Dual PARP / HDAC inhibitor for treating breast cancer, ovarian cancer, and prostate cancer

Abstract

A method for treating breast cancer, ovarian cancer, or prostate cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1- yl)pyrimidin-5-yl)-N-hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

Description

[0001] DUAL PARP / HDAC INHIBITOR FOR TREATING BREAST CANCER, OVARIAN CANCER, AND PROSTATE CANCER

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of US Patent Application No. 63 / 575232, filed April 5, 2024, expressly incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] Poly-(ADP-ribose)-polymerase (PARP) proteins catalyze PARylation of cellular proteins using an ADP (adenosine diphosphate)-ribose subunit of nicotinamide adenine dinucleotide (NAD+) as the donor. The human genome encodes 17 PARP enzymes where at least PARP 1-3 has critical functions in DNA repair, PARP1 being the best characterized. PARP1 is essential for the repair of single-strand DNA breaks (SSBs), which is the most common type of breakpoint lesion in cellular DNA. When cells encounter SSBs, PARP1 binds the lesion and initiates a PARylation cascade of itself and histones embedded in the chromatin surrounding the SSB lesion. This PARylation event serves as a signal to recruit the SSB repair machinery to patch the lesions before and during DNA replication in the S-phase of the cell cycle. Efficient SSB repair is important to prevent replication stress and the more severe double-strand break (DSB) lesions that occur in S-phase when unrepaired SSB lesions collide with the replication forks. DSB lesions in S-phase are mainly repaired by homologous recombination (HR) that relies on proteins such as BRCA1 and BRCA2. Deleterious mutations in BRCA1 / 2 are found in subsets of breast, ovarian, and prostate tumors, and sporadically in other solid tumor indications. These HR-deficient tumors are indirectly dependent on proficient PARP enzyme activity to avoid accumulation of catastrophic DSBs in S-phase and initiation of cell death. This dependency has paved the way for PARP inhibition as a therapeutic strategy to create synthetic lethality in tumor cells with BRCAl / 2-deficiencies.

[0006] There are currently four approved PARP inhibitors in the clinic being olaparib (approved in 2014), rucaparib (approved in 2016), niraparib (approved in 2017), and talazoparib (approved in 2018). These PARP inhibitors have been widely deployed in cancers with defects in HR DNA repair activity caused by BRCA1 / 2 mutations. Encouraged by the success of PARP inhibitors in BRCAl / 2-mutated cancers, research attention has been expanded towards cancer sub-types where HR repair is compromised due to molecular events other than BRCA1 / 2 mutations. For example, tumors with mutations in RAD51, an enzyme acting downstream of BRCA1 / 2 in the HR repair pathway, are also sensitive to PARP inhibition. This concept is commonly referred to as ‘BRCAness’ and includes all events that mimic BRCA1 / 2 loss in the context of HR repair.

[0007] In HR-proficient cancers, the state of BRCAness can be mimicked pharmacologically by inhibition of proteins that impact BRCA1 / 2 expression. This potentially invites opportunities to broaden the use of PARP inhibitors beyond current clinical practice. For example, impairing dynamic chromatin events related to DNA replication and repair such as histone acetylation can induce pharmacological BRCAness through indirect regulation of HR components. Recent studies in leukemia, breast cancer, liver cancer, glioblastoma, prostate cancer, and anaplastic thyroid cancer models demonstrated suppression of HR activity with HD AC inhibition that further supports the synergistic potential of HD AC and PARP inhibition.

[0008] Histone deacetylases (HDACs) play a major role in DNA repair and inhibition of HDACs has been shown to reduce tumor growth. PARPi combined with HDACi has shown synergistic efficacy in pre-clinical studies in various cancers, and a clinical trial of olaparib and vorinostat against metastatic breast cancer is ongoing. However, combination therapies can be limited clinically due to overlapping toxicities and differing pharmacokinetics.

[0009] Despite the advances noted above in the development of individual therapeutic agents for treating cancers, a need exists for improved therapeutic agents, including combination therapies, for treating cancers. The present invention seeks to fulfill this need and provides further related advantages.

