Bispecific PARP-HDAC inhibitors for treatment of ewing sarcoma

Abstract

A method for treating Ewing sarcoma 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] Cross - Reference to Related Applications

[0002] This application claims the benefit of applications having application numbers 63 / 496,633 filed on April 17, 2023, 63 / 490,477 filed on March 15, 2023, and 63 / 382,393 filed on November 4, 2022, each of which is hereby incorporated by reference in its entirety.

[0003] Background

[0004] Poly(ADP - ribose) polymerase (PARP) proteins catalyze the PARylation of cellular proteins using the ADP - ribose subunit of nicotinamide adenine dinucleotide (NAD+) as a donor. The human genome encodes 17 PARP enzymes, and at least PARP1 - 3 have key functions in DNA repair, with PARP1 being the most characterized. PARP1 is crucial for the repair of single - strand DNA breaks (SSBs), which are the most common breakpoint lesions in cellular DNA. When a cell encounters an SSB, PARP1 binds to the damage 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 damage before and during DNA replication in the S phase of the cell cycle. Effective SSB repair is important for preventing replication stress and more severe double - strand break (DSB) damage, which occurs in the S phase when unrepaired SSB lesions meet the replication fork. DSB damage in the S phase is mainly repaired by homologous recombination (HR), which relies on proteins such as BRCA1 and BRCA2. Harmful mutations in BRCA1 / 2 are present in subsets of breast, ovarian, and prostate tumors and occasionally in other solid - tumor indications. These HR - deficient tumors indirectly rely on normal PARP enzyme activity to avoid catastrophic DSB accumulation and trigger cell death in the S phase. This dependency has paved the way for PARP inhibition as a therapeutic strategy to generate synthetic lethality in tumor cells with BRCA1 / 2 deficiencies.

[0005] There are currently four approved PARP inhibitors in clinical practice, namely olaparib (approved in 2014), rucaparib (approved in 2016), niraparib (approved in 2017), and talazoparib (approved in 2018). These PARP inhibitors have been widely used in cancers with defective HR DNA repair activity caused by BRCA1 / 2 mutations. Encouraged by the success of PARP inhibitors in BRCA1 / 2-mutated cancers, the research focus has expanded to cancer subtypes with impaired HR repair due to molecular events other than BRCA1 / 2 mutations. For example, tumors with mutations in the enzyme RAD51, which acts downstream of BRCA1 / 2 in the HR repair pathway, are also sensitive to PARP inhibition. This concept is generally referred to as "BRCAness", including all events that mimic BRCA1 / 2 deficiency in the context of HR repair.

[0006] In cancers with normal HR, the state of BRCAness can be pharmacologically mimicked by inhibiting proteins that affect BRCA1 / 2 expression. This potentially presents an opportunity to broaden the use of PARP inhibitors beyond current clinical practice. For example, impairing dynamic chromatin events associated with DNA replication and repair, such as histone acetylation, can induce pharmacological BRCAness by indirectly regulating HR components. Recent studies in leukemia, breast cancer, liver cancer, glioblastoma, prostate cancer, and undifferentiated thyroid cancer models have demonstrated the inhibition of HR activity by HDAC inhibition, further supporting the synergistic potential of HDAC and PARP inhibition.

[0007] Ewing sarcoma is a highly metastatic bone and soft tissue tumor that mainly affects children and young adults, with a 5-year survival rate of only 15 - 30% for metastatic disease. Ewing sarcoma is defined by the presence of specific gene fusion events involving EWSR1 and the erythroblast transformation-specific (ETS) transcription factor FLI1 (85%) or other ETS family transcription factors (15%), most commonly ERG. These gene fusions encode chimeric oncoproteins (e.g., EWS-FLI1 or EWS-ERG) that drive the initiation and progression of Ewing sarcoma.

[0008] Ewing's sarcoma cells are sensitive to PARP inhibitors in vitro, and this sensitivity is dependent on EWS-FLI1. Xenografts derived from Ewing's sarcoma cell lines in mice exhibit sensitivity to FDA-approved PARP inhibitors, similar to the responses observed with the standard-of-care chemotherapy temozolomide. These observations prompted a phase II single-agent trial of olaparib in Ewing's sarcoma; however, despite encouraging preclinical data, these patients did not have a durable response to single-agent PARP inhibition. The suboptimal response of Ewing's sarcoma patients to PARP inhibitors is most likely due to insufficient synthetic lethality, and thus Ewing's sarcoma is an optimal candidate for exploring pharmacological BRCAness in the context of PARP inhibitors.

[0009] Despite the above progress in the development of therapeutic agents for treating Ewing's sarcoma, there remains a need for improved therapeutic agents. The present invention seeks to meet this need and provide further related advantages.

[0010] Overview

[0011] In one aspect, the present disclosure provides a method for treating Ewing's sarcoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) or a pharmaceutically acceptable salt thereof. In a related aspect, the present disclosure provides Compound A or a pharmaceutically acceptable salt thereof for treating Ewing's sarcoma in a subject.

[0012] In another aspect, the present disclosure provides a method for inhibiting PARP1, PARP2, and HDAC in a subject, comprising administering to the subject an effective amount of Compound A or a pharmaceutically acceptable salt thereof. In a related aspect, the present disclosure provides Compound A or a pharmaceutically acceptable salt thereof for inhibiting PARP1, PARP2, and HDAC in a subject.

[0013] In yet another aspect, the present disclosure provides a method for treating a disease or disorder in a subject that is treatable by inhibiting PARP1, PARP2, and HDAC, comprising administering to the subject a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof. In a related aspect, the present disclosure provides Compound A or a pharmaceutically acceptable salt thereof for treating a disease or disorder in a subject that is treatable by inhibiting PARP1, PARP2, and HDAC.

