Bispecific parp-hdac inhibitor for treating ewing sarcoma

Abstract

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] BISPECIFIC PARP-HDAC INHIBITOR FOR TREATING EWING SARCOMA

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of Application No. 63 / 496,633, filed April 17, 2023, Application No. 63 / 490,477, filed March 15, 2023, and Application No. 63 / 382,393, filed November 4, 2022, each 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 HDAC inhibition that further supports the synergistic potential of HDAC and PARP inhibition.

[0008] Ewing sarcoma is a highly metastatic bone and soft tissue tumor affecting mainly children and young adults, with a dire 5-year survival rate of 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 FLU (85%) or other ETS-family transcription factors (15%), most often ERG. These gene fusions encode chimeric oncoproteins (e.g., EWS-FLI1 or EWS-ERG) that drive Ewing sarcoma initiation and progression.

[0009] Ewing sarcoma cells are sensitive to PARP inhibitors in vitro and this sensitivity depends on EWS-FLH. Ewing sarcoma cell line-derived xenografts in mice display sensitivity to FDA-approved PARP inhibitors similar to the responses seen with the standard- of-care chemotherapy temozolomide. These observations prompted a phase II single-agent trial in Ewing sarcoma with olaparib, but despite encouraging pre-clinical data, these patients failed to produce durable responses to single agent PARP inhibition. The underwhelming response to PARP inhibitors in Ewing sarcoma patients is most likely due to insufficient synthetic lethality and Ewing sarcoma is therefore a prime candidate for exploring pharmacological BRCAness in the context of PARP inhibitors.

[0010] Despite the advances noted above in the development of therapeutic agents for treating Ewing sarcoma, a need exists for improved therapeutic agents. The present invention seeks to fulfill this need and provides further related advantages. SUMMARY

[0011] In one aspect, the disclosure provides 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-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 Compound A, or a pharmaceutically acceptable salt thereof, for use in the treatment of Ewing sarcoma in a subject.

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

[0013] In a further aspect, the disclosure provides a method for treating a disease or condition treatable by inhibiting PARP1, PARP2, and HD AC in a subject, comprising administering to a subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof. In a related aspect, the disclosure provides Compound A, or a pharmaceutically acceptable salt thereof, for use the treatment of disease or condition treatable by inhibiting PARP1, PARP2, and HD AC in a subject.

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

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A-1D compare dual activity of (E)-3-(2-(4-(2-fluoro-5-((4-oxo-3,4- dihydrophthalazin-l-yl)methyl)benzoyl)piperazin-l-yl)pyrimidin-5-yl)-N-hydroxy acrylamide (Compound A) against PARP1 / 2 and HD AC enzymes. FIG. 1A compares in vitro HD AC activity in HeLa nuclear extracts treated with Compound A or vorinostat. FIG. IB compares recombinant PARP1 activity in vitro after treatment with Compound A and olaparib. FIG. 1C compares recombinant PARP2 activity in vitro after treatment with Compound A and olaparib. FIG. ID compares PAR formation in CHLA10 cells treated with Compound A and olaparib. Values were normalized to control and ICso was calculated as the concentration required to produce 50% inhibition of activity from non-linear regression plots using GraphPad Prism8 software. Data shown are the mean values of n=3 replicates with representative graphs.

[0017] FIGS. 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 is compared to the activities of olaparib, niraparib, talazoparib, vorinostat, belinostat, and panobinostat. FIG. 2A compares cell viability of TC32 cells examined by IncuCyte® S3 live cell imaging system following three-day treatments with increasing concentrations of indicated compounds. ECso values were calculated as the concentration required for 50% cell viability, n=3. FIG. 2B compares ECso values of tested compounds determined in A673 cells using the same experimental condition as in FIG. 2A. FIG. 2C compares ECso values of indicated inhibitors determined using CellTiter-Glo® cell viability assays in CHLA10 cells using the same experimental condition as in FIG. 2A. Cells were exposed to ten-day treatments of increasing concentrations of the inhibitors, and the ECso values were calculated as the concentration required for 50% cell viability, n=3.

[0018] FIGS. 3A and 3B illustrate that Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells. FIG. 3A presents cell cycle analysis for TC32 cells synchronized at G0 / G1 phase by serum starvation for 24 h before treatments with Compound A, olaparib, or vorinostat as indicated in complete medium for 48 h. Then, cell cycle profiles were examined by propidium iodide (PI) staining followed by flow cytometric analysis. Cell cycle distribution is also shown. FIG. 3B presents cell cycle analysis for CHLA10 cells treated with Compound A, olaparib, or vorinostat as indicated for 24 h using the same experimental procedures as in FIG. 3A.