[0010] SUMMARY

[0011] In one aspect, the disclosure provides a method for treating breast cancer, ovarian cancer, or prostate cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4- dihydrophthalazin-l-yl)methyl)benzoyl)piperazin-l-yl)pyrimidin-5-yl)-N- hydroxyacrylamide (Compound A), or a pharmaceutically acceptable salt thereof. In one embodiment, the disclosure provides a method for treating breast cancer comprising administering to a subject in need thereof a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof. In another embodiment, the disclosure provides a method for treating ovarian cancer comprising administering to a subject in need thereof a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof. In a further embodiment, the disclosure provides a method for treating prostate cancer comprising administering to a subject in need thereof a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.

[0012] In a related aspect, the disclosure provides Compound A, or a pharmaceutically acceptable salt thereof, for use in the treatment of breast cancer, ovarian cancer, or prostate cancer in a subject. In one embodiment, the disclosure provides Compound A, or a pharmaceutically acceptable salt thereof, for use in the treatment of breast cancer in a subject. In another embodiment, the disclosure provides Compound A, or a pharmaceutically acceptable salt thereof, for use in the treatment of ovarian cancer in a subject. In a further embodiment, the disclosure provides Compound A, or a pharmaceutically acceptable salt thereof, for use in the treatment of prostate cancer in a subject.

[0013] In a further aspect, the disclosure provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and Compound A, or a pharmaceutically acceptable salt thereof, for treating breast cancer, ovarian cancer, or prostate cancer in a subject. In one embodiment, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and Compound A, or a pharmaceutically acceptable salt thereof, for treating breast cancer in a subject. In another embodiment, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and Compound A, or a pharmaceutically acceptable salt thereof, for treating ovarian cancer in a subject. In a further embodiment, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and Compound A, or a pharmaceutically acceptable salt thereof, for treating prostate cancer in a subject.

[0014] DESCRIPTION OF THE DRAWINGS

[0015] The foregoing aspects and attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

[0016] FIG. 1 is a schematic illustration of a proposed mechanism of action of dual PARP / HDAC inhibitor compounds (kt-3OOO compounds).

[0017] FIG. 2 illustrates PARP1 inhibitory activity of Compound A and shows that the IC50 value of Compound A against PARP1 is comparable or better than olaparib.

[0018] FIG. 3 illustrates the PARP2 inhibitory activity of Compound A and shows that the IC50 value of Compound A against PARP2 is comparable or better than olaparib.

[0019] FIG. 4 illustrates that Compound A inhibits PARylation (PAR formation) comparable to olaparib.

[0020] FIG. 5 illustrates that Compound A inhibits HD AC activity with an IC50 greater than approved HD AC inhibitor vorinostat alone. FIG. 6A compares cell viability for breast cancer cell line SUM149PT (BRCA- deficient) treated with Compound A and olaparib.

[0021] FIG. 6B compares cell viability for breast cancer cell line MDA-MB-231 (BRCA- proficient) treated with Compound A and olaparib.

[0022] FIG. 7A compares cell viability for ovarian cancer cell line UWB1.289 (BRCA- deficient) treated with Compound A and olaparib.

[0023] FIG. 7B compares cell viability for ovarian cancer cell line UWB1.289+BRCA1 (BRCA-proficient) treated with Compound A and olaparib.

[0024] FIG. 8A compares cell viability for prostate cancer cell line LNCaP treated with Compound A and olaparib.

[0025] FIG. 8B compares cell viability for prostate cancer cell line 22RV1 treated with Compound A and olaparib.

[0026] DETAILED DESCRIPTION

[0027] HD AC inhibition has been shown to induce pharmacological BRCAness in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors. The present disclosure provides a bifunctional PARP inhibitor, (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-l - yl)methyl)benzoyl)piperazin-l -yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) with dual activity towards PARP1 / 2 and HDAC enzymes in breast cancer cells, ovarian cancer cells, and prostate cancer cells. Compared to the FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, Compound A displayed enhanced cytotoxicity in breast cancer, ovarian cancer, and prostate cancer models.

