , R ₃ , and R ₄ is alkyl, haloalkyl, or cycloalkyl as defined herein, and only R ₄ is further substituted, for example, with F, Cl, Br, I, -OH, -CHO, -NH ₂ , or a combination thereof.

Exemplary compounds of formula (I) include, but are not limited to: 7-((4-(2-fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 1); 7-((4-(2-cyano-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 2); 7-((4-(2-fluoro-6-(cyclopropylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 3); 7-((4-(2-Fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3-trifluoromethyl-6-fluoropyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 4); 7-((4-(2-Methyl-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 5); 7-((4-(2-Fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 6); 7-((4-(2-Fluoro-6-(ethylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 7); 7-((4-(2-Fluoro-6-(2,2-difluoroethylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 8); 7-((4-(2-Fluoro-6-(cyclopropylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 9); 3-Fluoro-7-((4-(2-fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-chloropyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 10); 3-Fluoro-7-((4-(2-fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (Example 11); or a hydrate, isotope-substituted derivative, or pharmaceutically acceptable salt thereof.

Some of the compounds of the present invention may exist as stereoisomers, including optical isomers. The present invention includes all stereoisomers and racemic mixtures of such stereoisomers, as well as individual enantiomers that can be separated according to methods well known to those skilled in the art.

Examples of pharmaceutically acceptable salts include inorganic and organic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, citrates, lactates, tartrates, maleates, fumarates, mandelates and oxalates; and inorganic and organic salts formed from bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N-methylglucamine.

Examples of prodrugs of the compounds of the present invention include simple esters of compounds containing carboxylic acids (e.g., esters obtained by condensation with C _1-4 alcohols according to methods known in the art); esters of compounds containing hydroxy groups (e.g., esters obtained by condensation with C _1-4 carboxylic acids, C _3-6 diacids, or anhydrides thereof, such as succinic anhydride and fumaric anhydride according to methods known in the art); imines of compounds containing amino groups (e.g., imines obtained by condensation with C _1-4 aldehydes or ketones according to methods known in the art); carbamates of compounds containing amino groups, such as those described in Leu et al. ( J. Med. Chem. 42:3623-3628 (1999)) and Greenwald et al. ( J. Med. Chem. 42:3657-3667). (1999)); aldols or ketals of alcohol-containing compounds (e.g., those obtained by condensation with chloromethyl methyl ether or chloromethyl ethyl ether according to methods known in the art).

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 according to the exemplary reaction shown in Scheme 1. 4-Methyl-1H-pyrazole-5-carboxylic acid reacts with _SOCl₂ to obtain 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 obtain 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 ring-closure reaction under alkaline conditions (e.g., catalyzed by _K₂CO₃ ) to afford 7-bromo-6-fluoro _- 3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one. Reaction of ₇ -bromo-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one with (Bu) _₃SnCH₂OH over a palladium catalyst (e.g., Xphos Pd G2) affords 6-fluoro-7-(hydroxymethyl)-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one. 6-Fluoro-7-(hydroxymethyl)-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one reacts with HBr to afford 7-(bromomethyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one. 7-(Bromomethyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one then undergoes a substitution reaction with 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridinamide under alkaline conditions (e.g., catalyzed by DIEA and KI) to afford the target compound, 7-((4-(2-fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one. Reaction Scheme 1

Other related compounds can be prepared by similar methods. For example, 6-cyano-N-methyl-5-(piperazin-1-yl)pyridinamide is used instead of 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridinamine to prepare the target compound 7-((4-(2-cyano-6-(methylaminoformyl)pyridin-3-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one. 4-Fluoro-1H-pyrazole-5-carboxylic acid is used instead of 4-methyl-1H-pyrazine-5-carboxylic acid to prepare the target compound 7-( (4-(2-Fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one. Substituting 3-bromo-2-chloro-6-fluoroaniline for 3-bromo-2,6-difluoroaniline yields the target compound, 3-fluoro-7-((4-(2-fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-chloropyrazolo[1,5-a]quinoxalin-4(5H)-one.

The compounds of the present invention can be prepared as shown in the exemplary reaction in Reaction Scheme 2. The reaction of 4-fluoro-1H-pyrazole-5-carboxylic acid with _SOCl₂ affords 4-fluoro-1H-pyrazole-5-carbonyl chloride. The reaction of 4-fluoro-1H-pyrazole-5-carbonyl chloride with 3,5-dibromo-2,6-difluoroaniline under alkaline conditions (e.g., catalyzed by NaH or LiHMDS) affords 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 undergoes a ring-closure reaction to afford 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxalin-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]quinoxalin-4(5H)-one undergoes a selective dehalogenation reaction to afford 7-bromo-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one. 3,6-Difluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxaline-4(5H)-one reacts with HBr to give 7-(bromomethyl)-3,6-difluoropyrazolo[1,5-a]quinoxaline-4(5H)-one. Under alkaline conditions (such as catalysis 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)pyridinamide to give the target compound, 7-((4-(2-fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-ylmethyl)-3,3-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one. Reaction Scheme 2

Other related compounds can be prepared using similar methods. For example, replacing 4-fluoro-1H-pyrazine-5-carboxylic acid with 4-methyl-1H-pyrazole-5-carboxylic acid yields the target compound 7-((4-(2-fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one. Replacing 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridinamide with N,6-dimethyl-5-(piperazin-1-yl)pyridinamide yields the target compound 7-((4-(2-methyl-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one.

Pharmaceutically acceptable salts of the PARP1 inhibitors of the present invention are also 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 (e.g., 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 base, such as sodium hydroxide, potassium hydroxide, hydroquinoline, sodium carbonate, tris(hydroxymethyl)aminomethane, N-methylglucamine, etc.

The compounds of the present invention can be administered as a raw drug. 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 processing of the compound into a pharmaceutically acceptable formulation. In some embodiments, the pharmaceutical formulation, such as oral or other formulations, such as tablets, tablets, and capsules, and solutions suitable for injection or oral administration, contains from 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.

The PARP1 inhibitors of the present invention can be administered in a pharmaceutical composition containing a pharmaceutically acceptable carrier, wherein the pharmaceutical composition includes a pharmaceutical preparation containing a compound of the present invention in an amount effective to achieve its intended purpose. Although each individual's needs vary, those skilled in the art can determine the optimal dosage of each component of the pharmaceutical preparation. Generally, the compound, or its hydrate, isotopically substituted derivative, or pharmaceutically acceptable salt thereof, is orally administered to a mammal at a daily dose of about 0.0025 to 50 mg/kg body weight. In some embodiments, the oral dosage is about 0.01 to 10 mg/kg per kilogram. If a known anticancer drug is also administered, its dosage should be effective to achieve its intended purpose. The optimal dosage of these known anticancer drugs is well known to those skilled in the art.

The unit oral dosage may comprise about 0.01 to 50 mg, preferably about 0.1 to 10 mg, of a compound of the present invention. The unit dosage may be administered once or more, in one or more tablets per day, each tablet containing about 0.1 to 50 mg, conveniently about 0.25 to 10 mg, of a compound of the present invention or a solvate thereof.

In topical preparations, the concentration of the compound of the present invention may be about 0.01 to 100 mg per gram of carrier.

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 for use in the compositions disclosed herein are known to those skilled in the art and include, but are not limited to, those disclosed in, for example, the Handbook of Pharmaceutical Excipients (Raymond C. Rowe et al., 6th edition, 2009), the entire contents of which are incorporated herein by reference. In addition, exemplary dosage forms of compositions, such as the compositions disclosed herein, are known to those skilled in the art and include, but are not limited to, those disclosed in, for example, 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.

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 a 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 saline solutions, such as phosphates and salts of 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.

The pharmaceutical formulations disclosed herein can be administered to any mammal that can experience the therapeutic effects of the compounds disclosed herein. The most important of these mammals are humans and veterinarians, although the disclosure is not limited thereto.

The pharmaceutical formulations disclosed herein can be administered by any route that achieves their intended purpose. For example, administration can be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, intranasal, or topical. Alternatively, or concurrently, administration can be by oral route. The dosage administered will depend on the age, health, and weight of the subject, the type of concurrent treatment, the frequency of treatment, and the nature of the desired effect.

The pharmaceutical preparations of the present invention can be manufactured in known manners, for example, by conventional mixing, granulation, tableting, dissolution, or freeze-drying processes. For oral preparations, solid excipients and the active compound can be combined, and the mixture can be optionally ground. After adding appropriate auxiliary agents, if desired or necessary, the granular mixture can be processed to obtain tablets or tablet cores.