[0014] In other aspects, the present disclosure provides pharmaceutical compositions for the above uses. In these aspects, the pharmaceutical composition comprises Compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Brief Description of the Drawings

[0016] Figure 1A-1D The dual activities of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) against PARP1 / 2 and HDAC enzymes were compared. Figure 1A The in vitro HDAC activities in HeLa nuclear extracts treated with Compound A or vorinostat were compared. Figure 1B The in vitro activity of recombinant PARP1 after treatment with Compound A and olaparib was compared. Figure 1C The in vitro activity of recombinant PARP2 after treatment with Compound A and olaparib was compared. Figure 1D PAR formation in CHLA10 cells treated with Compound A and olaparib was compared. The values were normalized relative to the control, and the IC 50 was calculated as the concentration required to produce 50% inhibition of activity. The data shown are the mean of n = 3 replicates and are accompanied by representative curves.

[0017] Figures 2A - 2C illustrate that Ewing sarcoma cells are highly sensitive to dual PARP1 / 2 and HDAC inhibition. In the figures, the activity of Compound A was compared with the activities of olaparib, niraparib, talazoparib, vorinostat, belinostat, and panobinostat. Figure 2A compares the cell viability of TC32 cells examined by the Incucyte ® S3 live cell imaging system after treatment with increasing concentrations of the indicated compounds for three days. The EC 50 value was calculated as the concentration required for 50% cell viability, n = 3. Figure 2B compares the EC 50 values of the test compounds determined in A673 cells using the same experimental conditions as in Figure 2A. Figure 2C compares the EC ® values of the indicated inhibitors determined using the CellTiter-Glo 50 cell viability assay in CHLA10 cells using the same experimental conditions as in Figure 2A. The cells were exposed to increasing concentrations of the inhibitor for ten days, and the EC 50 value was calculated as the concentration required for 50% cell viability, n = 3.

[0018] Figures 3A and 3B illustrate that compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells. Figure 3A presents the cell cycle analysis of TC32 cells, which were synchronized at the G0 / G1 phase by serum starvation for 24 h and then treated with compound A, olaparib, or vorinostat as indicated in complete medium for 48 h. Then, the cell cycle profile was examined by propidium iodide (PI) staining and subsequent flow cytometry analysis. The cell cycle distribution is also shown. Figure 3B presents the cell cycle analysis of CHLA10 cells treated with compound A, olaparib, or vorinostat for 24 h using the same experimental procedure as in Figure 3A as indicated.

[0019] Figures 4A - 4F illustrate that compound A treatment induces DNA damage in Ewing sarcoma cells. Figure 4A compares γH2AX expression when using dulcitol (DAG), olaparib, vorinostat, or compound A by Western blot: TC32 cells were treated with 2.5 μM DAG or increasing doses of olaparib (0.35 - 13 μM), vorinostat (0.35 - 8 μM), or compound A (0.018 - 0.35 μM) for 48 h, and γH2AX expression was analyzed by Western blot. Figure 4B compares γH2AX expression when using DAG, olaparib, vorinostat, or compound A by Western blot: CHLA10 cells were treated with 5 μM dulcitol (DAG) or increasing doses of olaparib (1 - 37 μM), vorinostat (1 - 20 μM), or compound A (0.05 - 1 μM) for 48 h and analyzed as in (A). Figures 4C - 4E compare γH2AX focus analysis by immunofluorescence and confocal microscopy imaging: γH2AX foci in CHLA10 cells treated with DAG (2.5 μM) or increasing doses of compound A (0 - 1 μM) (Figure 4C), olaparib (1 - 37.5 μM) (Figure 4D), or vorinostat (1 - 18 μM) (Figure 4E) for 24 h were analyzed by immunofluorescence and confocal microscopy imaging. Scale bars represent 10 μm. Figure 4F Illustrates the results of the comet assay of CHLA10 cells treated with 1 μM compound A, olaparib, vorinostat, or olaparib + subsequent vorinostat. 5 μM DAG was included as a positive control. Scale bars represent 200 μm. ****p < 0.0001.

[0020] Figures 5A - 5F illustrate that compound A inhibits 3D spheroid growth and metastasis of Ewing sarcoma cells. Figure 5A compares as using IncuCyte ®Growth of TC32 spheroids after 4 days of treatment with compound A, olaparib, or vorinostat at increasing concentrations monitored by the spheroid analysis system. EC50 values were calculated as the concentration required for 50% growth inhibition according to the non-linear regression plot using GraphPad Prism 8 software. Representative images of TC32 spheroids at day 0 and day 4 with DMSO, 1 μM compound A, 1 μM olaparib, or 1 μM vorinostat are shown to represent a scale bar of 400 μm. *p < 0.05, **p < 0.01. Figure 5B compares the growth of CHLA10 spheroids after 4 days of treatment with compound A, olaparib, or vorinostat at increasing concentrations using the same experimental procedure as shown in Figure 5A. Representative images of CHLA10 3D spheroids at day 0 and day 4 with DMSO, 1 μM compound A, 1 μM olaparib, or 1 μM vorinostat are shown to represent a scale bar of 400 μm. *p < 0.05, ***p < 0.001. Figure 5C Lung tumor burden was compared after 14 days of treatment with vehicle, 5, 10, or 20 nM of compound A, n = 5 - 12. Representative fluorescence images of tdTomato TC32 cells in lung sections after 14 days of treatment with 5, 10, or 20 nM of compound A. Scale bar represents 1 mm. **p < 0.01. Figure 5D shows representative hematoxylin and eosin (H&E) staining and CD99 staining images of TC32 Ewing sarcoma cells in PuMA lung sections after 14 days of treatment with 5, 10, or 20 nM of compound A. Magnified images of the respective insets (upper panels) are shown below each image. Scale bar represents 50 μm. Figure 5E Lung tumor burden was compared after 14 days of treatment with vehicle, 5, 10, or 20 nM of compound A, n = 5 - 12. Representative fluorescence images of tdTomato A673 cells in lung sections after 14 days of treatment with 5, 10, or 20 nM of compound A. Scale bar represents 1 mm. **p < 0.01, ****p < 0.0001. Figure 5F shows representative H&E staining and CD99 staining images of A673 Ewing sarcoma cells in PuMA lung sections after 14 days of treatment with 5, 10, or 20 nM of compound A. Magnified images of the respective insets (upper panels) are shown below each image. Scale bar represents 50 μm.