[0028] In certain aspects, the disclosure provides methods for treating breast cancer, ovarian cancer, or prostate cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4- dihydrophthalazin-l-yl)methyl)benzoyl)piperazin-l-yl)pyrimidin-5-yl)-N- hydroxyacrylamide (Compound A), or a pharmaceutically acceptable salt thereof. In a related aspect, the disclosure provides (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4- dihydrophthalazin-l-yl)methyl)benzoyl)piperazin-l-yl)pyrimidin-5-yl)-N- hydroxyacrylamide (Compound A), or a pharmaceutically acceptable salt thereof, for use in the treatment of breast cancer, ovarian cancer, or prostate cancer in a subject.

[0029] In other aspects, the disclosure provides pharmaceutical compositions for the above-noted uses. In these aspects, the pharmaceutical composition comprises (E)-3-(2- (4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-l-yl)methyl)benzoyl)piperazin-l- yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0030] As used herein, “Compound A” refers to (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4- dihydrophthalazin-l-yl)methyl)benzoyl)piperazin-l-yl)pyrimidin-5-yl)-N- hydroxyacrylamide, having formula (I): or a tautomer thereof.

[0031] As described herein, the present disclosure provides methods for using Compound A, or pharmaceutically acceptable salts thereof. It will be appreciated that the methods described herein also include the use of prodrugs of Compound A. Prodrugs of Compound A include derivatives of Compound A that release Compound A after their administration.

[0032] FIG. 1 is a schematic illustration of a proposed mechanism of action of Compound A, a dual PARP / HDAC inhibitor compound.

[0033] The following describes the usefulness of Compound A for treating breast, ovarian, and prostate cancers.

[0034] Pharmacological BRCAness can potentially offer a path to take PARP inhibition beyond the BRCA1 / 2 mutation space and counter potential resistance to PARPi therapy. Epigenetic modifiers such as HDACs, as well as DNA and histone methyltransferases are attractive targets for induced BRCAness in BRCA1 / 2 proficient cancer scenarios. At present, four clinical trials using PARPi (PARP inhibitor) in combination with the HDACi (HDAC inhibitor) vorinostat (NCT03259503 and NCT03742245), the DNA methyltransferase inhibitor decitabine (NCT02878785), and the EZH2 histone methyltransferase inhibitor SHR2554 (NCT04355858), are currently ongoing.

[0035] FDA has approved three pan-HDACi drugs (vorinostat, belinostat, and panobinostat) and one HDACl / 2-selective HDACi (romidepsin) for treatment of hematological cancers. Histone acetylation attenuates chromatin structure and plays a critical role in recognition and repair of DNA lesions. HDACi-induced downregulation of key HR proteins including

[0036] BRCA1, BRCA2, and RAD51 has been established in a variety of cancer types, and HDACi treatment sensitizes cancer cells to PARPi. This corroborative activity of HDAC and PARP inhibition is particularly interesting in the context of HR-proficient cancer types where PARPi therapy has limited effect on its own. However, dose-limiting toxicity with HDACi therapy is not uncommon in solid tumor cancers and has been preventing some therapeutic effects as stand-alone and in treatment combinations, in for example breast cancer and sarcomas. The HDACi ingredient must be carefully adjusted to prevent overlapping toxicity events arising from the combination with other therapeutic moieties, which can be challenging when working with different pharmacokinetics profdes.

[0037] The present disclosure provides a bifunctional PARP-HDAC single-molecule inhibitor, Compound A, in breast, ovarian, and prostate cancer models to evaluate the potential benefit of combined PARP-HDAC inhibition over stand-alone PARPi or HDACi treatments. Compound A has similar PARPi activity as olaparib and slightly lower HDACi activity than vorinostat. However, the dual activity of Compound A is 7-170 times more cytotoxic to breast, ovarian, and prostate cancer cells than olaparib.

[0038] Combining PARP and HDAC inhibition into one single molecule offers a convenient way to prevent resistance to PARPi therapy. For example, breast, ovarian, and prostate cancers and many other solid tumor indications epigenetically suppress expression of the tumor suppressor gene Schlafen 11, which leads to resistance to DNA damageinducing agents, including PARPi therapy. Important here, HDACi treatment prompts reexpression of SLFN1 1 and re-sensitization to PARPi.