[0019] FIGS. 4A-4F illustrate that Compound A treatment induces DNA damage in Ewing sarcoma cells. FIG. 4A compares yH2AX expression by Western blot for dianhydrogalactitol (DAG), olaparib, vorinostat, or Compound A: TC32 cells were treated with 2.5 pM DAG or increasing doses of olaparib (0.35-13 pM), vorinostat (0.35-8 pM), or Compound A (0.018- 0.35 pM) for 48 h and analyzed for yH2AX expression by Western blot. FIG. 4B compares yH2AX expression by Western blot for DAG, olaparib, vorinostat, or Compound A: CHLA10 cells were treated with 5 pM dianhydrogalactitol (DAG) or increasing doses of olaparib (1-37 pM), vorinostat (1-20 pM), or Compound A (0.05-1 pM) for 48 h and analyzed as in (A). FIGS. 4C-4E compare yH2AX foci analysis by immunofluorescence and confocal microscopy imaging: CHLA10 cells treated with DAG (2.5 pM) or increasing doses of Compound A (0-1 pM) (FIG. 4C), olaparib (1-37.5 pM) (FIG. 4D), or vorinostat (1- 18 pM) (FIG. 4E) for 24 h were analyzed for yH2AX foci by immunofluorescence and confocal microscopy imaging. Scale bar represents 10 pm. FIG 4F illustrates comet assay results for CHLA10 cells treated with 1 pM Compound A, olaparib, vorinostat, or olaparib + vorinostat followed. 5 pM DAG was included as positive control. Scale bar represents 200 pm. ****p<0.0001.

[0020] FIGS. 5A-5F illustrate that Compound A inhibits 3D spheroid growth and metastasis of Ewing sarcoma cells. FIG. 5A compares TC32 spheroid growth following four days of treatment with increasing concentrations of Compound A, olaparib, or vorinostat, as monitored using the IncuCyte® Spheroid Analysis system. The EC50 values were calculated as the concentration required for 50% inhibition of growth from non-linear regression plots using GraphPad Prism8 software. Representative images of TC32 spheroids at day 0 and day 4 with DMSO,1 pM Compound A, 1 pM olaparib, or 1 pM vorinostat are shown with scale bars representing 400 pm. *p<0.05, **p<0.01. FIG. 5B compares CHLA10 spheroid growth following four-day treatment with increasing concentrations of Compound A, olaparib, or vorinostat using the same experimental procedures as noted in FIG. 5A. Representative images of CHLA10 3D spheroids at day 0 and day 4 with DMSO,1 pM Compound A, 1 pM olaparib, or 1 pM vorinostat are shown with scale bars representing 400 pm. *p<0.05, ***p<0.001. FIG. 5C compares lung tumour burden following 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 slices following 14 days of treatment with 5, 10, or 20 nM of Compound A. Scale bar represents 1 mm. **p<0.01. FIG. 5D shows representative hematoxylin and eosin (H&E) and CD99 staining images of TC32 Ewing sarcoma cells in PuMA lung sections following 14 days of treatment with 5, 10, or 20 nM of Compound A. The respective zoomed images from the insets (upper panel) are shown below each image. Scale bar represents 50 pm. FIG. 5E compares lung tumour burden following 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 slices following 14 days of treatment with 5, 10, or 20 nM of Compound A. Scale bar represents 1 mm. **p<0.01, ****p<0.0001. FIG. 5F shows representative H&E and CD99 staining images of A673 Ewing sarcoma cells in PuMA lung sections following 14 days of treatment with 5, 10, or 20 nM of Compound A. The respective zoomed images from the insets (upper panel) are shown below each image. Scale bar represents 50 pm.

[0021] FIG. 6 compares the induction of apoptosis by UT (dimethylsulfoxide (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] DETAILED DESCRIPTION

[0023] HDAC 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 PARP 1 / 2 and HDAC enzymes in Ewing sarcoma cells. Compared to the FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, Compound A displayed enhanced cytotoxicity in Ewing sarcoma models. Compound A-induced cytotoxicity was associated with a strong S and G2 / M cell cycle arrest in the nanomolar concentration range and elevated DNA damage as assessed by yH2AX tracking and comet assays. In three-dimensional spheroid models of Ewing sarcoma, Compound A showed efficacy in lower concentrations than olaparib and vorinostat.