[0021] Figure 6 compares the induction of apoptosis by UT (dimethyl sulfoxide (DMSO)), dianhydrogalactitol (DAG), olaparib (OLA), vorinostat (VOR), O+S (OLA+VOR), and compound A as determined by immunoblotting for cleaved caspase 3 using cell lysates.

[0022] Details

[0023] HDAC inhibition has been shown to induce pharmacological BRCAness in cancer cells with normal DNA repair activity. This provides a rationale for exploring combination therapy 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-dihydrophtalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A), which has dual activity against PARP1 / 2 and HDAC enzymes in Ewing sarcoma cells. Compared to FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, Compound A exhibits enhanced cytotoxicity in an Ewing sarcoma model. As evaluated by γH2AX tracking and comet assays, the cytotoxicity induced by Compound A is associated with strong S and G2 / M cell cycle arrest and elevated DNA damage in the nanomolar concentration range. In a three-dimensional spheroid model of Ewing sarcoma, Compound A shows efficacy at lower concentrations than olaparib and vorinostat.

[0024] In one aspect, the present disclosure provides a method for treating Ewing sarcoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) or a pharmaceutically acceptable salt thereof. A related aspect provides (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) or a pharmaceutically acceptable salt thereof for use in treating Ewing sarcoma in a subject.

[0025] In another aspect, the present disclosure provides a method for inhibiting PARP1, PARP2, and HDAC in a subject, comprising administering to the subject an effective amount of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) or a pharmaceutically acceptable salt thereof. A related aspect provides (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophtalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) or a pharmaceutically acceptable salt thereof for use in inhibiting PARP1, PARP2, and HDAC in a subject.

[0026] In another aspect, the present disclosure provides a method for treating a disease or disorder in a subject that is treatable by inhibiting PARP1, PARP2, and HDAC, which comprises administering to the subject 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 (Compound A) or a pharmaceutically acceptable salt thereof. In a related aspect, the present disclosure provides (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) or a pharmaceutically acceptable salt thereof for treating a disease or disorder in a subject that is treatable by inhibiting PARP1, PARP2, and HDAC.

[0027] In other aspects, the present disclosure provides pharmaceutical compositions for the above uses. In these aspects, the pharmaceutical composition comprises (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide (Compound A) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0028] As used herein, "Compound A" refers to (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide, which has the formula (I):

[0029] (I)

[0030] or a tautomer thereof.

[0031] As described herein, the present disclosure provides methods of using Compound A or a pharmaceutically acceptable salt thereof. It should be understood 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 upon administration.

[0032] The usefulness of Compound A for inhibiting PARP1, PARP2, and HDAC, and the effectiveness of Compound A for treating Ewing's sarcoma are described below.

[0033] Pharmacological BRCAness could potentially provide a way to extend PARP inhibition beyond the BRCA1 / 2 mutation space and counter potential resistance to PARPi therapies. Epigenetic modifiers, such as HDACs, as well as DNA and histone methyltransferases are attractive targets for inducing BRCAness in the context of BRCA1 / 2 wild-type cancers. Currently, four clinical trials are underway using PARPi (PARP inhibitors) in combination with HDACi (HDAC inhibitors) vorinostat (NCT03259503 and NCT03742245), the DNA methyltransferase inhibitor decitabine (NCT02878785), and the EZH2 histone methyltransferase inhibitor SHR2554 (NCT04355858).

[0034] The FDA has approved three pan-HDACi (pan-HDAC inhibitors) drugs (vorinostat, belinostat, and panobinostat) and one HDAC1 / 2-selective HDACi (romidepsin) for the treatment of blood cancers. Histone acetylation weakens chromatin structure and plays a key role in the recognition and repair of DNA damage. Downregulation of key HR proteins (including BRCA1, BRCA2, and RAD51) induced by HDACi has been demonstrated in multiple cancer types, and HDACi treatment renders cancer cells sensitive to PARPi. This established activity of HDAC and PARP inhibition is particularly interesting in the context of HR-proficient cancer types, such as Ewing sarcoma, where the effects of PARPi therapy alone are limited. However, dose-limiting toxicities of HDACi therapies are not uncommon in solid tumor cancers and have hindered some therapeutic effects, both alone and in treatment combinations, such as in breast cancer and sarcoma. Care must be taken to modulate the HDACi component to prevent toxicity-overlay events when combined with other treatment components, which can be challenging when acting with different pharmacokinetic profiles.

[0035] The current disclosure provides a bifunctional PARP-HDAC single molecule inhibitor, Compound A, in an Ewing sarcoma model to evaluate the potential benefits of combined PARP-HDAC inhibition relative to treatment with PARPi or HDACi alone. Compound A has PARPi activity similar to olaparib and HDACi activity slightly lower than vorinostat. However, the dual activity of Compound A is 30 to 80 times more cytotoxic to Ewing sarcoma cells than olaparib and 30 to 60 times more toxic than vorinostat alone. While panobinostat appears to be more efficacious in Ewing sarcoma cell lines, this may be attributable to the toxicity of panobinostat, as observed in clinical trials where dose-limiting toxicity limited its effective use in solid tumors. This is likely also the case for talazoparib. While talazoparib is the most potent FDA-approved PARPi to date, it also shows clinical toxicity more similar to other chemotherapeutic agents than other approved PARP inhibitors, including anemia, thrombocytopenia, and neutropenia.