[0039] Combination therapies can work in a synergistic or additive manner by simultaneously targeting different pathways in cells. Unfortunately, combination therapies that include chemotherapeutic agents can be toxic to patients and often have to be administered sequentially in clinical settings, sometimes with reduced biological efficacy. This offers a powerful rationale for the development of a dual-activity small molecule such as Compound A.

[0040] In summary, the present disclosure provides a single-molecule PARP-HDAC inhibitor, Compound A, with improved cytotoxicity and DNA damage activity as compared to PARPi and HDACi alone.

[0041] A bi-specific compound dual activity against PARP1 / 2 and HD AC enzymes

[0042] Through medicinal chemistry cycles, the present disclosure provides a smallmolecule inhibitor (Compound A) with dual activity against PARP1 / 2 and HDACs. In vitro activity assay kits were used to determine inhibition of PARPI, PARP2 and HD AC by Compound A compared to FDA-approved PARP inhibitor olaparib and HD AC inhibitor vorinostat. A wide concentration range of each compound was used to determine ICso values. The PARPI and PARP2 inhibitory activities of Compound A were comparable to olaparib, with ICso values for Compound A at 0.338 nM and 2.19 nM, respectively (FIGS. 2 and 3). To further validate the ability of Compound A to inhibit PARP1 / 2 activity, a cellular PAR synthesis assay was used to determine the level of PAR formation. Comparable to olaparib, an ICso of 1.39 nM was detected for the inhibition of PAR formation in cells treated with Compound A (FIG. 4). Compound A had an ICso value of 1.89 pM while vorinostat was about 40-fold lower at 0.05 pM (FIG. 5). These data indicate that Compound A is able to inhibit both PARP1 / 2 and HD AC enzymes. Breast. ovarian, and prostate cancer cells are highly sensitive to dual PARP1 / 2 and

[0043] HD AC inhibition

[0044] In order to investigate the effect of Compound A in cell growth, cell viability assays were performed in breast, ovarian, and prostate cancer cell lines.

[0045] FIG. 6A compares cell viability for breast cancer cell line SUM149PT (BRCA- deficient) treated with Compound A and olaparib. Compound A shows a 7-fold lower ICso compared to olaparib.

[0046] FIG. 6B compares cell viability for breast cancer cell line MDA-MB-231 (BRCA- proficient) treated with Compound A and olaparib. Compound A shows a 61 -fold lower ICso compared to olaparib.

[0047] FIG. 7A compares cell viability for ovarian cancer cell line UWB1.289 (BRCA- deficient) treated with Compound A and olaparib. Compound A shows a 113-fold lower ICso compared to olaparib.

[0048] FIG. 7B compares cell viability for ovarian cancer cell line UWB1.289+BRCA1 (BRCA-proficient) treated with Compound A and olaparib. Compound A shows a 170-fold lower ICso compared to olaparib.

[0049] FIG. 8A compares cell viability for prostate cancer cell line LNCaP treated with Compound A and olaparib. Compound A shows a 20-fold lower ICso compared to olaparib.

[0050] FIG. 8B compares cell viability for prostate cancer cell line 22RV1 treated with Compound A and olaparib. Compound A shows a 35-fold lower ICso compared to olaparib.

[0051] Taken together, the data demonstrates potent inhibitory effect of Compound A in breast, ovarian, and prostate cancer cells compared to FDA-approved PARP inhibitors.

[0052] Pharmaceutical Compositions

[0053] The pharmaceutical compositions of the present invention include Compound A as an active ingredient, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, and optionally other therapeutic ingredients.

[0054] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable acids including inorganic acids (e.g., mineral acids) and organic acids (carboxylic acids), known in the art. The “pharmaceutically acceptable salt” in the present invention means a salt that can be used as a medicament. When the compound has an acidic group, it can be reacted with a base to form a basic salt (also referred to as a “base addition salt”), and when it has a basic group, it can be reacted with an acid to form an acidic salt (also referred to as an “acid addition salt”).