[0024] In one aspect, the disclosure provides 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-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-dihydrophthal azin-1 -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 Ewing sarcoma in a subject.

[0025] In another aspect, the disclosure provides a method for inhibiting PARP1, PARP2, and HDAC in a subject, comprising administering to a subject an effective amount of (E)-3- (2-(4-(2-fluoro-5-((4-oxo-3, 4-dihydrophthal azin-1 -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-hydroxy acrylamide (Compound A), or a pharmaceutically acceptable salt thereof, for use in inhibiting PARP1, PARP2, and HDAC in a subject.

[0026] In a further aspect, the disclosure provides a method for treating a disease or condition treatable by inhibiting PARP1, PARP2, and HDAC in a subject, comprising administering to a subject 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-hydroxy acrylamide (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 the treatment of disease or condition treatable by inhibiting PARP1, PARP2, and HDAC in a subject.

[0027] In other aspects, the disclosure provides pharmaceutical compositions for the abovenoted uses. In these aspects, the pharmaceutical composition comprises (E)-3-(2-(4-(2- fluoro-5-((4-oxo-3, 4-dihydrophthal azin-1 -yl)methyl)benzoyl)piperazin-l-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-l-yl)methyl)benzoyl)piperazin-l-yl)pyrimidin-5-yl)-N- hydroxyacrylamide, having formula (I):

[0029] or a tautomer thereof.

[0030] 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.

[0031] The following describes the usefulness of Compound A for inhibiting PARP1, PARP2, and HD AC, and the effectiveness of Compound A for treating Ewing sarcoma.

[0032] 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 (HD AC inhibitor) vorinostat (NCT03259503 and NCT03742245), the DNA methyltransferase inhibitor decitabine (NCT02878785), and the EZH2 histone methyltransferase inhibitor SHR2554 (NCT04355858), are currently ongoing.

[0033] 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 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 HD AC and PARP inhibition is particularly interesting in the context of HR-proficient cancer types where PARPi therapy has limited effect on its own, such as Ewing sarcoma. However, doselimiting 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 over-lapping toxicity events arising from the combination with other therapeutic moieties, which can be challenging when working with different pharmacokinetics profiles.

[0034] The present disclosure provides a bifunctional PARP-HDAC single-molecule inhibitor, Compound A, in Ewing sarcoma 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 30-to-80 times more cytotoxic to Ewing sarcoma cells than olaparib, and 30-to-60 times more cytotoxic than vorinostat alone. While panobinostat appeared to have higher efficacy in Ewing sarcoma cell lines, this can be attributed to the toxicity of panobinostat, as seen in clinical trials where dose-limiting toxicity has restricted effective use in solid tumors. This is likely also the case with talazoparib. While talazoparib is the most potent FDA-approved PARPi to date, it also showed clinical toxicities more similar to other chemotherapeutic agents than the other approved PARP inhibitors, including anemia, thrombocytopenia and neutropenia.

[0035] Compound A also induces cell cycle arrest and DNA damage in Ewing sarcoma cells in much lower concentrations than olaparib and vorinostat. The cell cycle arrest pattern of Compound A was more similar to that of olaparib than the combination treatments as the PARP inhibitor activity of Compound A was stronger than the HD AC inhibitor portion of the drug. In TC32 cells, the combination of 0.7 pM olaparib and 0.7 pM belinostat showed some arrest in G0 / G1, consistent with the lower EC50 value for TC32 cells treated with belinostat. When compared in 3D spheroid models, Compound A showed efficacy at 30-to-40 times lower concentrations than olaparib and at 5-to-10 times lower concentrations than vorinostat. Spheroid models treated with talazoparib indicated equivalent efficacy to Compound A but 10 times lower efficacy by panobinostat compared to Compound A. This may be attributed to the overall toxicity of panobinostat as previously mentioned. Compound A also hinders metastatic growth of Ewing sarcoma cells in an ex vivo PuMA model, with a strong inhibitory effect using as little as 10 nM of inhibitor. As hematological toxicity of PARP and HD AC inhibitors is of concern, a pilot study of Compound A was performed in mice which showed no evidence of toxicity based on weight loss and blood cell counts. Combining PARP and HD AC inhibition into one single molecule offers a convenient way to prevent resistance to PARPi therapy. For example, Ewing sarcoma and many other solid tumor indications epigenetically suppress expression of the tumor suppressor gene Schlafen 11 (SLFN1 / ). which leads to resistance to DNA damage-inducing agents, including PARPi therapy. Important here, HDACi treatment prompts re-expression of SLFN11 and resensitization to PARPi.