[0036] Compound A also induces cell cycle arrest and DNA damage in Ewing sarcoma cells at much lower concentrations than olaparib and vorinostat. The cell cycle arrest pattern of Compound A is more similar to olaparib compared to combination therapy because the PARP inhibitor activity of Compound A is stronger than the HDAC inhibitor moiety of the drug. In TC32 cells, a combination of 0.7 μM olaparib and 0.7 μM belinostat showed some G0 / G1 arrest, consistent with the lower EC50 value of TC32 cells treated with belinostat. When compared in a 3D spheroid model, Compound A showed efficacy at concentrations 30 to 40 times lower than olaparib and 5 to 10 times lower than vorinostat. The spheroid model treated with talazoparib showed efficacy comparable to Compound A, but panobinostat was 10 times less efficacious compared to Compound A. This may be attributable to the overall toxicity of panobinostat mentioned previously. Compound A also impedes the metastatic growth of Ewing sarcoma cells in an ex vivo PuMA model, with strong inhibition using as little as 10 nM of the inhibitor. Due to concerns regarding the hematological toxicity of PARP and HDAC inhibitors, a preliminary study of Compound A was conducted in mice, which showed no evidence of toxicity based on weight loss and blood cell counts.

[0037] Combining PARP and HDAC inhibition into a single molecule provides a convenient way to prevent resistance to PARPi therapy. For example, Ewing sarcoma and many other solid tumor indications epigenetically suppress the expression of the tumor suppressor gene Schlafen 11 (SLFN11), which leads to resistance to DNA damage-inducing agents, including PARPi therapy. Importantly here, HDACi treatment prompts the re-expression of SLFN11 and resensitization to PARPi.

[0038] Combination therapies can act 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 must be administered sequentially in a clinical setting, sometimes with reduced biological efficacy. This provides a strong rationale for the development of dual-active small molecules such as Compound A.

[0039] In summary, the present disclosure provides a single-molecule PARP-HDAC inhibitor, Compound A, in Ewing sarcoma, which has improved cytotoxicity and DNA damage activity compared to PARPi and HDACi alone.

[0040] Dual activity of bispecific compounds against PARP1 / 2 and HDAC enzymes

[0041] Through medicinal chemistry cycles, the present disclosure provides a small molecule inhibitor (Compound A) with dual activity against PARP1 / 2 and HDAC. An in vitro activity assay kit was used to determine the inhibition of Compound A against PARP1, PARP2, and HDAC, in comparison to the FDA-approved PARP inhibitor olaparib and the HDAC inhibitor vorinostat. A wide concentration range of each compound was used to determine the IC 50 values. The IC 50 value of Compound A was 2.54 μM, which was approximately 50-fold lower than that of vorinostat at 0.05 μM ( Figure 1A ). The PARP1 and PARP2 inhibitory activities of Compound A were comparable to those of olaparib, with IC 50 values of 3.38 nM and 2.19 nM for Compound A, respectively ( Figure 1B and Figure 1C ). To further verify 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 IC 50 of 1.39 nM for inhibiting PAR formation was detected in cells treated with Compound A ( Figure 1D ). These data indicate that Compound A is capable of inhibiting both PARP1 / 2 and HDAC enzymes.

[0042] Ewing sarcoma cells are highly sensitive to dual PARP1 / 2 and HDAC inhibition

[0043] To investigate the role of Compound A in cell growth, a cell viability assay was performed in three Ewing sarcoma cell lines. Using the IncuCyte S3 live cell imaging system, we examined the EC 50Values. In TC32 cells, compound A showed higher efficacy than olaparib, niraparib, vorinostat, and belinostat in inhibiting cell viability, with an EC 50 of 0.0163 μM (Figure 2A). A similar effect of compound A was detected in A673 cells, with a much lower EC 50 value of 0.0365 μM compared to treatment with olaparib, niraparib, vorinostat, and belinostat alone (Figure 2B). However, treatment with talazoparib or panobinostat showed stronger inhibitory effects than compound A in both cell lines (Figure 2A and Figure 2B). To further verify the findings, CellTiter-Glo ® viability assays were performed to determine the EC 50 values of these tested compounds in CHLA10 cells. Consistent with the IncuCyte assay, the EC 50 value (0.053 μM) of compound A treatment in CHLA10 cells was also much lower than that of olaparib, niraparib, vorinostat, and belinostat (Figure 2C). In summary, the data demonstrated the potent inhibitory effect of compound A in Ewing sarcoma cells compared to FDA-approved PARP or HDAC inhibitors.

[0044] Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells

[0045] PARP inhibitors and HDAC inhibitors respectively continuously induce S / G2 / M and G0 / G1 cell cycle arrest because PARP regulates replication fork progression, and HDAC plays a major role in regulating the expression of cell cycle checkpoint proteins including cyclin-dependent kinases, cyclin D1, and p21. Examine the cell cycle profiles of CHLA10 and TC32 cells treated with compound A, olaparib, and vorinostat in both single and combination regimens. Serum-starved cells were treated with increasing concentrations of compound A in complete medium for 24 or 48 h and showed strong S and G2 / M arrest at and after 0.175 μM in TC32 cells and at and after 0.25 μM in CHLA10 cells. Similar cell cycle arrest was only observed when treating with olaparib at concentrations higher than 3 μM in TC32 and higher than 14.7 μM in CHLA10 (Figure 3A and Figure 3B). Compared to the control, combined treatment with olaparib and vorinostat / belinostat at equimolar concentrations to compound A (1 μM and 0.7 μM for CHLA10 and TC32 cells respectively) had little effect on the cell cycle phases (Figure 3A and Figure 3B). These data demonstrated that in Ewing sarcoma cells, compound A has a stronger potency in inducing S and G2 / M cell cycle arrest than olaparib alone or its combination with vorinostat or belinostat.

