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Discovery of Isaindigotone Derivatives as Novel Bloom's Syndrome Protein (BLM) Helicase Inhibitors that Disrupt the BLM/DNA Interactions and Regulate the Homologous Recombination Repair Qi-Kun Yin, Chen-Xi Wang, Yu-Qing Wang, Qian-Liang Guo, Zi-Lin Zhang, Tian-Miao Ou, ShiLiang Huang, Ding Li, Honggen Wang, Jia-Heng Tan, Shuo-Bin Chen, and Zhi-Shu Huang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00083 • Publication Date (Web): 02 Mar 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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Journal of Medicinal Chemistry

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Discovery of Isaindigotone Derivatives as Novel Bloom's Syndrome Protein (BLM) Helicase Inhibitors that Disrupt the BLM/DNA Interactions and Regulate the Homologous Recombination Repair

Qi-Kun Yin, Chen-Xi Wang, Yu-Qing Wang, Qian-Liang Guo, Zi-Lin Zhang, Tian-Miao Ou, Shi-Liang Huang, Ding Li, Hong-Gen Wang, Jia-Heng Tan, Shuo-Bin Chen* and Zhi-Shu Huang*

School of Pharmaceutical Sciences, Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, Sun Yat-sen University, Guangzhou 510006, China

* To whom correspondence should be addressed. *(S.-B. Chen) E-mail: [email protected]; *(Z.-S. Huang) E-mail: [email protected].

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Homologous recombination repair (HRR), a crucial approach in DNA damage repair, is an attractive target in cancer therapy and drug design. The Bloom syndrome protein (BLM) is a 3′-5′ DNA helicase that performs an important role in HRR regulation. However, limited studies about BLM inhibitors and their biological effects have been reported. Here, we identified a class of isaindigotone derivatives as novel BLM inhibitors by synthesis, screening and evaluating. Among them, compound 29 was found as an effective BLM inhibitor with a high binding affinity and good inhibitory effect on BLM. Cellular evaluation indicated that 29 effectively disrupted the recruitment of BLM at DNA double-strand break (DSB) site, promoted an accumulation of RAD51 and regulated the HRR process. Meanwhile, 29 significantly induced DNA damage response, as well as apoptosis and proliferation arrest in cancer cells. Our finding provides a potential anti-cancer strategy based on interfering with BLM via small molecules.

KEYWORDS isaindigotone derivatives; BLM helicase inhibitor; homologous recombination repair; DNA damage; proliferation inhibition


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INTRODUCTION DNA damage is generally caused by normal biological events and environmental factors, such as pollution and radiation. A single human cell experiences more than ten thousand DNA molecular lesions per day, such as DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), nucleotide mutations and other lesions.1 In response to such DNA damage, a series of DNA damage repair pathways are involved in to ensure genomic stability and prevent genomic diseases, and such pathways include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous DNA end joining (NHEJ) and HRR.2-4 Compared with normal cells, cancer cells harbor prevalent mutations and deficiencies in repair pathways, and in some cases, they even activate harmful repair pathways to drive tumor progression.5, 6 The replication stress and oxidative damage make repair systems more fragile and impressionable in cancer cells. Therefore, cancer cells are more reliant on certain repair pathways and sensitive to their specific inhibitors.7 These differences make intervening the process of the DNA damage repair pathways via small molecules an effective anti-cancer strategy. Impressively, several inhibitors aimed at repair pathways have already entered the clinical trial stage.8 HRR is performed by various DNA damage repair proteins, such as MRN protein complex, RPA, BRCA1, RAD51, BLM and so on.5 This repair is a primary means for the damaged DSBs, which represent the most serious damage in eukaryotes. In HRR pathway, a corresponding DNA sequence from an undamaged sister chromatid is used as a template. Due to its accurate and integral repair, HRR plays an essential role in genome stability.9, 10 And dysregulation of HRR usually causes repair dysfunctions, harmful genome rearrangements and loss of heterozygosity (LOH).7, 11, 12 Synthetic lethality originally means a genetic interaction between two individual viable mutations, resulting in a lethal phenotype.13 Because of the common mutations and abnormalities of HRR pathways in cancer cells, Hartwell and colleagues proposed the application of synthetic lethality in anti-cancer therapy.14 To expand the spectrum and enhance the therapeutic effect of anti-cancer drugs, combination therapy with drugs which target different DNA damage repair pathways has become a hot and potential field in research. For instance, RI-1 (RAD51 inhibitor) sensitizes cancer cells to chemotherapy drugs, and NSC19630 (WRN helicase inhibitor)



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increases cellular sensitivity to DNA damage agents.15, 16 Therefore, targeting repair-related proteins in HRR process is considered as an alternative anti-cancer therapeutic approach in combination therapy, and the development of inhibitors that target novel proteins in the core HRR pathways becomes necessary and urgent.17, 18 BLM, a 3′-5′ ATP-driven DNA helicase, can unwind multiple nucleic acid structures including duplexes, holliday junctions, G-quadruplexes, and DNA displacement loops (D-loops).19, 20 As a member of the highly conserved RecQ helicase family (RECQ1, BLM, WRN, RECQ4, RECQ5), BLM is highly expressed in cancer cells and considered to play a significant role in recombination.21,

22

Studies on the biological functions in cells present a detailed

characterization of BLM in each stage of the HRR process, for example, BLM displays a broad spectrum of activities that either negatively or positively regulate HRR: (i) it can facilitate the dissociation of the branch migration of holliday junctions and the unwinding of intermediates during DNA synthesis to promote HRR,23 and (ii) it can prevent recombinant related protein RAD51 binding to DNA strands to restrain the progress of repair.24 In addition, recent research showed that the deficiency of BLM induced a proliferation arrest and damage susceptibility in cancer cells, meanwhile BLM had a potential synthetic lethal effect with other repair-related proteins.25, 26 In brief, BLM is an attractive protein that has potential to be developed as an anti-cancer target. However, only ML216 is reported as a BLM inhibitor, and the pharmacology study of this inhibitor on cancer cells is still not clear.27 Thus, it is necessary and urgent to develop novel BLM inhibitors and explore the potential anticancer strategy by targeting BLM. To identify novel BLM inhibitors, we firstly performed a screening in our in-house small molecule library and found that isaindigotone derivatives showed inhibitory effects on BLM. Interestingly, we had found several isaindigotone compounds could effectively disrupt the protein/DNA interactions via specific binding on the DNA binding pocket of NM23-H2, a transcriptional factor that can activate c-MYC transcription by recognizing the Gquadruplex in the promoter of the gene, in the previous research.28 On this basis, we conducted an isaindigotone derivatives library containing 20 newly synthetic compounds (5 ~ 24, Table 1) and 35 reported compounds (Table S1) (Figure 1). In these new compounds, some closed-ring basic terminal groups, including N-methyl-piperazinyl, 4-fluorophenethyl, or 4-(pyridin-2-yl)-piperazinyl groups, were introduced at the 6-position, meanwhile the types


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of substituents at 4′-position were further expanded (such as isopropyl, isobutyl, and tertiary butyl groups) or the styryl portion was converted into more complicated aromatic structures (such as carbazolyl and benzofuranyl groups). Various experiments were carried out to make systematic evaluation for BLM inhibitors. Prominent compound 29 was then selected as the potential BLM inhibitor for further in-depth studies. Impressively, compound 29 exhibited remarkable activities on disrupting BLM from DSB sites, regulating HRR process and inducing DNA damage response. Furthermore, this compound could also induce cell apoptosis and inhibit cell proliferation in human colon cancer HCT116 cells. These results revealed that intervening repair factors via chemical molecules to affect important damage repair pathways might be an effective anti-cancer strategy.

Figure 1. The design and construction of isaindigotone derivatives library

RESULTS AND DISCUSSION Chemistry. The synthetic pathway for the isaindigotone derivatives was depicted in Scheme 1. According to our previous reports, the intermediate 2 was attained by 2-amino-4, 5-difluorobenzoic acid heated with pyrrolidin2-one in the presence of POCl3.28 Next the preparation of the key intermediates 3a-3h began with N-nucleophiles substituting the 6-position fluorine atom of intermediate 2, and meanwhile treatment of terephthalaldehyde or isophthalaldehyde with different substituted acetophenones produced the intermediates 4a-4d (Scheme S1). Finally, intermediates 3a-3h reacted with various aldehydes via the Knoevenagel reaction and provided the target compounds 5−24.



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Scheme 1. Synthesis route of isaindigotone derivatives. Reagents and conditions: (a) pyrrolidin-2-one, POCl3, reflux, 8 h; (b) R1NH2, 100 °C, 12 h; (c) aldehydes, DMF, TMSCl, 100 °C, 12−48 h. Table 1. Structures of the newly synthesized isaindigotone derivatives (5−24) Compd.

NR1

5

N

6

N

7

N

8

N

R2

R3

R4

R5

Compd.

N H

H

H

C(CH3)3

H

14

N H

H

H

CH2CH(CH3)2

H

15

N H

H

H

H

H

16

N H

H

H

F

H

17

NR1 N

N H

R2

R3

R4

R5

H

H

CH2CH(CH3)2

H

H

H

CH(CH3)2

H

H

H

C(CH3)3

H

H

H

CH(CH3)2

H

H

H

F N H

N N

N N N

O

9

N

N H

H

H

H

H

18

N O

N H

H

N O

O

10

N

N H

H

H

N(CH2CH3)2

H

19

N O

N H

H

H

H F

O

11

N

N H

OH

H

OCH2Ph

H

20

N O

N H

H

O

H

H

H

H

O

O

12

N

N H

H

H

CH(CH3)2

H

21

N O

N H

H

N O

13

N

N H

H

H

C(CH3)3

H


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O F N

O F

N

N H

N

22

O

N O

N H

O F

N N

N N

23

O

N H

N N

O

24

Screening and Evaluation of BLM Helicase Inhibitors. To screen BLM inhibitors, DNA helicase unwinding assay was performed to determinate the inhibitory effects of compounds on BLM unwinding DNA. We purified a truncated structure BLM that retained helicase activity in vitro and selected a duplex-forked DNA as a substrate.29 Meanwhile, the reported inhibitor, ML216, was used as a reference compound during the screening. 55 isaindigotone derivatives containing 20 newly synthetic compounds (Table 1) and 35 reported compounds (Table S1) were conducted as an isaindigotone compounds library. The library was then simultaneously evaluated following the process below (Figure 2A). The screening was performed at decreased concentrations (50, 10 and 1.0 μM) of compounds for three steps. First, we evaluated the inhibitory effects of 55 isaindigotone derivatives at the concentration of 50 μM (Figure S1). Twenty-nine candidate compounds that exhibited over 80% inhibitory effects on helicase unwinding were then selected for second-round screening treated with 10 μM of the compounds (Figure S1). The results showed that there were 12 isaindigotone derivatives that exhibited more than 80% inhibitory effects at 10 μM. Next, these 12 compounds were further evaluated at the concentration of 1.0 μM. As shown in Figure 2B, compound 29 showed a best inhibitory effect on BLM helicase among these compounds, and 3 compounds (5, 6, and 26) also showed better inhibitory effects than ML216. During the screening, some information about the structure-activity relationship was obtained and described below. Firstly, the size and type of the amine side chains (R1) at 6-position obviously impacted the inhibitory effects on BLM helicase. For example, the introduction of open-ring basic terminal groups, such as a diethylamino group (29), showed a remarkable improvement in the inhibitory effect of BLM compared with the corresponding closedring basic terminal groups, such as piperidinyl and 4-fluorophenethyl groups (12 and 15). Secondly, the role of a single substituent (R4) at the 4′-position was also investigated. 29, 5 and 6, which presented simple alkyl substituents (4′-isopropyl, 4′-tert-butyl and 4′-isobutyl group, respectively), exhibited better inhibitory effects than 27 and 28, which presented sterically hindered groups (4′-morpholinyl and 4′-piperidinyl group, respectively). Then, we



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quantified the inhibitory effect of 29 on BLM helicase. The IC50 value of 29 was 0.95 μM. Meanwhile, the IC50 value of ML216 was 2.15 μM, which was consistent with previous report (Figure S2).27 In conclusion, we found that compound 29 had a significantly inhibitory effect on BLM unwinding, stronger than reported inhibitor ML216. BLM was a critical repair factor in the HHR pathway, and the deficiency of BLM could induce DNA damage response and potential toxicity in cancer cells (Figure S3).30 We then determined the cytotoxicity and γ-H2AX expression (a DNA damage marker) on HCT116 cells upon treatment of 12 candidate compounds to evaluate their anti-cancer activities. The methyl thiazolyl tetrazolium (MTT) assay results showed most compounds had good cytotoxicity (IC50 < 10 μM) for HCT116 (Table S2). The cytotoxicity of compounds was also relative to their BLM inhibition activity. Among them, 29 induced remarkable increase of γ-H2AX level in cells, suggested that 29 was more likely to act as a BLM inhibitor in cells (Figure 2C). In conclusion, we selected 29 as a hit compound from the library for further study.