[0036] 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.

[0037] In summary, the present disclosure provides a single-molecule PARP-HDAC inhibitor, Compound A, in Ewing sarcoma with improved cytotoxicity and DNA damage activity as compared to PARPi and HDACi alone.

[0038] A bi-specific compound dual activity against PARP1 / 2 and HD AC enzymes

[0039] Through medicinal chemistry cycles, the present disclosure provides a small-molecule inhibitor (Compound A) with dual activity against PARP 1 / 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 HDAC inhibitor vorinostat. A wide concentration range of each compound was used to determine ICso values. Compound A had an ICso value of 2.54 pM while vorinostat was about 50-fold lower at 0.05 pM (FIGURE 1A). The PARPI and PARP2 inhibitory activities of Compound A were comparable to olaparib, with ICso values for Compound A at 3.38 nM and 2.19 nM, respectively (FIGURES IB and 1C). 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 (FIGURE ID). These data indicate that Compound A is able to inhibit both PARP 1 / 2 and HDAC enzymes.

[0040] Ewing sarcoma cells are highly sensitive to dual PARP 1 / 2 and HDAC inhibition In order to investigate the effect of Compound A in cell growth, cell viability assays were performed in three Ewing sarcoma cell lines. Using IncuCyte S3 live cell imaging system, we examined the ECso values in cell viability after three-day treatment with increasing concentrations of Compound A, three FDA-approved PARP inhibitors, or three FDA-approved HDAC inhibitors in TC32 and A673 cells. Compound A demonstrated higher efficacy in suppression of cell viability than olaparib, niraparib, vorinostat, and belinostat with ECso of 0.0163 pM in TC32 cells (FIGURE 2A). A similar effect of Compound A in A673 cells was detected with a much lower ECso value of 0.0365 pM compared to olaparib, niraparib, vorinostat, and belinostat treatment alone (FIGURE 2B). However, treatment with talazoparib or panobinostat showed stronger inhibitory effect in both cell lines compared with Compound A (FIGURES 2A and 2B). To further validate the findings, CellTiter-Glo® viability assay was performed to determine the ECso values of these tested compounds in CHLA10 cells. Consistent with the IncuCyte assay, the ECso value (0.053 pM) of Compound A treatment in CHLA10 cells was also much lower than olaparib, niraparib, vorinostat, and belinostat (FIGURE 2C). Taken together, the data demonstrates potent inhibitory effect of Compound A in Ewing sarcoma cells compared to FDA-approved PARP or HDAC inhibitors.

[0041] Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells

[0042] PARP inhibitors and HDAC inhibitors constantly induce S / G2 / M and G0 / G1 cell cycle arrest, respectively as PARP regulates replication fork progression and HDACs play a major role in regulating the expression of cell cycle checkpoint proteins including cyclin- dependent kinases, cyclin DI and p21. The cell cycle profiles of CHLA10 and TC32 cells upon treatment with Compound A, olaparib, and vorinostat in both single and combination regimens were examined. Serum-starved cells were treated with increasing concentrations of Compound A in complete medium for 24 or 48 h and displayed strong S and G2 / M arrest at and after 0.175 pM in TC32 and 0.25 pM in CHLA10 cells. Similar cell cycle arrest was only observed with olaparib treatment at concentrations higher than 3 pM in TC32 and 14.7 pM in CHLA10, respectively (FIGURES 3 A and 3B). Combination treatment with olaparib and vorinostat / belinostat at equimolar concentrations of Compound A (1 pM and 0.7 pM for CHLA10 and TC32 cells, respectively) had almost no influence on cell cycle phases compared to control (FIGURES 3A and 3B). These data demonstrate Compound A has stronger potency to induce S and G2 / M cell cycle arrest than olaparib alone or in combination with vorinostat or belinostat in Ewing sarcoma cells. Compound A treatment induces DNA damage in Ewing sarcoma cells

