Bispecific PARP-HDAC inhibitors for treating Ewing's sarcoma

Abstract

A method for treating Ewing's 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

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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 of which is expressly incorporated by reference herein in its entirety. [Background technology]

[0002] background Poly(ADP-ribose) polymerase (PARP) proteins catalyze the poly(ADP-ribosylation) of cellular proteins using the ADP (adenosine diphosphate)-ribose subunit of nicotinamide adenine dinucleotide (NAD+) as a donor. The human genome encodes 17 PARP enzymes, and at least PARP1-3 have important functions in DNA repair, with PARP1 being the most well-characterized. PARP1 is essential for the repair of single-strand DNA breaks (SSBs), the most common type of breakpoint lesion in cellular DNA. When cells encounter an SSB, PARP1 binds to the lesion and initiates a poly(ADP-ribosylation) cascade of itself and histones embedded in the chromatin surrounding the SSB lesion. This poly(ADP-ribosylation) event acts as a signal to recruit the SSB repair machinery to patch the lesion before and during DNA replication in the S phase of the cell cycle. Efficient SSB repair is critical to prevent replication stress and more severe double-strand break (DSB) lesions that occur in S phase when unrepaired SSB lesions collide with replication forks. DSB lesions in S phase are primarily repaired by homologous recombination (HR), which depends on proteins such as BRCA1 and BRCA2. Deleterious mutations in BRCA1 / 2 are found in a subset of breast, ovarian, and prostate tumors and sporadically in other solid tumor indications. These HR-deficient tumors indirectly rely on functional PARP enzymatic activity to avoid the accumulation of catastrophic DSBs in S phase and the initiation of cell death. This dependency has made PARP inhibition a therapeutic strategy to generate synthetic lethality in tumor cells with BRCA1 / 2 deficiency.

[0003] Currently, there are four approved PARP inhibitors in clinical practice: 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 HR DNA repair deficiencies caused by BRCA1 / 2 mutations. Encouraged by the success of PARP inhibitors in BRCA1 / 2-mutated cancers, research has focused on cancer subtypes in which HR repair is impaired due to molecular events other than BRCA1 / 2 mutations. For example, tumors with mutations in RAD51, an enzyme that acts 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.

[0004] In HR-competent cancers, BRCAness can be pharmacologically mimicked by inhibiting proteins that affect BRCA1 / 2 expression. This potentially provides 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 through indirect modulation of HR components. Recent studies in leukemia, breast cancer, liver cancer, glioblastoma, prostate cancer, and anaplastic thyroid cancer models have demonstrated suppression of HR activity by HDAC inhibition, further supporting the synergistic potential of HDAC and PARP inhibition.

[0005] Ewing sarcoma is a highly metastatic bone and soft tissue tumor that primarily affects children and young adults, with a 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 FLI1 (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 the initiation and progression of Ewing sarcoma.

[0006] Ewing sarcoma cells are sensitive to PARP inhibitors in vitro, and this sensitivity is dependent on EWS-FLI1. Ewing sarcoma cell line-derived xenografts in mice exhibit sensitivity to FDA-approved PARP inhibitors similar to the response seen with the standard-of-care chemotherapy temozolomide. These findings led to a phase II single-agent trial in Ewing sarcoma with olaparib, but despite encouraging preclinical data, these patients did not develop a durable response to single-agent PARP inhibition. The poor response to PARP inhibitors in Ewing sarcoma patients is most likely due to insufficient synthetic lethality, and thus Ewing sarcoma is a prime candidate for exploring pharmacological BRCAness in the context of PARP inhibitors. Summary of the Invention [Problem to be solved by the invention]

[0007] Despite the above advances in the development of therapeutic agents for treating Ewing's sarcoma, there remains a need for improved therapeutic agents. The present invention seeks to fulfill this need and provides further related advantages. [Means for solving the problem]

[0008] overview In one aspect, the disclosure provides a method for treating Ewing's 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 (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 treating Ewing's sarcoma in a subject.

[0009] In another aspect, the disclosure provides a method for inhibiting PARP1, PARP2, and HDAC in a subject, the method comprising administering to the 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 HDAC in a subject.

[0010] In a further aspect, the present disclosure provides a method for treating a disease or condition treatable by inhibiting PARP1, PARP2, and HDAC in a subject, 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 use in treating a disease or condition treatable by inhibiting PARP1, PARP2, and HDAC in a subject.

