Discovery of Novel Bromophenol–Thiosemicarbazone Hybrids as

two bromo atoms and methoxy groups was observed to stretch toward the back pocket. ... Hydrogen bonds are displayed as yellow dashed lines. ... Th...
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Discovery of novel bromophenol - thiosemicarbazone hybrids as potent selectve inhibitors of poly(ADPribose) polymerase-1 (PARP1) for use in cancer Chuanlong Guo, Lijun Wang, Xiuxue Li, Shuaiyu Wang, Xuemin Yu, kuo xu, Yue Zhao, Jiao Luo, Xiangqian Li, Bo Jiang, and Dayong Shi J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01946 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 8, 2019

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Journal of Medicinal Chemistry

Discovery of novel bromophenol-thiosemicarbazone hybrids as potent selective inhibitors of poly(ADPribose) polymerase-1 (PARP-1) for use in cancer Chuanlong Guo, †,‡,§,Δ,¶,∥ Lijun Wang, *,†,‡,§, ∥ Xiuxue Li, †,‡,§ Shuaiyu Wang, †,‡,§ Xuemin Yu, □ Kuo Xu, †,‡,§Yue Zhao, †,‡,§ Jiao Luo, †,‡,§, ¶ Xiangqian Li, †,‡,§Bo jiang, †,‡,§ Dayong Shi*,†,‡,§,# †Key

Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy

of Sciences, Qingdao, Shandong 266071, China ‡Laboratory

for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine

Science and Technology, Qingdao, Shandong 266071, China #State

Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, Shandong,

China §Center

for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, Shandong 266071,

China □Department

of Otorhinolaryngology, Qilu Hospital of Shandong University, Qingdao,

Shandong 266000, China Δ Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China

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¶University

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of Chinese Academy of Sciences, Beijing 100049, China

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KEYWORDS: selective PARP-1 inhibitor; bromophenol - thiosemicarbazone hybrids; DNA repair; autophagy; reactive oxygen species (ROS); multiple anticancer mechanisms

ABSTRACT: Poly(ADP-ribose) polymerase-1 (PARP-1) is a new potential target for anticancer-drug discovery. A series of bromophenol-thiosemicarbazone hybrids as PARP-1 inhibitors were designed, synthesized and evaluated for their antitumor activities. Among them, the most promising compound, 11, showed excellent selective PARP-1 inhibitory activity (IC50 = 29.5 nM) over PARP-2 (IC50 > 1000 nM) and potent anticancer activities toward the SK-OV-3, Bel-7402 and HepG2 cancer cell lines (IC50 = 2.39, 5.45 and 4.60 µM), along with inhibition of tumor growth in an in vivo SK-OV-3 cell xenograft model. Further study demonstrated that compound 11 played an anti-tumor role through multiple anticancer mechanisms, including the induction of apoptosis and cell-cycle arrest, cellular accumulation of DNA double-strand breaks, DNA repair alterations, inhibition of H2O2triggered PARylation, antiproliferative effects via the production of cytotoxic reactive oxygen species (ROS) and autophagy. In addition, compound 11 displayed good pharmacokinetic characteristics and favorable safety. These observations demonstrate that compound 11 may serve as a lead compound for the discovery of new anticancer drugs.

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■ INTRODUCTION In the pursuit of developing anticancer drugs, medicinal chemistry researchers have been concerned with how to cure cancer because of its current high incidence and lethality. Some new targeted drugs can prevent the growth of cancer cells based on molecular targeting, offering a promising treatment for cancer. Targeting tumor suppressor mechanisms such as DNA repair has been approved as a promising approach to sensitize tumor cells to cytotoxic treatments and overcome acquired resistance.1 The poly(ADP-ribose) polymerase-1 (PARP1), a 113-kDa nuclear protein, is associated with a variety of cellular functions such as DNA repair, transcriptional and posttranscriptional modulation of gene expression, inflammation, and regulation of cell death.2,

3, 4

Several studies have reported that PARP-1 inhibition is

protective in two ways: either targeting PARP-1 via its role in the DNA repair pathway or in transcriptional regulation.5 Thus, PARP-1 is a promising molecular target for the discovery of antitumor drugs.6-9 Nicotinamide analogues, the first generation of the typical PARP-1 inhibitors 3aminobenzamide (3-AB, Figure 1), were developed approximately 30 years ago. Secondgeneration PARP-1 inhibitors were developed based on quinazoline analogues, including isoquinolines, quinazolinediones, phthalazinones, and phenanthridinones. Third-generation PARP-1 inhibitors are currently being developed.10 Many effective PARP-1 inhibitors that have been used as single agents and combination with drugs targeting the DNA repair pathway have undergone preclinical and clinical evaluations. Four PARP-1 inhibitors

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(olaparib (2014.12), rucaparib (2016.12), niraparib (2017.3) and talazoparib (2018.10), Figure 1) have been approved to date for use in the clinic for cancer therapy (2018.11). However, Most PARP inhibitors have no significant selectivity, which might result in side effect profiling of these inhibitors in clinical trials.10, 11 Thus, the development of novel inhibitors with specificity for PARP-1 is a promising strategy to achieve efficient results. To our knowledge, a few potent PARP-1 inhibitors with high selectivity over PARP-2 (such as WD2000-012547, NMS-P118 and BYK204165, Figure 1) have been discovered; however, the need to develop selective potent PARP-1 inhibitors is still of pivotal importance.12-14 Thiosemicarbazones have many biological activities, including anticancer, antibacterial, antiviral, antifungal and antineoplastic effects.15 A variety of thiosemicarbazones have been developed and examined in vitro, in vivo and in clinical trials as anticancer agents, such as 3aminopyridine-2-carboxaldehyde

thiosemicarbazone

(3-AP)

and

5-hydroxypyridine

carboxaldehyde thiosemicarbazone (5-HP, Figure 1).16 The anticancer activities of thiosemicarbazones have been attributed to their ability to act as inhibitors of ribonucleotide reductase (RR), an enzyme involved in the rate-limiting step of DNA synthesis. Thiosemicarbazones can also induce antiproliferative effects via acting as transition metal chelators and leading to the production of deleterious reactive oxygen species (ROS).17-19 Bromophenols, a kind of natural marine product with the unique structure of a bromophenolic group (Figure 1), possess various potent activities, including anticancer, antioxidative, antimicrobial, anti-inflammatory protein tyrosine phosphatase 1B inhibitory,

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and antithrombotic activities.

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Molecular hybrids with two pharmacophores is an efficient

strategy for the design of novel antitumor drugs, which often lead to a synergistic activity,.21, 22

Based on our previous studies of bromophenol hybrids with other anticancer active

moieties,23,

24

a series of novel bromophenol-thiosemicarbazone hybrids was designed to

mimic the nicotinamide structure, which could form a critical H-bond network and undergo π−π stacking with PARP-1 in the nicotinamide binding sites, the common binding mode of most currently reported PARP inhibitors (Figure 2).5 Therefore, the designed hybrids contained multiple pharmacodynamic groups, including the bromophenol pharmacophore, thiosemicarbazone pharmacophore and common pharmacophore of most PARP-1 inhibitors. We propose that the combination of the thiosemicarbazone moiety in the bromophenol scaffold can afford a new series of bromophenol-thiosemicarbazone hybrids with not only inhibitory PARP-1 activity but also multiple anticancer mechanisms to increase their anticancer activities. Thus, a series of novel bromophenol hybrids was synthesized, evaluated for antitumor activities and characterized regarding their structure-activity relationships (SARs). The anticancer activities, pharmacokinetic (PK) characteristics and safety of the promising candidate compound 11 in vivo along with mechanistic studies were further investigated. ■ RESULTS AND DISCUSSION

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Chemistry. The general synthetic methods used for all compounds are illustrated in Scheme 1 and 2. First, a variety of aldehydes were synthesized per our previous report to explore the SARs of these hybrids and obtain potential lead compounds.25 Then, the reaction between semicarbazide and suitably substituted aldehydes was performed in ethanol with a

catalytic amount of CH3COOH to provide the desired compounds 1–36 in good yields, respectively. 26 The structures of these target compounds were characterized by spectroscopic means (1H, 13C NMR, MS and HRMS). Physicochemical properties, including the calculated cLogP (logarithm of the partition coefficient between n-octanol and H2O), cLogS (H2O solubility in mol/L), TPSA (polar surface area), Ha (hydrogen bond acceptor), Hd (hydrogen bond donor), toxicity profiles (including mutagenic effect, tumorigenic effect, irritating effect and reproductive effect) and drug-likeness scores of the target compounds, were calculated and predicted using OSIRIS Property Explorer software at the URL http://www.organic-chemistry.org/prog/peo/. The calculation results for the physicochemical properties and prediction of toxicity risks are shown in Table 1. Cell proliferation inhibition activity. The in vitro antitumor activities against human cancer cell lines including SK-OV-3, Caco2, HepG2, Bel-7402 and one human normal cell line HL-7702 were performed using the MTT assay. The results obtained for the inhibition rate of compounds 1–36 at the concentration of 20 µM and the IC50 values (compound 9, 11, 15, 20, 22, 28, 31, 33 and 35) were listed in Table 2 and Table 3 respectively.