[0055] Examples of the “basic salt” in the present invention include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as magnesium salt and calcium salt; organic base salts such as N-methylmorpholine salt, triethylamine salt, tributylamine salt, diisopropylethylamine salt, dicyclohexylamine salt, N-methylpiperidine salt, pyridine salt, 4-pyrrolidinopyridine salt, and picoline salt; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamic acid salt, and aspartic acid salt.

[0056] Examples of the “acidic salt” in the present invention include inorganic acid salts such as hydrohalide (e.g., hydrofluoride, hydrochloride, hydrobromide, hydroiodide), nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonate (e.g., methanesulfonate, trifluoromethanesulfonate, ethanesulfonate), arylsulfonate (e.g., benzenesulfonate, p- toluenesulfonate), organic acid salts such as acetate, malate, fumarate, succinate, citrate, ascorbate, tartrate, oxalate, and maleate; and amino acid salts such as glycinate, lysinate, argininate, omithinate, glutamate, and aspartate.

[0057] Compositions can include one or more carriers acceptable for the mode of administration of the preparation, be it by topical administration, lavage, epidermal administration, sub-epidermal administration, dermal administration, subdermal administration, transdermal administration, subcutaneous administration, systemic administration, injection, inhalation, oral, or any other mode suitable for the selected treatment. Topical administration includes administration to external body surfaces (e.g., skin) as well as to internal body surfaces (e.g., mucus membranes for vaginal or rectal applications by, for example, suppositories). Suitable carriers are those known in the art for use in such modes of administration. Suitable compositions can be formulated by means known in the art and their mode of administration and dose determined by a person of skill in the art. For parenteral administration, the compound can be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water-soluble compounds. For enteral administration, the compound can be administered in a tablet, capsule, or dissolved or suspended in liquid form. The tablet or capsule can be enteric coated, or in a formulation for sustained release. Many techniques for formulating compounds are known to one of skill in the art, such as described in Remington: the Science A Practice of Pharmacy by Alfonso Gennaro, 20th ed., Williams & Wilkins, (2000).

[0058] An "effective amount" of Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention as described herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.

[0059] It is to be noted that dosage values can vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges can be selected by a medical practitioner. The amount of Compound A, or a pharmaceutically acceptable salt thereof, in the composition can vary according to factors such as the disease state, age, sex, and weight of the subject.

[0060] Administration

[0061] In the methods described herein, the administration of Compound A, or a pharmaceutically acceptable salt thereof, can be a systemic administration, a local administration, or a topical administration.

[0062] The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects or patients include humans and non-human mammals, e.g., nonhuman primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In specific embodiments of the invention, the subject is a human.

[0063] The term "administering" includes any method of delivery of Compound A, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising Compound A, or a pharmaceutically acceptable salt thereof, into a subject's system or to a particular region in or on a subject. In certain embodiments, Compound A, or a pharmaceutically acceptable salt thereof, is administered intravenously or orally.

[0064] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms, diminishing the extent of a disorder, stabilized (i.e., not worsening) state of a disorder, amelioration or palliation of the disorder, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0065] MATERIALS AND METHODS Compound A was prepared by conventional synthetic organic techniques as shown below in Scheme 1.

[0066] Scheme 1

[0067] Compound X was prepared according to Menear, KA; et al.; J. Med. Chem. 2008, 51, 6581-6591.

[0068] The preparation of Compound A is described in PCT / CA2023 / 051456.