[0011] In other aspects, the present disclosure provides pharmaceutical compositions for the above uses. In these aspects, the pharmaceutical compositions comprise Compound A or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0012] [Figure 1A]Figure 1 compares the dual activity 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) against PARP1 / 2 and HDAC enzymes. In vitro HDAC activity is compared in HeLa nuclear extracts treated with Compound A or vorinostat. Values ​​were normalized to control, and IC50 was calculated as the concentration required to produce 50% inhibition of activity from nonlinear regression plots using GraphPad Prism8 software. Data shown are the mean of a representative graph and n=3 replicates. [Figure 1B] FIG. 1 compares the dual activity 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) against PARP1 / 2 and HDAC enzymes. Recombinant PARP1 activity in vitro after treatment with Compound A and olaparib is compared. Values ​​were normalized to control, and IC50 was calculated as the concentration required to produce 50% inhibition of activity from nonlinear regression plots using GraphPad Prism8 software. Data shown are the mean of a representative graph and n=3 replicates. [Figure 1C] Figure 1 compares the dual activity 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) against PARP1 / 2 and HDAC enzymes. Recombinant PARP2 activity in vitro after treatment with Compound A and olaparib is compared. Values ​​were normalized to control, and IC50 was calculated as the concentration required to produce 50% inhibition of activity from nonlinear regression plots using GraphPad Prism8 software. Data shown are the mean of a representative graph and n=3 replicates. [Figure 1D]Figure 1 compares the dual activity 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) against PARP1 / 2 and HDAC enzymes. PAR formation in CHLA10 cells treated with Compound A and olaparib is compared. Values ​​were normalized to control, and IC50 was calculated as the concentration required to produce 50% inhibition of activity from a nonlinear regression plot using GraphPad Prism8 software. Data shown are the mean of a representative graph and n=3 replicates. [Figure 2A] Figure 1 shows that Ewing's sarcoma cells are highly sensitive to PARP1 / 2 and HDAC dual inhibition. The activity of Compound A is compared with that of Olaparib, Niraparib, Talazoparib, Vorinostat, Belinostat, and Panobinostat. The cell viability of TC32 cells, as determined by an IncuCyte® S3 live cell imaging system, is compared after 3 days of treatment with increasing concentrations of the indicated compounds. EC50 values ​​were calculated as the concentration required for 50% cell viability (n=3). [Figure 2B] Figure 2 shows that Ewing's sarcoma cells are highly sensitive to PARP1 / 2 and HDAC dual inhibition. The activity of compound A is compared to that of olaparib, niraparib, talazoparib, vorinostat, belinostat, and panobinostat. The EC50 values ​​of the test compounds, determined in A673 cells using the same experimental conditions as in Figure 2A, are compared. Cells were exposed to 10-day treatments with increasing concentrations of inhibitors, and the EC50 values ​​were calculated as the concentration required for 50% cell viability (n=3). [Figure 2C]Figure 2 shows that Ewing sarcoma cells are highly sensitive to PARP1 / 2 and HDAC dual inhibition. The activity of Compound A is compared to that of olaparib, niraparib, talazoparib, vorinostat, belinostat, and panobinostat. The EC50 values ​​of the indicated inhibitors, determined using the CellTiter-Glo® cell viability assay in CHLA10 cells using the same experimental conditions as in Figure 2A, are compared. Cells were exposed to 10-day treatments with increasing concentrations of inhibitors, and the EC50 values ​​were calculated as the concentration required for 50% cell viability (n=3). [Figure 3A-1]

[0023] Figure 1 shows that Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells. Cell cycle analysis of TC32 cells synchronized in G0 / G1 phase by 24 hours of serum starvation prior to treatment with Compound A, olaparib, or vorinostat (as indicated) in complete medium for 48 hours. Cell cycle profiles were then examined by propidium iodide (PI) staining followed by flow cytometry analysis. Cell cycle distribution is also shown. [Figure 3A-2]

[0023] Figure 1 shows that Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells. Cell cycle analysis of TC32 cells synchronized in G0 / G1 phase by 24 hours of serum starvation prior to treatment with Compound A, olaparib, or vorinostat (as indicated) in complete medium for 48 hours. Cell cycle profiles were then examined by propidium iodide (PI) staining followed by flow cytometry analysis. Cell cycle distribution is also shown. [Figure 3A-3]

[0023] Figure 1 shows that Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells. Cell cycle analysis of TC32 cells synchronized in G0 / G1 phase by 24 hours of serum starvation prior to treatment with Compound A, olaparib, or vorinostat (as indicated) in complete medium for 48 hours. Cell cycle profiles were then examined by propidium iodide (PI) staining followed by flow cytometry analysis. Cell cycle distribution is also shown. [Figure 3B-1]Figure 3 shows that Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells. Figure 3 shows cell cycle analysis of CHLA10 cells treated with Compound A, olaparib, or vorinostat for 24 hours, as indicated, using the same experimental procedure as in Figure 3A. [Figure 3B-2] Figure 3 shows that Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells. Figure 3 shows cell cycle analysis of CHLA10 cells treated with Compound A, olaparib, or vorinostat for 24 hours, as indicated, using the same experimental procedure as in Figure 3A. [Figure 3B-3] Figure 3 shows that Compound A induces S and G2 / M cell cycle arrest in Ewing sarcoma cells. Figure 3 shows cell cycle analysis of CHLA10 cells treated with Compound A, olaparib, or vorinostat for 24 hours, as indicated, using the same experimental procedure as in Figure 3A. [Figure 4A] Figure 1 shows that Compound A treatment induces DNA damage in Ewing sarcoma cells. γH2AX expression is compared by Western blot for dianhydrogalactitol (DAG), olaparib, vorinostat, or Compound A. 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 hours and analyzed for γH2AX expression by Western blot. [Figure 4B] Figure 1 shows that compound A treatment induces DNA damage in Ewing sarcoma cells. γH2AX expression is compared by Western blot for DAG, olaparib, vorinostat, or compound A. CHLA10 cells were treated with 5 μM dianhydrogalactitol (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). [Figure 4C-1]Figure 1 shows that compound A treatment induces DNA damage in Ewing sarcoma cells. γH2AX foci analysis by immunofluorescence and confocal microscopy imaging is compared. CHLA10 cells treated for 24 h with DAG (2.5 μM) or increasing doses of compound A (0-1 μM) were analyzed for γH2AX foci by immunofluorescence and confocal microscopy imaging. Scale bar represents 10 μm. [Figure 4C-2] Figure 1 shows that compound A treatment induces DNA damage in Ewing sarcoma cells. γH2AX foci analysis by immunofluorescence and confocal microscopy imaging is compared. CHLA10 cells treated for 24 h with DAG (2.5 μM) or increasing doses of compound A (0-1 μM) were analyzed for γH2AX foci by immunofluorescence and confocal microscopy imaging. Scale bar represents 10 μm. [Figure 4D-1] Figure 1 shows that compound A treatment induces DNA damage in Ewing sarcoma cells. γH2AX foci analysis by immunofluorescence and confocal microscopy imaging is compared. CHLA10 cells treated with DAG (2.5 μM) or olaparib (1-37.5 μM) for 24 h were analyzed for γH2AX foci by immunofluorescence and confocal microscopy imaging. Scale bar represents 10 μm. [Figure 4D-2] Figure 1 shows that compound A treatment induces DNA damage in Ewing sarcoma cells. γH2AX foci analysis by immunofluorescence and confocal microscopy imaging is compared. CHLA10 cells treated with DAG (2.5 μM) or olaparib (1-37.5 μM) for 24 h were analyzed for γH2AX foci by immunofluorescence and confocal microscopy imaging. Scale bar represents 10 μm. [Figure 4E-1] Figure 1 shows that compound A treatment induces DNA damage in Ewing sarcoma cells. γH2AX foci analysis is compared by immunofluorescence and confocal microscopy imaging. CHLA10 cells treated with DAG (2.5 μM) or vorinostat (1–18 μM) for 24 h were analyzed for γH2AX foci by immunofluorescence and confocal microscopy imaging. Scale bar represents 10 μm. [Figure 4E-2] Figure 1 shows that compound A treatment induces DNA damage in Ewing sarcoma cells. γH2AX foci analysis is compared by immunofluorescence and confocal microscopy imaging. CHLA10 cells treated with DAG (2.5 μM) or vorinostat (1–18 μM) for 24 h were analyzed for γH2AX foci by immunofluorescence and confocal microscopy imaging. Scale bar represents 10 μm. [Figure 4F]