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As shown in Table 2, compounds 9, 11, 15, 20, 22, 28, 31, 33 and 35 exhibited potent anticancer activity against SK-OV-3, Bel-7402, HepG2 and CaCo-2 cancer cell lines at 20 µM, respectively. Compounds 1 and 12 containing two hydroxy groups on the phenol ring displayed weaker activity against four cancer cells at 20 µM. Methylation of the 3-(or 4-)hydroxyl group on the phenol ring of compounds 1, 4 and 12 could enhance their antitumor activity (2 vs. 1, 3 vs. 1, 13 vs. 12, 14 vs. 12, 5 vs. 4, 6 vs. 4), indicating that their anticancer activity was affected by the hydrophobic parameter. Introduction of the bromine atom on the phenol moiety could also enhance their antitumor activity resulting from the activity data of the brominated compounds (15-22) compared with compounds (1-3, 12-14). For example, the activities of compounds 9 and 20 were clearly superior to those of compounds 1 and 12, respectively. Among those compounds carrying a methylated hydroxyl group along with bromination of the phenol moiety, compound 11 showed significant activity against the SK-OV-3, Bel-7402, HepG2 and CaCo-2 cancer cell lines with an inhibition ratio of 100 %, 63.5 %, 71.6 % and 59.6 % at 20 µM, respectively. Furthermore, it is notable that most compounds carrying the introduction of the phenyl group in 1-position of amino group (12-20) showed potent activity comparing to those of compounds (1–9), indicating that the introduction of the phenyl group might be the useful moiety to enhance the hybrid anticancer activity. Based on the above findings, compounds 23-36 containing both methoxy and bromine atoms on the phenol moiety and different substituted phenyl groups in the 1-position of the amino group were designed and synthesized in order to obtain more effective hybrids and their information for the SARs. Most of them, especially

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compounds 28, 31, 33 and 35, exhibited potent anticancer activities inhibiting the four test cell lines. However, the anticancer activities of compounds 30, 34 and 36 did not significantly change, which indicated that the anticancer activities of those hybrids were related to many factors, such as the hydrogen-bonding capacity, electron density or steric effect. The cytotoxicity of the synthesized compounds 1-36 in human normal cell was evaluated in the HL-7702 cell line. Among of them, six compounds (3, 5, 7, 10, 13 and 21) showed a weak effect on the human normal HL-7702 cells, but potent inhibitory against the tested cancer cell lines or a portion of the cancer cell lines. For example, the rate of inhibition of compound 7 against the HepG2 cancer cell line and HL-7702 human normal cells was 61.1 % and 2.11 %, respectively, which indicated that compound 7 exhibited selective inhibition of HepG2 cancer cells. The common structural feature of these selective compounds is that most of the fragments of bromophenol contain one hydroxyl group, one methoxy group and one bromine atom, and thiourea fragments have an amino or a phenylamino group. To further study the cell proliferation inhibitory activity of potent compounds based on the data for the inhibition rate, nine compounds (9, 11, 15, 20, 22, 28, 31, 33 and 35) were selected to test their IC50 values against the SK-OV-3, Bel-7402, HepG2 and CaCo-2 cancer cell lines and HL-7702 human normal cell. The results are shown in Table 3. Among the potent compounds, compound 11 showed excellent antitumor activities, inhibiting SK-OV-3, Bel-7402 and HepG2 cancer cell lines with IC50 values of 2.39, 5.45 and 4.60 µM,

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respectively, and relatively weak inhibitory activity toward the human normal HL-7702 cells with an IC50 value of 13.3 µM, which was similar to that obtained for the positive control drug olaparib. Selective PARP-1 inhibitory activity. The PARP-1 inhibitory activities were investigated using the HT-F Homogeneous Inhibition Assay (Trevigen, Cat# 4690-096-K). The results for the inhibited ration of compounds 1–36 are listed in Table 4. Fourteen compounds showed potent inhibitory activity against PARP-1, with an inhibition ratio > 40 % at the concentration of 100 nM.

Among them, compounds 10, 11, 31 and 35 displayed excellent activities inhibiting PARP-1 with an inhibition ratio of 61.0 %, 84.6 %, 51.5 % and 72.2 %, respectively. Most of the current generation of PARP inhibitors have been shown to inhibit both PARP-1 and PARP2, which might result in side effect profiling of these inhibitors in clinical trials.11 Thus, further studies are warranted to examine whether they are the highly potent and selective inhibitor of PARP-1. Four compounds, 10, 11, 31 and 35, with potent PARP-1 activity were further

investigated for their PARP-2 enzyme activity using the PARP-2 Chemiluminescent Assay Kit (BPS Bioscience, Catalog #: 80552). As shown in Table 5, compounds 10, 11, 31 and 35 specifically and potently inhibited PARP-1 over PARP-2. Compound 11 in particular showed encouraging activity as a PARP-1 inhibitor displaying an IC50 value of 29.5 nM with exquisite selectivity over PARP-2 (selectivity index (SI) > 33.9). The olaparib in the clinical application, as a positive control drug, strongly inhibited PARP-1, but its selectivity toward PARP-2 was low (SI < 1.32).

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Molecular docking. To predict the possible mechanism of action against cancer of compound 11, molecular docking studies were performed to simulate a binding model derived from PARP-1 using the Auto-Dock tools. In this study, the cocrystal structure of BMN 673 with PARP-1 was selected as the docking model (PDB ID code: 4PJT).27 The docking orientation and interactions of compound 11 within the BMN 673 binding domain of PARP-1 are shown in Figure 3A and 3B. First, we found two hydrogen bonds between the O atom of GLU988 and the amino group of the thiosemicarbazone moiety (N-H…O: 2.1 Ǻ). The π−π stacking formed between the ring of bromophenol moiety and the TYR907 residue. The bromophenol moiety with two bromo atoms and methoxy groups were observed to stretch toward the back pocket. Based on the above results for the molecular docking studies, the structure of compound 11 could mimic the nicotinamide structure and bind competitively with nicotinamide adenine dinucleotide (NAD+) at the catalytic site of PARP1, which was similar to most currently reported PARP inhibitors. To predict the selectivity of compound 11 toward PARP-1 over PARP-2, the cocrystal structure of olaparib with PARP-2 was selected as the docking model (PDB ID code: 4TVJ).28 The key group of the thiosemicarbazone moiety of compound 11 stretched toward the back pocket without further interactions with the protein PARP-2, and the bromophenol moiety did not actively participate in the binding to PARP-2 (Figure 3C and 3D), which may result the selectivity observed in the enzyme activity. Unlike compound 11, the positive control drug olaparib could form strong interactions with the domain of PARP-1 and PARP-2, resulting potent

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inhibition of both PARP-1 and PARP-2.28 Of course, further studies were needed to clarify the effect of compound 11 and the responsive region. Bioinformatics analysis. The relative quantitation of proteins was divided into two categories. A quantitative ratio over 1.2 was considered upregulation, while a quantitative ratio less than 1/1.2 was considered downregulation. The amounts of the differentially expressed proteins are summarized in s-Table 1 (Supporting Information). We also summarized the gene ontology, domain, pathway, and subcellular localization in s-Table 2-5 (Supporting Information). Both identified and quantifiable proteins were annotated. As shown in s-Table 2 (Supporting Information), when analysis the GO distribution of downregulated proteins, we found that compound 11 had a significant inhibitory effect on the DNA repair protein domain of SK-OV-3 cells (Figure 4A). After treatment with compound 11, some biological processes, such as DNA metabolic processes, DNA replication, DNA strand elongation involvement in DNA replication, DNA strand elongation, DNA replication initiation, DNA-dependent DNA replication and cell cycle processes, were significantly suppressed (s-Table 3, Supporting Information). In the KEGG pathway analysis, we also found that compound 11 had a significant effect on DNA repair (s-Table 4, Supporting Information and Figure 4B). In this study, after treatment with compound 11, six minichromosome maintenance proteins (MCM 2-7) were significantly inhibited in SK-OV-3 cells (s-Figure 1, Supporting Information). Eukaryotic

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double-stranded DNA unwinding is carried out by the minichromosome maintenance protein (MCM). Its family members include MCM2-7.29,

30

The MCM proteins play an

essential role in eukaryotic DNA replication.31. Once the MCM complex is disrupted, the cells undergo limited replication and are hypersensitive to DNA replication stresses, resulting in DNA damage.32,

33

In this study, we also detected the expression of MCM2-7 after

treatment with compound 11. As shown in Figure 5F, the expression of MCM2-7 was decreased in SK-OV-3 cells after compound 11 treatment, which indicated that the MCM complex was disrupted by this compound. These data indicated that after treatment with compound 11, DNA repair was significantly inhibited in SK-OV-3 cells, likely due to PARP suppression by compound 11. In previous experiments, we found that compound 11 functioned a PARP-1 inhibitor, and further experiments were conducted in this study to verify whether the anticancer activity of compound 11 was due to its PARP-1 inhibitory activity. Compound 11 causes cellular accumulation of DNA double-strand breaks. DNA repair is a constitutive process in cells. It protects the genome from damage and mutations and is therefore important for cell survival. Poly-ADP ribosylation is a posttranslational modification catalyzed by poly-ADP ribose polymerase (PARP) and is one of the earliest cellular responses to DNA damage. Inhibition of PARP will lead to untimely DNA repair.2 γH2AX can be detected as soon as 20 s after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γ-H2AX occurs in one minute.34 In