[0069] Cell culture

[0070] Identities of all cell tines were confirmed by STR profiting at the Laboratory Corporation of America (Labcorp). All cell lines were confirmed free of mycoplasma and maintained at 37°C with 5% CO2 and 95% humidity. SUM149PT cells were maintained in Ham’s F-12 Medium (Thermo Fisher Scientific cat# 31765035) with 5% fetal bovine serum (FBS) (Gibco cat# A3160401), 5 pg / mL insulin (Sigma-Aldrich cat# 19278) and 1 pg / mL hydrocortisone (Sigma-Aldrich cat# H4001). MDA-MB-231 cells were maintained in Dulbecco’s Modified Eagle Medium (Gibco cat# 11995065) supplemented with lx GlutaMAX (Thermo Fisher Scientific cat# 35050061) and 0.1 mM non-essential amino acids (Thermo Fisher Scientific cat# 11140050). UWB1.289 andUWB1.289+BRCAl cells were maintained in 40% RPMI-1640 (Gibco cat# 11875119), 40% MEGM (Lonza Bioscience cat# CC-3150) and 10% FBS. CHLA10 cells were maintained in Iscove’s Modified Dulbecco’s Medium (Hyclone cat# SH30228.01) containing lx Insulin- Transferrin-Selenium (Thermo Fisher Scientific cat# 41400045) and 20% FBS. TC32 cells were maintained in RPMI-1640 with 10% FBS and lx GlutaMAX. A673 and PC3 cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS. LNCaP cells were maintained in RPMI-1640 with 10% FBS.

[0071] PARP1 and PARP2 activity assays

[0072] In vitro PARP1 activity was measured using the HT Universal Colorimetric PARP assay kit (R&D Systems cat# 4677-096-K) and PARP2 activity was measured using the PARP2 colorimetric assay kit (BPS Bioscience cat# 80581) following the manufacturer’s protocol. ICso values were calculated using a four-parameter variable slope non-linear regression in GraphPad Prism 8 (GraphPad Software Inc.).

[0073] Results are shown in FIGS. 2 and 3.

[0074] PAR formation assay

[0075] Cellular PAR formation assays were used to measure the ability of a tested compound to inhibit polymerization of PAR. CHLA10 cells were plated on a black, clearbottom 96-well plate and allowed to attach overnight. Cells were pre-treated with increasing concentrations of test inhibitors for 30 min at 37°C before H2O2 was added to a final concentration of 25 mM and incubated for 5 min at room temperature (RT). After two washes with 0.1% Tween-20 in PBS (PBS-T) and two washes with PBS, cells were fixed with pre-chilled 70:30 methanol: acetone for 15 min at -20°C. Cells were washed with PBS, twice with 3% BSA in PBS (BSA-PBS) and again with PBS and then blocked with 3% BSA-PBS for 30 minutes at RT. Following two washes with PBS and one wash with 3% BSA-PBS, cells were incubated for 1 h at RT with anti-PAR / pADPr monoclonal antibody (R&D Systems cat# 4335-MC-100) diluted 1:250 in 3% BSA-PBS. Plates were washed twice with 3% BSA-PBS, once with PBS, twice with PBS-T, twice with PBS and once with 3% BSA-PBS, then incubated with goat anti-mouse IgG-FITC (Thermo Scientific cat# F-2761) diluted 1:1000 in 3% BSA-PBS for 1 h at RT. After washing twice with 3% BSA-PBS, once with PBS, twice with PBS-T and thrice with PBS, 100 pL PBS per well was added and plates were imaged on an IncuCyte® S3 system (Sartorius). Fluorescence was quantified using the IncuCyte® analysis software. Values were normalized to no primary antibody control and then % PAR formation was calculated by normalizing to dimethylsulfoxide (DMSO) control. ICso values were then calculated using a four-parameter variable slope non-linear regression in GraphPad Prism 8 (GraphPad Software Inc.). The average ICso value ± SD of three biological replicates was calculated.

[0076] Results are shown in FIG. 4.

[0077] HD AC activity assay

[0078] In vitro HD AC activity was measured using the FLUOR DE LYS® HD AC fluorometric activity assay kit (Enzo Life Sciences cat# BML-AK500-0001) following the manufacturer’s protocol. ICso values were calculated using a four-parameter variable slope non-linear regression in GraphPad Prism 8 (GraphPad Software Inc.).

[0079] Results are shown in FIG. 5.