[0043] PARP inhibitors and HD AC inhibitors have been reported to induce DNA damage in cells. The effect of Compound A on DNA damage compared to olaparib and vorinostat treatment in Ewing sarcoma cells using western blot, immunofluorescence, and comet assay was investigated. Phosphorylated histone variant H2AX (yH2AX) is a surrogate marker for DSBs in DNA. Dianhydrogalactitol (DAG) was included as a positive control because DAG induces replication-dependent DNA damage in a variety of cancer cell lines. Treatment with Compound A, olaparib or vorinostat induced yH2AX expression in a dose-dependent manner in both CHLA10 and TC32 cells. In comparison to olaparib and vorinostat, Compound A was able to induce yH2AX expression at a much lower concentration range (FIGURES 4A and 4B). Moreover, CHLA10 cells treated with Compound A or olaparib also showed dosedependent yH2AX foci formation in immunofluorescence followed by confocal microscopy imaging with Compound A at a much lower concentration range (FIGURE 4C). However, vorinostat that induced G0 / G1 cell cycle arrest (FIGURES 3A and 3B) demonstrated milder DNA damage foci formation in CHLA10 cells (FIGURE 4C). To further consolidate the data, alkaline comet assay was employed as it can detect both SSBs and DSBs in cells. CHLA10 cells treated with 1 pM Compound A demonstrated significant amount of DNA damage but not with 1 pM olaparib or vorinostat treatment (FIGURE 4D). In summary, the data show Compound A is able to induce DNA damage in Ewing sarcoma cells at a much lower concentration range than olaparib or vorinostat.

[0044] PARP inhibitors and HD AC inhibitors have been reported to induce DNA damage in cells. The effect of Compound A on DNA damage compared to olaparib and vorinostat treatment in Ewing sarcoma cells was investigated using Western blot, immunofluorescence, and comet assay. Phosphorylated histone variant H2AX (yH2AX) is a surrogate marker for DSBs in DNA. Dianhydrogalactitol (DAG) was included as a positive control because previous studies showed that DAG induces replication-dependent DNA damage in a variety of cancer cell lines. Treatment with Compound A, olaparib or vorinostat induced yH2AX expression in a dose-dependent manner in both TC32 and CHLA10 cells. In comparison to olaparib and vorinostat, Compound A was able to induce yH2AX expression at a much lower concentration range (see FIGS. 4A and 4B). Moreover, CHLA10 cells treated with Compound A or olaparib also showed dose-dependent yH2AX foci formation in immunofluorescence followed by confocal microscopy imaging with Compound A at a much lower concentration range (see FIGS. 4C and 4D). However, vorinostat that induced G0 / G1 cell cycle arrest (See FIGS 3A and 3B) demonstrated milder DNA damage foci formation in CHLA10 cells (see FIG. 4E). Additionally, TC32 cells only showed increased yH2AX expression by western blot upon treatment with 0.35 pM of Compound A but not with 0.35 pM of olaparib, vorinostat, or olaparib + vorinostat. This observation was also confirmed using equimolar concentrations of these compounds in CHLA10 cells by western blot and immunofluorescence. To further consolidate the data, an alkaline comet assay was employed as the assay can detect both SSBs and DSBs in cells. There was significant DNA damage in CHLA10 cells treated with 1 pM Compound A but not with 1 pM olaparib or vorinostat or 1 pM olaparib + 1 pM vorinostat (See FIG. 4F). In summary, the data show Compound A is able to induce DNA damage in Ewing sarcoma cells at a much lower concentration range than olaparib or vorinostat.

[0045] Compound A inhibits 3D spheroid growth of Ewing sarcoma cells

[0046] Spheroids are three-dimensional (3D) cell aggregates that can mimic tumor behavior more accurately compared to two-dimensional cell cultures. To further validate the data, the effect of Compound A on the 3D spheroid model with Ewing sarcoma cells was investigated. CHLA10 and TC32 spheroids were established to 200-300 pm followed by treatment with increasing concentrations of Compound A, olaparib, or vorinostat. The growth of the spheroids was monitored and quantified for four days using the IncuCyte S3 imaging system. The ECso values of Compound A in suppression of spheroid growth were much lower than olaparib and vorinostat in both TC32 and CHLA10 cell models (FIGURES 5 A and 5B). These data suggest that Compound A is a potent inhibitor against 3D spheroid growth of Ewing sarcoma cells and demonstrates more effectiveness than olaparib or vorinostat alone.