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this study, we examined whether compound 11 could increase the levels of cellular γ-H2AX using immunofluorescence and western blotting assays. As shown in Figure 5A, 5C, treatment with compound 11 at various concentrations led to significantly increased expression levels of γ-H2AX. These results indicated that compound 11 caused DNA doublestrand breaks by inhibiting PARP-1. Compound 11 leads to the inhibition of H2O2-Triggered PARylation. The mechanism of PARP is to identify the SSB (single-strand DNA breaks) and then synthesis of a polymeric adenosine diphosphate ribose (poly (ADP-ribose) or PAR) chain..35, 36 In this study, we used H2O2 as an agonist to stimulate DNA single-strand breaks that could activate PARP and the synthesis of PAR.37 As shown in Figure 5D, the exposure of SK-OV-3 cells to H2O2 induced large amounts of PAR formation and PAR formation could be reduced by pretreatment with compound 11. Furthermore, intracellular PAR was also inhibited by compound 11 (Figure 5E). Our results demonstrate that compound 11 caused SSB in SK-OV-3 cells was performed by inhibiting of PARP-1. Compound 11 induces apoptosis and cell-cycle arrest in SK-OV-3 cells. PARP is a cleavage substrate for caspase, a core member of the apoptosis process. Therefore, it plays an important role in DNA damage repair and apoptosis. It has been reported that PARP inhibitors exert anti-tumor effects by inhibiting DNA damage repair and promoting tumor cell apoptosis.38-40 As shown in Figure 6A, 6B, compound 11 could induce cell apoptosis in SK-OV-3 cells. We also detected the expression changes of Bax, Bcl-2 and Caspase-3 in SK-

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OV-3 cells after treatment of compound 11. As shown in Figure 6C, the expression of the pro-apoptotic protein Bax increased, the anti-apoptotic protein Bcl-2 decreased, and Caspase3 was activated. In this study, compound 11 was shown to induce G2/M cell cycle arrest in SK-OV-3 cells in a concentration-dependent manner (Figure 6D). Consistently, compound 11 could also inhibit the expression of cyclin B1 and CDK1, which was important for cell cycle regulation (Figure 6E). These results indicated that compound 11 exhibited anti-tumor activities in vitro and that these effects might be achieved by inhibiting PARP-1. Compound 11 induces ROS generation in SK-OV-3 cells. PARP-1 was thought to be activated by DNA breaks. However, ROS could be produced during the process of DNA damage and was found to play an important role during the process of PARP-1 activation.41, 42

In this study, we detected intracellular ROS using DCFH-DA. As shown in Figure 7, the

ROS generation in SK-OV-3 cells was increased after treatment of compound 11. The DCF fluorescence in the compound 11 treatment groups increased by 1.76-fold, 2.51-fold and 3.04-flod compared with the control group. These results demonstrated that compound 11 could induce antiproliferative effects via the production of cytotoxic ROS, which catalyze oxidative damage as thiosemicarbazones. Compound 11 induces autophagy in SK-OV-3 cells. Autophagy, a lysosomal-dependent pathway for the degradation of redundant or damaged cell components, plays an important

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role in the treatment of cancer.43 Autophagy begins with the production of doublemembrane vacuoles (named autophagosomes), which eventually fuse with lysosomes and the contents degraded and recycled.44 It has been widely reported that transmission electron microscopy (TEM) observation is the gold standard technique for the detection of autophagy.45,

46

In this study, we observed autophagosomes by TEM, which indicated that

compound 11 could induce autophagy in SK-OV-3 cells (Figure 8A). The autophagosomes were characteristically marked by the presence of the protein LC3 on their membranes. When autophagy occurs, LC3-I (18 kD) is activated and forms LC3-II (16 kD).47 Therefore, we examined the activation of LC3 by GFP-LC3 infection and western blotting analyses. After infection of the cells with the GFP-LC3B adenovirus, GFP-LC3B was present in the cytoplasm in a diffuse form in the absence of autophagy, whereas in the case of autophagy, GFP-LC3B was accumulated on the autophagosome membrane, expressed as spots (LC3B dots or punctae). As shown in Figure 8B, we observed an obvious dot phenomenon in the compound 11 treatment group, indicating the occurrence of autophagy. The immunofluorescence assay showed a significant increase in fluorescence intensity in the compound 11 treatment group (Figure 8C), indicating that autophagy had occurred. Western blot analysis showed both the LC3-I (18 kDa) form and the active, autophagosome membrane-bound LC3-II (16 kDa) form (Figure 8D). Several studies have reported that Beclin 1 and Atg 14 play an important role in the regulation of autophagy.48 In the present study, our data showed that both Beclin 1 and Atg

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14 were activated after treatment with compound 11 (Figure 8D). These results indicated that compound 11 could induce autophagy in SK-OV-3 cells, which might represent one of the mechanisms by which compound 11 exerted its anti-tumor effects. Compound 11 inhibits tumor growth in vivo in a SK-OV-3 cell xenograft model. To study the anti-tumor properties of compound 11 in vivo, a SK-OV-3 xenografted athymic mice model was established. In this model, we used two doses (25 mg/kg and 50 mg/kg) to treat the mice for 21 days, adding the PARP inhibitor olaparib (25 mg/kg) as a positive control. As shown in Figure 9A, tumor growth was significantly inhibited in the compound 11-treated groups in a dose-dependent manner; the inhibition rate was 70.02 % and 58.87 %, respectively, while the inhibition in the olaparib group was 55.73 %. Furthermore, there was no significant change in athymic mice body weight during the experiments (Figure 9B). We further demonstrated that compound 11 induced PARP-1 inhibition in vivo. Immunohistochemistry with Ki67 (a cell proliferation marker) and γ-H2AX was examined in paraffin-embedded tumor sections. As shown in Figure 9C, compared with the control group, compound 11 treatment significantly decreased the expression of Ki67 and increased the expression of γ-H2AX. The western blotting results also indicated that compound 11 could increase the expression of γ-H2AX in the xenograft models (Figure 9D). These results indicated that compound 11 could exert anti-tumor effects in vivo, potentially through the inhibition of PARP-1.

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For histological analysis, the heart, liver and kidney were stained with H&E, and as shown in Figure 9E, there were no histological changes in these tissues after treatment with compound 11. These results could be considered to indicate a low toxicity of the anti-tumor activity of compound 11 in athymic mice. Pharmacokinetic (PK) Study of Compound 11 in vivo. In light of the excellent antitumor activities of compound 11 both in vitro and in vivo, compound 11 were further evaluated for its pharmacokinetic properties in male SD rats after a single intravenous injection (i.v., 2 mg/kg) and oral administration (p.o., 10 mg/kg), respectively. As shown in Table 6, compound 11 showed good oral bioavailability of 47.3 % with an AUC0→n of 886 nM∙h. The oral maximum plasma concentration (Cmax) was 19.9 nM and (Tmax) was 2.50 h. The elimination half-life (T1/2) was 2.60 h, and the mean residence times (MRT) were 3.76 h. In addition, the mean VSS of compound 11 was 6.10 L/kg with a mean CLtot,p of 5.32 L h−1 kg−1. These results indicated that compound 11 presented favorable pharmacokinetic properties. Safety of compound 11 in vivo. To investigate the safety of compound 11, acute toxicity and subacute toxicity tests were carried out in vivo in Kunming mice. Groups of healthy mice were orally administered single doses of 100, 500, or 1000 mg/kg of compound 11 in the acute toxicity study, respectively. No mortality or abnormalities were observed (including body weight, s-Table 6, Supporting Information) in the mice throughout the observation period for up to 14 days post-administration. The test results indicated that the LD50 of compound 11 was greater than 1000 mg/kg bw.

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In the subacute toxicity test, the treatment group was dosed by oral administration of 1000 mg/kg compound 11 for 14 days. No death and no obvious changes in clinical signs and body weights were detected during the 14-day feeding study (s-Table 7, Supporting Information). As shown in Figure 10, the H&E analysis of tissues of the mice also showed no significant changes after intragastric administration of compound 11. Taken together, the results of the acute toxicity and subacute toxicity testing demonstrated that compound 11 demonstrated a good safety in vivo and, thus, may be a promising antitumor agent. ■ CONCLUSIONS In summary, a series of bromophenol-thiosemicarbazone hybrids were designed, synthesized and their antitumor activities evaluated. One of the most promising compounds, 11, exhibited excellent selective PARP-1 inhibitory activity and anticancer activities in vitro and

in vivo , as further demonstrated through multiple anticancer mechanisms, including the induction of apoptosis and cell-cycle arrest, cellular accumulation of DNA double-strand breaks, effect on DNA repair, inhibition of H2O2-Triggered PARylation, antiproliferative effects via the production of cytotoxic ROS and autophagy. In addition, compound 11 displayed good pharmacokinetic characteristics and favorable safety. Compound 11 has been selected as a lead candidate and is currently undergoing further characterization of the preclinical profile in our follow-up studies.