Suitable excipients are, in particular, fillers, for example, sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate; and binders, for example, starch pastes including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone. If necessary, disintegrants may be added, such as the starches mentioned above, as well as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliary agents are, in particular, flow regulators and lubricants, for example, silica, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. If desired, the tablet cores can be provided with a suitable coating resistant to gastric juices. For this purpose, a concentrated sugar solution can be applied. This solution can contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. To prepare a coating resistant to gastric juices, suitable cellulose solutions can be used, for example, cellulose acetate phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments can be added to the coating of the tablet or tablet core. For example, to identify or characterize a combination of active ingredients.

Other pharmaceutical formulations for oral administration include push-fit capsules made of gelatin and sealed softgel capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules may contain the active compound in the form of granules mixed with a filler such as lactose; a binder such as starch; and/or a lubricant such as talc or magnesium stearate, and a stabilizer. In softgel capsules, the active compound is preferably dissolved or suspended in a suitable liquid such as oil or liquid paraffin, to which a stabilizer may be added.

Formulations suitable for parenteral administration include aqueous solutions of the active compound, such as solutions of water-soluble salts and alkaline solutions. In addition, suitable oily injection suspensions of the active compound may be administered. Suitable lipophilic solvents or carriers include oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides or polyethylene glycol 400, or hydrogenated castor oil, or cyclodextrins. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and/or dextran. They may also contain a suspension stabilizer.

According to one aspect of the present invention, the compounds of the present invention are used in topical and parenteral formulations for the treatment of skin cancer.

The topical formulations of the present invention can be formulated into oils, creams, lotions, ointments, and the like by preferably selecting a suitable carrier. Suitable carriers include plant or mineral oils, white mineral oil (white paraffin), branched fats 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 for imparting 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.

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

Ointments can be prepared by mixing a vegetable oil such as almond oil containing the active ingredient with warm soft wax and then allowing the mixture to cool. A typical ointment example includes about 30% by weight almond oil and 70% by weight white soft wax.

Another embodiment of the present invention relates to pharmaceutical compositions that are effective for treating 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, these compositions are used in combination with other anticancer drugs involved in DNA damage and repair mechanisms, such as the HDAC inhibitors vorinostat, romidepsin, panobinostat, and belinostat. These compositions are 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, and other targeted anticancer drugs, including USP1 inhibitors, PRMT5 inhibitors, Polθ inhibitors, RAD51 inhibitors, and the like. Other known anticancer drugs that can be used in combination anticancer therapy include, but are not limited to, alkylating agents such as busulfan, mefenamic acid, chlorambucil, cyclophosphamide, isocyclophosphamide, temozolomide, bendamustine, cisplatin, mitomycin C, bleomycin, and carboplatin; topoisomerase I inhibitors such as camptothecin, irinotecan, and topotecan; topoisomerase II inhibitors such as adriamycin, epididymoxetine, and cypermethrin; Adriamycin, aclacinomycin, mitoxantrone, methylhydroxyrosalpinol, and metoprolol; RNA/DNA anti-metabolites such as 5-azacytidine, gemcitabine, 5-fluorouracil, and methotrexate; DNA anti-metabolites such as 5-fluoro-2′-deoxyuridine, fludarabine, nelarabine, cytarabine, pralatrexate, pemetrexed, hydroxyurea, and thioguanine; anti-silk Splitting agents such as colchicine, vinblastine, vincristine, vinorelbine, paclitaxel, ixabepilone, cabazitaxel, and docetaxel; antibodies such as monoclonal antibodies, panitumumab, nexitozumab, nivolumab, pembrolizumab, ramucirumab, bevacizumab, pertuzumab, trastuzumab, cetuximab, otuzumab, ofatumumab, rituximab, alemtuzumab, ibritumomab tiuxetan, tositumomab, brentuximab, daratumumab, elotuzumab, ofatumumab, dinutuximab, blinatumomab, ipilimumab, Avastin, Herceptin, and rituximab; antibody-drug conjugates (ADCs) such as trastuzumab-emtansine conjugate T-DM1, humanized antibodies HER2 antibody-drug conjugates Trastuzumab Deruxtecan, Trastuzumab Emtansine, humanized anti-TROP2 monoclonal antibody-drug conjugates Datopotamab Deruxtecan, Gemtuzumab Ozogamicin, CD30-directed antibody-drug conjugates Brentuximab Vedotin, Inotuzumab Ozogamicin, Sacituzumab govitecan, Enfortumab Vedotin, and Belantamab Mafodotin; kinase inhibitors such as imatinib, gefitinib, erlotinib, ostinib, afatinib, ceritinib, alectinib, crizotinib, erlotinib, lapatinib, sorafenib, regorafenib, vemurafenib, dabrafenib, aflibercept, sunitinib, nilutinib, dasatinib, bosutinib, pralsetinib, ibrutinib, cabozantinib, lenvatinib, vandetanib, trametinib, cobimetinib, axitinib, temsirolimus, idelalisib, pazopanib, tecansibin, and everolimus. Other known anticancer drugs that can be used in combination therapy include tamoxifen, letrozole, fulvestrant, mitoguanidine, octreotide, retinoic acid, arsenic, zoledronic acid, bortezomib, carfilzomib, ixazomib, vismodegib, sonidegi, denosumab, thalidomide, lenalidomide, venetoclax, aldesleukin (recombinant human interleukin-2), and sipueucel-T (a prostate cancer vaccine).

In addition, in any embodiment of any of the methods and uses disclosed herein, or after administration of one or more additional anti-cancer therapies or drugs or in combination with one or more additional anti-cancer therapies or drugs, the one or more additional cancer 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 systemic radioisotopes), endocrine therapy, biological response modifiers (e.g., interferons, leukocytes), interleukins and tumor necrosis factor (TNF)), hyperthermia and cryotherapy, medications to mitigate any adverse effects (such as antiemetics) and other approved chemotherapy drugs, steroid poisons (such as vinblastine, vincristine, vinorelbine, and paclitaxel), podophyllotoxins (such as etoposide, irinotecan, and vopotescan), nitrosoureas (such as valproate and lomustine), inorganic ions (such as cisplatin and carboplatin), enzymes (such as asparaginase), hormones (such as tamoxifen, leuprorelin, flutamide, and megestrol acetate), Glivir™, doxorubicin, dexamethasone, and cyclophosphamide.

The present disclosure also provides the use of a PARP1 inhibitor in the preparation of a medicament for treating a central nervous system tumor (e.g., a brain tumor) in an individual. The central nervous system tumor can be selected from any of the tumors disclosed herein. The brain tumor can be selected from any of the tumors 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 or pharmaceutically acceptable salt thereof. The present disclosure also provides a PARP1 inhibitor or a pharmaceutical composition or pharmaceutical formulation containing one or more (e.g., two, three, four, or five) PARP1 inhibitors for use in the methods of treating a central nervous system tumor, such as a brain tumor, in a subject (e.g., a human, veterinarian, or zoo animal) disclosed herein. In some embodiments, the PARP1 inhibitor is a compound of formula (I), or a hydrate thereof as described herein, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof.

The present invention also provides the use of a 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 in need thereof. The present invention also provides the use of a PARP1 inhibitor or a pharmaceutical composition containing a PARP1 inhibitor in a method for treating a central nervous system tumor (e.g., a brain tumor) in a subject in need thereof. The present invention also provides a method for treating a central nervous system tumor, such as a brain tumor, in a subject in need thereof, 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, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof.

In another aspect, 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 in need of such treatment. In some embodiments, tumor regression is induced following administration of the compound. In some embodiments, induced tumor regression is assessed 7 to 84 days after administration of the compound, e.g., any day between 7 and 84 days, e.g., 8 days, 9 days, etc., or any time between any two days. In some embodiments, the 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 non-human animal, such as a veterinary animal or zoo animal, such as disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer therapies or drugs, such as those disclosed herein. In some embodiments, the volume of the CNS tumor, such as a brain tumor, at time t is less than the volume of the tumor 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 regression of a central nervous system tumor in a subject, the central nervous system tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinal In some embodiments of the method for inducing regression 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 central nervous system tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a 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 tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments of the method for inducing regression of a central nervous system tumor in a subject, the compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

In one aspect, the present invention provides a method for inducing increased 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 in need of such treatment. In some embodiments, increased tumor regression is induced following administration of the compound. In some embodiments, increased induced tumor regression is assessed 7 to 84 days after administration of the compound, e.g., any day between 7 and 84 days, e.g., 8 days, 9 days, etc., or any time between any two days. In some embodiments, the 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 non-human animal, such as a veterinary animal or zoo animal, such as disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer therapies or drugs, such as those disclosed herein. In some embodiments of the method for inducing increased regression of a central nervous system tumor in a subject, the central nervous system tumor is selected from the group consisting of meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinal Meningioblastoma, neurotheliomas, primary central nervous system lymphoma, brain stem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and dysembryonic neuroepithelial tumor. In some embodiments of the method for inducing increased regression 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 central nervous system tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a 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 tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments of the method for inducing increased regression of a central nervous system tumor in a subject, the compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable pharmaceutical composition thereof, is administered.