Figure 2. Identification of BLM inhibitors. (A) Diagram of BLM inhibitors identification and structure of hit compound 29. (B) The inhibitory effects on BLM unwinding at the concentration of 1.0 μM for 12 candidate compounds. (C) The expression level of γ-H2AX with the treatment of 12 candidate compounds in HCT116 cells for 24 h, the concentration of each compound used in the treatment was half of IC50 value in MTT assay, β-actin was used as a control.

Disrupting Effect of Compound 29 on the BLM/DNA Interaction. Besides unwinding DNA structures, BLM could play roles in HRR through recruiting to DSB sites.31 To investigate disruption of compound on the binding of BLM with DNA, we first evaluated the binding between BLM and DNA by ELISA and filter-


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binding assay. In ELISA assay, biotin-labeled DNA was immobilized on a plate and incubated with purified BLM upon treatment of compounds. The unbound BLM was removed after rinsing, and the BLM-bound DNA was quantified using BLM-antibody with horse radish peroxidase (HRP). In filter-binding assay, the BLM/DNA complex was separated using vacuum filtration. The BLM-bound DNA would retain in the upper membrane, and the lower membrane would hold the rest. The unbound DNA was quantified by counting the gray level of the dots in the lower membranes. Firstly, the EC50 value of BLM to DNA was determined. As shown in Figure S4, EC50 values were determined as 105.0 and 133.8 nM in ELISA and filter-binding assay, respectively. We then evaluated the potential disrupting effect of 29 on BLM/DNA interaction by ELISA and filter-binding assays. Upon treatment of 29, the percentage of unbound DNA in both assays was increased in a dose-dependent manner, and the IC50 value in ELISA was 3.95 μM, indicated 29 could effectively disrupt BLM binding to DNA (Figure 3A, 3B and Figure S5A). In addition, 29 showed a stronger ability than ML216 in disruption of BLM/DNA interaction. Afterwards, pull-down experiment was performed to investigate the disrupting effect of 29 on cellular BLM. Biotin-labeled DNA was immobilized on the magnetic beads, co-incubated with 29 and cell lysis solution for 1 h. Then, the quantity of BLM in both eluent and concentrate was detected using western blot. As shown in Figure 3C, 29 could disrupt binding of cellular BLM to DNA with efficiency up to 84.3% at the concentration of 1.0 μM (Figure S5B). In brief, the BLM inhibitor 29 could not only inhibit the unwinding activity of BLM but also disrupt the BLM binding to DNA.

Figure 3. The disrupting effect of 29 on the interaction of BLM and DNA. (A) Filter-binding dots of BLM with DNA upon addition of 29. (B) Dose-dependent disrupting of BLM/DNA binding upon addition of 29 and ML216 using ELISA assay, the IC50 value was fitted by Hill model. (C) The disrupting effect of 29 on cellular BLM binding to DNA determined by pull-


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down assay.

Regulation of Homologous Recombination by Compound 29 via Disrupting the Recruitment of BLM to DSB Sites. We then investigated the possible relation between cellular effect and BLM inhibition of 29. BLM is considered as a regulatory factor involved in HRR pathway, therefore we used HRR reporter assay to evaluate the possible effect of 29 on HRR level.32 The HRR reporter consists of two mutant green fluorescent protein (GFP) genes, SceGFP and iGFP. The SceGFP is disrupted by an 18-bp restriction site for ISceI endonuclease, and the iGFP is truncated at both 5′ and 3′ ends (Figure 4A). In the assay, a DSB site would firstly be generated by restrict cleavage of I-SceI nuclease on SceGFP. Secondly, the SceGFP could be repaired though HRR pathway with the iGFP gene as a homologous template and turned into a functional GFP gene. Thus, a percentage of GFP expressed cells could represent the level of HRR. As shown in Figure 4B, the level of HRR was upregulated in a 29-dependent manner and increased up to 3-fold at the concentration of 1.0 μM, indicating that 29 could improve the level of HRR in cells. ML216 also induced a similar effect, but the effective concentration (50μM) was much higher than 29.27 To obtain more details about the connection between cellular activity of 29 and BLM inhibition, we detected the HRR level in siBLM HCT116 cells which the BLM expression level was decreased by small interfering RNA (siRNA), and the efficiency of siRNA was shown in Figure S3. The results showed that the decrease of BLM could significantly upregulate the level of HRR (Figure 4C). This finding was consistent with the previous report that the hyper recombination level was prevalent in BLM deficiency cells.33 Then, we noticed that the HRR level in siBLM HCT116 cells was not altered upon treatment of 29. This result suggested that 29 regulated the level of HRR probably due to the inhibition of BLM. During the HRR process, many repair-related proteins including BLM are recruited to the DSB sites to perform repair functions.34 Because 29 could disrupt the interaction of BLM and DNA in vitro, it might also be able to disrupt BLM recruiting to the DSB sites in cells. To verify this hypothesis, the HRR reporter with DSB sites was used as DSB model to evaluate the recruitment of proteins. To validate the DSB model, the cleavage efficiency was quantified by fluorogenic quantitative PCR (qPCR) assay, and activation of damage response on DSB sites was


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confirmed by the expression level of γ-H2AX. The results showed that the cleavage efficiency reached approximately 60% and effective damage response (γ-H2AX) was observed after the cleavage (Figure S6A and Figure S6B). Based on the DSB model on HRR (-) reporter, chromatin immunoprecipitation (ChIP) assay was applied to evaluate the potential disrupting of 29 toward BLM on the DSB sites. The binding effects of BLM to DNA in different ranges (-100 to +2000 bp) around the DSB sites were shown in Figure 4D. BLM was recruited significantly near the DSB sites (in DNA fragments that -92 bp and +559 bp from the DSB sites). In addition, the recruitment of BLM on DSB sites was decreased significantly upon treatment of 29, but not treatment of ML216.35 These results revealed that 29 might disrupt the interaction of BLM and the plasmid reporter in cells more effective than the reported BLM inhibitor.

Figure 4. The HR regulation mediated by 29 via disrupting the interaction of BLM and DNA in cells. (A) Scheme of HRR and DSB model. (B) The relative HRR level in HCT116 cells after treated with 29 and ML216. (C) The relative HRR level in HCT116 and siBLM HCT116 cells after treated with 29. The HRR level was quantified by the percentage of GFP expressed cells and normalized to untreated samples. (D) The recruitment of BLM to the indicated DSB sites on HRR (-) reporters in 29treated and ML216-treated HCT116 cells. (E) The recruitment of BLM to the indicated DSB sites on chromosomes in 29treated HCT116 cells. BLM ChIP efficiency were determined by qPCR using specific primers and normalized to untreated


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samples. The data in experiment were made in triplicate and expressed as the mean ± SD: (*) P < 0.05, (**) P < 0.01 and (***) P < 0.001 compared to untreated groups.

In addition, I-SceI was also applied to cleavage DSB sites on chromosomes and generated chromosomic DSBs.36 Upon treatment of I-SceI alone, the expression level of γ-H2AX increased significantly, suggested I-SceI successfully induced DSBs on chromosomes (Figure S6C). To further evaluate the disrupting effect of 29, the ISceI induced DSB sites were figured out, and the binding effects of BLM to the I-SceI induced DSB sites were determined (Figure 4E and Figure S6D). Similar to the plasmid reporter, BLM was also recruited significantly near the DSB sites on chromosomes, and addition of 29 could effectively disrupt the recruitment. The data from these experiments indicated that 29 significantly disrupted the interaction of BLM and DNA near the DSBs sites both on the plasmid reporter and chromosomes. Hence, the transcription and translation levels of BLM were both upregulated upon treatment of 29, suggested that the decreased recruitment of BLM was not associated with its expression level but with the action of 29 (Figure S7). In brief, compound 29 could disrupt the interaction of BLM and DNA in cells.

Accumulation of RAD51 at DSB Sites Induced by Compound 29. RAD51, a single-strand DNA (ssDNA) binding protein, plays a crucial role in HRR process. During the repair, RAD51 binds to ssDNA and form a nucleoprotein filament to promote the progress of repair.5 Recent research suggested that BLM could bind to ssDNA and prevent the formation of the RAD51-ssDNA filaments in vitro.23 This evidence indicated us to consider whether 29 affect the behavior of RAD51 by inhibiting BLM. Firstly, RAD51 was purchased and its DNA binding ability was detected as shown in Figure S8A, the data revealed that RAD51 had a high binding ability to DNA substrate (EC50 = 108 nM). Secondly, DNA exchange activity assay was performed. As shown in Figure 5A, the quantities of RAD51-ssDNA filaments decreased in the presence of BLM, but this decrease was recovered with the increasing concentration of 29. These results indicated 29 could promote RAD51 binding to the ssDNA in vitro. Furthermore, ELISA assay was applied to discovery this promotion of RAD51 induced by 29. Interestingly, the


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consequence revealed that 29 could not facilitate RAD51 binding to ssDNA in vitro (Figure S8B). In this case, the promotion of RAD51 binding to DNA in vitro might be derived from the disruption of the BLM/DNA interaction caused by 29. Based on the DSB model, ChIP assay was performed to verify whether 29 could promote RAD51 binding to the DSB sites at the cellular level. As shown in Figure 5B, RAD51 was signiﬁcantly recruited near the DSB sites in siBLM HCT116 cells, indicating that the deficiency of BLM could promote RAD51 recruiting to the DSB sites. In addition, an increasing recruitment of RAD51 was also observed in a 29-dependent manner around the DSB sites on the plasmid reporter. Furthermore, similar results appeared around the DSB sites on chromosomes (Figure 5C). Based on the above results, 29 could significantly alter the behavior of RAD51 and promote RAD51 binding to DNA chains around the DSBs sites both on the plasmid reporter and chromosomes.