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■ EXPERIMENTAL SECTION Chemistry. Reaction reagents were obtained from commercial suppliers from J&K Scientific Ltd. (Beijing, China). Organic solvents (analytical reagent grade) were purchased from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China). Column chromatography (CC): silica gel (200–300 mesh; Qingdao Makall Group Co., Ltd; Qingdao, China) were used for purifying the crude product. Thin-layer chromatography (TLC) were used to monitor all reactions on silica gel plates. 1H and 13C nuclear magnetic resonance (NMR) measurements were recorded on a Bruker DRX 500 MHz spectrometer with tetramethylsilane (TMS) as the internal standard (Bruker, Bremerhaven, Germany). Chemical shifts are quoted in ppm downfield from TMS; coupling constants (J) are quoted in Hertz (Hz). A LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) were used to determine MS and HRMS spectra of target compounds. An SGW X-4 Melting Point Apparatus (Shanghai Precision Science Instrument Co., Ltd; Shanghai, China) were used to determine melting points. The names of synthesized compounds were named using ChemBioDraw Ultra software (v 12.0, PerkinElmer, MA, USA). The purities of all biologically evaluated compounds was determined to be >95% (s-Table 8) under two solvent conditions by analytical HPLC recorded on a Shimadzu LC-20A system. HPLC were equipped with SPD-20A detector and a Shimadzu InertSustain C-18 reverse phase column (4.6mm*250mm*5um). HPLC solvent conditions A: CH3OH/H2O with 0.1% trifluoroacetic acid 60−100% (25 min); UV detection, 254 nm; flow rate, 1.0 mL/min; temperature, 40 °C; injection volume, 1-15 μL. HPLC solvent

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conditions B: CH3CN/H2O with 0.1% trifluoroacetic acid 40−100% (25 min); UV detection, 254 nm; flow rate, 1.0 mL/min; temperature, 40 °C; injection volume, 1-15 μL. General Procedures for the Preparation of Compounds 1-36. A solution of thiosemicarbazide (2.2 mmol, 1.1 equiv) in ethanol (95 %, 20 mL) was refluxed with various aldehydes (2.0 mmol, 1. equiv) and a few drops of glacial acetic acid for 8 h - 12 h . TLC analysis indicated when the reaction was complete. The solid was collected by filtration after cooling to room temperature and washed with ethanol. Then, the solid were dried in a vacuum to afford the title compounds 1–36 (the crude product was purified by a silica gel column if no solid precipitated). 2-(4,5-dihydroxybenzylidene)hydrazine-1-carbothioamide(1). Red solid; Yield: 65.7 %; mp 261-262 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.21(1H, s), 9.49(1H, s), 9.01(1H, s), 8.05(1H, s), 7.88(1H, s), 7.73(1H, s), 7.18(1H, s), 7.00(1H, d, J=8.4 Hz), 6.75(1H, d, J=8.4 Hz);

13C-NMR

(150 MHz, DMSO-d6)δ: 177.9, 148.3, 146.1, 143.8, 126.1, 120.7, 116.1, 114.4; ESIMS: m/z 210 [M-H]‐ HRESIMS: calc for C8 H9 N3 O2 S [M-H]‐ 210.0343, found 210.0350. 2-(4-hydroxy-3-methoxybenzylidene)hydrazine-1-carbothioamide (2). White solid; Yield: 83.6 %; mp 235-236 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.24(1H, s), 9.49(1H, s), 8.09(1H, s), 7.95(1H, s), 7.94(1H, s), 7.46(1H, s), 7.04(1H, d, J=8.4 Hz), 6.77(1H, d, J=8.4 Hz), 3.82(3H, s); 13C-NMR

(150 MHz, DMSO-d6)δ: 177.9, 149.4, 148.6, 143.6, 126.1, 122.9, 115.8, 109.9,56.3;

ESIMS: m/z 224 [M-H]‐ HRESIMS: calc for C9 H11 N3 O2 S [M-H]‐224.0499, found 224.0487.

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2-(3-hydroxy-4-methoxybenzylidene)hydrazine-1-carbothioamide (3). Light yellow solid, Yield: 81.5 %; mp 177-178 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.28(1H, s), 9.06(1H, s), 8.10(1H, s), 7.92(1H, s), 7.83(1H, s), 7.27(1H, d, J=1.8 Hz), 7.10(1H, dd, J=1.8, 8.4 Hz), 6.92(1H, d, J=8.4 Hz), 3.79(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 178.1, 150.1, 147.2,

143.4, 127.6, 120.7, 113.8, 112.3,56.2; ESIMS: m/z 224 [M-H]‐ HRESIMS: calc for C9 H11 N3 O2 S [M-H]‐224.0499, found224.0489. 2-(3-bromo-4,5-dihydroxybenzylidene)hydrazine-1-carbothioamide(4). Red solid; Yield: 76.3 %; mp 215-216 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.28(1H, s), 10.00(1H, s), 9.61(1H, s), 8.11(1H, s), 7.86(1H, s), 7.52(1H, s), 7.04(1H, s); 13C-NMR (150 MHz, DMSO-d6)δ: 178.2, 146.7, 143.4, 142.2, 126.9, 122.5, 114.3, 110.9; ESIMS: m/z 224 [M-H]‐ HRESIMS: calc for C8 H8 N3 O2 SBr [M-H]‐287.9448, found287.9423. 2-(3-bromo-4-hydroxy-5-methoxybenzylidene)hydrazine-1-carbothioamide (5). Light yellow solid, Yield: 90.6 %; Purity 97.8% (HPLC); mp 232-234 ˚C; 1H-NMR (600 MHz, DMSOd6)δ:11.36(1H,

s), 9.91(1H, s), 8.17(1H, s), 8.10(1H, s), 7.90(1H, s), 7.48(1H, s), 7.43(1H, s),

3.88 (3H, s); 13C-NMR (150 MHz, DMSO-d6)δ: 178.2, 149.1, 146.0, 141.9, 127.1, 124.9, 109.9, 109.6, 57.0; ESIMS: m/z 224 [M-H]‐ HRESIMS: calc for C9 H10 N3 O2 S Br [M-H]‐301.9604 found301.9588. 2-(2-bromo-3-hydroxy-4-methoxybenzylidene)hydrazine-1-carbothioamide (6). Light yellow solid, Yield: 88.9 %; mp 247-248 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.51(1H, s), 9.59(1H, s), 8.40(1H, s), 8.18(1H, s), 7.95(1H, s), 7.72(1H, d, J=8.4 Hz), 6.99(1H, d, J=8.4 Hz), 3.87 (3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 178.3, 150.0, 144.2, 142.3, 126.3, 118.8, 112.6, 111.4,

56.9;[M-H]‐ HRESIMS: calc for C9 H10 N3 O2 S Br [M-H]‐301.9604 found301.9585.

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2-(3-bromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (7). White solid; Yield: 92.1 %; mp 236-238 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ: 7.62(1H, s), 7.23(1H, d, J=1.8 Hz), 7.16(1H, s), 7.01(1H, d, J=1.8 Hz), 3.73 (3H, s), 3.70 (3H, s); 13C-NMR (150 MHz, DMSO-d6)δ: 174.0, 149.9, 144.3, 138.6, 126.5, 120.5, 113.9, 105.6, 56.7, 52.1; [M-H]‐ HRESIMS: calc for C10 H12 N3 O2 S Br [M-H]‐315.9761 found 315.9748. 2-(2-bromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (8). White solid; Yield: 91.5 %; mp 262-263 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ: 11.5(1H, s), 8.36(1H, s), 8.27(1H, s), 8.14(1H, s), 7.68(1H, s), 7.14(1H, s), 3.83 (3H, s), 3.80 (3H, s); d6)δ:

13C-NMR

(150 MHz, DMSO-

178.3, 151.6, 149.2, 141.8, 125.6, 115.8, 115.7, 109.8, 56.6, 56.5 ; [M-H]‐ HRESIMS:

calc for C10 H12 N3 O2 S Br [M-H]‐315.9761 found 315.9746. 2-(2,3-dibromo-4,5-dihydroxybenzylidene)hydrazine-1-carbothioamide (9). White solid; Yield: 80.3 %; mp 245-246 ˚C;1H-NMR (600 MHz, DMSO-d6)δ: 11.56(1H, s),10.14(2H, s), 8.38(1H, s), 8.23(1H, s), 7.76(1H, s), 7.56(1H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 178.5,

147.6, 145.9, 143.2, 126.1, 116.8, 113.8, 113.1;ESIMS: m/z 365 [M-H]‐HRESIMS: calc for C8 H7 N3 O2 S Br2 [M-H]‐ 365.8553, found 365.8528. 2-(2,3-dibromo-4-hydroxy-5-methoxybenzylidene)hydrazine-1-carbothioamide (10). White solid; Yield: 88.6 %; mp 243-244 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ: 11.54(1H, s) , 10.40(1H, s) , 8.44(1H, s), 8.28(1H, s), 8.15(1H, s), 7.75(1H, s), 3.92 (3H, s);

13C-NMR

(150

MHz, DMSO-d6)δ: 178.4, 148.2, 147.8, 143.0, 126.0, 118.7, 113.4, 109.3, 57.3 ; ESIMS: m/z 379 [M-H]‐ HRESIMS: calc for C9H9N3O2SBr2 [M-H]‐379.8709, found 379.8693.