[0023] Figure 1 shows that Compound A treatment induces DNA damage in Ewing sarcoma cells. Shown are comet assay results for CHLA10 cells after treatment with 1 μM Compound A, olaparib, vorinostat, or olaparib followed by vorinostat. 5 μM DAG was included as a positive control. Scale bar represents 200 μm. ****p<0.0001. [Figure 5A] Figure 1 shows that Compound A inhibits 3D spheroid growth and metastasis of Ewing sarcoma cells. TC32 spheroid growth after 4 days of treatment with increasing concentrations of Compound A, olaparib, or vorinostat is compared, as monitored using the IncuCyte® Spheroid Analysis System. EC50 values ​​were calculated from nonlinear regression plots using GraphPad Prism8 software as the concentration required for 50% inhibition of growth. 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, with the scale bar representing 400 μm. *p<0.05, **p<0.01. [Figure 5B] Figure 5A shows that Compound A inhibits 3D spheroid growth and metastasis of Ewing sarcoma cells. Using the same experimental procedure as shown in Figure 5A, CHLA10 spheroid growth is compared after 4 days of treatment with increasing concentrations of Compound A, olaparib, or vorinostat. Representative images of CHLA10 3D spheroids at days 0 and 4 with DMSO, 1 μM Compound A, 1 μM olaparib, or 1 μM vorinostat are shown with the scale bar representing 400 μm. *p<0.05, ***p<0.001. [Figure 5C] Figure 1 shows that Compound A inhibits 3D spheroid growth and metastasis of Ewing sarcoma cells. Compare lung tumor burden after 14 days of treatment with vehicle, 5 nM, 10 nM, or 20 nM Compound A, n=5-12. Representative fluorescence images of tdTomato TC32 cells in lung sections after 14 days of treatment with 5 nM, 10 nM, or 20 nM Compound A. Scale bar represents 1 mm. **p<0.01. [Figure 5D]

[0023] Figure 1 shows that Compound A inhibits 3D spheroid growth and metastasis of Ewing sarcoma cells. Representative hematoxylin and eosin (H&E) and CD99 staining images of TC32 Ewing sarcoma cells in PuMA lung slices after 14 days of treatment with 5 nM, 10 nM, or 20 nM Compound A are shown. Magnified images of each from the inset (top panel) are shown below each image. Scale bar represents 50 μm. [Figure 5E] Figure 1 shows that Compound A inhibits 3D spheroid growth and metastasis of Ewing sarcoma cells. Compare lung tumor burden after 14 days of treatment with vehicle, 5 nM, 10 nM, or 20 nM Compound A, n=5-12. Representative fluorescence images of tdTomato A673 cells in lung sections after 14 days of treatment with 5 nM, 10 nM, or 20 nM Compound A. Scale bar represents 1 mm. **p<0.01, ****p<0.0001. [Figure 5F]

[0023] Figure 1 shows that Compound A inhibits 3D spheroid growth and metastasis of Ewing sarcoma cells. Representative H&E and CD99 staining images of A673 Ewing sarcoma cells in PuMA lung slices after 14 days of treatment with 5 nM, 10 nM, or 20 nM Compound A are shown. Enlarged images of each from the inset (top panel) are shown below each image. Scale bar represents 50 μm. [Figure 6]FIG. 1 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. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description HDAC inhibition has been shown to induce pharmacological BRCAness in cancer cells with functional DNA repair activity. This provides a rationale for exploring combination treatments using HDAC and PARP inhibition in cancer types 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-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 demonstrated enhanced cytotoxicity in Ewing sarcoma models. Compound A-induced cytotoxicity, as assessed by γH2AX tracking and comet assays, was associated with potent S and G2 / M cell cycle arrest and increased DNA damage in the nanomolar concentration range. In a 3D spheroid model of Ewing sarcoma, Compound A demonstrated efficacy at lower concentrations than olaparib and vorinostat.

[0014] In one aspect, the disclosure provides a method for treating Ewing's sarcoma in a subject, the method 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 (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-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's sarcoma in a subject.

[0015] In another aspect, the disclosure provides a method for inhibiting PARP1, PARP2, and HDAC in a subject, the method comprising 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 (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-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.

[0016] 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, the method comprising 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 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 use in treating a disease or condition treatable by inhibiting PARP1, PARP2, and HDAC in a subject.

[0017] In other aspects, the present disclosure provides pharmaceutical compositions for the above uses. In these aspects, the pharmaceutical compositions include (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.

[0018] As used herein, "compound A" refers to a compound of formula (I):

[0019] [ka]

[0020] or a tautomer thereof.

[0021] As described herein, the present disclosure provides methods for using Compound A or a pharmaceutically acceptable salt thereof. It will 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 after administration.