In one aspect, 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 in need of such treatment. In some embodiments, after administration of the compound, tumor growth is inhibited. In some embodiments, inhibition of central nervous system tumor growth is assessed 7 to 84 days after administration of the compound, e.g., any day between 7 and 84 days, e.g., 8 days, 9 days, etc., or any time between any two days. In some embodiments, tumor growth is inhibited by 10% to 150%, such as 30% to 150% or 50% to 120%, such as any integer value between 10% and 150%, such as 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 non-human animal, such as a veterinary animal or a zoo animal, such as disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer 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 the group consisting of meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, craniopharyngioma, retinal 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 central nervous system tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a 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 tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments of the method for inhibiting the growth of a central nervous system tumor in a subject, the compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

In one aspect, the present invention provides a method for inhibiting PARP1 activity, for example, 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 a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is in need of such treatment. In some embodiments, after administration of the compound, inhibition of PARP1 activity in the subject is greater than inhibition of PARP2 activity. In some embodiments, the ratio of inhibition of PARP1 activity to inhibition of PARP2 activity in the subject is at least 50, at least 750, at least 1500, at least 2500, at least 5000, at least 7500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, or a range between 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, such as between 15000 and 25000. In some embodiments, the subject is a human or non-human animal, such as a veterinary animal or a zoo animal, such as disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer therapies or drugs, such as those disclosed herein. In some embodiments, the compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

In one aspect, the present invention provides a method for treating a subject in need thereof, 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 stroke or a neurodegenerative disease.

In another aspect, the present invention provides a method for treating cancer 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 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 non-human animal, such as a veterinary animal or zoo animal, such as those disclosed herein. In some embodiments, the compound is administered prior to, concurrently with, or after administration of one or more additional anti-cancer therapies or drugs (e.g., the anti-cancer 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, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, 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 dysembryoplastic neuroepithelial tumor. In some embodiments, the CNS tumor is any CNS tumor disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a medulloblastoma), an ependymoma, a glioblastoma, a primary CNS lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments, the compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, 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 a compound, such as a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof, is administered.

In one aspect, the present invention provides the use of a compound of formula (I) disclosed herein (e.g., an effective amount of the compound), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament. In one aspect, the present invention provides the use of a compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating any condition, such as cancer, such as any CNS tumor, for example, a brain tumor as disclosed or described herein.

In one aspect, the present invention provides a compound of formula (I) disclosed herein (e.g., an effective amount of the compound), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, for use in treating a disease or for a purpose disclosed herein.

In one aspect, the present invention provides a compound of formula (I) disclosed herein (e.g., an effective amount of the compound), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, for use in a method of treating a disease disclosed herein or for a purpose disclosed herein.

In one aspect, the present invention provides a method for treating a central nervous system (CNS) disease or condition (e.g., a CNS tumor, such as a brain tumor), 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 BBB. In some embodiments, the CNS disease or condition is a brain tumor. In some embodiments, the CNS disease or condition is a CNS tumor. In some embodiments, the central nervous system (CNS) disease or disorder is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, glioblastoma multiforme, medulloblastoma, primary central nervous system lymphoma, brain stem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma, and dysembryoplastic neuroepithelial tumor. In some embodiments, the CNS tumor is any CNS tumor disclosed herein. In some embodiments, the CNS tumor is a brain tumor, such as a brain tumor disclosed herein. In some embodiments, the brain tumor is a high-grade (fast-growing) tumor. In some embodiments, the brain tumor is a low-grade (slow-growing) tumor. In some embodiments, the brain tumor is a neuroglioma, an embryonal brain tumor (e.g., a medulloblastoma), an ependymoma, a glioblastoma, a primary CNS lymphoma, a pineal region tumor (e.g., a germ cell tumor or a pineal cell tumor), a pituitary tumor, a meningioma, or an acoustic neuroma (vestibular schwannoma). In some embodiments, a therapeutically effective amount of a compound, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered. In some embodiments, a therapeutically effective amount of the compound, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises the compound, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents, carriers, or excipients.

In one aspect, 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.

In some embodiments of the methods disclosed herein, the method comprises administering a therapeutically effective amount of a compound of Formula (I) as described herein, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments of the methods disclosed herein, the method comprises administering a therapeutically effective amount of a compound of Formula I as described herein, or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof. In various embodiments, the pharmaceutically acceptable salt is an inorganic or organic acid salt, such as a 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 hydroxylate, tris(hydroxymethyl)aminomethane (TRIS, tromethamine), or N-methyl-glucamine salt.

In any embodiment of any method and use disclosed herein, the compound of formula (I), or a hydrate thereof, or an isotopically substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is administered before, simultaneously with, or after, 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 systemic radioisotopes), endocrine therapy, biologic response modifiers (such as interferons, interleukins, and tumor necrosis factor), hyperthermia and cryotherapy, medications to mitigate any adverse effects (such as antiemetics) and other approved chemotherapy drugs, steroid toxins (such as vincristine, vinpoxetine, vinorelbine, and paclitaxel), podophyllotoxins (such as etofoside, irinotecan, and vopotex), nitrosoureas (such as vastatin and lomustine), inorganic ions (such as cisplatin and carboplatin), enzymes (such as asparaginase), hormones (such as tamoxifen, leucine, flutamide, and megestrol acetate), Glivir™, doxorubicin, dexamethasone, and cyclophosphamide.

In any of the treatment methods disclosed herein, in some embodiments, the subject is a subject in need of such treatment. In various embodiments, the subject is a human. In some embodiments, the subject is a non-human, such as a veterinary animal (e.g., pigs, cows, sheep, horses, dogs, and cats) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

In any use of the compounds disclosed herein for treating a condition in a subject, in some embodiments, the subject is a subject in need of such treatment. In various embodiments, the subject is a human. In some embodiments, the subject is a non-human, such as a veterinary animal (e.g., pigs, cows, sheep, horses, dogs, and cats) or a zoo or laboratory animal (e.g., any non-human primate or mammal).

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

The following examples are provided to illustrate, but not to limit, the methods and formulations of the present invention. Other modifications and adaptations that are obvious to those skilled in the art and that are normally encountered in clinical treatment, are within the spirit and scope of the present invention.

Embodiment

All reagents used were of commercial quality, and solvents were dried and purified according to standard methods. Mass spectroscopy was performed using an electrospray single-quadrupole mass spectrometer. ^1H NMR spectra were recorded at 400 MHz. Chemical shifts are reported in ppm downfield from TMS (0.00 ppm) as the internal standard, and coupling constants (J ) are reported in Hz.

Example 1 7-((4-(2-Fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one

a) Preparation of 4-Methyl-1H-pyrazole-5-carbonyl chloride: Dissolve 4-methyl-1H-pyrazole-5-carboxylic acid (2.0 g, 15.9 mmol) in _SOCl₂ (20 mL) and stir at 80°C under nitrogen for 16 hours. Concentrate under reduced pressure to obtain the desired product (2.2 g, off-white solid, 91.7% yield).

b) Preparation of N-(3-bromo-2,6-difluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide: Dissolve 3-bromo-2,6-difluoroaniline (2.5 g, 16.7 mmol) in THF (20 mL). Add NaH (60% in mineral oil, 2.776 g, 69.4 mmol) at 0°C and stir under nitrogen at 0°C for 30 minutes. Then, add 4-methyl-1H-pyrazole-5-carbonyl chloride (2.0 g, 13.9 mmol) at 0°C. After the reaction is complete, slowly pour the reaction mixture into ice water (100 mL) and extract with ethyl acetate (200 mL × 3). The combined organic phases were washed with saturated brine (100 mL x 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 desired product (2.7 g, gray solid, yield: 67.0%). MS (ESI): 315.90 [M+H] ⁺ .