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2-(2,3-dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (11). White solid; Yield: 93.0 %; mp 273-274 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ: 11.62(1H, s) , 8.44(1H, s), 8.34(1H, s), 8.20(1H, s), 7.80(1H, s), 3.91 (3H, s), 3.76 (3H, s); d6)δ:

13C-NMR

(150 MHz, DMSO-

178.5, 153.0, 148.9, 142.1, 131.2, 121.6, 117.5, 110.6, 60.7, 57.1 ; ESIMS: m/z 393 [M-

H]‐ HRESIMS: calc for C10H11N3O2SBr2 [M-H]‐ 393.8866, found 393.8846. 2-(3,4-dihydroxybenzylidene)-N-phenylhydrazine-1-carbothioamide

(12).

White

solid;

Yield: 77.4 %; mp 213-214 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.62(1H, s), 9.95(1H, s), 9.57(1H, s), 9.04(1H, s), 8.00(1H, s), 7.59(1H, d, J=8.4 Hz), 7.33-7.37(3H, m), 7.19 (1H, m), 7.10 (1H, dd, J=2.4,8.4 Hz), 6.78(1H, d, J=8.4 Hz);

13C-NMR

(150 MHz, DMSO-d6)δ: 175.9,

148.6, 146.1, 144.4, 139.7, 128.6(2C), 126.0, 125.9, 125.6(2C), 121.2, 116.0, 114.6; ESIMS: m/z 286 [M-H]‐ HRESIMS: calc for C14 H13 N3 O2 S [M-H]‐ 286.0656, found 286.0629. 2-(4-hydroxy-3-methoxybenzylidene)-N-phenylhydrazine-1-carbothioamide

(13).

Brown

solid; Yield: 90.1 %; mp 181-182 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.70(1H, s), 10.01(1H, s), 9.59(1H, s), 8.09(1H, s), 7.55-7.59(3H, overlap), 7.38(2H, m), 7.20-7.22 (2H, m), 6.84(1H, d, J=8.4 Hz), 3.85(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 175.9, 149.6, 148.6, 144.3, 139.7,

128.7(3C), 126.6, 125.9(2C), 123.2, 116.0, 110.6, 56.4; ESIMS: m/z 300 [M-H]‐ HRESIMS: calc for C15 H15 N3 O2 S [M-H]‐ 300.0812, found 300.0788. 2-(3-hydroxy-4-methoxybenzylidene)-N-phenylhydrazine-1-carbothioamide

(14).

Brown

solid; Yield: 88.6 %; mp 174-176 ˚C;1H-NMR (600 MHz, DMSO-d6)δ:11.69(1H, s), 10.02(1H, s), 9.10(1H, s), 8.05(1H, s), 7.59(1H, d, J=8.4 Hz), 7.46(1H, d, J=1.8 Hz), 7.38(2H, m), 7.187.21 (2H, m), 6.96(1H, d, J=8.4 Hz), 3.82(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 176.1,

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150.3, 147.2, 144.0, 139.7, 128.7(3C), 127.4, 126.2, 125.7, 121.3, 114.0, 112.2, 56.2; ESIMS: m/z 300 [M-H]‐ HRESIMS: calc for C15 H15 N3 O2 S [M-H]‐ 300.0812, found 300.0790. 2-(3-bromo-4,5-dihydroxybenzylidene)-N-phenylhydrazine-1-carbothioamide (15). Light yellow solid; Yield: 68.9 %; mp 182-183 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.68(1H, s), 10.06(1H, s), 10.02(1H, s), 9.68(1H, s), 7.97(1H, s), 7.65(1H, d, J=1.8 Hz), 7.56(2H, d, J=7.8 Hz), 7.37 (2H, m), 7.20 (1H, m), 7.16(1H, d, J=2.0 Hz);

13C-NMR

(150 MHz, DMSO-d6)δ:

176.2, 146.7, 145.6, 142.8, 139.7, 128.6(2C), 126.8, 126.4, 125.9(2C), 122.8, 114.6, 110.9 ; ESIMS: m/z 363 [M-H]‐ HRESIMS: calc for C14 H12 N3 O2 SBr [M-H]‐ 363.9761, found 363.9727. 2-(3-bromo-4-hydroxy-5-methoxybenzylidene)-N-phenylhydrazine-1-carbothioamide (16). White solid; Yield: 91.6 %; mp 205-207 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.78(1H, s), 10.09(1H, s), 9.99(1H, s), 8.04(1H, s), 7.67(1H, d, J=1.8 Hz), 7.54(2H, d, J=7.8 Hz), 7.46(1H, d, J=1.8 Hz), 7.38 (2H, m), 7.22 (1H, m); 13C-NMR (150 MHz, DMSO-d6)δ: 176.4, 149.0, 146.2, 142.6, 139.7, 128.7(2C), 127.0, 126.8, 126.1 (2C), 124.9, 110.4, 110.2; ESIMS: m/z 377 [MH]‐ HRESIMS: calc for C15 H14 N3 O2 SBr [M-H]‐ 377.9917, found 377.9888. 2-(2-bromo-3-hydroxy-4-methoxybenzylidene)-N-phenylhydrazine-1-carbothioamide (17). White solid; Yield: 90.8 %; mp 232-233 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.92(1H, s), 10.08(1H, s), 9.64(1H, s), 8.54(1H, s), 7.89(1H, d, J=8.4 Hz), 7.57(2H, d, J=7.8 Hz), 7.38(2H, m), 7.20 (1H, m), 7.02(1H, d, J=8.4 Hz), 3.88(3H, s); 13C-NMR (150 MHz, DMSO-d6)δ: 176.1, 150.3, 147.2, 144.0, 139.7, 128.7(2C), 127.4, 126.2(2C), 125.7, 121.3, 114.0, 112.2, 56.2 ;

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ESIMS: m/z 377 [M-H]‐ HRESIMS: calc for C15 H14 N3 O2 SBr [M-H]‐ 377.9917, found 377.9899. 2-(3-bromo-4,5-dimethoxybenzylidene)-N-phenylhydrazine-1-carbothioamide (18). White solid; Yield: 94.6 %; mp 173-174 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.91(1H, s), 10.17(1H, s), 8.07(1H, s), 7.81(1H, d, J=1.8 Hz), 7.52(2H, d, J=7.8 Hz), 7.50(1H, d, J=1.8 Hz), 7.39 (2H, m), 7.23 (1H, m), 3.90(3H, s), 3.77(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 176.8, 154.1,

147.5, 141.8, 139.7, 132.0 128.7(2C), 127.1, 126.2 (2C), 123.8, 117.8, 112.1, 60.8, 57.0 ; ESIMS: m/z 392[M-H]‐ HRESIMS: calc for C16 H16 N3 O2 SBr [M-H]‐ 392.0074, found 392.0061. 2-(2-bromo-4,5-dimethoxybenzylidene)-N-phenylhydrazine-1-carbothioamide (19). White solid; Yield: 93.1 %; mp 237-239 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.95(1H, s), 10.12(1H, s), 8.50(1H, s), 7.80(1H, s), 7.58(2H, d, J=7.8 Hz), 7.39 (2H, m), 7.23 (1H, m), 3.90(3H, s), 7.19(1H, s), 3.86(3H, s), 3.83(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 176.5, 151.9, 149.2,

142.5, 139.7, 132.0 128.7(2C), 126.8, 126.1 (2C), 125.4, 116.1, 115.9, 110.4, 56.7, 56.6 ; ESIMS: m/z 392 [M-H]‐ HRESIMS: calc for C16 H16 N3 O2 SBr[M-H]‐ 392.0074, found 392.0058. 2-(2,3-dibromo-4,5-dihydroxybenzylidene)-N-phenylhydrazine-1-carbothioamide

(20).

White solid; Yield: 67.2 %; mp 214-216 ˚C;1H-NMR (600 MHz, DMSO-d6)δ: 11.94(1H, s) , 10.16(2H, s), 10.03(1H, s), 8.50(1H, s), 7.73(1H, s), 7.60(2H, d, J = 7.8 Hz), 7.37 (2H, dd, J = 7.8, 6.0 Hz), 7.19 (1H, dd, J = 7.2, 7.2 Hz);;

13C-NMR

(150 MHz, DMSO-d6)δ: 176.4, 147.7,

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145.9, 143.6, 128.7(2C), 126.0, 125.8(3C), 117.0, 113.8, 113.4 ; ESIMS: m/z 441 [M-H]‐ HRESIMS: calc for C14 H11 N3 O2 S Br2 [M-H]‐ 441.8866, found 441.8848. 2-(2,3-dibromo-4-hydroxy-5-methoxybenzylidene)-N-phenylhydrazine-1-carbothioamide (21). White solid; Yield: 90.8 %; mp 231-233 ˚C;1H-NMR (600 MHz, DMSO-d6)δ: 11.96 (1H, s), 10.49 (1H, s), 10.12(1H, s), 8.58(1H, s), 7.87(1H, s), 7.57(2H, d, J = 7.8 Hz), 7.38 (2H, m), 7.23 (1H, m), 3.94(3H, s); 13C-NMR (150 MHz, DMSO-d6)δ: 176.5, 148.2, 148.1, 143.6, 139.7, 128.7(2C), 126.8(2C), 126.1, 125.8, 119.0, 113.5, 109.8, 57.4 ; ESIMS: m/z 455[M-H]‐ HRESIMS: calc for C15 H13 N3 O2 S Br2 [M-H]‐ 455.9022, found 455.9015. 2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-phenylhydrazine-1-carbothioamide

(22).