[0046] Treatment with Compound A induces DNA damage in Ewing sarcoma cells

[0047] It has been reported that PARP inhibitors and HDAC inhibitors induce DNA damage in cells. The effect of compound A on DNA damage was investigated in Ewing sarcoma cells using Western blot, immunofluorescence, and comet assays compared to olaparib and vorinostat treatments. Phosphorylated histone variant H2AX (γH2AX) is a surrogate marker for DSBs in DNA. Didehydrogalactitol (DAG) was included as a positive control because DAG induces replication-dependent DNA damage in multiple cancer cell lines. Treatment with compound A, olaparib, or vorinostat induced γH2AX expression in a dose-dependent manner in both CHLA10 and TC32 cells. Compound A was able to induce γH2AX expression in a much lower concentration range compared to olaparib and vorinostat (Figures 4A and 4B). Additionally, CHLA10 cells treated with compound A or olaparib also showed dose-dependent γH2AX foci formation in immunofluorescence and subsequent confocal microscopy imaging, with a much lower concentration range for compound A (Figure 4C). However, vorinostat, which induces G0 / G1 cell cycle arrest (Figures 3A and 3B), showed milder DNA damage foci formation in CHLA10 cells (Figure 4C). To further integrate the data, an alkaline comet assay was performed as it can detect both SSBs and DSBs in cells. CHLA10 cells treated with 1 μM compound A showed a significant amount of DNA damage, but not with 1 μM olaparib or vorinostat (Figure 4D). In summary, the data show that compound A is able to induce DNA damage in Ewing sarcoma cells in a much lower concentration range than olaparib or vorinostat.

[0048] It has been reported that PARP inhibitors and HDAC inhibitors induce DNA damage in cells. The effects of compound A on DNA damage in Ewing sarcoma cells were investigated using Western blot, immunofluorescence, and comet assays, compared with olaparib and vorinostat treatments. Phosphorylated histone variant H2AX (γH2AX) is a surrogate marker for DSBs in DNA. Didehydrogalactitol (DAG) was included as a positive control because previous studies have shown that DAG induces replication-dependent DNA damage in multiple cancer cell lines. Treatment with compound A, olaparib, or vorinostat induced γH2AX expression in a dose-dependent manner in both TC32 and CHLA10 cells. Compound A was able to induce γH2AX expression in a much lower concentration range compared with olaparib and vorinostat (see Figures 4A and 4B). In addition, CHLA10 cells treated with compound A or olaparib also showed dose-dependent γH2AX foci formation in immunofluorescence and subsequent confocal microscopy imaging, with a much lower concentration range for compound A (see Figures 4C and 4D). However, vorinostat, which induces G0 / G1 cell cycle arrest (see Figures 3A and 3B), showed milder DNA damage foci formation in CHLA10 cells (see Figure 4E). Additionally, by Western blot, TC32 cells showed increased γH2AX expression only after treatment with 0.35 μM of compound A, but not after treatment with 0.35 μM of olaparib, vorinostat, or olaparib + vorinostat. This observation was also confirmed by Western blot and immunofluorescence using equimolar concentrations of these compounds in CHLA10 cells. To further integrate the data, an alkaline comet assay was performed because this assay can detect both SSBs and DSBs in cells. There was significant DNA damage in CHLA10 cells treated with 1 μM of compound A, but not when treated with 1 μM of olaparib or vorinostat or 1 μM of olaparib + 1 μM of vorinostat (see Figure 4F ) In summary, the data show that compound A is able to induce DNA damage in Ewing sarcoma cells in a much lower concentration range compared with olaparib or vorinostat.

[0049] Compound A inhibits 3D spheroid growth of Ewing sarcoma cells

[0050] Spheroids are three-dimensional (3D) cell aggregates that can more precisely mimic tumor behavior compared to two-dimensional cell cultures. To further validate the data, the effect of compound A on a 3D spheroid model with Ewing sarcoma cells was investigated. CHLA10 and TC32 spheroids were constructed to 200 - 300 μm and then treated with increasing concentrations of compound A, olaparib, or vorinostat. The growth of the spheroids was monitored and quantified over four days using an IncuCyte S3 imaging system. In both the TC32 and CHLA10 cell models, the EC 50 value of compound A for inhibiting spheroid growth was much lower than that of olaparib and vorinostat (Figure 5A and Figure 5B). These data indicate that compound A is a potent inhibitor of 3D spheroid growth in Ewing sarcoma cells and is demonstrated to be more effective than olaparib or vorinostat alone.

[0051] The spheroid assays using both TC32 and CHLA10 cells demonstrated equivalent activity of compound A and talazoparib, but the efficacy of panobinostat was 10-fold lower compared to compound A. The effect of compound A on the metastatic growth of Ewing sarcoma cells was examined in an ex vivo pulmonary metastasis assay (PuMA). Here, compound A at a concentration as low as 10 nM inhibited the colonization of tdTomato-expressing TC32 and A673 cells in the lungs of mice (see Figure 5C and Figure 5E ). Parallel hematoxylin and eosin (H&E) staining and CD99 IHC staining (see Figure 5D and Figure 5F) confirmed that the tdTomato fluorescence signal in the lung tissue was indeed Ewing sarcoma cells. Fluorescence microscopy confirmed that the fluorescence of each cell in both tdTomato-expressing TC32 and A673 cells was not affected by treatment with compound A. A study in nude mice showed that after four days of intraperitoneal treatment with 30 mg / kg compound A (B.I.D.), body weight and blood cell counts indicated no signs of toxicity. These data suggest that compound A is a potent inhibitor of Ewing sarcoma lung metastasis and is more effective than olaparib or vorinostat alone in inhibiting the 3D growth of Ewing sarcoma spheroids.