c) Preparation of 7-bromo-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one: To a solution of N-(3-bromo-2,6-difluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide (2.7 g, 8.6 mmol) in DMSO (27 mL) was added _K₂CO₃ (2.4 g, 17.2 mmol). The _mixture was stirred at 120°C overnight under nitrogen. After the reaction was complete, the reaction mixture was poured into ice-cold brine and filtered to obtain the desired product (2.5 g, white solid, yield: 86.8%). MS (ESI): 293.95 [M-1] ^- .

d) Preparation of 6-fluoro-7-(hydroxymethyl)-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one: To a solution of 7-bromo-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (500.0 mg, 1.7 mmol) in dioxane (10 mL) were added (Bu) ₃ SnCH ₂ OH (816.3 mg, 2.55 mmol) and Xphos Pd G2 (133.2 mg, 0.2 mmol) at room temperature. The resulting mixture was stirred at 90 ^° C under nitrogen for 3 hours. After the reaction, aqueous KF solution (1 M, 20 mL) was added at room temperature, and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was filtered and extracted with EA (500 mL × 3). The collected organic phase was washed with saturated brine (100 mL × 2), dried over anhydrous _Na₂SO₄ , _and concentrated under reduced pressure to remove the solvent. The crude product was slurried with a mixture of PE and EA (v/v = 1:1), and 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] ^- .

e) Preparation of 7-(Bromomethyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one: A solution of 6-fluoro-7-(hydroxymethyl)-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (500.0 mg, 2.0 mmol) in HBr (48% aqueous solution, 5 mL) was stirred at 80 ^° C for 1 hour. After the reaction, the reaction mixture 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] ^- .

f) Preparation of 7-((4-(2-fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one: 7-(Bromomethyl)-6-fluoro-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (200.0 mg, 0.67 mmol), 6-fluoro-N-methyl-5-(piperazin-1-yl)pyridinamide (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, and 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%).

Implementation Examples Compounds MW LC-MS (ESI) ¹ H NMR (400 MHz) 1 467.48 468.20 [M+H] ⁺ DMSO-d ₆ : δ 8.36 (d, J = 4.9 Hz, 1H), 7.89 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.55 – 7.49 (m, 1H), 7.30 – 7.24 (m, 1H), 3.63 (s, 2H), 3.18 – 3.06 (m, 4H), 2.72 (d, J = 4.8 Hz, 3H), 2.60 – 2.51 (m, 4H), 2.40 (s, 3H).

Examples 2-5

The compounds of Examples 2-5 were prepared using a synthetic method similar to that of Example 1 (Reaction Scheme 1). The results are shown below. Embodiment Compound MW LC-MS (ESI) ¹ H NMR (400 MHz) 2 474.5 475.15 [M+H] ⁺ DMSO-d ₆ : δ 11.76 (s, 1H), 8.59-8.52 (m, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.90 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.27 (t, J = 7.7 Hz, 1H), 3.66 (s, 2H), 3.29-3.19 (m, 4H), 2.74 (d, J = 4.8 Hz, 3H), 2.65 – 2.53 (m, 4H), 2.40 (s, 3H). 3 493.52 494.20 [M+H] ⁺ DMSO-d ₆ : δ 11.75 (br, 1H), δ 8.33 (d, J = 5.2 Hz, 1H), 7.91 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.53 (dd, J = 8.0,8.0 Hz, 1H), 7.28 (t, J = 6.8 Hz, 1H), 3.65 (s, 2H), 3.14-3.11 (m, 4H), 2.83-2.82 (m, 1H), 2.59-2.57 (m, 4H), 2.42 (s, 3H), 0.64-0.62 (m, 4H). 4 521.45 522.35 [M+H] ⁺ DMSO-d ₆ : δ 8.50 (s, 1H),8.40-8.33 (m, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.52 (dd, J = 10.6, 8.0 Hz, 1H), 7.36 (dd, J = 8.6, 6.7 Hz, 1H), 3.67 (s, 2H), 3.15 – 3.12 (m, 4H), 2.72 (d, J = 4.8 Hz, 3H), 2.59-2.54 (m, 4H). 5 467.48 468.10 [M+H] ⁺ DMSO-d ₆ : δ 11.98 (s, 1H), 8.45 – 8.38 (m, 1H), 8.22 (d, J = 3.8 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.35 (t, J = 8.1 Hz, 1H), 3.70 (s, 2H), 2.99 – 2.89 (m, 4H), 2.79 (d, J = 4.1 Hz, 3H), 2.66 – 2.56 (m, 4H), 2.46 (s, 3H).

Example 6 7-((4-(2-Fluoro-6-(methylaminoformyl)pyridin-3-yl)piperazin-1-yl)methyl)-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one

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 afford the desired product (22.6 g, white solid, yield: 66%).

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

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

d) Preparation of 7-bromo-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one: To a solution of 7,9-dibromo-3,6-difluoropyrazolo[1,5-a]quinoxalin-4(5H)-one (44.0 g, 116.1 mmol) in DMF (420 mL) and _H₂O (70 mL) was added 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) under nitrogen. The mixture was stirred at 120°C overnight. The mixture was then cooled to room temperature. Water (600 mL) was added, and the resulting mixture was filtered. The solid was washed three times with (DCM/MeOH = 15/1, 200 mL) and concentrated under reduced pressure. The crude product was extracted with EA (2000 mL x 3). The organic phase was collected and concentrated to obtain the desired product (17 g, yellow solid, yield: 49%). MS (ESI): 299.90 [M+H] ⁺ .

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

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

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

Implementation Examples Compounds MW LC-MS (ESI) ¹ H NMR (400 MHz) 6 471.44 472.15 [M+H] ⁺ DMSO-d ₆ : δ 8.40 (q, J = 4.8 Hz, 1H), 8.22 (d, J = 3.7 Hz, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 7.0 Hz, 1H), 7.56 (dd, J = 10.4, 8.2 Hz, 1H), 7.34 (dd, J = 8.4, 7.1 Hz, 1H), 3.68 (s, 2H), 3.21 – 3.11 (m, 4H), 2.76 (d, J = 4.8 Hz, 3H), 2.63 – 2.55 (m, 4H).

Example 7-11

The compounds of Examples 7-11 were prepared using a synthetic method similar to that of Example 1 (Reaction Scheme 1). The results are shown below. Embodiment Compound MW LC-MS (ESI) ¹ H NMR (400 MHz) 7 485.47 486.35 [M+H] ⁺ DMSO-d ₆ : δ 8.47 (t, J = 5.8 Hz, 1H), 8.22 (d, J = 3.7 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.56 (dd, J = 10.4, 8.1 Hz, 1H), 7.34 (dd, J = 8.8, 7.2 Hz, 1H), 3.68 (s, 2H), 3.27 - 3.23 (m, 2H), 3.19 - 3.13 (s, 4H), 2.63 - 2.48 (s, 4H), 1.08 (t, J = 7.2 Hz, 3H). 8 521.45 522.05 [M+H] ⁺ DMSO-d ₆ : δ 8.74 (t, J = 6.1 Hz, 1H), 8.22 (d, J = 3.8 Hz, 1H), 7.91-7.86 (m, 2H), 7.61 – 7.52 (m, 1H), 7.38 – 7.30 (m, 1H), 6.10 (tt, J = 56.6, 4.2 Hz, 1H), 3.68 (s, 2H), 3.62 (dt, J = 15.3, 5.1 Hz, 2H), 3.22-3.14 (m, 4H), 2.62-2.57 (m, 4H). 9 497.48 498.60 [M+H] ⁺ DMSO-d ₆ : δ8.38 (d, J = 4.8 Hz, 1H), 8.21 (d, J = 3.8 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.56 (dd, J = 10.4, 8.3 Hz, 1H), 7.36 - 7.29 (m, 1H), 3.68 (s, 2H), 3.18 - 3.12 (m, 4H), 2.88 - 2.80 (m, 1H), 2.64 - 2.56 (m, 4H), 0.67 - 0.62 (m, 4H). 10 487.90 488.55 [M+H] ⁺ DMSO-d ₆ : δ 8.39 (q, J = 5.0 Hz, 1H), 8.18 (d, J = 3.8 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.83 – 7.77 (m, 1H), 7.53 (dd, J = 10.7, 8.0 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 3.68 (s, 2H), 3.20-3.08 (m, 4H), 2.72 (d, J = 4.8 Hz, 3H), 2.64-2.52 (m, 4H). 11 467.48 468.05 [M+H] ⁺ DMSO-d ₆ : δ 11.04 (s, 1H), 8.42 (q, J = 5.3 Hz, 1H), 8.18 (d, J = 3.7 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.55 (dd, J = 10.6, 8.0 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 3.59 (s, 2H), 3.20-3.08 (m, 4H), 2.75 (d, J = 4.7 Hz, 3H), 2.60-2.56 (m, 4H), 2.47 (s, 3H).

