DOI:
10.1039/D3MD00648D
(Research Article)
RSC Med. Chem., 2024,
15, 937-962
Molecular editing of NSC-666719 enabling discovery of benzodithiazinedioxide-guanidines as anticancer agents†
Received
17th November 2023
, Accepted 25th January 2024
First published on 30th January 2024
Abstract
DNA polymerase β (Polβ) is crucial for the base excision repair (BER) pathway of DNA damage repair and is an attractive target for suppressing tumorigenesis as well as chemotherapeutic intervention of cancer. In this study, a unique strategy of scaffold-hopping-based molecular editing of a bioactive agent NSC-666719 was investigated, which led to the development of new molecular motifs with Polβ inhibitory activity. NSC compound and its analogs (two series) were prepared, focusing on pharmacophore-based molecular diversity. Most compounds showed higher activities than the parent NSC-666719 and exhibited effects on apoptosis. The inhibitory activity of Polβ was evaluated in both in vitro reconstituted and in vivo intact cell systems. Compound 10e demonstrated significant Polβ interaction and inhibition characteristics, including direct, non-covalent, reversible, and comparable binding affinity. The investigated approach is useful, and the discovered novel analogs have a high potential for developing as anticancer therapeutics.
1 Introduction
The cellular genome is continuously damaged by endogenous and exogenous agents.1 An elaborate set of different DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), and homologous recombination (HR)2–4 has evolved to maintain the integrity of the cellular genome and to prevent genetic instability and carcinogenesis. BER corrects genomic DNA of damaged nucleotides, abasic sites, and single-strand breaks.5–8 The BER pathway blocking can be an important strategy for cancer chemotherapy and the use of DNA polymerase β (Polβ) inhibitors is one approach that can efficiently block the BER pathway.9–11 Highly specific Polβ inhibitors have been identified that dramatically increase colon cancer cell sensitivity to temozolomide.12,13 Polβ belongs to the X family of DNA polymerases.14 The enzyme has an 8 kDa N-terminal domain (90 amino acids) and a 31 kDa C-terminal domain (250 amino acids).15 Nucleotidyl transferase activity (gap-filling step in BER) is found in the C-terminal domain,16 whereas dRP-lyase activity is present in the N-terminal domain (gap-tailoring step in BER).17 The BER activity of Polβ is altered by several interacting proteins, such as adenomatous polyposis coli (APC),18 X-ray repair cross-complementing protein-1 (XRCC1),19 poly(ADP-ribose)polymerase-1 (PARP-1),20 flap endonuclease-1 (FEN-1),21 apurinic/apyrimidinic endonuclease-1 (APE-1),22 proliferating cell nuclear antigen (PCNA),23 and high-mobility group protein-1 (HMGB1).24,25
Polβ expression has been extensively studied in cancer tissues. Expression arrays have revealed increased levels of Polβ mRNA in about 30% of all screened tumour samples, mostly solid tumours (gastric, uterine, prostate, thyroid and ovarian) and in chronic myeloid leukemia.26,27 An increased level of Polβ has been found in several cancer cells.28,29 Its protein level has also been shown to be increased in response to cytotoxic agents.30 Polβ upregulation contributes to increased resistance against anticancer drugs, such as cisplatin, melphalan and mechlorethamine.31 However, the substantial evidence demonstrates that deregulation of Polβ expression has a detrimental effect on genomic integrity that leads to mutation and cancer.32,33 These signify that a balanced moderate level of inhibition of Polβ function, especially by a combination therapy, would kill the growth of susceptible as well as resistant cancer cells, without mutation.
Many alkylating agents induce DNA damage. If the DNA damages are not repaired before cell division, then the DNA damage accumulates and triggers apoptosis.34 In this study, our aim is to develop Polβ inhibitors which can block the BER pathway. In recent decades, various Polβ inhibitors have been identified from both natural sources and synthetic studies. Specific Polβ inhibitor is useful as a probe to unravel Polβ functions in the various sub-pathways of BER and the crosstalk between repair pathways in vivo.25 Some Polβ inhibitors discovered by us12,13,35 and others are NSC-666715 and NSC-666719, rhodanine scaffold,36 cloretazine, and DNA lesion analogous covalent molecules 3a (ref. 37) and 14 (ref. 38) (Fig. 1).
 |
| Fig. 1 Various DNA Polβ inhibitors. | |
NSC-666719 was designed first for inhibition of viral integration and explored as an anti-HIV compound.39 In our previous study of drug or bioactive compound repurposing using structure-based drug design, we discovered NSC-666719 as a potential Polβ inhibitor.35NSC-666719 molecule interacts with Polβ that induces unique effect in multiple biological pathways. It blocks both single nucleotide (SN)- and long-patch (LP)-BER activities, but does not interfere with Fen1 activity, and enhances the efficacy of temozolomide in cancer cells.12 Interestingly, NSC-666719 is more potent than temozolomide. In a recent study, we also showed that Polβ small molecular inhibitors pamoic acid and NSC-666719 were selectively toxic to BRCA2 deficient cells and associated with double-strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis.40
In this study, we have considered molecular modulation of the hit compound NSC-666719 and investigated to develop its analogs as potent anticancer agents and Polβ inhibitors. We analysed the structures of previously known Polβ inhibitors and their structure–activity relationship (SAR) and found that the common pharmacophores for inhibition of Polβ are bicyclic aromatic/heteroaromatic ring motif and polyphenolic aromatic/heteroaromatic ring motif and carboxylic acid functionality. These pharmacophores indicate that the arene sulphonamide (ring A) and 1,2,4-triazole (ring B) of the NSC compounds are better to be preserved in the new investigational analogs,41,42 however, the naphthalene (ring C) can be modified (Fig. 2). In the modification of ring C, we have considered a scaffold hopping strategy with a wide range of ring C-hopped motifs (Fig. 2).43 With this molecular editing, the structural components of the NSC compound are preserved, but in the form of a new molecular chemotype. Thus, the new analogs were expected to possess similar biofunctional properties.