[0080] Cell viability assay

[0081] Cells were plated on a 96-well plate (1000-5000 cells per well) in 100 pL appropriate medium and allowed to attach overnight. 100 pL medium containing DMSO or increasing concentration of test compound was added to each well. Cells were maintained at 37°C with 5% CO2 and 95% humidity for ten days for CHLA10, SUM149PT, MDA-MB-231, UWB1.289, UWB1.289+BRCA1, LNCaP and PC3, and three days for TC32 and A673. Cell-Titer-Glo® viability assay was carried out for ten-day assays.150 pL media per well was removed and plates were equilibrated at RT for 30 min, then CellTiter-Glo® assay reagent was added to the wells. The plates were gently shaken on an orbital shaker for 2 min and incubated at RT for 10 min in the dark. Luminescence was measured using a Tecan Infinite M200Pro microplate reader. All measurements were carried out in triplicate. For TC32 and A673, the plates were imaged on an Incucyte® S3 live cell imaging system after the treatments and % confluency was measured using the Incucyte® software. Values were normalized to media-only and DMSO controls to calculate % cell survival. ECso values were calculated using a four-parameter variable slope non-linear regression in GraphPad Prism 8 (GraphPad Software Inc.). The mean ECso value ± SD was calculated using three biological replicates.

[0082] Results for breast cancer cell lines are shown in FIGS. 6A and 6B.

[0083] Results for ovarian cancer cell lines are shown in FIGS. 7A and 7B.

[0084] Results for prostate cancer cell lines are shown in FIGS. 8 A and 8B.

[0085] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for treating breast cancer, ovarian cancer, or prostate cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-l - yl)methyl)benzoyl)piperazin-l -yl)pyrimidin-5-yl)-N-hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

2. (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-l- yl)methyl)benzoyl)piperazin-l -yl)pyrimidin-5-yl)-N-hydroxyacrylamide, or a pharmaceutically acceptable salt thereof, for use in the treatment of breast cancer, ovarian cancer, or prostate cancer.

3. A pharmaceutical composition for treating breast cancer, ovarian cancer, or prostate cancer comprising a pharmaceutically acceptable carrier and (E)-3-(2-(4-(2- fluoro-5-((4-oxo-3, 4-dihydrophthal azin-1 -yl)methyl)benzoyl)piperazin-l-yl)pyrimi din-5- yl)-N-hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

4. A method for treating breast cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5- ((4-oxo-3, 4-dihydrophthal azin- 1 -yl)methyl)benzoyl)piperazin- 1 -yljpyrimi din-5-yl)-N- hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

5. (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-l- yl)methyl)benzoyl)piperazin-l -yl)pyrimidin-5-yl)-N-hydroxyacrylamide, or a pharmaceutically acceptable salt thereof, for use in the treatment of breast cancer.

6. A pharmaceutical composition for treating breast cancer comprising a pharmaceutically acceptable carrier and (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4- dihydrophthalazin-1 -yl)methyl)benzoyl)piperazin-l -yljpyrimi din-5-yl)-N- hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

7. A method for treating ovarian cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5- ((4-oxo-3,4-dihydrophthalazin- 1 -yl)methyl)benzoyl)piperazin- 1 -yl)pyrimidin-5-yl)-N- hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

8. (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-l- yl)methyl)benzoyl)piperazin-l -yl)pyrimidin-5-yl)-N-hydroxyacrylamide, or a pharmaceutically acceptable salt thereof, for use in the treatment of ovarian cancer.

9. A pharmaceutical composition for treating ovarian cancer comprising a pharmaceutically acceptable carrier and (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4- dihydrophthalazin-l-yl)methyl)benzoyl)piperazin-l-yl)pyrimidin-5-yl)-N- hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

10. A method for treating prostate cancer in a subj ect, comprising administering to a subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5- ((4-oxo-3,4-dihydrophthalazin- 1 -yl)methyl)benzoyl)piperazin- 1 -yl)pyrimidin-5-yl)-N- hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

11. (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-l- yl)methyl)benzoyl)piperazin-l -yl)pyrimidin-5-yl)-N-hydroxyacrylamide, or a pharmaceutically acceptable salt thereof, for use in the treatment of prostate cancer.

12. A pharmaceutical composition for treating prostate cancer comprising a pharmaceutically acceptable carrier and (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4- dihydrophthalazin-l-yl)methyl)benzoyl)piperazin-l-yl)pyrimidin-5-yl)-N- hydroxyacrylamide, or a pharmaceutically acceptable salt thereof.

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