Example 12

PARP1 and PARP2 chemiluminescent detection

A dilution of recombinant poly (ADP-ribosyltransferase) 1 and 2 (PARP1 and PARP2) (40 ng enzyme/well) and the test compound were added to a 96-well plate coated with the 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 incubated at room temperature for 30 minutes. Finally, ELISA ECL substrate was added, and the plate was read using an EnviSion instrument. The chemiluminescent signal was recorded, and the inhibitory rate of the test compound on PAPP1 and PARP2 enzymatic activity was calculated according to the following formula. 100%

XL Fit software was used to fit a sigmoidal dose-response curve equation to obtain the IC ₅₀ value. The curve equation was Y = 100 / (1 + 10^(logC - logIC ₅₀ )), where C is the compound concentration.

Table 1 summarizes the inhibitory effects (IC ₅₀ ) of the compounds of the present invention on PARP1 and PARP2 enzyme activities.

Table 1 Embodiment IC ₅₀ (nM) Ratio of PARP1 inhibition to PARP2 inhibition PARP1 PARP2 1 1.38 >10000 >7246 2 0.48 2504 5217 3 0.48 731.1 1523 4 0.50 67.13 134 5 1.06 2821.75 2662 6 0.53 462.44 873 7 0.24 431 1796 8 0.25 587 2348 9 0.23 314.97 1369 10 0.98 >10000 ＞10204 11 0.25 188 752

Compared with PARP2 enzyme, the compound of the present invention has a good selective inhibitory effect on PARP1 enzyme activity.

Example 13

The compound of the present invention inhibits the growth of BRCA mutant human breast cancer cells MDA-MB-436

After recovery, cells were cultured and passaged in complete medium (DMEM + 10% FBS + insulin + glutathione). When the cells reached approximately 80% confluency, gently aspirate the cells from the bottom of the culture dish using a 1 mL pipette, collect the cell suspension, and centrifuge at 500 rpm for 3 minutes. Discard the supernatant, resuspend the cells in complete medium, plate the appropriate number of cells onto culture dishes, and incubate in a 37°C, 5% _CO2 incubator. Cells were passaged until they were well-grown and approximately 80% confluent, at which point they were used for experiments. Centrifuge cells in the logarithmic growth phase and remove the supernatant. Resuspend the cells in fresh complete medium and count them. Seed the resuspended cells at 3000/well in a 96-well plate and culture overnight in a 37°C, 5% CO ₂ incubator. Compounds were prepared by adding 5 μL of 1000 μL of 120 μL of culture medium. Prepare 40 test compound solutions (25-fold dilution) Solution of the test compound. Mix the solution by vortexing. 0.1% dimethyl sulfoxide was used as a control.

The next day, take out the 96-well plate seeded with cells from the incubator and remove the culture supernatant. Then add 195 μL of fresh culture medium to each well, and then add 5 μL of the above-mentioned 40 The test compound solution was then incubated in a 37°C, 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 plates were shaken for 5 minutes before continuing to incubate. The absorbance was read on a multi-functional reader at either 450 nm or 650 nm (OD = absorbance at _{450 nm} - absorbance at _{650 nm} ).

Data were analyzed using GraphPad Prism 6.0. The inhibitory activity of the compounds on cell proliferation was plotted against cell viability and compound concentration. Cell viability (%) = (OD _compound - OD _background ) / (OD _DMSO - OD _background ) × 100. _{IC 50} values were fitted to a sigmoidal dose-response curve: Y = 100 / (1 + 10^(LogC - LogIC ₅₀ )), where C is the compound concentration.

Table 2 summarizes the inhibitory effect data (IC ₅₀ ) of the compounds on the growth of human breast cancer MDA-MB-436 cells.

Table 2 Embodiment IC ₅₀ (nM) 1 2.99 2 1.58 3 4.50 4 4.49 5 1.72 6 1.32 7 1.24 8 0.62 9 1.90 10 5.58 11 4.98

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

Example 14

Pharmacokinetic study of compounds in plasma and brain of mice after single oral administration

The compound of the present invention was prepared as a homogenous suspension in 0.5% methylcellulose/water and administered orally at 10 mg/kg to CD-1 (ICR) mice. Mice were euthanized by _CO₂ inhalation 0.5, 4, and 8 hours after administration, and blood and brain were collected from each mouse. Blood (approximately 0.1 mL per time point) was collected from the venous vein or other appropriate site of each mouse into a pre-chilled EDTA-K2 tube. After collection, the brain was washed with cold saline, wiped dry, 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, a dilution ratio of 5). Tissue homogenates will be kept at -60°C or lower prior to LC-MS/MS analysis. Compound concentrations in plasma and brain will be determined by LC-MS/MS.

The compound concentrations, calculated AUC _0-8h , and B/P ratios are summarized in Table 3.

Table 3 Embodiment Time point (h) Concentration in plasma (ng/mL) Concentration in brain (ng/g) AUC _0-8h in brain (h·ng/mL) * B/P ratio 1 0.5 4650 2087 16389 0.47 4 3970 2705 0.68 8 1793 1228 0.69 2 0.5 4097 1260 6037 0.31 4 3817 900 0.24 8 981 234 0.24 3 0.5 2313 1450 11359 0.64 4 2173 1982 0.91 8 872 722 0.83 4 0.5 18587 537 8541 0.03 4 36100 1377 0.04 8 38567 1158 0.03 5 0.5 18733 2128 14877 0.12 4 26567 2252 0.09 8 13967 1198 0.09 6 0.5 17002 1556 15031 0.09 4 14265 1966 0.14 8 10844 2273 0.21 7 0.5 4625 1864 12616 0.40 4 4887 1758 0.38 8 4910 1186 0.25 8 0.5 5909 1406 7610 0.24 4 3685 943 0.29 8 2983 673 0.26 9 0.5 1133 204 3490 0.16 4 2593 455 0.17 8 3897 688 0.18 10 0.5 4625 1864 12616 0.40 4 4887 1758 0.38 8 4910 1186 0.25 11 0.5 775 551 4656 0.71 4 1046 666 0.66 8 1313 533 0.44 Note: *Compound concentration-time relationship data were analyzed using WinNonlin software using a non-compartmental analysis, and AUC _0-8h was calculated using the linear-log trapezoidal method.

The compounds of the present invention have good exposure in the brain of mice.

Example 15

K _p,uu of compounds in mice

The ratio of unbound drug in the brain to unbound drug in plasma (Kp,uu) was determined using the following method.

Determination of free components in mouse plasma

On the day of the experiment, thaw mouse plasma under running cold tap water and centrifuge at 3220 × g for 5 minutes to remove any clots. Check and record the pH. Only use plasma within the pH range of 7.0 to 8.0.

Test compounds or the positive control, warfarin, were spiked into frozen mouse plasma at a final concentration of 2 μM. A 150 μL aliquot of the compound-spiked plasma sample was added to one side of a chamber in a 96-well balanced dialysis plate (HTD dialysis), and an equal volume of dialysis buffer was added to the other side. An aliquot of the plasma sample was collected before incubation and used as the _T0 sample for recovery calculation. Three incubations were performed. The plates were then incubated for 4 hours at 37°C in a humidified incubator with 5% _CO₂ . Following incubation, 50 μL samples were collected from both the plasma and buffer sides. Plasma samples were mixed with an equal volume of blank buffer; buffer samples were mixed with an equal volume of blank plasma. Matrix-matched samples were quenched with a stop solution containing an internal standard (IS). Samples were analyzed by LC-MS/MS. In the absence of a standard curve, test compound concentrations in plasma and buffer samples were determined based on the peak area ratio of the analyte to the IS.

Determination of free components in mouse brain homogenate

On the day of the experiment, CD-1 mouse brain homogenates were thawed in a warm water bath 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.

A 100 µL aliquot of compound-spiked brain homogenate was added to one side of a 96-well balanced dialysis plate (HTD dialysis), and an equal volume of dialysis buffer (100 mM sodium phosphate and 150 mM NaCl, pH 7.4 ± 0.1) was added to the other side. An aliquot of the brain homogenate sample was harvested before incubation and used as the _T0 sample for recovery calculation. Three incubations were performed.