[0022] Described below is the utility of Compound A for inhibiting PARP1, PARP2, and HDAC, as well as the efficacy of Compound A for treating Ewing's sarcoma.

[0023] Pharmacological BRCAness may result in PARP inhibition beyond the BRCA1 / 2 mutation region, potentially countering 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-normal cancer scenarios. Currently, four clinical trials are underway using PARPi inhibitors in combination with the HDACi inhibitor vorinostat (NCT03259503 and NCT03742245), the DNA methyltransferase inhibitor decitabine (NCT02878785), and the EZH2 histone methyltransferase inhibitor SHR2554 (NCT04355858).

[0024] The FDA has approved three pan-HDACi drugs (vorinostat, belinostat, and panobinostat) and one HDAC1 / 2-selective HDACi (romidepsin) for the treatment of hematologic cancers. Histone acetylation attenuates chromatin structure and plays a key role in DNA damage recognition and repair. HDACi-induced downregulation of key HR proteins, including BRCA1, BRCA2, and RAD51, has been established in various cancer types, and HDACi treatment sensitizes cancer cells to PARPi. This confirmed activity of HDAC and PARP inhibition is particularly interesting in the context of HR-normal cancer types, such as Ewing's sarcoma, where PARPi therapy itself has limited efficacy. However, dose-limiting toxicities from HDACi therapy are not uncommon in solid tumor cancers, preventing some therapeutic efficacy both as stand-alone agents and in combination treatments, for example, in breast cancer and sarcoma. HDACi components must be carefully tailored to prevent overlapping toxicity events resulting from combination with other therapeutic moieties, which can be difficult when dealing with different pharmacokinetic profiles.

[0025] This disclosure provides Compound A, a bifunctional PARP-HDAC single-molecule inhibitor, in a Ewing sarcoma model to evaluate the potential benefit of combined PARP-HDAC inhibition over standalone PARPi or HDACi treatment. Compound A has PARPi activity similar to olaparib and slightly lower HDACi activity than vorinostat. However, the dual activity of Compound A makes it 30-80 times more cytotoxic to Ewing sarcoma cells than olaparib and 30-60 times more cytotoxic than vorinostat alone. While panobinostat appeared to have greater efficacy in Ewing sarcoma cell lines, this may be due to its toxicity, as seen in clinical trials where dose-limiting toxicities have limited its effective use in solid tumors. This is likely also true for talazoparib. While talazoparib is the most potent FDA-approved PARPi to date, it also exhibited clinical toxicities more similar to other chemotherapeutic agents than other approved PARP inhibitors, including anemia, thrombocytopenia, and neutropenia.

[0026] Compound A also induces cell cycle arrest and DNA damage in Ewing sarcoma cells at concentrations much lower than those of olaparib and vorinostat. Because the PARP inhibitor activity of Compound A is stronger than the HDAC inhibitor portion of the drug, the cell cycle arrest pattern of Compound A was more similar to that of olaparib than the combination treatment. In TC32 cells, the combination of 0.7 μM olaparib and 0.7 μM belinostat resulted in a lower EC200 of belinostat-treated TC32 cells. 50 Consistent with the values, compound A showed some arrest in G0 / G1. When compared in 3D spheroid models, compound A showed efficacy at concentrations 30-40 times lower than olaparib and 5-10 times lower than vorinostat. While spheroid models treated with talazoparib showed comparable efficacy to compound A, the efficacy of panobinostat was 10-fold lower compared to compound A. This may be due to the overall toxicity of panobinostat, as previously mentioned. Compound A also prevented metastatic growth of Ewing sarcoma cells in an ex vivo PuMA model, with potent inhibitory effects using as little as 10 nM of inhibitor. Due to concerns about the hematological toxicity of PARP and HDAC inhibitors, a pilot study of compound A was conducted in mice that showed no evidence of toxicity based on weight loss and blood counts.

[0027] Combining PARP and HDAC inhibition into a single molecule offers a convenient way to prevent resistance to PARPi therapy. For example, Ewing's sarcoma and many other solid tumor indications epigenetically suppress the expression of the tumor suppressor gene Schlafen11 (SLFN11), resulting in resistance to DNA damage-inducing agents, including PARPi therapy. Importantly, HDACi treatment promotes re-expression of SLFN11 and resensitization to PARPi therapy.

[0028] Combination therapies can act synergistically or additively by simultaneously targeting different intracellular pathways. Unfortunately, combination therapies involving chemotherapeutic agents can be toxic to patients and often require sequential administration in clinical settings, sometimes with reduced biological efficacy. This provides a strong rationale for the development of dual-active small molecules such as Compound A.

[0029] In summary, the present disclosure provides a single molecule PARP-HDAC inhibitor, Compound A, with improved cytotoxic and DNA damaging activity compared to PARPi and HDACi alone in Ewing's sarcoma.

[0030] Dual specificity compound with dual activity against PARP1 / 2 and HDAC enzymes Through a medicinal chemistry cycle, the present disclosure provides a small molecule inhibitor (Compound A) with dual activity against PARP1 / 2 and HDAC. Using an in vitro activity assay kit, the inhibition of PARP1, PARP2, and HDAC by Compound A was determined compared to the FDA-approved PARP inhibitor olaparib and the HDAC inhibitor vorinostat. IC values ​​were measured using a wide range of concentrations of each compound. 50 Compound A had an IC value of 2.54 μM. 50 Compound A had an IC value of 0.05 μM, whereas vorinostat had an IC value of 0.05 μM, approximately 50-fold lower (Figure 1A). The PARP1 and PARP2 inhibitory activity of Compound A was comparable to that of Olaparib, and the IC value of Compound A was 50 The IC values ​​were 3.38 nM and 2.19 nM, respectively (Figures 1B and 1C). To further validate Compound A's ability to inhibit PARP1 / 2 activity, a cellular PAR synthesis assay was used to determine the level of PAR formation. Similar to olaparib, Compound A showed an IC of 1.39 nM for inhibition of PAR formation in cells treated with Compound A. 50 was detected (Figure 1D). These data indicate that Compound A can inhibit both PARP1 / 2 and HDAC enzymes.