[0052] Materials and methods

[0053] Cell culture

[0054] The identification of all human Ewing's sarcoma cell lines was confirmed by STR profiling at Laboratory Corporation of America (Labcorp). All cell lines were confirmed to be mycoplasma-free and maintained at 37 °C, 5% CO2, and 95% humidity. CHLA10 cells were maintained in Iscove's Modified Dulbecco's Medium (Hyclone, catalog number SH30228.01) containing 1x insulin-transferrin-selenium (Thermo Fisher Scientific, catalog number 41400045) and 20% fetal bovine serum (FBS) (Gibco, catalog number A3160401). TC32 cells were maintained in RPMI-1640 (Gibco, catalog number 11875119) containing 10% FBS and 1x GlutaMAX supplement (Thermo Fisher Scientific, catalog number 35050061). A673 cells were maintained in Dulbecco's Modified Eagle Medium (Gibco, catalog number 11995065) supplemented with 10% FBS.

[0055] HDAC activity assay

[0056] Using FLUOR DE LYS ® HDAC Fluorescent Activity Assay Kit (Enzo Life Sciences, catalog number BML-AK500-0001), the in vitro HDAC activity was measured according to the manufacturer's protocol. The IC 50 values were calculated using four-parameter variable slope nonlinear regression in GraphPad Prism 8 (GraphPad Software Inc.).

[0057] PARP1 and PARP2 activity assay

[0058] The in vitro PARP1 activity was measured using the HT Universal Colorimetric PARP Assay Kit (R&D Systems, catalog number 4677-096-K), and the PARP2 activity was measured using the PARP2 Colorimetric Assay Kit (BPS Bioscience, catalog number 80581) according to the manufacturer's protocol. The IC 50 values were calculated using four-parameter variable slope nonlinear regression in GraphPad Prism 8 (GraphPad Software Inc.).

[0059] PAR formation assay

[0060] The ability of a test compound to inhibit PAR polymerization was measured using a cellular PAR formation assay. CHLA10 cells were plated on a 96-well plate with a black clear bottom and allowed to adhere overnight. Cells were pretreated with increasing concentrations of the test inhibitor for 30 min at 37 °C, after which H2O2 was added to a final concentration of 25 mM and incubated for 5 min at room temperature (RT). After washing twice with PBS containing 0.1% Tween-20 (PBS-T) and twice with PBS, cells were fixed with pre-chilled 70:30 methanol:acetone for 15 min at -20 °C. Cells were washed with PBS, washed twice with PBS solution containing 3% BSA (BSA-PBS), washed again with PBS, and then blocked with 3% BSA-PBS for 30 min at RT. After washing twice with PBS and once with 3% BSA-PBS, cells were incubated with an anti-PAR / pADPr monoclonal antibody (R&D Systems, catalog number 4335-Mc-100) diluted 1:250 in 3% BSA-PBS for 1 h at RT. The plate was washed twice with 3% BSA-PBS, once with PBS, twice with PBS-T, twice with PBS, and once with 3% BSA-PBS, and then incubated with goat anti-mouse IgG-FITC (Thermo Scientific, catalog number 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 three times with PBS, 100 μL of PBS was added to each well and the plate was imaged on an IncuCyte® S3 system (Sartorius). Fluorescence was quantified using IncuCyte ® analysis software. Values were normalized relative to the no-primary-antibody control and then the % PAR formation was calculated by normalizing relative to the dimethyl sulfoxide (DMSO) control. The IC 50 value was then calculated using four-parameter variable slope nonlinear regression in GraphPad Prism 8 (GraphPad Software Inc.). The mean IC 50 value ± SD of three biological replicates was calculated.

[0061] Cell viability assay

[0062] Seed cells in 100 μL of appropriate medium in a 96-well plate (1000 - 5000 cells / well) and allow them to adhere overnight. Add 100 μL of medium containing DMSO or the test compound at increasing concentrations to each well. Maintain the cells at 37 °C, 5% CO2, and 95% humidity. Maintain CHLA10 cells for ten days and TC32 and A673 cells for three days. Perform Cell-Titer-Glo ® viability assay on CHLA10. Remove 150 μL of medium from each well and equilibrate the plate at RT for 30 min, then add CellTiter-Glo ® assay reagent to the wells. Gently shake the plate on an orbital shaker for 2 min and incubate in the dark at RT for 10 min. Measure luminescence using a Tecan Infinite M200Pro microplate reader. All measurements are repeated three times. For TC32 and A673, after treatment, image the plate on an Incucyte ® S3 live cell imaging system and measure the confluence % using Incucyte ® software. Normalize the values relative to the medium-only and DMSO controls to calculate the % cell survival. Calculate the EC 50 values using four-parameter variable slope nonlinear regression in GraphPad Prism 8 (GraphPad Software Inc.). Calculate the mean EC 50 values ± SD using three biological replicates.

[0063] Cell cycle analysis

[0064] Evaluate the cell cycle profiles via propidium iodide (PI) staining and flow cytometry. Seed CHLA10 and TC32 cells at cell densities of 1.5×10 6 cells / plate and 2.0×10 6 cells / plate, respectively, in 10 cm plates. The next day, change the medium to serum-free medium and culture for 24 h. Treat the cells with olaparib, vorinostat, and compound A in a dose-escalating manner. Treat TC32 for 24 h and CHLA10 for 48 h. Evaluate the combination treatment of olaparib and vorinostat and the combination treatment of olaparib and belinostat at equimolar concentrations to compound A (0.7 and 1 μM for TC32 and CHLA10 cells, respectively). Harvest the cells and take 1.0×10 6The cells were fixed overnight at -30 °C in 70% ethanol. Then the cell suspension was washed with cold PBS and stained with PI solution (PBS containing 50 µg / mL PI, 0.1 mg / mL RNase A, 0.05% Triton X-100), and incubated in the dark at 37 °C for 40 min. Then the cells were washed with PBS, filtered through a 40-μm filter, and resuspended in 500 μL PBS. Then the samples were examined by flow cytometry and analyzed in FlowJo v10.