Figure 5. The accumulation of RAD51 at the DSB sites mediated by 29. (A) 29 reversed the decrease of RAD51-ssDNA filaments induced by BLM in vitro. (B) The recruitment of RAD51 to the indicated DSB sites on HRR (-) reporters in 29treated and siBLM HCT116 cells, same as Figure 4D. (C) The recruitment of RAD51 to the indicated DSB sites on chromosomes in 29-treated HCT116 cells, same as Figure 4E. (D) The distribution and co-localization of RAD51 and γ-H2AX in HCT116 cells treated with 29 for 24 h using immunofluorescence assay.



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Since 29 induced an increase in the level of γ-H2AX in cells, and the behavior of RAD51 is related to DNA damage response and repair as reported.37 Then, we verified whether the occurrence of damage was related to the inhibition of BLM by 29. Immunofluorescence assay was performed to detect the distribution and localization of RAD51 and DNA damages (DSBs were represented by γ-H2AX foci in cells), and then we observed an increasing RAD51 foci (green) accumulated on the γ-H2AX foci (red) in 29-treated cells (Figure 5D), suggested that 29 could induce DNA damage response and this damage might be associated with RAD51. Furthermore, the expression level of RAD51 was not significantly changed under the treatment with 29, indicating that it was probably behavior not expression of RAD51 affecting this process (Figure S8C). Taken together, the results revealed that 29 could induce DNA damage and promote the accumulation of RAD51 to the damage sites in cells, and this process might be related with the disrupting effect of 29 on BLM.

DNA Damage Response and Apoptosis Induced by Compound 29 in Cells. Because compound 29 have a significant impact on the DNA damage, then we evaluated its effect on the DNA damage pathway.38 As shown in Figure 6A, the level of major damage sensors in damage response, such as p-ATM and pATR, was obviously upregulated with the treatment of 29. Afterwards, the expression of other damage-related proteins in ATM-CHK2-p53 and ATR-CHK1-p53 pathways was remarkably upregulated as the concentration of 29 increased. These results demonstrated that 29 could activate two DNA damage response pathways. Since the deficiency of BLM in Prostate cancer (PCa) cells from patients could induce apoptosis, which suggested us that 29 might also induce a cellular apoptosis.39 As shown in Figure 6B, we found that the ratio of apoptosis cells (staining by Hoechst and PI) increased from 1.09% to 71.59% in a concentration-dependent fashion of 29 (from 0 to 2.0 μM). Further, the expression level of cleaved caspase-3, cleaved caspase-7 and cleaved PARP was clearly upregulated in 29-treated cells (Figure 6C). Taken together, the BLM inhibitor 29 could significant active DNA damage pathways and induce a p53-dependent apoptosis (Figure 6D). The mechanism of 29 was also consistent with appearances in BLM-deficient cells, suggested that the damage and apoptosis might be induced by the suppression of BLM via 29.


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Figure 6. DNA damage response and apoptosis in HCT116 cells induced by 29. (A) The expression level of major signaling proteins in response to DNA damage in HCT116 cells treated with 29 for 24 h, β-actin was used as a control. (B) An apoptosis evaluation of HCT116 cells treated with 29 for 48 h. (C) The expression level of apoptosis pathway-related proteins in HCT116 cells treated with 29 for 48 h, β-actin was used as a control. (D) An action pathway diagram of 29 in HCT116cells.

Evaluation of Proliferation Arrest, and Combination Treatment by Compound 29 in Cells. Since the deficiency of BLM induced a proliferation arrest in cancer cells, we then verified whether 29 had the similar anti-proliferation effect.39 Firstly, we used a real-time cellular analysis (RTCA) to investigate the impact of BLM on cellular proliferation. The result showed an obvious proliferative inhibition in HCT116 cells but not in human fibroblast BJ cells treated with siBLM for 48 h, indicating that the absence of BLM inhibited the proliferation of cancer cells (Figure S9). In this case, we evaluated the cytotoxicity of 29 in different cells and found 29 showed a stronger anti-proliferative effect in cancer cells (Table S3). Then, the expression level of BLM in different cells was detected using western blot, and the results showed that the cytotoxicity of 29 increased in a BLM dependent fashion (Figure S10). To gain more details evidences, we further evaluated the cytotoxicity of 29 in siBLM HCT116 cells. As shown in Figure 7A, a significant proliferation arrest appeared in a dose-dependent manner of 29 in HCT116 cells, however the growth of siBLM HCT116 cells was not inhibited with the same treatment of 29 (Figure 7B). Meanwhile, a long-term proliferation assay was performed and shown the similar results, indicating that the arrest of cell proliferation induced by 29 might due to the inhibition of BLM (Figure 7C and Figure 7D).



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Figure 7. Proliferation arrest and synthetic interaction induced by 29. (A, B) The proliferative ability in HCT116 and siBLM HCT116 cells treated with 29 for 48 h. (C, D) The proliferative ability in HCT116 and siBLM HCT116 cells treated with 29 in a long-term proliferation assay. (E, F) The anti-proliferative effect of 29 in combined therapy with a RAD51 inhibitor (RI1) and a DNA damage agent (Cisplatin) in HCT116 cells. The data were derived from three experiments and expressed as the mean ± SEM: (*) P < 0.05, (**) P < 0.01 and (***) P < 0.001 compared with untreated groups.

In the previous research, Mao proposed that the decrease of BLM enhanced the chemotherapy sensitivity of cancer cells.25 In this case, we selected Cisplatin (a traditional DNA cross-linking agent) for the combination treatment.40 As shown in Figure 7E, the results showed that HCT116 cells became more sensitive to Cisplatin in the presence of 29 (1.25 μM), and the anti-proliferative effect of Cisplatin (100 μM) increased from 15.4% to 71.2%. In addition, 29 induced an obvious DNA damage, and the potential synergistic lethal effect of 29 is of


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interest. Recent research indicated that BLM deficiency cells increased cell death associated with RAD51 deficiency.26 Herein, RI-1, a RAD51 inhibitor, was applied to evaluate the anti-proliferative effect in the synthetic interaction.15 As shown in Figure 7F, a strong anti-proliferative effect emerged under the combined administration of 29 and RI-1, such as the proliferative effect of RI-1 (50 μM) in the combined medicine group increased up to 4fold higher than the simple medicine group. Furthermore, the anti-proliferative effects of RI-1 and Cisplatin were also enhanced by siBLM treatment in HCT116 cells, suggested these sensitization effects in cancer cells were related to the decrease of BLM (Figure S11A and Figure S11B). Then, 29 was added, and there were not significant differences between combined and simple medicine groups yet, indicating that the enhanced anti-proliferative effects by 29 was due to the inhibition of BLM (Figure S11C and Figure S11D). In brief, compound 29 could cause proliferation arrest, sensitize cancer cells to chemotherapeutic agents and damage repair inhibitors.

Binding of Compound 29 to BLM Protein. Previous research showed that 29 effectively inhibited the activity of BLM and displayed great cellular activity. To further illuminate the interaction mode between 29 and BLM, binding studies were performed. First, an isothermal titration microcalorimeter assay (ITC) was applied to detect the binding affinity of 29 to BLM. The disassociated constant (KD) value of compound 29 to BLM was determined as 1.81 μM, suggested that 29 perfectly bound to BLM (Figure 8A).



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Figure 8. The binding mode of 29 to BLM. (A) An ITC curve and fitted affinity of 29 to wild-type BLM, the top panel showed the heat change upon ligand titration, the bottom panel showed the integrated data and ITC isotherm (solid line) fitted by a single-site binding model. (B) Predicted binding mode of 29 with BLM (PDB code 4CGZ), the diagram showed the interaction between ligand 29 (cyan) and predicted residues (green). (C) Binding affinity of 29 to wild-type and mutant BLM using ITC assay. “N.D.” in the table meant the KD value was not obtained by fitting.

The active core of BLM consists of three functional domains: helicase core domain capable of ATP hydrolysis, RecQ C-terminal domain (RQC) for DNA binding, and HRDC domain with unwinding activity.41 To further evaluate the binding sites, we performed a molecular docking simulation of 29 with all these domains in BLM based on the reported X-ray structure (PDB code 4CGZ) using Schrodinger software.42 As shown in Figure S12, 29 preferred to bind to the 3′-tailedduplex DNA binding pocket of the RQC domain. To figure out the reliable binding geometry of 29 with BLM, molecular dynamics studies were then carried out.43 The top-ranked predict binding mode in MD simulation was shown in Figure 8B. In this binding mode, 29 had π-π stacking interactions with Tyr995 and His996, hydrophobic interactions with Met1111 and Ile1168,44, 45 as well as hydrogen-bonding interactions with Glu1143, implying that these amino acids might be the crucial residues in 29 binding to BLM (Figure 8B and Table S4 and Figure S12). In addition, slight conformation changes of the DNA binding pocket were observed in the presence of 29 in the MD simulations (Table S5 and Figure S13). To further confirm this binding mode, the binding affinity of compound 29 with five single-point amino acid mutants (Y995A, H996A, M1111A, E1143A, I1168A) was determined by ITC. As shown in Figure 8C and Figure S14, the binding constants of 29 for mutants were all beyond that of 29 with the wild-type BLM, indicating that these five amino acids were involved in the binding of 29 with BLM. In addition, His996 seemed most important for the binding because no binding between 29 and BLM occurred when it was mutated. Above all, this result was consistent with our predicted binding mode in which 29 bound to the pocket along the DNA binding pocket in RQC domain. In addition, the binding mode explained the quinazolinone portion of 29 had a dominant role in binding to BLM, meanwhile the amine side chains and styryl portion also had partial effects, and these findings roughly correspond to the results of the SAR analysis in preliminary screening. Moreover, the ELISA results (Figure S15) showed that the disrupting effect of 29


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significantly decreased in the five mutants compared with wild-type protein, further indicating that these residues were vital for the interaction between 29 and BLM. Additionally, no binding of 29 to DNA was observed using ITC assay (Figure S16). Furthermore, the ATP hydrolysis activity of BLM was evaluated upon treatment of 29, and the result suggested that 29 had no effect on BLM using ATP in vitro (Figure S17). Taken together, 29 might competitively bind to the DNA binding pocket in RQC domain of BLM, disrupt BLM/DNA interaction and inhibit the DNA unwinding activity of BLM.

CONCLUSION In this study, we screened our in-house library and firstly found that isaindigotone derivatives had inhibitory effects of BLM. On this basis, we newly synthesized and screened a series of derivatives to explore an optimized compound and discovery the influence of structural diversity on the inhibitory effects of BLM. The results from SAR analysis indicated that long amine side chains (R1) at 6-position and simple alkyl substituents (R4) at 4-position might be the important structural units. After screening, we identified compound 29 possessed the best inhibitory effect on BLM for further study. A panel of in vitro assays, including ELISA, filter-binding assay, pull-down assay were performed to support that 29 could disrupt the interaction of BLM and DNA in vitro. In cells, subsequent biological assays, including HRR reporter assay, ChIP, western blot and immunofluorescence assay, were applied and confirmed that 29 altered the level of HRR, disrupted the interaction of BLM/DNA, induced DNA damage and promoted the accumulation of RAD51 to the DSB sites. At present, BLM and RAD51 are considered to play crucial roles in repair of DSBs in cells. In this case, the hyper-recombination caused by 29 via inhibiting BLM might break the balance of repair and induce DNA damage response. In addition, 29 activated the DNA damage response pathways, induced cellular apoptosis, caused proliferation arrest and enhanced the sensitizing effects of RI-1 and Cisplatin in cancer cells. Compared with the reported inhibitor ML216, compound 29 showed a lower effective concentration in inducing DNA damage and stronger cell cytotoxicity to cancer cells. As both compounds could uptake into cells with high



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efficiency (Figure S18), the better disruption of 29 on BLM/DNA interaction both in vitro and in cells might be the reason of its better inhibitory effect on cancer cells. To further elucidate the interaction between 29 and BLM, we performed ITC study and demonstrated that 29 had a high binding affinity to BLM. Molecular docking suggested 29 was suitable to the DNA binding pocket in RQC domain of BLM. The binding mode was confirmed by ITC studies of 29 with five single-point mutants with predict binding amino acids (Y995, H996, M1111, E1143 and I1168A). This binding mode might be valuable to design more effective BLM inhibitors in the feature. In summary, we identified a novel BLM inhibitor, isaindigotone derivative 29, and firstly determined fairly complete interaction studies and pharmacological evaluations of present BLM inhibitor. The different cellular effect of 29 and ML216 suggested that the disrupting ability of compounds on BLM/DNA interaction might be more important in impacting BLM functions in cells. Based on this, the identification of 29 provides an important tool in the research of BLM functions, and detailed evaluation also gives a new perspective on the development of BLM in anti-cancer treatment.