[0031] Ewing sarcoma cells are highly sensitive to dual inhibition of PARP1 / 2 and HDAC To investigate the effect of Compound A on cell proliferation, we performed cell viability assays in three Ewing's sarcoma cell lines. Using an IncuCyte S3 live-cell imaging system, we measured the EC20 values ​​in cell viability after 3 days of treatment with increasing concentrations of Compound A, three FDA-approved PARP inhibitors, or three FDA-approved HDAC inhibitors in TC32 and A673 cells. 50 Compound A had an EC value of 0.0163 μM in TC32 cells. 50 Compound A demonstrated greater efficacy in inhibiting cell viability than olaparib, niraparib, vorinostat, and belinostat (Figure 2A). Similar efficacy of Compound A in A673 cells was observed with a much lower EC of 0.0365 μM compared to olaparib, niraparib, vorinostat, and belinostat treatment alone. 50 However, treatment with talazoparib or panobinostat showed stronger inhibitory effects in both cell lines compared to Compound A (Figures 2A and 2B). To further validate the findings, a CellTiter-Glo® viability assay was performed to assess the EC values ​​of these test compounds in CHLA10 cells. 50 Consistent with the IncuCyte assay, the EC values ​​for Compound A treatment in CHLA10 cells were determined. 50 The value (0.053 μM) was also much lower than that of olaparib, niraparib, vorinostat, and belinostat (Figure 2C). Collectively, the data demonstrate the potent inhibitory effect of Compound A compared to FDA-approved PARP or HDAC inhibitors in Ewing sarcoma cells.

[0032] Compound A induces S and G2 / M cell cycle arrest in Ewing's sarcoma cells PARP inhibitors and HDAC inhibitors consistently induce S / G2 / M and G0 / G1 cell cycle arrest, respectively, because PARP regulates replication fork progression and HDACs play a key role in regulating the expression of cell cycle checkpoint proteins, including cyclin-dependent kinases, cyclin D1, and p21. We examined the cell cycle profile of CHLA10 and TC32 cells treated with Compound A, olaparib, and vorinostat in both single-agent and combination regimens. Treatment of serum-starved cells with increasing concentrations of Compound A in complete medium for 24 or 48 hours resulted in potent S and G2 / M arrest in TC32 cells at or after 0.175 μM and in CHLA10 cells at or after 0.25 μM. Similar cell cycle arrest was only observed with olaparib treatment at concentrations above 3 μM for TC32 and 14.7 μM for CHLA10, respectively (Figures 3A and 3B). Treatment with equimolar concentrations of Compound A (1 μM and 0.7 μM for CHLA10 and TC32 cells, respectively) in combination with olaparib and vorinostat / belinostat had little effect on cell cycle phase compared to controls (Figures 3A and 3B). These data demonstrate that Compound A has a stronger potency in inducing S and G2 / M cell cycle arrest in Ewing sarcoma cells than olaparib alone or in combination with vorinostat or belinstat.

[0033] Compound A treatment induces DNA damage in Ewing sarcoma cells PARP inhibitors and HDAC inhibitors have been reported to induce DNA damage in cells. Western blot, immunofluorescence, and comet assays were used to examine the effect of Compound A on DNA damage in Ewing sarcoma cells compared with olaparib and vorinostat treatment. Phosphorylated histone variant H2AX (γH2AX) is a surrogate marker for DSBs in DNA. Dianhydrogalactitol (DAG) was included as a positive control because it induces replication-dependent DNA damage in various cancer cell lines. Treatment with Compound A, olaparib, or vorinostat dose-dependently induced γH2AX expression in both CHLA10 and TC32 cells. Compared to olaparib and vorinostat, Compound A was able to induce γH2AX expression at a much lower concentration range (Figure 4A and Figure 4B). Furthermore, CHLA10 cells treated with compound A or olaparib also demonstrated dose-dependent γH2AX foci formation in immunofluorescence, followed by confocal microscopy imaging with compound A at a much lower concentration range (Figure 4C). However, vorinostat, which induced G0 / G1 cell cycle arrest (Figure 3A and Figure 3B), demonstrated milder DNA damage foci formation in CHLA10 cells (Figure 4C). To further strengthen the data, we used the alkaline comet assay, which can detect both single-substrate and double-substrate (SSB) in cells. CHLA10 cells treated with 1 µM compound A demonstrated significant amounts of DNA damage, whereas 1 µM olaparib or vorinostat treatment did not (Figure 4D). In summary, the data indicate that compound A can induce DNA damage in Ewing sarcoma cells at a much lower concentration range than olaparib or vorinostat.

[0034] PARP inhibitors and HDAC inhibitors have been reported to induce DNA damage in cells. Western blot, immunofluorescence, and comet assays were used to examine the effect of Compound A on DNA damage in Ewing sarcoma cells compared with olaparib and vorinostat treatment. Phosphorylated histone variant H2AX (γH2AX) is a surrogate marker for DSBs in DNA. Dianhydrogalactitol (DAG) was included as a positive control because previous studies have shown it induces replication-dependent DNA damage in various cancer cell lines. Treatment with Compound A, olaparib, or vorinostat dose-dependently induced γH2AX expression in both TC32 and CHLA10 cells. Compared to olaparib and vorinostat, Compound A was able to induce γH2AX expression at a much lower concentration range (see Figures 4A and 4B). Furthermore, CHLA10 cells treated with compound A or olaparib also showed dose-dependent γH2AX foci formation in immunofluorescence, followed by confocal microscopy imaging with compound A at a much lower concentration range (Figures 4C and 4D). However, vorinostat, which induced G0 / G1 cell cycle arrest (Figures 3A and 3B), demonstrated milder DNA damage foci formation in CHLA10 cells (Figure 4E). Furthermore, TC32 cells only showed increased γH2AX expression by Western blot when treated with 0.35 μM compound A, but not with 0.35 μM 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 strengthen the data, we used the alkaline comet assay, which can detect both SSBs and DSBs in cells. There was significant DNA damage in CHLA10 cells treated with 1 μM Compound A, but not with 1 μM olaparib or vorinostat or 1 μM olaparib + 1 μM vorinostat (see Figure 4F).In summary, the data show that compound A is able to induce DNA damage in Ewing sarcoma cells at a much lower concentration range than olaparib or vorinostat.