[0047] Spheroid assays with both TC32 and CHLA10 cells demonstrate equivalent activity of Compound A and talazoparib but ten times lower efficacy of panobinostat compared to Compound A. The effect of Compound A on metastatic growth of Ewing sarcoma cells was examined in an ex vivo pulmonary metastasis assay (PuMA). Here, colonization of td- Tomato expressing TC32 and A673 cells in mouse lungs was inhibited by Compound A at concentrations as low as 10 nM (See FIGS. 5C and 5E). Parallel hematoxylin and eosin (H&E) and CD99 IHC staining (see FIGS. 5D and 5F) confirm that the tdTomato fluorescent signals in the lung tissues were indeed Ewing sarcoma cells. Fluorescence per cell of both tdTomato expressing TC32 and A673 cells were confirmed not to be affected by treatment with Compound A by fluorescence microscopy. A study in nude mice showed no sign of toxicity as indicated by weight and blood cell counts after intraperitoneal treatment with 30 mg / kg Compound A (B.I.D.) for four days. These data suggest that Compound A is a potent inhibitor of Ewing sarcoma lung metastasis and that it is more effective than olaparib or vorinostat alone in inhibiting 3D growth in Ewing sarcoma spheroids.

[0048] Materials and Methods

[0049] Cell culture

[0050] Identities of all human Ewing sarcoma cell lines were confirmed by STR profiling 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. 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% fetal bovine serum (FBS) (Gibco cat# A3160401). TC32 cells were maintained in RPMI-1640 (Gibco cat# 11875119) with 10% FBS and lx GlutaMAX supplement (Thermo Fisher Scientific cat# 35050061). A673 cells were maintained in Dulbecco’s Modified Eagle Medium (Gibco cat# 11995065) supplemented with 10% FBS.

[0051] HD AC activity assay

[0052] 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. IC50 values were calculated using a four-parameter variable slope non-linear regression in GraphPad Prism 8 (GraphPad Software Inc.).

[0053] PARP1 and PARP2 activity assay

[0054] 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. IC50 values were calculated using a four-parameter variable slope non-linear regression in GraphPad Prism 8 (GraphPad Software Inc.).

[0055] PAR formation assay

[0056] 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, clear-bottom 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 IC50 value ± SD of three biological replicates was calculated.

[0057] Cell viability assay

[0058] 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 and three days for TC32 and A673. Cell-Titer-Glo® viability assay was carried out for CHLA10.150 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.

[0059] Cell cycle analysis

[0060] Cell cycle profdes were evaluated via propidium iodide (PI) staining and flow cytometry. CHLA10 and TC32 cells were plated in 10 cm plates with a cell density of 1.5xl06cells / plate and 2.0x106cells / plate, respectively. The media was replaced the following day with serum-free media for 24 h. Cells were treated with olaparib, vorinostat and Compound A in a dose escalating manner for 24 h for TC32 and 48 h for CHLA10. Combination treatments of olaparib with vorinostat and olaparib with belinostat were evaluated at equimolar concentration of Compound A (0.7 and 1 pM for TC32 and CHLA10 cells, respectively). Cells were harvested and l.OxlO6cells from each treatment were fixed in 70% ethanol overnight at -30°C. The cell suspension was then washed with cold PBS and stained with a PI solution (50 pg / mL PI, O.lmg / mL RNase A, 0.05% Triton X-100 in PBS) and incubated at 37°C for 40 min in the dark. Cells were then washed with PBS, filtered through a 40 pm strainer and resuspended with 500 pL PBS. Samples were then examined by flow cytometry and analyzed in FlowJo vlO.

[0061] Alkaline comet assay

[0062] Cells were plated in a 6-well plate at a density of 1x106cells / well and allowed to settle overnight. Cell media was replaced with serum-free media for 24 h, then treated with DMSO or test compound for 24 h at 37°C with 5% CO2 and 95% humidity. Cells were harvested as instructed in Trevigen’s CometAssay® protocol and combined with molten LMAgarose at a ratio of 1:10 and pipetted onto CometSlides®. Cells were placed in the dark for 30 min and immersed in lysis solution overnight at 4°C. Slides were immersed in Alkaline Unwinding solution for 1 h at 4°C in the dark, then placed in a gel electrophoresis tray and immersed in Alkaline Electrophoresis solution with an applied voltage of 25V for 30 min. Samples were washed with dfbO and 70% ethanol before staining with SYBR® Gold. Samples were then viewed using a fluorescence microscope. Acquired images were analyzed using OpenComet on ImageJ (NIH).