[0065] Alkaline comet assay

[0066] The cells were seeded in 6-well plates at a density of 1×10 6 cells / well and allowed to stand overnight. The cell medium was replaced with serum-free medium and cultured for 24 h, then treated with DMSO or the test compound at 37 °C, 5% CO2 and 95% humidity for 24 h. Cells were harvested according to the instructions in Trevigen’s CometAssay ® protocol and the cells were combined with molten LMAgarose at a ratio of 1:10, and transferred to CometSlides ® using a pipette. The cells were placed in the dark for 30 min and immersed in the lysis solution overnight at 4 °C. The slides were immersed in the alkaline unwinding solution and placed in the dark at 4 °C for 1 h, then placed in a gel electrophoresis tray and immersed in the alkaline electrophoresis solution, and a voltage of 25 V was applied for 30 min. The samples were washed with dH2O and 70% ethanol, and then stained with SYBR ® Gold. Then the samples were observed using a fluorescence microscope. The obtained images were analyzed using OpenComet on ImageJ (NIH).

[0067] Immunofluorescence

[0068] CHLA10 cells were seeded at a density of 3.5×10 5 cells / well on glass coverslips in 24-well plates and allowed to stand overnight. The medium was replaced with serum-free medium and cultured for 24 h, then the cells were treated with DMSO or the test compound at increasing concentrations for 24 h. The cells were fixed with 4% paraformaldehyde at RT for 30 min and permeabilized with PBS containing 0.5% Triton-X, and then probed overnight at 4 °C with rabbit antibody against phosphorylated histone H2AX (Ser 139) (Cell Signaling Technology, catalog number 2577). Then the cells on the coverslips were washed with PBS and incubated with goat anti-rabbit IgG AlexaFluor ®Cells were detected with 488 (Abcam, catalog number ab150077), and then the cells were mounted onto microscope slides with VECTASHIELD™ Antifade Mounting Medium containing DAPI solution. The cells were then observed on a confocal microscope (Olympus FV3000). The acquired images were analyzed by quantifying the foci using ImageJ (NIH).

[0069] Western blot analysis

[0070] Cells were seeded in 6-well plates to 70 - 80% confluence. After allowing the cells to settle overnight, the medium was changed to serum-free medium and cultured for 24 h. The cells were treated with DMSO or increasing concentrations of the test compound for 24 h. The cells were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer combined with protease and phosphatase inhibitors. Pierce TM BCA Protein Assay Kit (Thermofisher, catalog number 23225) was used to evaluate the protein yield and quantified at 562 nm using a spectrophotometric microplate reader (TECAN). A total of 20 μg of protein extract was loaded per well in a 4 - 15% Mini-PROTEAN ® TGX TM precast protein gel (Bio-Rad, catalog number 4561084). After electrophoresis, the proteins were transferred onto a 0.2 μm nitrocellulose membrane. The membrane was blocked with PBS containing LICOR ® Odyssey blocking buffer. After blocking, the membrane was incubated overnight at 4 °C with anti-phospho-histone H2AX (Ser 139) rabbit antibody (Cell Signaling Technology, catalog number 2577) and H2AX rabbit antibody (Abcam, catalog number ab11175), and incubated with donkey anti-rabbit IRDye ® 800CW secondary antibody (LI-COR, catalog number 926-32213) for 1 h at RT. The membrane was washed with 1X tris-buffered saline with 1% Tween-20 (TBS-T), and then scanned with an Odyssey scanner (LI-COR).

[0071] Spheroid formation assay

[0072] CHLA10-tdTomato or TC32-tdTomato cells (2,500 cells / well) were added to a 96-well clear round-bottom ultra-low attachment microplate (Corning, catalog number 7007) and allowed to form spheroids for 24 h or until their diameter reached 200 - 300 μm. Media containing DMSO or increasing concentrations of the test compound were added to each well, and spheroid growth was monitored for four days post-treatment using an IncuCyte ® Spheroid Analysis System (Sartorius). Images from day 0 and day 4 post-treatment were analyzed using the IncuCyte ® Spheroid Analysis Software Module. Day 4 values were normalized using the normalization factor derived from day 0 values, and EC 50 values were calculated using four-parameter variable slope nonlinear regression in GraphPad Prism 8 (GraphPad Software Inc.). Mean EC 50 values ± SD were calculated using three biological replicates.

[0073] Lung metastasis assay (PuMA)

[0074] Procedures involving mice were approved by the local Animal Care Committee of the University of British Columbia. TC32 and A673 cells expressing tdTomato (1 × 10 6Cells / 100 μl normal saline) were injected into the tail vein of 6-8-week-old immunocompromised NSG female mice (Jax Laboratories). After injection, the mice were euthanized by isoflurane and CO2 asphyxiation according to the local animal care standard operating procedures. As previously described (Scopim-Ribeiro R, Lizardo MM, Zhang HF, Dhez AC, Hughes CS, Sorensen PH. NSG Mice Facilitate ex vivo Characterization of Ewing Sarcoma Lung Metastasis Using the PuMA Model. Front Oncol 2021; 11: 645757 doi 10.3389 / fonc.2021.645757), the lungs were blown into a 1:1 mixture of pre-warmed (37°C) fully supplemented PneumaCult™-ALI medium (STEMCell, catalog number 05001) and 1.2% low melting point agarose (Lonza) by gravity perfusion. The viscera (heart and lungs) were carefully removed and placed in ice-cold PBS (supplemented with 1X penicillin / streptomycin) for 20 min to allow the agarose to solidify. Small lung slices (approximately 2 mm × 4 mm) were obtained by manual cutting with sterile surgical scissors, and 5-12 slices were selected for each condition at 0 and 14 days after injection / processing for serial imaging. In vitro, the lung slices were maintained in a 6-well plate partially immersed on gelatin sponges with 2 mL of PneumaCult™ medium + / - compound; the medium + / - compound was renewed every three days. On the day of imaging, the lung slices from each group were transferred to a 35 mm glass-bottomed Petri dish (IBIDI) to allow sterile wide-field fluorescence imaging. The lung slices were imaged using a 2.5X objective on an inverted Zeiss Observer.Z1 Colibri microscope. As previously described (Lizardo MM, Sorensen PH. Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay. J Vis Exp 2018(133) doi 10.3791 / 56332), the lung tumor burden (tumor burden %) of the lung slices was calculated as the total area of tdTomato lesions divided by the total area of the lung slices, multiplied by 100. Image processing was performed using ImageJ software.This calculation was performed for all lung sections (n = 5 - 12 lung sections) of each experimental group. The mean percentage of lung tumor burden for each group was compared and analyzed in GraphPrism 8 (GraphPad Software Inc.).