[0035] Compound A inhibits 3D spheroid growth of Ewing sarcoma cells Spheroids are three-dimensional (3D) cell aggregates that can more accurately mimic tumor behavior compared to 2D cell cultures. To further validate the data, we investigated the effect of Compound A on a 3D spheroid model containing Ewing sarcoma cells. CHLA10 and TC32 spheroids were established at 200–300 μm and subsequently treated with increasing concentrations of Compound A, olaparib, or vorinostat. Spheroid growth was monitored and quantified for 4 days using an IncuCyte S3 imaging system. The EC of Compound A in inhibiting spheroid growth was 0.01. 50 The values ​​were much lower than those of olaparib and vorinostat in both the TC32 and CHLA10 cell models (Figure 5A and Figure 5B). These data suggest that compound A is a potent inhibitor of 3D spheroid growth of Ewing sarcoma cells and demonstrate greater efficacy than olaparib or vorinostat alone.

[0036] Spheroid assays using both TC32 and CHLA10 cells demonstrate comparable activity for Compound A and talazoparib, but the efficacy of panobinostat is 10-fold lower than that of Compound A. The effect of Compound A on the metastatic growth of Ewing sarcoma cells was examined using an ex vivo pulmonary metastasis assay (PuMA). Here, colony formation of td-Tomato-expressing TC32 and A673 cells in mouse lungs was inhibited by Compound A at concentrations as low as 10 nM (Figures 5C and 5E). Parallel hematoxylin-eosin (H&E) and CD99 IHC staining (Figures 5D and 5F) confirmed that the tdTomato fluorescent signal in the lung tissue was indeed Ewing sarcoma cells. Fluorescence microscopy confirmed that the per-cell fluorescence of both td-Tomato-expressing TC32 and A673 cells was unaffected by treatment with Compound A. Studies in nude mice showed no signs of toxicity as indicated by weight and blood counts after 4 days of intraperitoneal treatment with 30 mg / kg Compound A (BID). 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 3D growth in Ewing sarcoma spheroids.

[0037] material and method cell culture The identity 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 with 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's medium (Gibco catalog number 11995065) supplemented with 10% FBS.

[0038] HDAC activity assay In vitro HDAC activity was measured using the FLUOR DE LYS® HDAC Fluorescent Activity Assay Kit (Enzo Life Sciences Catalog No. BML-AK500-0001) according to the manufacturer's protocol. 50 Values ​​were calculated using four-parameter variable slope nonlinear regression in GraphPad Prism8 (GraphPad Software Inc.).

[0039] PARP1 and PARP2 activity assays In vitro PARP1 activity was measured using the HT Universal Colorimetric PARP Assay Kit (R&D Systems catalog number 4677-096-K) and PARP2 activity was measured using the PARP2 Colorimetric Assay Kit (BPS Bioscience catalog number 80581) according to the manufacturer's protocol. 50Values ​​were calculated using four-parameter variable slope nonlinear regression in GraphPad Prism8 (GraphPad Software Inc.).

[0040] PAR formation assay A cellular PAR formation assay was used to measure the ability of test compounds to inhibit PAR polymerization. CHLA10 cells were seeded into black, clear-bottom 96-well plates and allowed to adhere overnight. Cells were pretreated with increasing concentrations of test inhibitors for 30 min at 37°C, after which HO was added to a final concentration of 25 mM and incubated for 5 min at room temperature (RT). After washing twice with 0.1% Tween-20 in PBS (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, twice with 3% BSA in PBS (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, the cells were incubated for 1 hour at room temperature with anti-PAR / pADPr monoclonal antibody (R&D Systems catalog no. 4335-MC-100) diluted 1:250 in 3% BSA-PBS. 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 for 1 hour at room temperature with goat anti-mouse IgG-FITC (Thermo Scientific catalog no. F-2761) diluted 1:1000 in 3% BSA-PBS. 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 per well, and the plate was imaged on an IncuCyte® S3 system (Sartorius). Fluorescence was quantified using IncuCyte® analysis software. Values ​​were normalized to a control without primary antibody, and then % PAR formation was calculated by normalizing to a dimethyl sulfoxide (DMSO) control. 50Values ​​were calculated using four-parameter variable slope nonlinear regression in GraphPad Prism 8 (GraphPad Software Inc.). Mean IC of three biological replicates 50 Values ​​± SD were calculated.

[0041] Cell viability assay Cells were seeded in 96-well plates (1,000–5,000 cells per well) in 100 μL of appropriate medium and allowed to adhere overnight. 100 μL of medium containing DMSO or increasing concentrations of test compound was added to each well. Cells were maintained at 37°C with 5% CO2 and 95% humidity for 10 days for CHLA10 and 3 days for TC32 and A673. A Cell-Titer-Glo® assay was performed for CHLA10. 150 μL of medium per well was removed, the plate was equilibrated at RT for 30 minutes, and then CellTiter-Glo® assay reagent was added to the wells. The plate was gently shaken on an orbital shaker for 2 minutes and incubated in the dark at RT for 10 minutes. Luminescence was measured using a Tecan Infinite M200Pro microplate reader. All measurements were performed in triplicate. For TC32 and A673, plates were imaged with an Incucyte® S3 live cell imaging system after treatment and % confluency was measured using Incucyte® software. Values ​​were normalized to media only and DMSO controls to calculate % cell viability. EC 50 Values ​​were calculated using four-parameter variable slope nonlinear regression in GraphPad Prism8 (GraphPad Software Inc.). Mean EC 50 Values ​​± SD were calculated.