EXPERIMENTAL SECTION Chemical Synthesis. All chemicals were purchased from commercial sources unless otherwise specified. All the solvents were of analytical reagent grade and were used without further purification. 1H and 13C NMR spectra were recorded using tetramethylsilane (TMS) as the internal standardin DMSO-d6 or CDCl3 with a Bruker BioSpin GmbH spectrometer at 400 and 100 MHz, respectively. Chemical shifts are expressed in parts per million downfield from tetramethylsilane as an internal standard and coupling constants in hertz. Mass spectra (MS) were recorded on a Shimadzu LCMS-2010A instrument with an ESI or ACPI mass selective detector and high resolution mass spectra (HRMS) on Shimadzu LCMSITTOF. Flash column chromatography was performed with silica gel (200−300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The purities of synthesized compounds were confirmed to be higher than 95% by analytical HPLC performed with a dual pump Shimadzu LC-20AB system equipped with a Ultimate XB-C18 column (4.6 mm × 250 mm, 5 μm) and eluted with methanol/water (15:85) containing 0.1% TFA


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at a flow rate of 0.5 mL/min. Procedure for the Synthesis of Intermediate 6,7-Difluoro-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (2). 45 mL of POCl3 was carefully added to a solution of 2-amino-4,5-difluorobezoic acid (1 equiv.) and pyrrolidin-2-one (2 equiv.) at room temperature and then stirred at 120 °C for 4 h and monitored by TLC. The crude product was adsorbed on silica and purified by flash chromatography. Yield: 3.9 g, 64%, white solid. 1H NMR (400 MHz, CDCl3) δ 8.02 (t, J = 9.2 Hz, 1H), 7.49-7.34 (m, 1H), 4.20 (t, J = 7.1 Hz, 2H), 3.17 (t, J = 7.8 Hz, 2H), 2.37-2.23 (m, 2H). General Procedure for the Synthesis of Intermediates 3a-3h. The mixture of intermediate 2 (1 equiv.) and aliphatic amines (2 equiv.) stirring in a 38 mL sealed tube was heated at 100 °C for 12 h and monitored by TLC. After cooling to room temperature, the crude product was adsorbed on silica and purified by flash chromatography. 6-((3-(diethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (3a). According to the general procedure above, the intermediate 3a was obtained. Yield: 3.5 g, 74%, pale yellow solid. 1H

NMR (400 MHz, CDCl3) δ 7.72 (d, J = 11.7 Hz, 1H), 6.72 (s, 1H), 6.67 (d, J = 7.7 Hz, 1H), 4.19−4.12 (m, 2H),

3.31 (dd, J = 10.6, 5.8 Hz, 2H), 3.11 (t, J = 7.9 Hz, 2H), 2.63−2.59 (m, 2H), 2.55 (q, J = 7.1 Hz, 4H), 2.30−2.19 (m, 2H), 1.89−1.81 (m, 2H), 1.06 (t, J = 7.1 Hz, 6H). 7-fluoro-6-((3-morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one

(3b).

According to the general procedure above, the intermediate 3b was obtained. Yield: 2.7 g, 67%, milk white solid. 1H

NMR (400 MHz, CDCl3) δ 7.67 (d, J = 11.7 Hz, 1H), 6.62 (d, J = 7.7 Hz, 1H), 4.13−4.05 (m, 2H), 3.72−3.67

(m, 2H), 3.66−3.61 (m, 2H), 3.25 (dd, J = 11.1, 5.8 Hz, 2H), 3.04 (t, J = 7.9 Hz, 4H), 2.51−2.46 (m, 3H), 2.24−2.12 (m, 4H), 1.86−1.75 (m, 2H). 7-fluoro-6-((3-(pyrrolidin-1-yl)propyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (3c). According to the general procedure above, the intermediate 3c was obtained. Yield: 2.3 g, 61%, pale yellow solid. 1H

NMR (400 MHz, CDCl3) δ 7.74 (d, J = 11.7 Hz, 1H), 6.70 (d, J = 7.7 Hz, 1H), 4.22−4.12 (m, 2H), 3.33 (dd, J

= 11.1, 6.0 Hz, 2H), 3.13 (t, J = 7.9 Hz, 2H), 2.68 (t, J = 6.2 Hz, 2H), 2.56 (t, J = 5.9 Hz, 5H), 2.31−2.22 (m, 2H), 1.90 (dt, J = 12.4, 6.2 Hz, 2H), 1.85− 1.80 (m, 4H).



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6-((2-(dimethylamino)ethyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (3d). According to the general procedure above, the intermediate 3d was obtained. Yield: 2.1 g, 59%, milk white solid. 1H

NMR (400 MHz, CDCl3) δ 7.71 (d, J = 11.5 Hz, 1H), 6.61 (d, J = 7.4 Hz, 1H), 4.22−4.01 (m, 2H), 3.44 (dd, J

= 12.6, 6.5 Hz, 2H), 3.14 (t, J = 7.8 Hz, 2H), 2.47 (t, J = 6.5 Hz, 4H), 2.18 (s, 6H). 7-fluoro-6-((2-(piperidin-1-yl)ethyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one

(3e).

According to the general procedure above, the intermediate 3e was obtained. Yield: 2.6 g, 79%, milk white solid. 1H

NMR (400 MHz, CDCl3) δ 7.75 (dd, J = 11.6, 4.6 Hz, 1H), 6.71 (d, J = 7.5 Hz, 1H), 5.32 (s, 1H), 4.16 (t, J =

7.0 Hz, 2H), 3.33−3.20 (m, 2H), 3.12 (dd, J = 9.6, 5.9 Hz, 2H), 2.64 (t, J = 5.6 Hz, 2H), 2.42 (s, 4H), 2.30−2.21 (m, 2H), 1.59 (d, J = 4.8 Hz, 4H), 1.46 (d, J = 3.3 Hz, 2H). 7-fluoro-6-(4-methylpiperazin-1-yl)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (3f). According to the general procedure above, the intermediate 3f was obtained. Yield: 1.8 g, 54%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 12.7 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 3.43 (t, J = 8.2 Hz, 4H), 3.17 – 3.05 (m, 4H), 2.34 (t, J = 8.2 Hz, 4H), 2.31 – 2.22 (m, 2H), 2.21 (s, 3H). 7-fluoro-6-(4-(pyridin-2-yl)piperazin-1-yl)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one

(3g).

According to the general procedure above, the intermediate 3g was obtained. Yield: 3.6 g, 87%, pale yellow solid. 1H

NMR (400 MHz, CDCl3) δ 8.03 (dd, J = 12.0, 2.4 Hz, 1H), 7.54 (m, 1H), 7.42 (d, J = 12.7 Hz, 1H), 7.22 (m,

1H), 6.79 (d, J = 8.1 Hz, 1H), 6.72 (m, 1H), 3.63 – 3.45 (m, 8H), 3.22 – 3.02 (m, 4H), 2.36 – 2.18 (m, 2H). 7-fluoro-6-((4-fluorophenethyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one

(3h).

According to the general procedure above, the intermediate 3h was obtained. Yield: 2.2 g, 69%, pale yellow solid. 1H

NMR (400 MHz, CDCl3) δ 7.51 (d, J = 12.7 Hz, 1H), 7.30 – 7.09 (m, 4H), 6.74 (d, J = 7.9 Hz, 1H), 3.41 (m,

3H), 3.17 – 3.06 (m, 4H), 2.93 (t, J = 8.3 Hz, 2H), 2.35 – 2.15 (m, 2H). General Procedure for the Synthesis of Intermediates 4a-4d. The mixture of terephthalaldehyde or isophthalaldehyde (1 equiv.) and different substituted acetophenones (2 equiv.) in a 38 mL sealed tube was stirred at 0 °C for 2 h first and then at room temperature for another 4 h, which was monitored by TLC. The crude product was adsorbed on silica and purified by flash chromatography.


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(E)-4-(3-(4-morpholinophenyl)-3-oxoprop-1-en-1-yl)benzaldehyde (4a). According to the general procedure above, the intermediate 4a was obtained. Yield: 1.2 g, 49%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 10.05 (s, 1H), 8.02 (d, J = 9.0 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.80 (m, 3H), 7.66 (d, J = 15.7 Hz, 1H), 6.93 (d, J = 9.0 Hz, 2H), 3.92 – 3.83 (m, 4H), 3.40 – 3.32 (m, 4H). (E)-4-(3-(4-fluorophenyl)-3-oxoprop-1-en-1-yl)benzaldehyde (4b). According to the general procedure above, the intermediate 4b was obtained. Yield: 2.1 g, 55%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.88 (s, 1H), 8.05 (d, J = 24.2 Hz, 1H), 7.96 – 7.88 (m, 2H), 7.79 – 7.68 (m, 4H), 7.59 (d, J = 24.1 Hz, 1H), 7.53 – 7.42 (m, 2H). (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-3-oxoprop-1-en-1-yl)benzaldehyde (4c). According to the general procedure above, the intermediate 4c was obtained. Yield: 2.5 g, 59%, pale yellow solid. 1HNMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 8.11 – 7.82 (m, 4H), 7.69 (m, 1H), 7.58 (d, J = 24.2 Hz, 1H), 7.45 (t, J = 2.4 Hz, 1H), 7.33 (d, J = 2.5 Hz, 1H), 7.15 (d, J = 12.0 Hz, 1H), 6.05 (s, 2H). (E)-3-(3-(4-morpholinophenyl)-3-oxoprop-1-en-1-yl)benzaldehyde (4d). According to the general procedure above, the intermediate 4d was obtained. Yield: 1.9 g, 51%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 10.08 (s, 1H), 8.19 – 7.99 (m, 3H), 7.93 – 7.76 (m, 3H), 7.70 – 7.50 (m, 2H), 6.93 (d, J = 9.0 Hz, 2H), 3.95 – 3.77 (m, 4H), 3.43 – 3.29 (m, 4H). General Procedure for the Synthesis of Final Compounds 5-24. The mixture of intermediates 3a-3h (1 equiv.) and different aldehydes (1.2 equiv.) was dissolved in DMF and catalyzed by TMSCl in a 38 mL sealed tube at 100 °C for 12-48 h and monitored by TLC. The crude product was adsorbed on silica and purified by flash chromatography. (E)-3-(4-(tert-butyl)benzylidene)-6-((3-(diethylamino)propyl)amino)-7-fluoro-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (5). According to the general procedure above, the compound 5 was obtained. Yield: 115 mg, 38%, white solid. 1H NMR (400 MHz, CDCl3) δ 7.73 (m, 2H), 7.47 (q, J = 8.5 Hz, 4H), 6.77 (d, J = 7.7 Hz, 1H), 6.65 (s, 1H), 4.21 (t, J = 7.3 Hz, 2H), 3.34 (dd, J = 10.7, 5.6 Hz, 2H), 3.24 (m, 2H), 2.61 (t, J = 5.9 Hz, 2H), 2.55 (q, J = 7.1 Hz, 4H), 1.89 – 1.80 (m, 2H), 1.35 (s, 9H), 1.06 (t, J = 7.1 Hz, 6H). 13C