[0075] Immunohistochemistry and histopathology

[0076] Fresh-cut formalin-fixed paraffin-embedded (FFPE) lung tissue sections of PuMA were analyzed for the immunoreexpression of CD99 using a Ventana Discovery Ultra automated stainer (Ventana Medical Systems, Tucson, Arizona). Briefly, baked and dewaxed tissue sections were incubated in a Tris-based buffer (CC1, Ventana) at 95 °C for 64 min to retrieve antigenicity, followed by incubation with a rabbit polyclonal anti-CD99 antibody (Abcam, catalog number ab27271) at RT for 1 h. Bound primary antibody was visualized using an UltraMap DAB anti-Rb detection kit (Ventana). All stained slides were digitized using a Leica scanner (Aperio AT2, Leica Microsystems; Concord, Ontario, Canada) at a magnification equivalent to 40X. Subsequently, the images were stored in Aperio eSlide Manager (Leica Microsystems) located at the Vancouver Prostate Centre. IHC-positive areas, along with their corresponding hematoxylin and eosin (H&E) sections, were reviewed by a study pathologist (HZO) to confirm the presence of Ewing sarcoma cells.

[0077] Statistical analysis

[0078] Data are presented as mean ± SD. Statistical analysis of cell cycle profiles, comet assays, spheroid assays, and PuMA assays was performed using GraphPad Prism 8.0 (GraphPad Software, Inc.). Statistical analysis of cell cycle curves was performed using a multiple t-test, and only curves showing a change in the cell cycle curve greater than 5% were analyzed, as these were considered biologically relevant changes. The Mann-Whitney nonparametric test was used to determine the statistics of the comet assay. Unpaired t-tests were used to analyze the statistics of the spheroid assay, and the Kruskal-Wallis nonparametric test was used for the PuMA assay. n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0079] Apoptosis assay

[0080] The induction of apoptosis by UT (dimethyl sulfoxide (DMSO)), diaziquone (DAG), olaparib (OLA), vorinostat (VOR), O+S (OLA+VOR), and Compound A was determined by immunoblotting for cleaved caspase 3 using cell lysates. CHLA10 cells were treated with 1 μM of the indicated compounds for 24 hours, then the cells were collected and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein yield was evaluated using the Pierce BCA Protein Assay Kit (Thermofisher, catalog number 23225), and 20 μg of the lysate was run on a 4-15% mini-PROTEAN TGX gel (BioRad, catalog number 4561084) at 100 V for 1 hour. The gel was transferred to a 0.2 μm nitrocellulose membrane (BioRad, catalog number 1620112) using the TransBlot Turbo Transfer System (BioRad, catalog number 1704150). After transfer, the membrane was blocked with LI-COR Odyssey blocking buffer and incubated overnight at 4 °C in anti-cleaved caspase 3 antibody (Cell Signaling Technology, catalog number 9661S) and anti-caspase 3 antibody (Cell Signaling Technology, catalog number 9662S), followed by incubation for 1 hour at room temperature in donkey anti-rabbit Alexa Fluor 680 (LI-COR, catalog number 926-68073). The membrane was then scanned with an Odyssey scanner (LI-COR). The results are shown in Figure 6.

[0081] Synthesis of Compound A

[0082] Compound A was prepared by conventional synthetic organic techniques as shown in Scheme 1 below.

[0083]

[0084] Compound X was prepared according to Menear, KA; et al.; J. Med. Chem. 2008, 51, 6581–6591.

[0085] Although the exemplary embodiments have been illustrated and described, it should be understood that various changes can be made thereto without departing from the spirit and scope of the invention.

Claims

1. A method for treating Ewing's sarcoma in a subject, which comprises 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.

2. (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, which is used for treating Ewing's sarcoma.

3. A pharmaceutical composition for treating Ewing's sarcoma, which comprises a pharmaceutically acceptable carrier and (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.

4. A method for inhibiting PARP1, PARP2 and HDAC in a subject, which comprises administering to the subject an 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.

5. (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, which is used for inhibiting PARP1, PARP2 and HDAC in a subject.

6. A pharmaceutical composition for inhibiting PARP1, PARP2 and HDAC, which comprises a pharmaceutically acceptable carrier and (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.

7. A method for treating a disease or disorder in a subject that is treatable by inhibiting PARP1, PARP2 and HDAC, which comprises administering to the subject an 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-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide or a pharmaceutically acceptable salt thereof, which is used for treating a disease or disorder in a subject that is treatable by inhibiting PARP1, PARP2, and HDAC.

9. A pharmaceutical composition for treating a disease or disorder in a subject that is treatable by inhibiting PARP1, PARP2, and HDAC, which comprises a pharmaceutically acceptable carrier, and (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.

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