[0042] Cell cycle analysis Cell cycle profiles were assessed by propidium iodide (PI) staining and flow cytometry. CHLA10 and TC32 cells were cultured at 1.5 × 10 6 cells / plate and 2.0 x 106 Cells were seeded in 10 cm plates with a cell density of 1.0 x 10 cells / plate. The next day, the medium was replaced with serum-free medium for 24 hours. Cells were treated with olaparib, vorinostat, and dose-escalating Compound A for 24 hours for TC32 and 48 hours for CHLA10. Combination treatments of olaparib and vorinostat and olaparib and belinostat were evaluated at equimolar concentrations of Compound A (0.7 μM and 1 μM for TC32 and CHLA10 cells, respectively). Cells were harvested and 1.0 x 10 cells from each treatment were harvested. 6 Cells were fixed in 70% ethanol overnight at -30°C. The cell suspension was then washed with cold PBS, stained with PI solution (50 μg / mL PI, 0.1 mg / mL RNase A, 0.05% Triton X-100 in PBS), and incubated in the dark at 37°C for 40 minutes. Cells were then washed with PBS, filtered through a 40 μm strainer, and resuspended in 500 μL of PBS. Samples were then examined by flow cytometry and analyzed using FlowJo v10.

[0043] Alkaline comet assay 1 x 10 cells in a 6-well plate 6 Cells were seeded at a density of 100 cells / well and allowed to stand overnight. The cell culture medium was replaced with serum-free medium for 24 hours, and then treated with DMSO or test compounds for 24 hours at 37°C with 5% CO2 and 95% humidity. Cells were harvested as instructed in Trevigen's CometAssay® protocol, combined with molten LMAgarose at a 1:10 ratio, and pipetted onto CometSlides®. The cells were placed in the dark for 30 minutes and then immersed in lysis solution overnight at 4°C. The slides were immersed in alkaline unwinding solution in the dark at 4°C for 1 hour, then placed in a gel electrophoresis tray and immersed in alkaline electrophoresis solution at an applied voltage of 25V for 30 minutes. The samples were washed with dH2O and 70% ethanol and then stained with SYBR® Gold. The samples were then observed using a fluorescent microscope. The acquired images were analyzed using OpenComet on ImageJ (NIH).

[0044] Immunofluorescence CHLA10 cells, 3.5 x 10 5 Cells were seeded onto glass coverslips in 24-well plates at a density of 10 cells / well and allowed to rest overnight. The medium was replaced with serum-free medium for 24 hours, and then cells were treated with DMSO or increasing concentrations of test compound for 24 hours. Cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0.5% Triton-X in PBS, and then probed with anti-phosphohistone H2AX (Ser139) rabbit antibody (Cell Signaling Technology catalog no. 2577) overnight at 4°C. Cells on the coverslips were then washed with PBS, probed with goat anti-rabbit IgG Alexa Fluor® 488 (Abcam catalog no. ab150077), and mounted on microscope slides in VECTASHIELD antifade mounting medium containing DAPI solution. Cells were then visualized using a confocal microscope (Olympus FV3000). Acquired images were analyzed by quantifying foci using ImageJ (NIH).

[0045] Western blot analysis Cells were seeded at 70-80% confluency in 6-well plates. After allowing the cells to settle overnight, the medium was replaced with serum-free medium for 24 hours. Cells were treated with DMSO or increasing concentrations of test compound for 24 hours. Cells were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer combined with protease and phosphatase inhibitors. Protein yield was assessed using the Pierce™ BCA Protein Assay Kit (Thermofisher catalog number 23225) and quantified at 562 nm using a spectrophotometer plate reader (TECAN). A total of 20 μg of protein extract was loaded per well onto a 4-15% Mini-PROTEAN® TGX™ precast protein gel (Bio-Rad catalog number 4561084). After electrophoresis, proteins were transferred to a 0.2 μm nitrocellulose membrane. The membrane was blocked with LICOR® Odyssey blocking buffer in PBS. After blocking, the membrane was incubated with anti-phospho-histone H2AX (Ser139) rabbit antibody (Cell Signaling Technology catalog no. 2577) and H2AX rabbit antibody (Abcam catalog no. ab11175) overnight at 4°C, and then incubated with donkey anti-rabbit IRDye® 800CW secondary antibody (LI-COR catalog no. 926-32213) for 1 hour at RT. After washing with 1x Tris-buffered saline containing 1% Tween-20 (TBS-T), the membrane was scanned on an Odyssey scanner (LI-COR).

[0046] Spheroid formation assay CHLA10-tdTomato or TC32-tdTomato cells (2500 cells per well) were added to a 96-well clear round-bottom ultra-low attachment microplate (Corning catalog no. 7007) and allowed to form spheroids for 24 hours or until they reached a diameter of 200-300 μm. Media containing DMSO or increasing concentrations of test compound was added to each well, and spheroid growth was monitored for 4 days after treatment using an IncuCyte® Spheroid Analysis System (Sartorius). Images taken on days 0 and 4 after treatment were analyzed using the IncuCyte® Spheroid Analysis software module. Day 4 values ​​were normalized using a normalization factor from day 0 values ​​to obtain EC values. 50 Values ​​were calculated using four-parameter variable slope nonlinear regression in GraphPad Prism8 (GraphPad Software Inc.). Mean EC 50 Values ​​± SD were calculated.