The cells were then rotated in a humidified incubator at approximately 100 rpm at 37°C and 5% _CO₂ for 4 hours. Following incubation, 50 µL samples were collected from both the brain homogenate and buffer sides. The brain homogenate samples were mixed with an equal volume of buffer; the buffer samples were mixed with an equal volume of blank brain homogenate. Matrix-matched samples were quenched with a stop solution containing an internal standard.

Samples were analyzed by LC-MS/MS. Compound concentrations in brain homogenate and buffer samples were determined based on the peak area ratio of the analyte to the IS, without a standard curve.

Kp _,uu values were calculated using the following formula: Kp _,uu = ( _AUCbrain / _AUCplasma ) × (fu _{, brain} /fu _{, plasma} ), where _AUCbrain and _AUCplasma represent the _{AUC0-8h values} in plasma and brain, respectively, obtained in Example 14; fu _{, plasma} and fu _{, brain} represent the unbound percentage (%) in plasma and brain, respectively. The Kp _,uu values of these compounds in mice are summarized in Table 4.

Table 4 Embodiment f _{u, plasma} f _{u, brain} AUC _plasma (h·ng/mL) AUC _brain (h·ng/mL) K _p,uu 1 2.0 2.2 27171 16389 0.663 5 0.5 3.8 162344 14877 0.699 6 1.1 4.3 108733 15031 0.540 10 1.2 1.8 37396 12616 0.505

The tested compounds have good K _p,uu .

Example 16

Pharmacokinetic study of compounds after single oral administration in mice

An exemplary compound of the present invention was prepared as a homogeneous suspension in 0.5% methylcellulose/water and administered to CD-1 (ICR) mice by oral gavage at 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 after administration. Blood (approximately 0.1 mL per time point) was collected from the venous vein or other appropriate site of each mouse and placed into pre-chilled EDTA-K2 tubes. Compound concentrations were determined by LC-MS/MS.

The plasma concentration and time data were analyzed using WinNonlin software using a non-compartmental model, and pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. The pharmacokinetic parameters for the compound in mice are shown in Table 5, where the half-life (t½ ₎ represents the time required for half of the drug concentration to be eliminated; _Cmax represents the maximum drug concentration after administration; AUC0 _-t represents the area under the plasma concentration-time curve from time 0 to the final time; and AUC0 _-inf represents the area under the plasma concentration-time curve from time 0 to infinity.

Table 5 Embodiment t _1/2 (h) C _max (ng/mL ⁾ AUC _0-t (h·ng/mL) AUC _0-inf (h·ng/mL) 1 3.43 4520 37212 37529 3 8.05 618 6593 7998 5 26.6 17867 311006 677057 6 8.80 25678 371983 446442 8 5.26 6530 46656 48717 10 9.45 4793 57428 70053

The tested compounds showed good exposure in mouse plasma.

Example 17

Study on antitumor efficacy in the MDA-MB-436 subcutaneous human breast cancer model

Human breast cancer MDA-MB-436 cells were purchased from ATCC (HTB-130). Cells were cultured in RPMI-1640 medium supplemented with 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 were harvested during the exponential growth phase, counted, and plated.

0.2 mL (1 × 10 ⁷ cells) of tumor cell suspension containing 50% artificial Matrigel was subcutaneously inoculated into the right back of each mouse. Mice were randomized and administered drugs when the average tumor volume reached ~150 mm ³ .

After group dosing, animals were monitored daily. Body weight and tumor size were measured three times a week.

Monitor animal weight regularly as an indirect measure of toxicity.

Measure the tumor diameter three times a week using a vernier caliper. Tumor volume (mm ³ ) is calculated as: V = 0.5 × a × b ² , where a and b represent the major and minor diameters of the tumor, respectively.

Tumor growth inhibition rate (TGI) (%) was calculated based on tumor size, and the antitumor efficacy of the compound was evaluated using TGI (%). TGI (%) = [1-(Vdrug _{Group -} t - _Vdrug Group _-1 )/(Vvehicle _{Group -t} - _{Vvehicle Group -1} )] × 100, where _{Vdrug Group -1} and _{Vvehicle Group -1} refer to the average tumor volume of the compound-treated and vehicle-controlled groups, respectively, at the time of dosing (i.e., on the first day of dosing); Vdrug _{Group -t} and _{Vvehicle Group -t} refer to the average tumor volume of the compound-treated and vehicle-controlled groups, respectively, after dosing. When the TGI (%) value is greater than 100%, the tumor elimination rate (%) is calculated using the formula (Vdrug _{Group -1} - _{Vdrug Group -t} )/Vdrug _{Group -1} × 100.

Research -1

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

The grouping and dosing schedule are shown in Table 6.

Table 6 Group ^Na Compound therapy Dosage (mg/kg) Dosage volume (µL/g) ^{c Route} of administration Dosing ^frequency 1 8 solvent ^b -- 10 po QD Í28D 2 8 Compound 1 0.2 10 po QD Í 28D 3 8 Compound 1 1 10 po QDÍ28D 4 8 Compound 6 0.2 10 po QDÍ28D 5 8 Compound 6 1 10 po QDÍ28D Note: a. N is the number of animals per group, with 8 tumor-bearing nude mice per group; b. The vehicle was a 0.5% MC aqueous solution; c. The dosing volume was adjusted based on the mouse's body weight at 10 μL/g; if the body weight loss exceeded 15%, the dosing regimen was adjusted; po refers to oral administration; e. QD refers to once-daily dosing; dosing was continued for 28 days. NA means not applicable.

Results: 1) Mortality, Morbidity, and Weight Gain or Loss No significant weight loss, mortality, or any signs of morbidity were observed throughout the study. 2) Tumor Growth Inhibition and Tumor Regression Based on data from day 28, tumor growth inhibition, including tumor volume and TGI values, was observed in the treatment group compared to the control group (see Table 7).

Table 7 Group Compound therapy Tumor volume on day 28 (mm ³ ) ^a TGI (%) Tumor regression (%) 1 solvent 1161.61±99.42 NA NA 2 Compound 1, 0.2 mg/kg 771.55±37.64 38.57 NA 3 Compound 1, 1 mg/kg 405.52±31.89 74.89 NA 4 Compound 6, 0.2 mg/kg 139.03±7.42 101.32 9 5 Compound 6, 1 mg/kg 92.95±8.76 105.89 39 Notes: a. Mean ± SD; NA means not applicable.

In summary, all treatment groups demonstrated significant tumor growth inhibition compared to the vehicle control group. Compound 1 exhibited a good dose-dependent response 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.

During the study period, no significant weight loss was observed in any group, the animals behaved normally, and there were no deaths or any signs of morbidity, indicating that the compound was well tolerated in mice at the dose levels tested.

Research -2

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

The grouping and dosing schedule are shown in Table 8.

Table 8 Group ^Na Compound therapy Dosage (mg/kg) Dosage volume (µL/g) ^{c Route} of administration Dosing ^frequency 1 8 solvent ^b NA 10 po QD Í28D 2 8 Compound 5 0.1 10 po QD Í 28D 3 8 Compound 6 0.1 10 po QDÍ28D 4 8 Compound 6 1 10 po QDÍ28D 5 8 Compound 6 10 10 po QDÍ28D Note: a. N is the number of animals per group, with 8 tumor-bearing nude mice per group; b. The vehicle was a 0.5% MC aqueous solution; c. The dosing volume was adjusted based on the mouse's body weight at 10 μL/g; if the body weight loss exceeded 15%, the dosing regimen was adjusted; po refers to oral administration; e. QD refers to once-daily dosing; dosing was continued for 28 days. NA means not applicable.

Results: 1) Mortality, Morbidity, and Weight Gain or Loss No significant weight loss, mortality, or any signs of morbidity were observed throughout the study. 2) Tumor Growth Inhibition and Tumor Regression Based on data from day 28, tumor growth inhibition, including tumor volume and TGI values, was observed in the treatment group compared to the control group (see Table 9).

Table 9 Group Compound therapy Tumor volume on day 28 (mm ³ ) ^a TGI (%) Tumor regression (%) 1 solvent 948.08±90.41 NA NA 2 Compound 5, 0.1 mg/kg 256.12±15.74 86.64 NA 3 Compound 6, 0.1 mg/kg 188.2±19.47 95.20 NA 4 Compound 6, 1 mg/kg 120.71±6.81 103.67 19 5 Compound 6, 10 mg/kg 104.13±10.35 105.73 31 Notes: a. Mean ± SD; NA means not applicable.

In summary, all treatment groups demonstrated significant tumor growth inhibition compared to the vehicle control group. Compounds 5 and 6 demonstrated similar effects at a dose of 0.1 mg/kg. Compound 6 exhibited tumor regression at both 1 mg/kg and 10 mg/kg doses.