White solid; Yield: 91.3 %; mp 221-222 ˚C;1H-NMR (600 MHz, DMSO-d6)δ: 12.07 (1H, s), 10.18 (1H, s), 8.59(1H, s), 7.94(1H, s), 7.57(2H, d, J = 7.8 Hz), 7.39 (2H, m), 7.23 (1H, m), 3.94(3H, s), 3.79(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 176.8, 153.1, 149.2, 142.8, 139.6,

131.2, 128.8(2C), 126.8(2C), 126.2, 121.8, 117.9, 111.3, 60.9, 57.4; ESIMS: m/z 471 [M+H]+ HRESIMS: calc for C16 H15 N3 O2 S Br2 [M+H]+ 471.9324, found 471.9314. 2-(3-bromo-4,5-dimethoxybenzylidene)-N-methylhydrazine-1-carbothioamide (23). White solid; Yield: 82.6 %; mp 208-210 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.55(1H, s), 8.56(1H, q, J=4.2 Hz), 7.95(1H, s), 7.70(1H, d, J=1.8 Hz), 7.40(1H, d, J=1.8 Hz), 3.89(3H, s), 3.76(3H, s), 3.03(3H, d, J=4.2 Hz);

13C-NMR

(150 MHz, DMSO-d6)δ: 178.3, 154.1, 147.3, 140.6, 132.3,

123.2, 117.8, 111.9, 60.8, 57.0, 31.4 ESIMS: m/z 331 [M+H]+ HRESIMS: calc for C11H14N3O2SCBr [M+H]+ 332.0063, found 331.9980.

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2 - (3 - bromo-4, 5-dimethoxybenzylidene) - N - (3 - morpholinopropyl) hydrazine – 1 carbothioamide (24). Light yellow solid; Yield: 77.9 %; mp 161-163 ˚C; 1H-NMR (500 MHz, DMSO-d6)δ:11.51(1H, s), 8.57(1H, t, J=4.8 Hz), 7.96(1H, s), 7.69(1H, d, J=1.5 Hz), 7.36(1H, d, J=1.5 Hz), 3.89(3H, s), 3.76(3H, s), 3.60(2H, m), 3.56(4H, t, J=3.8 Hz), 2.30-2.35(6H, overlap), 1.76(2H, m);

13C-NMR

(125 MHz, DMSO-d6)δ: 177.4, 154.1, 147.3, 140.8, 132.2,

123.0, 117.8, 112.2, 66.7(2C), 60.8(2C), 56.9, 56.5, 53.9, 42.7, 26.3; ESIMS: m/z 445 [M-H]‐ HRESIMS: calc for C17 H25 N4 O3 S Br [M-H]‐ 445.0887, found 445.0903. 2-(3-bromo-4,5-dimethoxybenzylidene)-N-(4-fluorophenyl)hydrazine-1-carbothioamide (25). White solid; Yield: 92.6 %; mp 179-181 ˚C; 1H-NMR (500 MHz, DMSO-d6)δ:11.93(1H, s), 10.16(1H, s), 8.07(1H, s), 7.81(1H, d, J=1.5 Hz), 7.56(2H, m), 7.22 (2H, m), 3.94(3H, s), 3.79(3H, s); 13C-NMR (125 MHz, DMSO-d6)δ: 177.2, 160.4(d, J=201 Hz), 153.1, 149.2, 143.0, 136.0, 131.2, 129.3(2C, d, J=6.8 Hz), 123.8, 117.9, 115.3(2C, d, J=18.5 Hz), 112.2, 60.8, 57.0; ESIMS: m/z 412 [M+H]+ HRESIMS: calc for C16 H15 N3 O2F S Br [M+H]+ 412.0125, found 412.0105. 2-(3-bromo-4,5-dimethoxybenzylidene)-N-(4-chlorophenyl)hydrazine-1-carbothioamide (26). White solid; Yield: 89.1 %; mp 186-188 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.98(1H, s), 10.19(1H, s), 8.07(1H, s), 7.82(1H, s), 7.60(2H, d, J=8.4 Hz), 7.50(1H, s), 7.43(2H, d, J=8.4 Hz), 3.90(3H, s), 3.77(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 176.9, 154.1, 147.6, 142.1, 131.9,

130.2(2C), 128.6(2C), 128.5, 123.8, 117.8, 112.3, 60.8, 57.0; ESIMS: m/z 427 [M+H]+ HRESIMS: calc for C16H15N3O2SClBr [M+H]+ 427.9830, found 427.9797. 2-(3-bromo-4,5-dimethoxybenzylidene)-N-(3-(trifluoromethyl)phenyl)hydrazine-1carbothioamide (27). White solid; Yield: 92.7 %; mp 190-192 ˚C; 1H-NMR (500 MHz, DMSO-

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d6)δ:12.09(1H,

s), 10.34(1H, s), 8.10(1H, s), 8.01(1H, s), 7.89(1H, d, J=6.5 Hz), 7.83(1H, s),

7.62(1H, m), 7.57(1H, d, J=6.5 Hz), 7.50(1H, s), 3.90(3H, s), 3.78(3H, s); 13C-NMR (125 MHz, DMSO-d6)δ: 176.7,154.1, 147.6, 142.4, 140.5, 131.8, 130.7, 129.8, 129.4, 123.7(J=225.3 Hz), 123.8, 123.0, 122.4, 117.8, 112.4, 60.8, 57.0; ESIMS: m/z 462 [M+H]+ HRESIMS: calc for C17 H15 N3 O2F 3S Br [M+H]+ 462.0093, found 462.0072. Ethyl

-4-(2-(3-bromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamido)benzoate

(28). White solid; Yield: 91.2 %; mp 223-225 ˚C; 1H-NMR (500 MHz, DMSO-d6)δ:12.08(1H, s), 10.33(1H, s), 8.10(1H, s), 7.96(2H, d, J=7.0 Hz), 7.84(2H, d, J=7.0 Hz), 7.51(1H, s), 4.31(2H, q, J=6.0 Hz), 3.91(3H, s), 3.78(3H, s), 1.33(3H, t, J=6.3 Hz);

13C-NMR

(125 MHz, DMSO-d6)δ:

176.3, 165.9, 154.1, 147.6, 144.1, 142.4, 131.8, 129.7(2C), 126.8, 125.7(2C), 123.9, 117.8, 112.3, 61.2, 60.8, 57.0, 14.8; ESIMS: m/z 466 [M+H]+ HRESIMS: calc for C19 H2O N3 O4S Br [M+H]+ 466.0431, found 466.0418. 2-(3-bromo-4,5-dimethoxybenzylidene)-N-(4-phenoxyphenyl)hydrazine-1-carbothioamide (29). White solid; Yield: 81.8 %; mp 196-198 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:11.91(1H, s), 10.16(1H, s), 8.08(1H, s), 7.81(1H, s), 7.51-7.53(3H, overlap), 7.40 (2H, m), 7.14 (1H, m), 7.017.04(4H, overlap),3.90(3H, s), 3.77(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 177.0, 157.4,

154.7, 154.1, 147.5, 141.8, 135.2, 132.0 130.6(2C), 128.9, 124.0 (2C), 123.8, 119.0(2C), 118.9(2C), 117.8, 112.1, 60.8, 57.0; ESIMS: m/z 486 [M+H]+ HRESIMS: calc for C22 H19 N3 O3S Br2 [M+H]+ 486.0481, found 486.0468. 2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-methylhydrazine-1-carbothioamide

(30).