[0063] Immunofluorescence

[0064] CHLA10 cells were seeded on glass coverslips in a 24-well plate at a density of 3.5xl05cells / well and allowed to settle overnight. Media was replaced with serum-free media for 24 h, then cells were treated with DMSO or increasing concentrations of test compound for 24 h. Cells were fixed with 4% paraformaldehyde for 30 min at RT and permeabilized with 0.5% Triton-X in PBS before probing with anti -phospho-histone H2AX (Ser 139) rabbit antibody (Cell Signaling Technology cat# 2577) overnight at 4°C. Cells on coverslips were then washed with PBS and probed with goat anti -rabbit IgG Alexa Fluor® 488 (Abeam cat# abl 50077) and mounted with VECTASHIELD antifade mounting medium with DAPI solution onto microscope slides. Cells were then visualized on a confocal microscope (Olympus FV3000). Acquired images were analyzed by quantifying the foci using ImageJ (NIH).

[0065] Western blot analysis

[0066] Cells were plated in a 6-well plate to 70-80% confluency. After allowing cells to settle overnight, media was replaced with serum-free media for 24 h. Cells were treated with DMSO or increasing concentrations of test compound for 24 h. Cells were harvested in Radioimmunoprecipitation Assay (RIPA) lysis buffer combined with protease and phosphatase inhibitors. Protein yield was assessed using Pierce™ BCA Protein Assay Kit (Thermofisher cat# 23225) and quantified using a spectrophotometer plate reader (TECAN) at 562 nm. A total of 20 pg protein extracts were loaded per well in 4-15% Mini- PROTEAN® TGX™ Precast Protein gels (Bio-Rad cat# 4561084). After electrophoresis, proteins were transferred onto a 0.2 pm nitrocellulose membrane. The membrane was blocked with LICOR® Odyssey Blocking Buffer in PBS. After blocking, the membrane was incubated with anti-phospho-histone H2AX (Ser 139) rabbit antibody (Cell Signaling Technology cat# 2577) and H2AX rabbit antibody (Abeam cat# abll l75) at 4°C overnight and incubated with donkey anti-rabbit IRDye®800CW secondary antibody (LI-COR cat# 926-32213) for 1 h at RT. The membrane was washed with IX tris-buffered saline with 1% tween-20 (TBS-T) before being scanned with Odyssey scanner (LI-COR).

[0067] Spheroid formation assay

[0068] CHLAlO-tdTomato or TC32-tdTomato cells (2500 per well) were added to a 96-well clear round bottom ultra-low attachment microplate (Coming cat# 7007) and allowed to form spheroids for 24 h or until they reached 200-300 pm in diameter. Medium containing DMSO or increasing concentration of test compound was added to each well and spheroid growth was monitored for four days post-treatment using the 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 a normalization factor from day 0 values and 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.

[0069] Pulmonary metastasis assay (PuMA)

[0070] Procedures involving mice were approved by local animal care committee, University of British Columbia. tdTomato-expressing TC32 and A673 cells (1 x 106cells / 100 pl saline) were injected tail-vein into 6-8-week-old immune-compromised NSG female mice (Jax Laboratories). After injection, mice were euthanized via isoflurane and CO2 asphyxiation according to local animal care standard operating procedures. Lungs were insufflated via gravity perfusion with a pre-warmed (37°C) 1:1 mixture of fully supplemented PneumaCult™-ALI media (STEMCell cat #05001) and 1.2% low-melting point agarose (Lonza) 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;! 1:645757 doi 10.3389 / fonc.202L 645757). The pluck (heart & lungs) was carefully removed and placed in ice-cold PBS (supplemented with IX penicillin / streptomycin) for 20 min to allow for agarose solidification. Small lung slices (about 2 mm x 4 mm) were obtained by manual cutting with sterile surgical scissors, and 5-12 slices per condition were chosen for serial imaging at 0 and 14 days post-injection / treatment. Lung slices were maintained in vitro on gelatine sponges partially submerged in 2 mL PneumaCult™ media + / - compound in a 6-well plate; media + / - compound was refreshed every three days. On the day of imaging, lung slices from each group were transferred to a small 35 mm petri dish with a glass coverslip bottom (IBIDI) to permit aseptic widefield fluorescence imaging. The lung slices were imaged on an inverted Zeiss Observer. / 1 Colibri microscope using 2.5X objective. Lung tumour burden (% tumor burden) for a lung slice is calculated as the quotient of the summed area of tdTomato lesions and total area of lung slice, multiplied by 100, 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). ImageJ software was used for image processing. This calculation was performed for all lung slices (n = 5-12 lung slices) per experimental group. Average values of percent lung tumour burden per group were compared and analyzed in Graph Prism 8 (GraphPad Software Inc.).