During the study period, no significant weight loss was observed in any group, the animals behaved normally, and there were no deaths or any signs of morbidity, indicating that compounds 5 and 6 were well tolerated in mice at the dose levels tested.

Example 18

Study on the antitumor efficacy of DLD-1 in a BRCA2 ^-/- human colorectal cancer subcutaneous epithelial model

The antitumor activity of compound 6 was evaluated in the DLD-1 BRCA2 ^-/- human colorectal cancer epithelial subcutaneous xenograft tumor model.

The laboratory of Beijing Aisiyipu Biotechnology Co., Ltd. used CRISPR/Cas9 technology to knock out BRCA2 in wild-type DLD-1 cells (purchased from ATCC) to generate DLD-1 BRCA2 ^-/- cells.

DLD-1 BRCA2 ^-/- cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 100 μg/mL Hygromycin B at 37°C in a 5% CO ₂ incubator. Cells were harvested during the exponential growth phase, counted, and plated.

Each mouse was subcutaneously inoculated with 0.1 mL (1×10 ⁵ cells) of a tumor cell suspension containing 50% artificial basement membrane at the right anterior shoulder site. Mice were randomly divided into groups and administered drugs when the average tumor volume reached ~175 mm ³ . Grouping and dosing schedules are shown in Table 10.

Table 10 Group ^Na Compound therapy Dosage (mg/kg) Dosage volume (µL/g) ^{c Route} of administration Dosing ^frequency 1 8 solvent ^b NA 10 po QD Í28D 2 8 Compound 6 0.2 10 po QD Í 28D 3 8 Compound 6 1 10 po QDÍ28D Note: a. N is the number of animals per group, with 8 tumor-bearing nude mice per group; b. The vehicle was a 0.5% MC aqueous solution; c. The dosing volume was adjusted based on the mouse's body weight at 10 μL/g; if the body weight loss exceeded 15%, the dosing regimen was adjusted; po refers to oral administration; e. QD refers to once-daily dosing; dosing was continued for 28 days. NA means not applicable.

After group dosing, animals were monitored daily. Body weight and tumor size were measured twice a week.

Monitor animal weight regularly as an indirect measure of toxicity.

Measure the tumor diameter three times a week using a vernier caliper. Tumor volume (mm ³ ) is calculated as: V = 0.5 × a × b ² , where a and b represent the major and minor diameters of the tumor, respectively.

Tumor growth inhibition rate (TGI) (%) was calculated based on tumor size, and the antitumor efficacy of the compound was evaluated using TGI (%). TGI (%) = [1-(Vdrug _{Group -} t - _Vdrug Group _-1 )/(Vvehicle _{Group -t} - _{Vvehicle Group -1} )] × 100, where _{Vdrug Group -1} and _{Vvehicle Group -1} refer to the average tumor volume of the compound-treated and vehicle-controlled groups, respectively, at the time of dosing (i.e., on the first day of dosing); Vdrug _{Group -t} and _{Vvehicle Group -t} refer to the average tumor volume of the compound-treated and vehicle-controlled groups, respectively, after dosing. When the TGI (%) value is greater than 100%, the tumor elimination rate (%) is calculated using the formula (Vdrug _{Group -1} - _{Vdrug Group -t} )/Vdrug _{Group -1} × 100.

Results: 1) Mortality, Morbidity, and Weight Gain or Loss No significant weight loss, mortality, or any signs of morbidity were observed throughout the study. 2) Tumor Growth Inhibition and Tumor Regression Based on the data on day 28, the tumor growth inhibition effect, including tumor volume and TGI values, compared to the control group is shown in Table 11.

Table 11 Group Compound therapy Tumor volume on day 28 (mm ³ ) ^a TGI (%) Tumor regression (%) 1 solvent 1100.61±100.63 NA NA 2 Compound 6, 0.2 mg/kg 174.20±31.06 100.05 0.27 3 Compound 6, 1 mg/kg 49.23±6.23 113.43 72 Notes: a. Mean ± SD; NA means not applicable.

In summary, compared with the vehicle control group, the compound 6-treated groups showed significant tumor growth inhibition and exhibited good dose dependence at doses of 0.2 mg/kg and 1 mg/kg.

During the study period, no significant weight loss was observed in any group, the animals behaved normally, and there were no deaths or any signs of morbidity, indicating that the compounds were well tolerated in mice at the current dosage levels.

Example 19

Study on the anti-tumor efficacy of MDA-MB-436-luc in the intracranial human breast cancer model

MDA-MB-436-luc tumor cells were cultured in vitro in DMEM supplemented with 10% fetal bovine serum and 1200 ng/mL puromycin at 37°C in a 5% _CO2 incubator. The medium was changed every 1-2 days. When the cells reached 80%-90% confluence, they were passaged using 0.25% trypsin-EDTA, with a maximum of 4-5 passages. Tumor cells in the logarithmic growth phase were used for inoculation of in vivo tumors.

After anesthesia with an intramuscular injection of Zotacil, the animals were positioned prone on an operating table. The skin on the top of their heads was disinfected with iodine and 75% alcohol, respectively. An approximately 0.5 cm incision was made along the midline of the head to expose the coronal and sagittal lines. A brain locator was used to locate the site 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 with a 25-gauge syringe needle. The microinjector needle was inserted vertically into the site to a depth of 3 mm. A 2 × 10 ⁵ / 2 μL suspension of MDA-MB-436-luc tumor cells was slowly injected (over approximately 1 minute). After the injection, the needle was retained for 1 minute. After removal, the needle hole was quickly sealed with medical bone wax and the wound was sutured.

After surgery, the analgesic meloxicam was administered subcutaneously at a dose of 5 mg/kg per animal. The antibiotic levofloxacin (0.1 mg/mL) was added to the animals' drinking water for 3-7 days.

Tumor growth was monitored by image analysis. Mice were randomly divided into groups and drug administration began when the average optical signal intensity (BLI, bioluminescent imaging) at the tumor site reached 20-30 photons/ ^sÍ106 .

Imaging analysis of tumor development

Mice were intraperitoneally injected with D-luciferin (15 mg/mL, 5 μL/g depending on animal weight) and anesthetized weekly with 1%-2% isoflurane. Approximately 10 minutes after D-luciferin injection, the animals were imaged using an IVIS Lumina III. Images were analyzed using Living Image software (Perkin Elmer), and the optical signal intensity within each animal's ROI (region of interest) was calculated. The bioluminescent imaging signal (photons/s) from the ROI was quantified and used as an indicator of tumor growth and anti-tumor activity.

The reduction in bioluminescence imaging (BLI) signal was calculated using the following formula (% reduction): BLI % reduction = (1-BLI _{treatment group} /BLI _{control group} ) × 100%, where BLI _{treatment group} and BLI _{control group} are the average BLI values of the treatment group and control group on the day after tumor cell inoculation, respectively.

Research -1

The antitumor activity of compound 6 was evaluated in the MDA-MB-436-luc human breast cancer intracranial xenograft model. The grouping and dosing schedule are shown in Table 12.

Table 12 Group ^Na Compound therapy Dosage (mg/kg) Dosage volume (µL/g) ^{c Route} of administration Dosing ^frequency 1 8 solvent ^b -- 10 po QD Í39D 2 8 Compound 6 1 10 po QD Í 42D 3 8 Compound 6 3 10 po QDÍ42D 4 8 Compound 6 10 10 po QDÍ42D Note: a. N is the number of animals per group, with 8 tumor-bearing nude mice per group; b. The vehicle was a 0.5% aqueous solution of MC; c. The dosing volume was adjusted based on the mouse body weight at 10 μL/g. Animals were euthanized if: 1) the animal showed a significant decrease in weight (emaciation); a significant decrease in weight > 20% with no recovery after drug withdrawal; or 2) the animal did not have adequate access to food and water. dpo refers to oral administration; e. Qd refers to once daily dosing; the vehicle control group and the treatment group were administered for 39 and 42 consecutive days, respectively. NA means not applicable.

Results: 1) Mortality, Morbidity, and Weight Gain or Loss Mice in the vehicle control group experienced significant weight loss and mortality on day 34 after tumor cell inoculation, likely due to intracranial tumor burden. No significant weight loss, mortality, or any signs of morbidity were observed during the 42-day treatment with Compound 6. 2) Tumor Growth Inhibition Based on the data from day 42, antitumor activity data, including tumor BLI values and % BLI reduction compared to the control group, are shown in Table 13.