White solid; Yield: 94.1 %; mp 245-247 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ: 11.73(1H, s), 8.59(1H, q, J=4.2 Hz), 8.46(1H, s), 7.80(1H, s), 3.95 (3H, s), 3.79 (3H, s), 3.05(3H, d, J=4.2

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Hz);

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(150 MHz, DMSO-d6)δ: 178.4, 153.1, 149.0, 141.7, 131.5, 121.8, 117.6, 110.9,

60.8, 57.3, 31.6 ; ESIMS: m/z 409 [M+H]+ HRESIMS: calc for C11H13N3O2SBr2 [M+H]+ 409.9168, found 409.9142. 2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-(3-morpholinopropyl)hydrazine-1carbothioamide (31). White solid; Yield: 92.4 %; mp 206-208 ˚C; 1H-NMR (500 MHz, DMSOd6)δ:11.70(1H,

s), 8.60(1H, t, J=4.8 Hz), 8.45(1H, s), 7.76(1H, s), 3.94(3H, s), 3.78(3H, s),

3.60(2H, m), 3.54(4H, t, J=3.8 Hz), 2.30-2.34(6H, overlap), 1.76(2H, m); 13C-NMR (125 MHz, DMSO-d6)δ: 177.6, 153.1, 149.0, 141.9, 131.5, 121.8, 117.6, 111.1, 66.7(2C), 60.8(2C), 57.2, 56.5, 53.9, 42.8, 26.2; ESIMS: m/z 523 [M+H]+ HRESIMS: calc for C17 H24 N4 O3 S Br2 [M+H]+ 523.0009, found 523.0009. 2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-(4-fluorophenyl)hydrazine-1-carbothioamide (32). White solid; Yield: 90.2 %; mp 219-221 ˚C; 1H-NMR (500 MHz, DMSO-d6)δ:12.09 (1H, s), 10.17(1H, s), 8.59(1H, s), 7.92(1H, s), 7.50-7.53(2H, overlap), 7.21 (2H, m), 3.90(3H, s), 3.77(3H, s); 13C-NMR (125 MHz, DMSO-d6)δ: 177.2, 160.4(d, J=200.8 Hz), 154.1, 147.5, 141.9, 136.0, 132.0, 129.0(2C, d, J=6.8 Hz), 121.8, 117.8, 115.4(2C, d, J=18.5 Hz), 111.3, 60.8, 57.4; ESIMS: m/z 489 [M+H]+ HRESIMS: calc for C16 H14 N3 O2F S Br2 [M+H]+ 489.9230, found 489.9237. N-(4-chlorophenyl)-2-(2,3-dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (33). White solid; Yield: 88.9 %; mp 247-249 ˚C; 1H-NMR (600 MHz, DMSO-d6)δ:12.13(1H, s), 10.20(1H, s), 8.59(1H, s), 7.93(1H, s), 7.63(2H, d, J=8.4 Hz), 7.43(2H, d, J=8.4 Hz), 3.94(3H, s), 3.80(3H, s);

13C-NMR

(150 MHz, DMSO-d6)δ: 176.8, 153.1, 149.3, 143.1, 138.6, 131.1 ,

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130.1(2C), 128.7(2C), 128.2, 121.8, 118.0, 111.5, 60.9, 57.4; ESIMS: m/z 505 [M+H]+ HRESIMS: calc for C16H14N3O2SClBr2 [M+H]+ 505.8935, found 505.8926. 2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-(3-(trifluoromethyl)phenyl)hydrazine-1carbothioamide (34). Light yellow solid; Yield: 89.7 %; mp 206-208 ˚C; 1H-NMR (500 MHz, DMSO-d6)δ:12.24 (1H, s), 10.36(1H, s), 8.61(1H, s), 8.07(1H, s), 7.91-7.93(2H, overlap), 7.62 (1H, m), 7.57(1H, d, J=6.5 Hz), 3.90(3H, s), 3.77(3H, s);

13C-NMR

(125 MHz, DMSO-d6)δ:

176.8, 153.1, 149.3, 143.4, 140.4, 131.1, 130.3, 129.9, 129.4, 123.7(q, J=225.3 Hz), 122.7, 122.5, 121.8, 118.0, 111.5, 60.9, 57.3; ESIMS: m/z 539 [M+H]+ HRESIMS: calc for C17 H14 N3 O2F 3S Br2 [M+H]+ 539.9198, found 539.9188. Ethyl-4-(2-(2,3-dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamido)benzoate (35). White solid; Yield: 92.6 %; mp 222-224 ˚C; 1H-NMR (500 MHz, Py-d5)δ:12.25 (1H, s), 9.86(1H, s), 6.98(2H, d, J=8.5 Hz), 6.92(2H, d, J=8.5 Hz), 6.47(1H, s), 6.36(1H, s), 3.11(2H, q, J=7.0 Hz), 2.65(3H, s), 2.29(3H, s), 0.03(3H, t, J=7.0 Hz); 13C-NMR (125 MHz, Py-d5)δ: 176.2, 164.6, 151.7, 148.8, 142.9, 141.4, 129.4, 128.7(2C), 126.0, 123.6(2C), 121.0, 117.3, 109.2, 59.6, 59.1, 54.6, 13.0; ESIMS: m/z 543 [M+H]+ HRESIMS: calc for C19 H19 N3 O4S Br2 [M+H]+ 543.9536, found 543.9525. 2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-(4-phenoxyphenyl)hydrazine-1carbothioamide (36). White solid; Yield: 91.2 %; mp 193-195 ˚C; 1H-NMR (600 MHz, DMSOd6)δ:12.06(1H,

s), 10.16(1H, s), 8.59(1H, s), 7.94(1H, s), 7.54(2H, d, J=8.4 Hz), 7.41 (2H, m),

7.15 (1H, m), 7.02-7.04(4H, overlap), 3.94(3H, s), 3.80(3H, s); d6)δ:

13C-NMR

(150 MHz, DMSO-

177.0, 157.4, 154.7, 154.1, 153.2, 149.2, 142.9, 135.1, 131.2, 130.6(2C), 128.6, 124.0 (2C),

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121.8, 119.0(4C), 117.8, 111.3, 60.9, 57.4; ESIMS: m/z 563 [M+H]+ HRESIMS: calc for C22 H19 N3 O3S Br2 [M+H]+ 563.9587, found 563.9550. Biology. The HT-F Homogeneous Inhibition Assay (Cat# 4690-096-K) was from Trevigen. The PARP-2 Chemiluminescent Assay Kit (Cat# 80552) was from BPS Bioscience. Antibodies against γ-H2AX and PAR were from Santa Cruz Biotechnology. Antibodies against Bcl-2, Bax, Caspase-3, Cyclin B1, CDK 1, LC3, Atg14 and Beclin 1 were from Cell Signaling Technology. The antibody against GAPDH was from Wanleibio. Goat anti-mouse/antirabbit IgG horseradish peroxidase antibodies were from the Proteintech Group. The Alexa Fluor 488/647 Antibodies were from Beyotime Biotechnology. The apoptosis assay kit and ROS assay kit were from Beyotime Biotechnology. Cell culture. All cell lines were purchased from Cell bank, Chinese Academy of Sciences (Shanghai, China). SK-OV-3 (human ovarian cancer skov-3 cell line) cells were cultured in McCOY's 5A, Bel-7402 (human hepatocellular carcinoma cell line) and HL-7702 (human hepatocyte cell line) cells were cultured in RPMI-1640, HepG2 (human hepatocellular carcinoma cell line) cells were cultured in DMEM, and Caco-2 (human colonic epithelial cell line) cells were cultured in MEM. Cells were cultured in a humidified environment with CO2 (5 %) at 37 ◦C. All the media were supplemented with 10 % FBS, 100 U/mL penicillin and 100 µg/mL streptomycin.

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Cytotoxicity assays. Cells were seeded in 96-well plates and exposed to the compounds for 48 h. The cytotoxicity was assessed by the MTT assay.49 The experiments were repeated three times. PARP-1 enzyme assay. The enzymatic assay was carried out using a highly sensitive fluorescence assay (HT-F Homogeneous Inhibition Assay; Trevigen, Cat# 4690-096-K) according to the manufacturer’s instructions..50 PARP-2 enzyme assay. PARP-2 enzyme activity was detected using the PARP-2 Chemiluminescent Assay Kit (BPS Bioscience, Catalog #: 80552) according to the manufacturer’s instructions.51 Immunofluorescence analyses of γ-H2AX. The levels of cellular γ-H2AX were detected using the immunofluorescence assay as previously described.37, 52, 53 Poly(ADP-ribose) (PAR) synthesis inhibition analyses. SK-OV-3 cells were harvested and seeded in 96-well plates. After 24 h of incubation, cells were treated with compound 11 for 4 h and then treated with 10 mM H2O2 for 5 min. After treatment, cells were fixed and incubated with primary antibody against PAR overnight at 4 ◦C. ADP-ribose polymers were visualized with Alexa Fluor 488-labeled second antibody. Nuclei were stained with 1 % DAPI, and images were captured using a confocal microscope. Flow cytometry assays for cell cycle and apoptosis analyses. For the cell cycle analyses, SKOV-3 cells were harvested and seeded in 6-well plates. After 24 h of incubation, cells were

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treated with compound 11 for 48 h. Cells were harvested and fixed. Then cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Bioinformatics analysis. In this study, we used TMT labeling and LC-MS/MS to quantify the dynamic changes in the whole proteome of the human species in SK-OV-3 (PTM-Biolabs (HangZhou) Co., Ltd). The general work flow is indicated below: (1) protein extraction; (2) trypsin digestion; (3) labeling; (4) HPLC fractionation; (5) LC-MS/MS analysis; (6) database search. The fold-change cutoff was set when proteins with quantitative ratios above 1.2 or below 1/1.2 were deemed significant.