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NMR (101 MHz, CDCl3) δ 160.51 (d, J = 3.5 Hz), 155.33 (d, J = 1.6 Hz), 152.11, 151.94, 149.51, 148.79, 143.39 (d, J = 14.0 Hz), 132.94, 131.14, 129.56 (2C), 125.76 (2C), 109.64 (d, J = 20.9 Hz), 109.31 (d, J = 7.7 Hz), 105.51 (d, J = 4.1 Hz), 52.82, 46.90 (2C), 43.86, 43.70, 34.80, 31.19 (3C), 25.60, 25.26, 11.71 (2C). ESI-HRMS [M + H]+ m/z = 477.3024, calcd for C29H37N4OF, 477.3029. Purity: 100.0% by HPLC. (E)-6-((3-(diethylamino)propyl)amino)-7-fluoro-3-(4-isobutylbenzylidene)-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (6). According to the general procedure above, the compound 6 was obtained. Yield: 193 mg, 47%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.76 – 7.70 (m, 2H), 7.48 – 7.42 (m, 2H), 7.23 – 7.17 (m, 2H), 6.79 – 6.74 (m, 1H), 6.63 (s, 1H), 4.20 (m, 2H), 3.34 (dd, J = 10.1, 5.1 Hz, 2H), 3.22 (m, 2H), 2.61 (t, J = 5.9 Hz, 2H), 2.53 (m, 6H), 1.95 – 1.81 (m, 3H), 1.06 (t, J = 7.1 Hz, 6H), 0.92 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.51 (d, J = 2.5 Hz), 155.33, 151.92, 149.50, 148.80, 143.37 (d, J = 14.1 Hz), 142.80, 133.22, 130.98, 129.62 (d, J = 11.4 Hz)(2C), 109.63 (d, J = 20.8 Hz), 109.30 (d, J = 7.6 Hz), 105.51 (d, J = 4.1 Hz), 52.80, 46.90 (2C), 45.28, 43.86, 43.67, 30.16, 25.63, 25.25, 22.37 (2C), 11.69 (2C). ESI-HRMS [M + H]+ m/z = 477.3024, calcd for C29H37N4OF, 477.3028. Purity: 100.0% by HPLC. (E)-3-benzylidene-6-((3-(diethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1b]quinazolin-9(1H)-one (7). According to the general procedure above, the compound 7 was obtained. Yield: 141 mg, 52%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.75 (dd, J = 7.1, 4.6 Hz, 2H), 7.54 (d, J = 7.6 Hz, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 6.78 (s, 1H), 6.76 (s, 1H), 4.26 – 4.18 (m, 2H), 3.34 (dd, J = 10.8, 5.6 Hz, 2H), 3.28 – 3.20 (m, 2H), 2.65 – 2.59 (m, 2H), 2.55 (q, J = 7.1 Hz, 4H), 1.90 – 1.81 (m, 2H), 1.06 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.50 (d, J = 3.4 Hz), 155.17 (d, J = 1.7 Hz), 151.99, 149.57, 148.73, 143.51, 143.37, 135.67, 132.09, 129.65 (d, J = 5.8 Hz) (2C), 128.76 (d, J = 7.6 Hz)(2C), 109.66 (d, J = 20.8 Hz), 109.29 (d, J = 7.7 Hz), 105.46 (d, J = 4.2 Hz), 52.87, 46.85 (2C), 43.88, 43.76, 25.64, 25.08, 11.69 (2C). ESIHRMS [M + H]+ m/z = 421.2398, calcd for C25H29N4OF, 421.2394. Purity: 99.9% by HPLC. (E)-7-fluoro-3-(4-fluorobenzylidene)-6-((3-(pyrrolidin-1-yl)propyl)amino)-2,3-dihydropyrrolo[2,1b]quinazolin-9(1H)-one (8). According to the general procedure above, the compound 8 was obtained. Yield: 177 mg, 52%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.79 – 7.70 (m, 2H), 7.53 (dd, J = 8.3, 5.6 Hz, 2H), 7.13 (t, J = 8.5 Hz, 2H), 6.78 (d, J = 7.7 Hz, 1H), 6.36 (s, 1H), 4.25 (t, J = 7.1 Hz, 2H), 3.37 (t, J = 6.1 Hz, 2H),


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3.23 (t, J = 5.9 Hz, 2H), 2.76 (t, J = 6.3 Hz, 2H), 2.66 (m, 4H), 2.00 – 1.92 (m, 2H), 1.86 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 163.94, 161.45, 160.48 (d, J = 3.3 Hz), 155.12, 150.71 (d, J = 244.3 Hz), 148.67, 143.38 (d, J = 14.0 Hz), 131.75 (dd, J = 32.2, 2.8 Hz), 131.47 (d, J = 8.3 Hz)(2C), 128.51, 115.98 (d, J = 21.7 Hz)(2C), 109.78 (d, J = 20.8 Hz), 109.45 (d, J = 7.6 Hz), 105.51 (d, J = 4.0 Hz), 54.63, 53.92 (2C), 43.88, 42.83, 26.40, 25.51, 23.45 (2C). ESI-HRMS [M + H]+ m/z = 437.2147, calcd for C25H26N4OF2, 437.2143. Purity: 99.3% by HPLC. (E)-3-benzylidene-6-((2-(diethylamino)ethyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1b]quinazolin-9(1H)-one (9). According to the general procedure above, the compound 9 was obtained. Yield: 140 mg, 55%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.82 (d, J = 11.0 Hz, 1H), 7.60 (d, J = 7.4 Hz, 2H), 7.47 (t, J = 7.3 Hz, 2H), 7.44 – 7.38 (m, 1H), 7.09 (s, 1H), 6.58 (s, 1H), 4.37 – 4.29 (m, 2H), 3.96 – 3.87 (m, 2H), 3.33 (m, 4H), 3.28 – 3.12 (m, 4H), 1.43 (t, J = 7.1 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 159.74 (d, J = 3.3 Hz), 155.70, 151.26, 149.09 (d, J = 61.3 Hz), 143.05 (d, J = 13.7 Hz), 135.86, 133.68, 130.08 (2C), 129.36 (2C), 129.16, 128.65, 109.56 (d, J = 20.4 Hz), 109.07 (d, J = 7.1 Hz), 106.12 (d, J = 3.9 Hz), 51.09, 46.91 (2C), 44.31, 40.77, 25.60, 12.34 (2C). ESI-HRMS [M + H]+ m/z= 407.2242, calcd for C24H27N4OF, 404.2237. Purity: 99.9% by HPLC. (E)-3-(4-(diethylamino)benzylidene)-6-((2-(dimethylamino)ethyl)amino)-7-fluoro-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (10). According to the general procedure above, the compound 10 was obtained. Yield: 237 mg, 66%, testaceous solid. 1H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 11.5 Hz, 1H), 7.68 (m, 1H), 7.45 (d, J = 8.3 Hz, 2H), 6.80 (d, J = 7.5 Hz, 1H), 6.70 (d, J = 8.2 Hz, 2H), 5.20 (s, 1H), 4.24 (t, J = 7.1 Hz, 2H), 3.42 (dd, J = 13.7, 6.8 Hz, 4H), 3.34 (m, 2H), 3.27 – 3.21 (m, 2H), 2.70 (m, 2H), 2.34 (s, 6H), 1.21 (t, J = 6.7 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.69 (d, J = 3.3 Hz), 156.42 (d, J = 1.3 Hz), 151.48, 149.00 (d, J = 11.8 Hz), 148.04, 142.66 (d, J = 13.8 Hz), 131.80 (2C), 130.54, 125.42, 122.80, 111.31 (2C), 109.97 (d, J = 20.6 Hz), 109.71 (d, J = 7.5 Hz), 105.85 (d, J = 3.6 Hz), 57.31, 45.03 (2C), 44.43 (2C), 43.96, 40.03, 25.60, 12.63 (2C). ESI-HRMS [M + H]+ m/z = 450.2664, calcd for C26H32N5OF, 450.2652. Purity: 99.5% by HPLC. (E)-3-(4-(benzyloxy)-2-hydroxybenzylidene)-7-fluoro-6-((2-(piperidin-1-yl)ethyl)amino)-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (11). According to the general procedure above, the compound 11 was obtained. Yield: 112 mg, 43%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.76 (d, J =



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11.3 Hz, 1H), 7.40 (m, 2H), 7.33 – 7.26 (m, 4H), 6.83 (d, J = 6.9 Hz, 1H), 6.55 (d, J = 7.4 Hz, 1H), 6.44 (s, 1H), 5.20 (s, 1H), 4.94 (s, 2H), 4.25 – 4.10 (m, 2H), 3.17 (m, 4H), 2.57 (m, 2H), 2.39 (s, 4H), 1.57 (m, 4H), 1.44 (s, 2H). 13C

NMR (101 MHz, CDCl3) δ 160.94, 151.61, 143.09 (d, J = 13.7 Hz), 136.59, 129.88, 128.52 (2C), 127.90, 127.10

(2C), 125.62, 116.92, 110.06 (d, J = 21.2 Hz), 107.60, 105.36, 102.94, 100.00, 70.01, 56.61, 54.27 (2C), 44.09, 39.53, 25.85 (2C), 25.53, 24.29. ESI-HRMS [M + H]+ m/z = 541.2609, calcd for C32H33N4O3F, 541.2591. Purity: 95.8% by HPLC. (E)-7-fluoro-3-(4-isopropylbenzylidene)-6-((2-(piperidin-1-yl)ethyl)amino)-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (12). According to the general procedure above, the compound 12 was obtained. Yield: 77 mg, 32%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.76 (m, 2H), 7.48 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 6.81 (d, J = 7.7 Hz, 1H), 5.31 (s, 1H), 4.23 (t, J = 7.2 Hz, 2H), 3.34 – 3.19 (m, 4H), 3.00 – 2.87 (m, 1H), 2.67 (t, J = 6.0 Hz, 2H), 2.44 (m, 4H), 1.61 (dt, J = 10.7, 5.4 Hz, 4H), 1.52 – 1.40 (m, 2H), 1.28 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.55 (d, J = 3.1 Hz), 155.50, 151.79, 149.97, 149.37, 148.68, 143.00 (d, J = 13.5 Hz), 133.27, 130.90, 129.86 (2C), 126.95 (2C), 109.96, 109.72 (d, J = 6.1 Hz), 106.13 (d, J = 3.6 Hz), 56.64, 54.27 (2C), 43.94, 39.46, 34.04, 25.96 (2C), 25.61, 24.38, 23.82 (2C). ESI-HRMS [M + H]+ m/z = 461.2711, calcd for C28H33N4OF, 461.2703. Purity: 99.8% by HPLC. (E)-3-(4-(tert-butyl)benzylidene)-7-fluoro-6-((2-(piperidin-1-yl)ethyl)amino)-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (13). According to the general procedure above, the compound 13 was obtained. Yield: 164 mg, 50%, milk white solid. 1H NMR (500 MHz, CDCl3) δ 7.77 (m, 2H), 7.48 (dd, J = 19.8, 8.5 Hz, 4H), 6.81 (d, J = 7.7 Hz, 1H), 5.33 (s, 1H), 4.29 – 4.19 (m, 2H), 3.28 (m, 4H), 2.68 (t, J = 5.8 Hz, 2H), 2.46 (s, 4H), 1.68 – 1.57 (m, 4H), 1.48 (s, 2H), 1.35 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 160.53 (d, J = 3.3 Hz), 155.51 (d, J = 1.8 Hz), 152.21, 151.56, 149.62, 148.69, 142.97 (d, J = 13.6 Hz), 132.89, 131.04, 129.74, 129.61 (2C), 125.82 (2C), 110.02 – 109.76 (m), 106.16 (d, J = 3.7 Hz), 56.61 (2C), 54.26 (2C), 43.94, 39.41, 34.84, 31.21 (3C), 25.91, 25.61, 24.35. ESI-HRMS [M + H]+ m/z = 475.2868, calcd for C29H35N4OF, 475.2874. Purity: 100.0% by HPLC. (E)-7-fluoro-3-(4-isobutylbenzylidene)-6-((2-(piperidin-1-yl)ethyl)amino)-2,3-dihydropyrrolo[2,1b]quinazolin-9(1H)-one (14). According to the general procedure above, the compound 14 was obtained. Yield:


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198 mg, 71%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.81 – 7.72 (m, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.1 Hz, 2H), 6.80 (d, J = 7.7 Hz, 1H), 5.52 (s, 1H), 4.29 – 4.20 (m, 2H), 3.43 (m, 2H), 3.30 – 3.21 (m, 2H), 2.81 (m, 2H), 2.61 (s, 4H), 2.52 (d, J = 7.2 Hz, 2H), 2.00 – 1.81 (m, 1H), 1.73 (m, 4H), 1.52 (m, 2H), 0.93 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.50 (d, J = 3.3 Hz), 155.56, 151.80, 149.37, 148.65, 142.99, 142.66 (d, J = 13.9 Hz), 133.14, 130.86, 129.96 (2C), 129.63 (2C), 110.11 (d, J = 8.9 Hz), 109.97 (d, J = 3.1 Hz), 106.06 (d, J = 3.7 Hz), 56.52, 54.29 (2C), 45.29, 43.95, 39.08, 30.22 (2C), 25.65, 25.23, 23.85, 22.41 (2C). ESI-HRMS [M + H]+ m/z = 475.2868, calcd for C29H35N4OF, 475.2858. Purity: 100.0% by HPLC. (E)-7-fluoro-6-((4-fluorophenethyl)amino)-3-(4-isopropylbenzylidene)-2,3-dihydropyrrolo[2,1b]quinazolin-9(1H)-one (15). According to the general procedure above, the compound 15 was obtained. Yield: 103 mg, 47%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.90 – 7.74 (m, 2H), 7.51 (m, 2H), 7.31 (m, 2H), 7.21 (m, 2H), 7.03 (t, J = 8.0 Hz, 2H), 6.96 (s, 1H), 4.56 (s, 1H), 4.26 (s, 2H), 3.55 (s, 2H), 3.28 (s, 2H), 3.09 – 2.86 (m, 3H), 1.28 (d, J = 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 163.00, 160.57, 160.30, 155.78, 151.60, 150.25, 149.18, 142.41 (d, J = 13.3 Hz), 134.13 (d, J = 3.1 Hz), 133.16, 130.24, 130.16, 130.07 (2C), 127.04 (2C), 115.72 (2C), 115.51 (2C), 110.16 (d, J = 21.2 Hz), 105.92, 44.37, 44.25, 34.24, 34.07, 25.72, 23.83 (2C). ESI-HRMS [M + H]+ m/z = 472.2195, calcd for C29H27N3OF2, 472.2182. Purity: 97.1% by HPLC. (E)-3-(4-(tert-butyl)benzylidene)-7-fluoro-6-(4-methylpiperazin-1-yl)-2,3-dihydropyrrolo[2,1b]quinazolin-9(1H)-one (16). According to the general procedure above, the compound 16 was obtained. Yield: 109 mg, 57%, milk white solid. 1H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 12.9 Hz, 1H), 7.77 (m, 1H), 7.49 (q, J = 8.5 Hz, 4H), 7.17 (d, J = 7.8 Hz, 1H), 4.26 (t, J = 7.2 Hz, 2H), 3.30 (m, 6H), 2.66 (m, 4H), 2.39 (s, 3H), 1.35 (s, 9H). 13C NMR (126 MHz, CDCl3) δ 160.32 (d, J = 3.0 Hz), 155.70 (d, J = 1.8 Hz), 153.98 (d, J = 249.1 Hz), 152.42, 147.74, 146.24 (d, J = 10.0 Hz), 132.74, 130.67, 130.22, 129.67 (2C), 125.87 (2C), 115.19 (d, J = 3.3 Hz), 114.51 (d, J = 8.4 Hz), 112.01 (d, J = 23.8 Hz), 54.91 (2C), 49.88, 46.08 (2C), 44.03, 34.87, 31.20 (3C), 25.57. ESI-HRMS [M + H]+ m/z = 447.2555, calcd for C27H31N4OF, 447.2553. Purity: 100.0% by HPLC. (E)-7-fluoro-3-(4-isopropylbenzylidene)-6-(4-(pyridin-2-yl)piperazin-1-yl)-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (17). According to the general procedure above, the compound 17 was obtained. Yield: 181 mg, 62%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.23 (dd, J = 4.9, 1.1 Hz,



Page 28 of 44

1H), 7.85 (dd, J = 12.8, 1.4 Hz, 1H), 7.77 (m, 1H), 7.56 – 7.45 (m, 3H), 7.31 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 7.8 Hz, 1H), 6.73 – 6.64 (m, 2H), 4.25 (t, J = 7.2 Hz, 2H), 3.78 – 3.69 (m, 4H), 3.43 – 3.37 (m, 4H), 3.27 (t, J = 6.8 Hz, 2H), 3.02 – 2.87 (m, 1H), 1.28 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.27 (d, J = 2.7 Hz), 159.36, 155.74 (d, J = 1.8 Hz), 155.18, 152.70, 150.18, 147.87 (d, J = 20.9 Hz), 146.16 (d, J = 10.3 Hz), 137.65, 133.14, 130.47 (d, J = 18.9 Hz), 129.91 (2C), 126.99 (2C), 115.22 (d, J = 3.0 Hz), 114.68 (d, J = 8.3 Hz), 113.78, 112.20, 111.97, 107.22, 49.88 (d, J = 4.3 Hz), 45.26 (2C), 44.01, 34.04 (2C), 25.57, 23.78 (2C). ESI-HRMS [M + H]+ m/z = 496.2507, calcd for C30H30N5OF, 496.2517. Purity: 99.3% by HPLC. 7-fluoro-3-((E)-4-((E)-3-(4-morpholinophenyl)-3-oxoprop-1-en-1-yl)benzylidene)-6-((3morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (18). According to the general procedure above, the compound 18 was obtained. Yield: 62 mg, 29%, yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 8.9 Hz, 2H), 7.78 (m, 3H), 7.69 (d, J = 8.2 Hz, 2H), 7.59 (m, 3H), 6.92 (d, J = 8.9 Hz, 2H), 6.81 (d, J = 7.7 Hz, 1H), 6.48 (s, 1H), 4.26 (t, J = 7.1 Hz, 2H), 3.92 – 3.83 (m,4H), 3.84 – 3.75 (m, 4H), 3.43 – 3.32 (m, 6H), 3.31 – 3.25 (m, 2H), 2.58 (m, J = 16.8, 11.1 Hz, 6H), 1.92 (m, J = 11.5, 5.8 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 187.75, 160.43 (d, J = 3.6 Hz), 155.06 (d, J = 1.9 Hz), 154.27, 152.05, 149.62, 148.69, 143.32 (d, J = 13.9 Hz), 142.14, 137.31, 135.58, 133.25, 130.67, 130.13, 128.77 (d, J = 18.8 Hz), 128.71, 122.67, 113.40, 109.83 (d, J = 21.1 Hz), 109.61 (d, J = 7.6 Hz), 105.65 (d, J = 3.8 Hz), 66.84 (2C), 66.57 (2C), 58.05, 53.77 (2C), 47.47 (2C), 43.93, 43.37, 25.79, 24.03. ESI-HRMS [M + H]+ m/z = 650.3137, calcd for C38H40N5O4F, 650.3139. Purity: 98.8% by HPLC. 7-fluoro-3-((E)-4-((E)-3-(4-fluorophenyl)-3-oxoprop-1-en-1-yl)benzylidene)-6-((3morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (19). According to the general procedure above, the compound 19 was obtained. Yield: 114 mg, 40%, yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.08 (m, 2H), 7.89 – 7.67 (m, 5H), 7.57 (m, 3H), 7.19 (t, J = 7.9 Hz, 2H), 6.81 (d, J = 7.3 Hz, 1H), 6.52 (s, 1H), 4.33 – 4.20 (m, 2H), 3.80 (s, 4H), 3.38 (m, 2H), 3.30 (m, 2H), 2.58 (m, 6H), 1.93 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 188.55, 166.97, 164.44, 160.42 (d, J = 2.9 Hz), 155.01, 148.66, 143.89 (2C), 137.82, 134.99, 134.43 (d, J = 2.7 Hz), 133.63, 131.13 (d, J = 9.3 Hz), 130.19 (2C), 128.91 (2C), 128.71, 122.20 (2C), 115.96, 115.74, 109.85 (d, J = 21.2 Hz), 109.61 (d, J = 6.9 Hz), 105.63 (d, J = 3.7 Hz), 66.81 (2C), 58.06, 53.75 (2C), 43.94, 43.37,