Table 13 Group Compound therapy Average BLI (photons/sÍ10 ⁶ ) ^a BLI reduction (%) ^b 1 solvent 807 ± 180 NA 2 Compound 6, 1 mg/kg 8 ± 3 99.0 3 Compound 6, 3 mg/kg 6 ± 2 99.3 4 Compound 6, 10 mg/kg 3 ± 0 99.6 Notes: a. Mean ± SD; b. Compared to the control group (i.e., vehicle group). NA means not applicable.

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

Research -2

The anti-tumor effects of Compounds 5 and 6 were evaluated in the MDA-MB-436-luc human breast cancer intracranial xenograft model. The groups and dosing schedules are shown in Table 14.

Table 14 Group ^Na Compound therapy Dosage (mg/kg) Dosage volume (µL/g) ^{c Route} of administration Dosing ^frequency 1 8 solvent ^b -- 10 po QD Í28D 2 8 Compound 5 1 10 po QD Í 28D 3 8 Compound 6 0.2 10 po QDÍ28D 4 8 Compound 6 0.5 10 po QDÍ28D 5 8 Compound 6 1 10 po QDÍ28D Note: a. N is the number of animals per group, with 8 tumor-bearing nude mice per group; b. The vehicle was 0.5% MC aqueous solution; c. The dosing volume was adjusted based on the mouse body weight at 10 μL/g. Animals were euthanized if 1) the animal showed a significant decrease in weight (emaciation); a significant decrease in weight > 20% with no recovery after drug withdrawal; or 2) the animal did not have adequate access to food and water. dpo refers to oral administration; e. Qd refers to once daily dosing; dosing was continued for 28 days. NA means not applicable.

Results: 1) Mortality, Morbidity, and Weight Gain or Loss One mouse in the vehicle group experienced a significant weight loss (>20%), and two mice experienced significant weight loss on days 24 and 28, respectively. These three mice were euthanized. One mouse in Group 3 (0.2 mg/kg Compound 6-treated group) died. Autopsy revealed severe food accumulation in the intestine and no brain tumors. No significant weight loss, mortality, or any signs of morbidity were observed in Groups 2, 4, and 5. 2) Tumor Growth Inhibition Based on the data from day 28, antitumor activity data, including tumor BLI values and BLI reduction (%) compared to the control group, are shown in Table 15.

Table 15 Group Compound therapy Average BLI (photons/sÍ10 ⁶ ) ^a BLI reduction (%) ^b 1 solvent 642±140 NA 2 Compound 5, 1 mg/kg 10±1 101.9% 3 Compound 6, 0.2 mg/kg 11±2 102.7% 4 Compound 6, 0.5 mg/kg 12±3 102.6% 5 Compound 6, 1 mg/kg 15±4 102.5% Notes: a. Mean ± SD; b. Compared to the control group (i.e., vehicle group). NA means not applicable.

In summary, all treatment groups showed significant tumor growth inhibition compared with the vehicle group.

Example 20

Brain permeability of SD rats after oral administration of radiolabeled compound 6

Male and female Sprague Dawley (Sprague Dawley) rats were administered [ ¹⁴ C] Compound 6 by oral gavage at 5 mg/100 µCi/kg. The distribution of radiolabeled Compound 6 in the blood, plasma, and tissues was measured 1, 6, 12, and 24 hours after administration. The total radioactivity in plasma and whole brain is shown in the table below.

Table 16 male organization Total radioactivity (ng Eq./g) Parameters 001h 006h 012h 024h _Tmax (h) C _max (ng Eq./g) AUC _0-last (h∙ng Eq./g) average value average value average value average value plasma 1587 1948 1139 206 6 1948 26962 Whole Brain 961 1066 650 120 6 1066 15316 Sexuality organization Total radioactivity (ng Eq./g) Parameters 001h 006h 012h 024h _Tmax (h) C _max (ng Eq./g) AUC _0-last (h∙ng Eq./g) average value average value average value average value plasma 3340 2250 2210 530 1 3340 45465 Whole Brain 1519 1024 907 186 1 1519 19468

The results showed that compound 6 had good brain penetration.

Example 21

Pharmacokinetic study of compound 6 in plasma and brain of rats after single oral administration

Compound 6 was prepared as a homogenous suspension in 0.5% methylcellulose/water and administered to SD rats by oral gavage at 5 mg/kg. Blood (approximately 0.2 mL at each time point) was collected from the cervical vein or other appropriate site of each rat into pre-chilled EDTA-K2 tubes. Rats were euthanized by _CO₂ inhalation 1, 4, and 8 hours after administration, then perfused with saline, and the brain was collected from each rat. After collection, the brain was washed with cold saline, wiped dry, 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, a dilution ratio of 5). Tissue homogenates were kept at -60 ^° C or lower until analysis by LC-MS/MS. The concentrations of compound 6 in plasma and brain were determined by LC-MS/MS.

The concentrations, calculated AUC _0-8h , and B/P ratios of Compound 6 are summarized in the table below.

Table 17 Time point (h) Concentration in plasma (ng/mL) Concentration in brain (ng/g) AUC _0-8h in brain (h·ng/g) * B/P ratio 1 2497 338 4003 0.14 4 3057 562 0.18 8 2773 680 0.25 Note: *Compound concentration-time relationship data were analyzed using WinNonlin software using a non-compartmental analysis, and AUC _0-8h was calculated using the linear-log trapezoidal method.

The results showed that compound 6 had good exposure in the rat brain.

Although the present invention has been fully described, it will be understood by those skilled in the art that the same may be practiced within a wide range of equivalent conditions, formulations, and other parameters without affecting the scope of the present invention or any of its embodiments. All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

without

without.

Claims (10)

A compound having the following structure (I) or a pharmaceutically acceptable salt thereof: (I) wherein _R1 is selected from halogen, alkyl and halogenated alkyl; _R2 is selected from halogen and alkyl; _R3 is selected from halogen, alkyl and cyano; _R4 is selected from alkyl, halogenated alkyl and cycloalkyl. The compound or pharmaceutically acceptable salt thereof as described in claim 1, wherein: (a) R ₁ is a halogen, a C _1-3 alkyl group, or a halogenated C _1-3 alkyl group, or (b) R ₂ is a halogen or a C _1-3 alkyl group, or (c) R ₃ is a halogen, a C _1-3 alkyl group, or a cyano group, or (d) R ₄ is a C _1-3 alkyl group, a halogenated C _1-3 alkyl group, or a C _3-4 cycloalkyl group, or any combination of (a) to (d). The compound or pharmaceutically acceptable salt thereof as described in claim 1, wherein: (e) R ₁ is F, methyl or trifluoromethyl, or (f) R ₂ is F, Cl or methyl, or (g) R ₃ is F, methyl or cyano, or (h) R ₄ is C _1-3 alkyl, C _1-3 haloalkyl or C _3-4 cycloalkyl, or any combination of (e) to (h). The compound or a pharmaceutically acceptable salt thereof as described in claim 1, wherein all of R ₁ , R ₂ , R ₃ and R ₄ in the compound are defined in any one of rows 1 to 11 of the following table: R ₁ R ₂ R ₃ R ₄ 1 CH ₃ F F CH ₃ 2 CH ₃ F CN CH ₃ 3 CH ₃ F F Cyclopropyl 4 CF ₃ F F CH ₃ 5 F F CH ₃ CH ₃ 6 F F F CH ₃ 7 F F F CH ₂ CH ₃ 8 F F F _{CH2CHF2 ​} 9 F F F Cyclopropyl 10 F Cl F CH ₃ 11 F CH ₃ F CH ₃ The compound or pharmaceutically acceptable salt thereof as described in 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 . Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in a subject. The use as described in claim 6, wherein the subject has a central nervous system (CNS) tumor, and the central nervous system tumor is selected from meningioma, meningeal sarcoma, ependymoma, astrocytoma, neuroglioma, glioblastoma, pinealoma, invasive pituitary tumor, pituitary carcinoma, germ cell tumor, sarcoma, primary central nervous system lymphoma, brain stem glioma, pituitary adenoma, anaplastic astrocytoma, mixed glioma, primitive neuroectodermal tumor, hemangioblastoma, vestibular schwannoma, chordoma, spinal neurofibroma, lymphoma, optic glioma and dysembryonic neuroepithelial tumor. Use of the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof for preparing a medicament for selectively inhibiting PARP1 activity relative to PARP2 activity in a subject. The use of claim 8, wherein the ratio of PARP1 activity inhibition to PARP2 activity inhibition in the subject is between 750 and 15,000. A pharmaceutically acceptable composition comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable diluent, carrier or formulator.