[0071] Immunohistochemistry and histopathology

[0072] Formalin-fixed paraffin-embedded (FFPE) PuMA lung tissue sections were freshly cut and analyzed for CD99 immunoexpression using Ventana Discovery Ultra autostainer (Ventana Medical Systems, Tucson, Arizona). In brief, baked and deparaffmized tissue sections were incubated in Tris-based buffer (CC1, Ventana) at 95°C for 64 min to retrieve antigenicity, followed by incubation with anti-CD99 rabbit polyclonal antibody (Abeam cat# ab27271) at RT for 1 h. Bound primary antibody was visualized with the UltraMap DAB anti-Rb Detection Kit (Ventana). All stained slides were digitized with Leica scanner (Aperio AT2, Leica Microsystems; Concord, Ontario, Canada) at magnification equivalent to 40X. The images were subsequently stored in the Aperio eSlide Manager (Leica Microsystems) at the Vancouver Prostate Centre. The IHC positive areas were reviewed by a research pathologist (HZO), along with their corresponding hematoxylin and eosin (H&E) sections to confirm the presence of Ewing sarcoma cells.

[0073] Statistical analysis

[0074] Data are presented as the mean ± SD. Statistical analysis for cell cycle profiling, comet assay, spheroid assay and PuMA assay were performed using GraphPad Prism 8.0 (GraphPad Software, Inc.). Statistical analysis of the cell cycle profiles was done using a multiple t-test and was only performed on the profiles that displayed a change in cell cycle profile greater than 5% as these were considered to be biologically relevant changes. Statistics for the comet assay were determined using a Mann-Whitney nonparametric test. Statistical analysis of the spheroid assay was analyzed using unpaired t-test, and PuMA assay was done using Kruskal-Wallis nonparametric test. n.s.=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0075] Apotosis Assay

[0076] Induction of apoptosis by UT (dimethylsulfoxide (DMSO)), dianhydrogalactitol (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 indicated compounds at 1 pM for 24 hours, then cells were collected and lysed in RIPA buffer with protease and phosphatase inhibitors. Protein yield was assessed using Pierce BCA Protein Assay Kit (Thermofisher cat. no. 23225) and 20 pg of lysate was run on a 4-15% mini-PROTEAN TGX gel (BioRad cat. no. 4561084) at 100V for 1 hour. The gel was transferred to 0.2 pm nitrocellulose membrane (BioRad cat. no. 1620112) using the TransBlot Turbo Transfer System (BioRad cat. no. 1704150). After transfer, the membrane was blocked with LI-COR Odyssey blocking buffer and incubated in anti-cleaved caspase 3 antibody (Cell Signaling Technology cat. no. 9661 S) and anti-caspase 3 antibody (Cell Signaling Technology cat. no. 9662S) overnight at 4°C followed by donkey anti-rabbit Alexa Fluor 680 (LI-COR cat. no. 926-68073) for 1 hour at room temperature. The membrane was then scanned with an Odyssey scanner (LI-COR). The results are illustrated in FIG. 6.

[0077] Synthesis of Compound A

[0078] Compound A was prepared by conventional synthetic organic techniques as shown below in Scheme 1.

[0079] Scheme 1

[0080] Compound X was prepared according to Menear, KA; et al.; J. Med. Chem. 2008, 51, 6581-6591.

[0081] 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 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-dihy drophthalazin- 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-l- yl)methyl)benzoyl)piperazin-l -yl)pyrimidin-5-yl)-N-hydroxyacrylamide, or a pharmaceutically acceptable salt thereof, for use in the treatment of Ewing sarcoma.

3. A pharmaceutical composition for treating Ewing sarcoma, 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.

4. A method for inhibiting PARP1, PARP2, and HD AC in a subject, comprising administering to a subject an 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.

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 inhibition of PARP1, PARP2, and HD AC in a subject.

6. A pharmaceutical composition for use in inhibiting PARP1, PARP2, and HD AC in a subject, 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.

7. A method for treating a disease or condition treatable by inhibiting PARP1, PARP2, and HD AC in a subject, comprising administering to a subject an 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.

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 a disease or condition treatable by inhibiting PARP1, PARP2, and HD AC in a subject.

9. A pharmaceutical composition for use in the treatment of a disease or condition treatable by inhibiting PARP1, PARP2, and HD AC in a subject, 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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