In vivo anticancer activity experiments. 6-8-weed old female BALB/c nude (nu/nu) mice were obtained from the Model Animal Research Center of Nanjing University. Mice were housed in a specific pathogen-free room with controlled temperature (24 ± 2°C), humidity (60-80%) and lighting (12 h light/dark cycle), provided with sterile food and water. SK-OV-3 xenografts were established by inoculating 5×106 cells s.c. in the nude mice. When the tumor reached a volume of 80–120 mm3, mice were divided into four groups (n=6) and administered p.o. at doses of 50 mg/kg and 25 mg/kg (dissolved in 0.5 % CMC-Na); olaparib (25 mg/kg) served as a positive control. Tumor volumes were assessed by bilateral Vernier caliper measurement every three days and calculated according to the following equation: [tumor volume = X × (Y2/2)], where X represents the longer and Y represents the shorter of the two dimensions. Body weight was measured every three days, and clinical symptoms were observed daily. The animals were sacrificed at day 21, the tumors were removed, and

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the tumors were frozen in liquid nitrogen or fixed in 10 % neutral-buffered formalin. Western blotting, hematoxylin-eosin (H&E) and immunohistochemical staining were conducted to test related proteins. All experiments with mice were approved by Institute of Oceanology, Chinese Academy of Sciences Laboratory Animal Care and Ethics Committee (Qingdao, China) in accordance with the animal care and use guidelines. Electron Microscopy. SK-OV-3 cells were exposed to compound 11 (10 µM) for 48 h and then fixed, dehydrated, and embedded in resin. Ultrathin sections (200 nm) were collected on formvar-coated grids, counterstained with uranil acetate and lead citrate at room temperature and visualized with Transmission Electron Microscopy Facility (JEM-1200EX, Japan). Analysis of cell with GFP-LC3. SK-OV-3 cells were grown on glass coverslips and then infected with Ad-mCherry-GFP-LC3B for 24 h. After infection, cells were treated with compound 11 (10 µM) for 48 h. Then, cells were fixed with 4 % paraformaldehyde and examined under a fluorescence microscope. Western blotting analyses. SK-OV-3 cells were seeded in 6-well plates and treated with compound 11 for 48 h. Proteins were harvested and separated by 8-15 % SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked in blocking solution (containing 5 % nonfat milk) and subsequently probed with primary antibodies at 4 °C overnight. After 15-min washes in TBST, the membranes were incubated with a

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secondary antibody for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence system BeyoECL Plus (Beyotime, Nanjing, China).

In Vivo Pharmacokinetic (PK) Profile of compound 11. The pharmacokinetics analysis of compound 11 was conducted in male Sprague-Dawley rats. All experiments with animals were approved by Institute of Oceanology, Chinese Academy of Sciences Laboratory Animal Care and Ethics Committee (Qingdao, China) in accordance with the animal care and use guidelines. The rats were randomly assigned to two groups consisting of 4 males per group. The groups of rats were administered the agent orally and intravenously, respectively. The oral dose of compound 11 was 10 mg/kg, and the intravenous dose was 2 mg/kg. Briefly, blood samples were collected for plasma through the saphenous vein of the thigh. The compound concentrations in the plasma were determined using LC-MS-MS. The plasma compound 11 concentration-time data were analyzed using the noncompartmental method (Phoenix, version 1.3; Pharsight, Mountain View, CA) to derive the pharmacokinetic parameters. Noncompartmental analysis with Kinetica software (version 5.0; Thermo Scientific, Philadelphia, PA, USA) and pharmacokinetic parameters are shown as the means ± standard deviation. Safety of compound 11 in vivo. In the acute toxicity testing, twenty-four SPF Kunming mice (18 - 22 g), half males and half females, were randomly assigned to 4 groups consisting of 3 males and 3 females per group. The groups of mice were orally administered compound 11 at single doses of 0 (blank group, the same volume of physiological saline), 100 mg/kg, 500

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mg/kg and 1000 mg/kg. The mice were housed with free access to water and food in stainless cages in a room with a controlled temperature (25 ± 1 °C) and a 12-h light/dark cycle. Mouse survival and body weights were monitored and recorded up to 14 days post-treatment. In the subacute toxicity test, twenty SPF Kunming mice (18 - 22 g), half males and half females, were randomly assigned to 2 groups consisting of 5 males and 5 females per group. The treatment group was orally treated with 1000 mg/kg once a day for 14 days. After 14 days of treatment, the mice were anesthetized with isoflurane and killed by decapitation, and the tissues were collected for further analysis. All experiments with animals were approved by Institute of Oceanology, Chinese Academy of Sciences Laboratory Animal Care and Ethics Committee (Qingdao, China) in accordance with the animal care and use guidelines. Statistical analysis. Statistical analyses were performed using GraphPad Prism 5.0 (San Diego, CA, USA), and the data are presented as the mean ± SD. P < 0.05 was considered statistically significant. Statistical comparisons were performed using one-way analysis of variance. Molecular docking. The 3D structure of compound 11 was built using Chemdraw12.0 followed by MM2 energy minimization. The molecular docking study was performed using Auto-Dock Tools version 1.5.6 in combination with PyMol software. The cocrystal structures of BMN 673 with PARP-1 (PDB ID code: 4PJT) and olaparib with PARP-2 (PDB ID code: 4TVJ) were used as the docking model. The protein target was prepared for the molecular docking simulation by removing the water molecules and bound ligands. Hydrogen atoms and Kollman charges were added to each protein atom. The site sphere was defined based on

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the binding site of BMN 673 and olaparib in their crystal pose in 4PJT and 4TVJ, respectively. The grid map in Autodock that defined the interaction of protein and ligands in the binding pocket was defined. Each docking experiment was performed 200 times, yielding 200 docked conformations. All the other parameters used in the docking process were the default values of the system. The best model was picked based on the best stabilization energy. ■ ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge at the ACS Publications website at DOI: XXXX. S-tables, S-figures, 1H and 13C NMR spectra of compounds 1−36 and HPLC analysis of compounds 1-36 (PDF) Molecular formula strings (CSV) 4JPT−11 complex (PDB) 4TVJ−11 complex (PDB) ■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected](L.W.).

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*E-mail: [email protected] (D.S.). ORCID Li-jun Wang: 0000-0002-0462-7033 Author Contributions ∥C.G.

and L.W. contributed equally to this work.

Notes The authors declare no competing financial interests. ■ ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 81773586, 81703354, 81600782), Shandong Provincial Natural Science Foundation for Distinguished Young Scholars (JQ201722), Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC014), the Project of Discovery, Evaluation and Transformation of Active Natural Com-pounds, Strategic Biological Resources Service Network Program of Chinese Academy of Sciences (ZSTH-026), the National Program for Support of Top-notch Young Professionals, and the Taishan scholar Youth Project of Shandong province. ■ ABBREVIATIONS PARP-1, poly(ADP-ribose) polymerase-1; NAD+, nicotinamide adenosine dinucleotide; TLC, thin layer chromatography; SK-OV-3, human ovarian cancer skov-3 cell line; Caco2, Human

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Figure captions Figure 1. Structures of 3-AB, PARP inhibitors in clinical marketed drugs, selective PARP-1 inhibitors, thiosemicarbazone anticancer drugs and bromophenols Figure 2. Design of multifunctional bromophenol-thiosemicarbazone hybrids Figure 3. The cocrystal structures of BMN 673 with PARP-1 (PDB ID code: 4PJT) and olaparib with PARP-2 (PDB ID code: 4TVJ) were selected as the docking model. (A) Presumed interaction surface of 11 with PARP-1 (PDB code: 4PJT). (B) Model of 11 bound to PARP-1 (PDB code: 4PJT). (C) Presumed interaction surface of 11 with PARP-2 (PDB code: 4TVJ). (D) Model of 11 bound to PARP-2 (PDB code: 4TVJ). The surface of PARP-1/-2 is colored according to the atom color. The carbon atoms of 11 and selected key residues in PARP-1/-2 were colored in green and red, respectively. Hydrogen bonds are displayed as yellow dashed lines. Figure 4. Functional classification of the differentially quantified proteins. (A) GO-based enrichment analysis. (B) KEGG pathway-based enrichment analysis. Figure 5. Compound 11 inhibits PARP-1. (A) Immunofluorescence analysis of the changes in the formation of γ-H2AX foci in SK-OV-3 cells induced by compound 11. (B) PARP-1 inhibition of compound 11 measured by the PARP-1 enzyme assay. (C) Western Blotting analysis of the changes of the protein levels of γ-H2AX in SK-OV-3 cells induced by compound 11. (D) Compound 11 prevented H2O2-triggered formation of PAR in SK-OV-3 cells. (E) Western blotting analysis of PAR. (F) Western blotting analysis of MCM2-7. All data are expressed as the mean ± SD or representative images from 3 independent experiments. Figure 6. Compound 11 induces cell apoptosis and cell cycle arrest. (A, B) SK-OV-3 cells were treated with compound 11 for 48 h, cells were harvested and stained with Annexin V/PI and analyzed by FACS. (C) SK-OV-3 cells were treated with compound 11 for 48 h, and then apoptosis-related proteins, including Bcl-2, Bax and Caspase-3, were analyzed by western blotting. (D) SK-OV-3 cells were treated with compound 11 for 48 h, cells were harvested, fixed, stained with PI and analyzed by FACS. (E) SK-OV-3 cells were treated with compound 11 for 48 h, and then cell cycle-related proteins, including Cyclin B1 and CDK1, were analyzed by western blotting. Data are expressed as the mean ± SD (n=3). *P1000

>33.9

31

186

>1000

>5.37

35

58.3

>1000

>17.2

Olaparibd

2.96