[0047] Pulmonary metastasis assay (PuMA) Procedures involving mice were approved by the local animal care committee, University of British Columbia. tdTomato-expressing TC32 and A673 cells (1 × 10 6100 μl of cells / 100 μl saline) were injected into the tail vein of 6-8 week-old immunodeficient NSG female mice (Jax Laboratories). After injection, mice were euthanized by isoflurane and CO asphyxiation according to local animal care standard operating procedures. Lungs were ventilated by gravity perfusion with a prewarmed (37°C) 1:1 mixture of fully supplemented PneumaCult™-ALI medium (STEMCell catalog no. 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;11:645757 doi 10.3389 / fonc.2021.645757). Internal organs (heart and lungs) were carefully removed and placed in ice-cold PBS (supplemented with 1x penicillin / streptomycin) for 20 minutes to allow the agarose to solidify. Small lung sections (approximately 2 mm x 4 mm) were obtained by manual cutting with sterile surgical scissors, and 5 to 12 sections per condition were selected for serial imaging on days 0 and 14 after injection / treatment. Lung sections were maintained in vitro on gelatin sponges partially soaked in 2 mL of PneumaCult™ medium + / - compound in 6-well plates. The medium + / - compound was refreshed every 3 days. On the day of imaging, lung sections from each group were transferred to small 35 mm Petri dishes with glass cover slip bottoms (IBIDI) for sterile wide-field fluorescent imaging. Lung sections were imaged with an inverted Zeiss Observer.Z1 Colibri microscope using a 2.5x objective.The lung tumor burden (% tumor burden) per lung section was calculated by multiplying the total area of ​​the tdTomato lesion by the total area of ​​the lung section 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). Image processing was performed using ImageJ software. This calculation was performed for all lung sections (n ​​= 5–12 lung sections) per experimental group. Mean values ​​of percent lung tumor burden per group were compared and analyzed using Graph Prism 8 (GraphPad Software Inc.).

[0048] Immunohistochemistry and histopathology Formalin-fixed, paraffin-embedded (FFPE) PuMA lung tissue slices were freshly cut and analyzed for CD99 immunoexpression using a Ventana Discovery Ultra autostainer (Ventana Medical Systems, Tucson, AZ). Briefly, baked and deparaffinized tissue slices were incubated in a Tris-based buffer (CC1, Ventana) at 95°C for 64 minutes to retrieve antigenicity, followed by incubation with anti-CD99 rabbit polyclonal antibody (Abcam catalog no. ab27271) at room temperature for 1 hour. Bound primary antibody was visualized using the UltraMap DAB term Rb detection kit (Ventana). All stained slides were digitized at a magnification equivalent to 40x using a Leica scanner (Aperio AT2, Leica Microsystems; Concord, Ontario, Canada). Images were then saved in the Vancouver Prostate Centre's Aperio eSlide Manager (Leica Microsystems). 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's sarcoma cells.

[0049] statistical analysis Data are shown as mean ± SD. Statistical analysis for cell cycle profiling, comet assay, spheroid assay, and PuMA assay was performed using GraphPad Prism 8.0 (GraphPad Software, Inc.). Statistical analysis of cell cycle profiles was performed using a multiple t-test and was only performed on profiles showing a cell cycle profile change of more than 5%, as these were considered biologically relevant. Statistics for comet assays were determined using the Mann-Whitney nonparametric test. Statistical analysis of spheroid assays was analyzed using an unpaired t-test, and PuMA assays were performed using the Kruskal-Wallis nonparametric test. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0050] Apoptosis assay Induction of apoptosis by UT (dimethyl sulfoxide (DMSO)), dianhydrogalactitol (DAG), olaparib (OLA), vorinostat (VOR), O+S (OLA+VOR), and compound A was determined by immunoblotting of cell lysates for cleaved caspase 3. CHLA10 cells were treated with 1 μM of the indicated compounds for 24 h, then harvested and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein yield was assessed using the Pierce BCA Protein Assay Kit (Thermofisher catalog no. 23225), and 20 μg of lysate was run on a 4-15% mini-PROTEAN TGX gel (BioRad catalog no. 4561084) at 100 V for 1 h. The gel was transferred to a 0.2 μm nitrocellulose membrane (BioRad catalog no. 1620112) using a TransBlot Turbo transfer system (BioRad catalog no. 1704150). After transfer, the membranes were blocked with LI-COR Odyssey blocking buffer and incubated overnight at 4°C in anti-cleaved caspase 3 antibody (Cell Signaling Technology catalog no. 9661S) and anti-caspase 3 antibody (Cell Signaling Technology catalog no. 9662S), followed by incubation at room temperature for 1 hour in donkey anti-rabbit Alexa Fluor 680 (LI-COR catalog no. 926-68073). The membranes were then scanned with an Odyssey scanner (LI-COR). The results are shown in Figure 6.

[0051] Synthesis of Compound A Compound A was prepared by conventional synthetic organic techniques as shown in Scheme 1 below.

[0052] [ka]

[0053] Compound X was prepared according to Menear, KA; et al.; J. Med. Chem. 2008, 51, 6581-6591.

[0054] While exemplary embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

[0055] The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

Claims

1. 1. A method for treating Ewing's 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.

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 for use in the treatment of Ewing's sarcoma.

3. A pharmaceutical composition for treating Ewing's sarcoma, comprising 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. 1. 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-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 for use in inhibiting PARP1, PARP2 and HDAC in a subject.

6. 1. A pharmaceutical composition for use in inhibiting PARP1, PARP2 and HDAC in a subject, comprising 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 condition treatable by 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-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)pyrimidin-5-yl)-N-hydroxyacrylamide or a pharmaceutically acceptable salt thereof.

8. 1. (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 for use in treating a disease or condition treatable by inhibiting PARP1, PARP2 and HDAC in a subject.

9. 1. A pharmaceutical composition for use in treating a disease or condition treatable by inhibiting PARP1, PARP2, and HDAC in a subject, comprising 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.

Images