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25.83, 23.95. ESI-HRMS [M + H]+ m/z = 583.2515, calcd for C34H32N4O3F2, 583.2514. Purity: 97.7% by HPLC. 3-((E)-3-((E)-3-(benzo[d][1,3]dioxol-5-yl)-3-oxoprop-1-en-1-yl)benzylidene)-7-fluoro-6-((3morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (20). According to the general procedure above, the compound 20 was obtained. Yield: 136 mg, 37%, yellow solid. 1H NMR (500 MHz, DMSO) δ 8.04 (s, 1H), 7.93 (d, J = 19.6 Hz, 1H), 7.85 (m, 3H), 7.74 (d, J = 15.6 Hz, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.62 (m, 2H), 7.56 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.1 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.14 (s, 2H), 4.16 (t, J = 7.0 Hz, 2H), 3.93 (m, 4H), 3.38 (t, J = 6.5 Hz, 2H), 3.34 (m, 2H), 3.24 – 3.15 (m, 2H), 2.89 (s, 2H), 2.74 (s, 2H), 2.17 – 2.08 (m, 2H). 13C NMR (126 MHz, DMSO) δ 187.72, 162.74, 159.58, 155.83, 152.08, 148.55, 143.21, 143.04 (d, J = 13.9 Hz), 136.56, 136.08, 134.25, 132.93, 131.34, 130.42, 129.87, 129.22, 129.07, 125.49, 123.68, 109.85 (d, J = 20.9 Hz), 108.58, 108.45, 105.85 (d, J = 3.8 Hz), 102.50, 63.61 (2C), 54.63, 51.64 (2C), 44.57, 36.17, 31.31, 25.60, 22.70. ESI-HRMS [M + H]+ m/z = 609.2508, calcd for C35H33N4O5F, 609.2503. Purity: 99.3% by HPLC. 7-fluoro-3-((E)-3-((E)-3-(4-morpholinophenyl)-3-oxoprop-1-en-1-yl)benzylidene)-6-((3morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (21). According to the general procedure above, the compound 21 was obtained. Yield: 123 mg, 34%, yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 8.8 Hz, 2H), 7.79 (m, 4H), 7.68 – 7.55 (m, 3H), 7.49 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 6.81 (d, J = 7.7 Hz, 1H), 6.53 (s, 1H), 4.27 (t, J = 7.1 Hz, 2H), 3.92 – 3.84 (m, 4H), 3.84 – 3.76 (m, 4H), 3.43 – 3.33 (m, 6H), 3.30 (m, 2H), 2.58 (m, 6H), 1.96 – 1.87 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 187.82, 160.47 (d, J = 3.1 Hz), 155.05 (d, J = 1.6 Hz), 154.30, 150.82 (d, J = 244.3 Hz), 148.67, 143.35 (d, J = 13.9 Hz), 142.55, 136.33, 135.93, 133.02, 130.87, 130.72 (2C), 129.81, 129.40, 129.00, 128.58, 128.05, 122.69, 113.41 (2C), 109.82 (d, J = 21.0 Hz), 109.57 (d, J = 7.5 Hz), 105.62 (d, J = 4.0 Hz), 66.86 (2C), 66.58 (2C), 58.09, 53.77 (2C), 47.46 (2C), 43.96, 43.42, 25.67, 23.98. ESI-HRMS [M + H]+ m/z = 650.3137, calcd for C38H40N5O4F, 650.3153. Purity: 99.1% by HPLC. (E)-3-(benzofuran-2-ylmethylene)-6-((3-(diethylamino)propyl)amino)-7-fluoro-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (22). According to the general procedure above, the compound 22 was obtained. Yield: 230 mg, 46%, yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 11.7 Hz, 1H), 7.63



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(t, J = 2.7 Hz, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.36 – 7.30 (m, 1H), 7.25 (m, 1H), 6.92 (s, 1H), 6.76 (d, J = 7.7 Hz, 1H), 6.71 (s, 1H), 4.30 – 4.23 (m, 2H), 3.47 (td, J = 7.9, 2.6 Hz, 2H), 3.34 (dd, J = 10.3, 5.7 Hz, 2H), 2.62 (t, J = 5.9 Hz, 2H), 2.56 (q, J = 7.1 Hz, 4H), 1.87 (m, 2H), 1.07 (t, J = 7.1 Hz,6H). 13C NMR (101 MHz, CDCl3) δ 160.48 (d, J = 3.3 Hz), 155.61, 154.58 (d, J = 1.9 Hz), 153.87, 150.90 (d, J = 244.5 Hz), 148.69, 143.46 (d, J = 14.0 Hz), 133.24, 128.41, 125.72, 123.35, 121.51, 116.62, 111.29, 110.09, 109.75 (d, J = 21.0 Hz), 109.40 (d, J = 7.6 Hz), 105.48 (d, J = 4.1 Hz), 52.79, 46.87 (2C), 43.94, 43.67, 25.62, 25.12, 11.64 (2C). ESI-HRMS [M + H]+ m/z = 461.2347, calcd for C27H29N4O2F, 461.2344. Purity: 97.6% by HPLC. (E)-3-((9-ethyl-9H-carbazol-3-yl)methylene)-7-fluoro-6-((3-morpholinopropyl)amino)-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (23). According to the general procedure above, the compound 23 was obtained. Yield: 101 mg, 40%, pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.96 (s, 1H), 7.76 (d, J = 11.7 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.43 (m, 2H), 7.26 (m, 1H), 6.80 (d, J = 7.7 Hz, 1H), 6.41 (s, 1H), 4.37 (q, J = 7.1 Hz, 2H), 4.26 (t, J = 7.2 Hz, 2H), 3.80 (s, 4H), 3.36 (m, 4H), 2.66 – 2.48 (m, 6H), 1.98 – 1.88 (m, 2H), 1.46 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 160.63 (d, J = 3.3 Hz), 155.92, 151.76, 149.35, 148.93, 143.17 (d, J = 13.9 Hz), 140.44, 140.03, 131.04, 128.32, 128.06, 126.79, 126.27, 123.40, 122.82, 122.08, 120.51, 119.50, 109.75 (d, J = 20.8 Hz), 109.41 (d, J = 7.6 Hz), 108.82 (d, J = 8.4 Hz), 105.51 (d, J = 3.9 Hz), 66.82, 58.04, 53.75 (2C), 43.99, 43.33, 37.75, 25.78, 24.04, 13.89 (2C). ESI-HRMS [M + H]+ m/z = 552.2769, calcd for C33H34N5O2F, 552.2757. Purity: 96.1% by HPLC. (E)-3-(benzofuran-2-ylmethylene)-7-fluoro-6-((3-morpholinopropyl)amino)-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (24). According to the general procedure above, the compound 24 was obtained. Yield: 211 mg, 63%, yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 11.7 Hz, 1H), 7.65 (t, J = 2.7 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.39 – 7.33 (m, 1H), 7.29 (m, 1H), 6.94 (s, 1H), 6.81 (d, J = 7.7 Hz, 1H), 6.49 (s, 1H), 4.33 – 4.25 (m, 2H), 3.81 (t, J = 4.3 Hz, 4H), 3.55 – 3.45 (m, 2H), 3.39 (t, J = 5.6 Hz, 2H), 2.60 (m, 6H), 1.93 (dt, J = 11.4, 5.6 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 160.45 (d, J = 3.2 Hz), 155.62, 154.68 (d, J = 2.0 Hz), 153.82, 150.85 (d, J = 244.2 Hz), 148.68, 143.28 (d, J = 14.0 Hz), 133.18, 129.87 (d, J = 206.9 Hz), 128.40, 125.76, 123.37, 121.52, 116.68, 111.30, 110.16, 109.86 (d, J = 21.0 Hz), 105.62 (d, J = 3.9 Hz), 66.83 (2C), 58.04, 53.76 (2C), 43.96, 43.35, 25.61, 24.02. ESI-HRMS [M + H]+ m/z = 475.2140,


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calcd for C27H27N4O3F, 475.2154. Purity: 97.0% by HPLC. DNA Preparation. All Oligonucleotides used in experiments were shown in Table S6 and S7. Plasmid Construction. A truncated protein BLM (BLM642-1296) protein expression plasmid was a gift from Zheng Tan.46 BLM were constructed by digesting and inserting the corresponding cDNA into the pET28b plasmid using the Bam HI and XhoI restriction enzyme. In HRR reporter assay, pDRGFP (HRR (+) reporter) was a gift from Maria Jasin (Addgene plasmid # 26475), pCBASceI was a gift from Maria Jasin (Addgene plasmid # 26477). To conduct HRR (-) reporter, the reverse GFP template was digested using Hind III restriction enzyme, and the recombinant sequences were spliced and inserted into PGK-puro plasmid. Protein Preparation. In brief, BLM and mutant variants were cloned into the pET28b plasmids containing the His-tag and transformed into E. coli strain BL21 (DE3) cells. The positive-clones were enlarged overnight at 37 °C and 0.1 μM IPTG were added to induce protein expression at 16 °C for 24 h. Then, cells were collected and lysed using lysozyme (MP Biomedicals). The purification of protein was harvested by HisTrap HP (Roche), and the purified protein were stored in buffer of 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 2 mM MgCl2, 2 mM DTT and 50% (v/v) glycerol conditions. RAD51 protein was purchased from Abcam (ab63808, Abcam). Helicase Unwinding Assay. A 5′-biotin labeled forked-DNA was used as a DNA substrate. The DNA substrate was diluted in buffer of 10 mM Tris-HCl (pH 7.4), 20 mM NaCl, and heated to 95 °C for 5 min, cooling down to 25 °C slowly. The purified BLM protein was incubated with a series of different concentration of compounds (1.0, 10 and 50 μM) at 4 °C for 1 h with no more than 1% DMSO. After 1 h incubation, the duplex forked-DNA was added. Finally, the reaction sample contained 8 nM duplex forked-DNA, 32 nM purified BLM, 2 mM ATP (Sigma), 0.1% BSA and compounds. After 1 h co-incubation, the reaction was stopped by DNA loading buffer (Takara), and samples were run on the 20% native polyacrylamide gels at 8V·cm-1 and 4 °C in 0.5×TB buffer for 180 min. After the electrophoresis, the gels were transferred to nylon membranes. The membranes were crosslinked under UV irradiation at 265 nm for 10 min and the visualization of DNA was performed using Nucleic Acid Detection Module Kit (Thermo Scientific). The upper bands were duplex DNA and the lower bands were single strand DNA after unwinding. The image analysis was performed using ImageJ 2.0, the untreated group was set as



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100%, and the other bands were quantified to gain the inhibitory effect of each compound. The half maximal inhibitory concentration of BLM helicase (IC50 value) was calculated via a non-linear regression model in GraphPad software. MTT Assay. Various cells (5×103) were planted in per well of 96-well plate and cultured for 24 h. Then, a series concentration of 29 (from 0 to 50 μM) were added into cells and incubated for different times. For BLM RNA interference, cells were pre-treated with 50 nM BLM small interfering RNA for 24 h and treated with a series concentration of 29 (from 0 to 50 μM) for indicated times. Then, cells were incubated with 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide for 4 h at 37 °C at the final concentration of 0.5 mg/mL. Finally, removing the medium and dissolving the blue crystal in 100 μL of DMSO. The absorbance was measured at 470 nm by microplate reader (BioTek. USA). The IC50 was calculated via Hill Model in GraphPad software. Enzyme-Linked Immunosorbent Assay (ELISA). A 5′-biotin labeled forked-DNA and the streptaWell High Bind plates (Roche) were used in ELISA assay. Plates were hydrated with PBS for 30 min and DNA substrates were diluted to 50 nM in Tris-HCl buffer (10 mM, pH 7.4) and immobilized on the plate at 4 °C overnight. The purified BLM and mutant variants were final diluted to 100 nM. BLM variants and half-diluted 29 were blend in the blocking buffer (3% BSA in ELISA buffer) with DMSO no more than 1%. After 1h incubation on ice, the plates were washed by ELISA buffer (50 mM KH2PO4, 100 mM KCl, pH 7.4) for three times; blocked in blocking buffer for 3 h at room temperature, incubated with protein-ligand complexes at 37 °C for 1 h, repeat washed three times by washing buffer (0.1 % Tween-20 in ELISA buffer), incubated with primary antibody (ab86664, Abcam) at 4 °C overnight, washed by washing buffer for three times, incubated with secondary HRP-conjugated antibody (no. 7074S Cell Signaling Technology) at 25 °C for 2 h, repeat washed with washing buffer for three times and added 100 μL of TMB Chromogen Solution (Life Technologies), incubated for 5 min and then stopped by 50 μL of 2M H2SO4. Absorbance was measured at 450 nm and the disrupting effect of 29 was obtained using GraphPad software via Hill fitting. Filter-Binding Assay. The nitrocellulose membrane was activated with 0.5 M KOH for 10 min at 25 °C and washed three times with 0.5×TB buffer. The nylon membrane was placed under the nitrocellulose membrane to


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arrest the free DNA that not bound to the upper layer. 8 nM biotin-labeled duplex forked-DNA was incubated with increasing concentration of BLM for 1 h at 37 °C. The protein-DNA complexes were penetrated the membrane under vacuum and washed three times with 0.5×TB buffer. The membranes were cross-linked under UV irradiation at 265 nm for 10 min and the visualization of DNA was performed using Nucleic Acid Detection Module Kit (Thermo Scientific). The image analysis was applied using ImageJ 2.0, we set the grey level of the left dot (total of the dots in upper and lower membranes) as 100 %, and the subsequent dots were quantified to obtain the relative level of binding status, of which the upper dots were protein-DNA complex and the lower were free DNA. The effective binding concentration (EC50) was calculated via Hill model using GraphPad software. In addition, BLM (100 nM) was incubated with two concentrates of 29 (0.5 μM and 1.0 μM) for 1 h at 37 °C before mixing with duplex forked-DNA and then following the above steps to quantify the disrupting effect of 29. Pull-Down Assay. Streptavidin magnetic beads (Thermo Scientific) were performed in this experiment. After pre-washing, excessive biotin-labeled duplex forked-DNA were added to be incubated with streptavidin magnetic beads at 4 °C overnight. HCT116 cells were harvested with cold PBS. The cell pellets were lysed by RIPA lysis buffer and centrifugated at 12000 rpm for 10 min to obtain the supernatant. Then, the cell lysate was incubated with labeled streptavidin magnetic beads at 25 °C for 1 h with the treatment of 29. After immunoprecipitation, the beads and supernatant were collected respectively and the washing steps following the manufacturer’s protocol. Finally, both the elution and supernatant were heated to 95 °C for 5 min and complemented with loading buffer. The protein was separated using SDS-PAGE and detected by western-blot. Cell Culture and Treatment. Colon cancer HCT116 cells were grown in GIBCO PPMI 1640 medium complemented with 10% fetal bovine serum (FBS) at 37°C constant temperature incubator with 5% CO2. For the drug treatment experiments, cells were treated with 29 and collected for various times in different experiments.

HRR Reporter Assay. HCT116 cells (2×105) were seeded at culture dishes and cultured for 24 h. Then, cells were co-transfected with HRR reporters and I-SceI plasmids following the manufacturer’s protocol of lipo3000 (Invitrogen). After 24 h co-transfection, the medium was discarded and a series concentration of 29 (from 0 to 2 μM) and ML216 (from 0 to 50 μM) were added into per well. Finally, the cells were harvested after 24 h treatment.



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The cell pellets were washed three times and resuspended in cold PBS. The quantities of GFP expressed cells were detected using flow cytometry (EPICS XL). Chromatin Immunoprecipitation Assay. A PierceTM Magnetic ChIP Kit (Thermo Scientific) was used to perform the Chromatin immunoprecipitation (ChIP) experiments according to the manufacturer’s protocol. HCT116 cells were co-transfected with DSB models and I-SceI plasmids. After 24 h co-transfection, a series concentration of 29 (from 0 to 2 μM) and ML216 (50 μM) were added, and cells were harvested and fixed with 1% formaldehyde for 15 min and lysed after 24 h treatment. The chromatins were dissociated into an average size of 0.2-0.5 kb using micrococcal nuclease (10 U/μL) provided in kit. Then, 10% of the lysis was used as input, 2 μg primary antibody of BLM (ab2179, Abcam) and RAD51 (ab133534, Abcam) was used for immunoprecipitation, and 2 μg of normal rabbit IgG (no. 7074S Cell Signaling Technology) was used as negative control. IP reactions were performed overnight at 4 °C. The antigen-antibody complexes were harvested using ChIP Grade Protein A/G M magnetic beads supplied in the kit. After the elution and DNA recovery, the purified DNA were amplified and detected by qPCR. DNA Exchange Activity Assay. Strepta-Well High Bind plates (Roche) were used in this experiment. Final concentration of 50 nM biotin-labeled duplex forked-DNA was planted on the plate at 4 °C overnight. A series concentration of purified RAD51 (from 0 to 500 nM) was added following the ELISA. The absorbance was measured at 450 nm and the effective binding concentration (EC50) of RAD51 with duplex forked-DNA was obtained using GraphPad software via Hill fitting. Then a series of increasing concentration of purified BLM (from 10 to 1000 nM) was incubated with 250 nM of RAD51 at 37 °C for 1 h following the procedure of ELISA. The amount of RAD51 was measured in 450 nm absorbance to present the level of the formation of RAD51-ssDNA filaments. For ligand intervention, BLM and different concentration of 29 were pre-treated at 37 °C for 1 h, and the complex was added to RAD51 solution following the procedure of ELISA. Immunofluorescence Assay. Cells grown on glass coverslips were fixed in 4% paraformaldehyde/PBS for 15 min, then permeabilized with 0.5% Triton-X100/PBS at 37 °C for 20 min, and finally blocked with 5% goat serum/PBS at 37 °C for 3 h. The protocol was followed with theγ-H2AX antibody (no.9718, Cell Signaling


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Technology) and RAD51 antibody (ab133534, Abcam) at 4 °C overnight. The glass coverslips were washed six times with blocking buffer and were then incubated with anti-rabbit Alexa 488-conjugated antibody (A21206, Life Technology), anti-mouse Alexa 647-conjugated antibody (A21235, Life Technology), and 2 μg/mL of 4,6diamidino-2-phenylindole (DAPI, Invitrogen) was diluted in 5% goat serum/PBS at 37 °C for 3 h. The glass coverslips were again washed six times with blocking buffer, and then, digital images were recorded using an LSM710 microscope (Zeiss, GER) and analyzed with ZEN software. Protein Extract and Western Blot Assay. The HCT116 cells were washed with cold PBS and lysed by RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, pH 7.4) at 4 °C for 30 min. The lysis of cells was centrifuged at 12000 rpm at 4 °C for 5 min. The supernatant was diluted to BCA determination and denatured at 95 °C for 5 min with addition of loading buffer (50 mM TrisHCl, 6 M urea, 6% 2-mercaptoethanol, 3% SDS, 0.003% bromophenol blue, pH 6.8), 40 μg of protein was loaded on each lane, run on the SDS-PAGE and transferred to a microporous polyvinylidene difluoride (PVDF) membranes and then detected by western blot. The primary antibodies used in this experiment were β-actin (no. 4970S, Cell Signaling Technology), BLM (ab2179, Abcam), RAD51 (ab133534, Abcam), DNA Damage Antibody Sampler Kit (no. 9947T Cell Signaling Technology), Apoptosis Antibody Sampler Kit (no. 9915T Cell Signaling Technology). And secondary antibodies were horseradish peroxidase-conjugated anti-mouse (no. 7076S Cell Signaling Technology) and anti-rabbit (no. 7074S Cell Signaling Technology). The visualization of protein bands replied on the chemiluminescence substrate. Annexin V-FITC / PI Apoptosis Detection. HCT116 cells (2×105) were planted in culture dish and treated with 29 for 48 h. The apoptosis detection was applied using the FITC Annexin V/PI apoptosis Detection Kit (KeyGEN). The collected cells were resuspended in 500 μL binding buffer and added with 5 μL of FITC Annexin V and PI dyes. Slowly blending the samples and incubating at 25 °C for 30 min away from light. Finally, the fluorescence-positive cells were quantified by flow cytometry (EPICS XL). ITC Studies. The interactions between 29, BLM variants (wild-type, Y995A, H996A, M1111A, E1143A, I1168A) and DNA were measured using isothermal titration calorimetry (ITC) with a VP-isothermal titration



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calorimeter from Microcal, Inc. (Northampton, USA). Calorimetric titrations of 29 (0.6 mM in the syringe), BLM variants (0.04 mM in the cuvette) and DNA (0.04 mM in the cuvette) were carried out at 25 °C in 2 mM MgCl2, 10 mM KH2PO4, pH 7.5. 29 was titrated into BLM variants/DNA in 10 μL injections with a spacing of 300 s between injections. Calorimetric data were analyzed by integrating heat effects normalized to the amount of injected protein and curve-fitting based on a 1:1 binding model using the Origin software package (Microcal). The dissociation constant was derived from data using standard procedures. Molecular Modelling Studies. Docking studies were performed using the Schrodinger Maestro software. The crystal structure of BLM (code ID: 4CGZ) was obtained from the Protein Data Bank. The duplex DNA and ADP in the crystal structure were removed. The structure of 29 was generated using LigPrep module. Possible binding pockets over all the domains in BLM were predicted by SiteMap module. Docking analysis was first performed using the Glide module. 200 poses were generated for each pocket, and post-docking minimization were performed. The binding pose with strong predicted docking score were obtained and clustered. Simulations of molecular dynamics (MD) were performed using the sander module of the AMBER 12 program suite using the parm99bsc0 parameters.47, 48 Partial atomic charges for compound 29 were derived using the HF/6-31G* basis set, followed by an RESP calculation, while force-field parameters were obtained from the generalized Amber force field (GAFF) using the ANTECHAMBER module.49 The complexes of BLMs and compounds were solvated in a rectangular box of TIP3P water molecules with solvent layers of 8 Å. The solvated structures were subjected to initial minimization to equilibrate the solvent and counter cations. The system was then heated from 0 to 300 K in a 100 ps simulation, followed by a 100 ps simulation to equilibrate the density of the system. Subsequently, a constant pressure MD simulation of 20 ns was performed in an NPT ensemble at 1 atm and 300 K. The complete trajectory analysis was performed with the Ptraj module in the Amber 10 suite. The molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method implemented in the AMBER 12 suite was applied to calculate the binding free energy between the BLM and compound 29.50 The models were visualized using the Discovery Studio software package.

ACKNOWLEDGMENTS


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This work was supported by the National Natural Science Foundation of China (81872732, 21708053, 81330077, and 21672265), the Natural Science Foundation of Guangdong Province (2017A030308003 and 2017A030313040), the Ministry of Education of China (No. IRT-17R111), the Fundamental Research Funds for the Central Universities (17ykpy18), the Guangdong Provincial Key Laboratory of Construction Foundation (2017B030314030).

AUTHOR INFORMATION Corresponding Author *S.-B.C.: phone, 8620-39943068; e-mail, [email protected] * Z.-S. H.: phone, 8620-39943056; e-mail, [email protected]. ORCID Tian-Miao Ou: 0000-0002-8176-4576 Jia-Heng Tan: 0000-0002-1612-7482 Shuo-Bin Chen: 0000-0001-9118-2185 Zhi-Shu Huang: 0000-0002-6211-5482

Notes The authors declare no competing financial interests.

ABBREVIATIONS BLM, bloom’s syndrome protein; HRR, homologous recombination repair; SSBs, single-strand breaks; DSBs, double-strand breaks; BER, base excision repair; NER, nucleotide excision repair; MMR, mismatch repair; NHEJ, non-homologous DNA end joining; LOH, loss of heterozygosity; D-loops, displacement loops; MTT, methyl thiazolyl tetrazolium; HRP, horse radish peroxidase; GFP, green fluorescent protein; siRNA, small interfering RNA; qPCR, fluorogenic quantitative PCR; ssDNA, single-strand DNA; PCa, Prostate cancer; RTCA, real-time cellular activity assay; RQC, RecQ C-terminal.


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ANCILLARY INFORMATION Supporting Information Additional experimental results, 1H NMR, 13C NMR, HPLC and HRMS spectra for final compounds, molecular formula strings and coordinates information for structure representation. The Supporting Information is available free of charge on the ACS Publications website. We provide the molecular information for a model (29/4CGZ) in this study. The authors will release the atomic coordinates and experimental data upon article publication.

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(BLM) Helicase Inhibitors That Disrupt the BLM ... - ACS Publications

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