 |
| Fig. 2 Design of NSC-666719 investigated analogs as potential Polβ inhibitors (series I). | |
In another series, a strategy of molecular editing by ring-opening and ring closing scaffold hopping (2°) was taken. This strategy involves cleavage of 1,2,4-triazole converting into its acyclic motif, and the conversion of benzene-sulfonamide-thiol into its cyclic motif. Both conversions build a new molecular motif, i.e., benzodithiazinedioxide-guanidine (Fig. 3). The benzodithiazinedioxide scaffold is known for its exceptional anticancer efficacy.44–49 The guanidine has a unique feature of being a hydrogen bond donor/acceptor.
 |
| Fig. 3 Design of benzodithiazinedioxide-guanidine derivatives as potential DNA polymerase β inhibitors (series II). | |
In the benzodithiazinedioxide-guanidine series molecules, the naphthyl motif (ring C) has been bioisosterically replaced with a wide range of bicyclic aromatic/heteroaromatic ring motifs.43,50
2 Results and discussion
2.1 Chemistry
Based on previously known reactions,39,51,52 we prepared the compound NSC-666719 and its analogs (Schemes 1 and 2). The compound benzodithiazinedioxide-SMe 6 was prepared by a known synthetic route (Scheme 1a).44 It involves a sequence of reactions, including chlorosulfonylation of arene, sulfonamidation, intramolecular cyclization with carbon disulfide, and S-methylation. The compound 2-naphthylguanidine hydrochloride salt 8a was prepared following a reported reaction51 of naphthylamine with cyanamide and then converted into its non-protonated form 9a by treatment with sodium carbonate (Scheme 1b). The benzodithiazinedioxide-guanidine 10a was synthesized by a SNAr-reaction52 of benzodithiazinedioxide-SMe 6 with naphthylguanidine 9a (Scheme 1c). A reaction39 of the benzodithiazinedioxide-guanidine 10 with hydrazine that undergoes a pathway of cleavage of dithiazinedioxide ring and subsequent formation of an aminotriazole-sulfonamide motif provided the NSC-666719 (Scheme 1c). The thiol and amine groups present in the product were preliminarily identified by staining with TLC visualization reagent, p-anisaldehyde–ethanol solution, which gave dark pink color. The structure of the NSC-666719 was well characterized by various spectroscopic and spectrometric techniques, 1H NMR, 13C NMR, IR, and HRMS. This synthetic route for access to NSC-666719 involves eleven reaction steps and is a convergent process. The overall yield of the NSC-666719 in the process was 12.7%.
 |
| Scheme 1 Synthetic scheme for the synthesis of NSC-666719. Reagents and conditions: (a) ClSO3H, 0 °C to 30 °C, 4 h, 74%; (b) aq. NH4OH (25%), 80 °C, 15 min, 78%; (c) CS2, KOH, EtOH, 30 °C, 6 h then 90 °C, 48 h, 68%; (d) aq. HCl, 25 °C, 3 h, 92%; (e) Me2SO4, NaOH, H2O, 0–20 °C, 4 h, 91%; (f) CuI, DMEDA, NH4OH–DMSO (3 : 1), 150 °C, 12 h, Ar, 75%; (g) cyanamide, Sc(OTf)3, H2O, 100 °C, 24 h, 48%; (h) sat. Na2CO3 soln, 60 °C, 5 h, 96%; (i) anhyd. toluene, 120 °C, 48 h, Ar, 76%; (j) NH2NH2·H2O, EtOH, 35 °C, 48 h, 51%. | |
 |
| Scheme 2 Synthesis of NSC-666719 scaffold-hopped analogs 10b–z, 10za and 10zb, and 11b–s. Reagents and conditions: (a) cyanamide, AlCl3, EtOH, μW, 130 °C, 10 min, 69–95%; (b) Pd2(dba)3, xantphos, Cs2CO3, anhyd. toluene, 100 °C, 6–24 h, Ar, 60–92%; (c) 6 M HCl, H2O/acetic acid, 100 °C, 8–24 h, 34–81%; (d) Na2CO3, anhyd. toluene, 60 °C, 3–7 h; (e) 120 °C, 22–67 h, 22–71%; (f) NH2NH2·H2O, EtOH, 35 °C, 48 h, 16–75%. | |
In the synthetic route for access to NSC compound skeleton, naphthyl guanidine as a substrate introduces the naphthyl motif in the product. We were interested to obtain the scaffold-hopped analogs of NSC skeleton with structurally-varied arenes and heteroarenes in place of naphthyl motif. Therefore, we planned next to synthesize N-aryl and heteroaryl-guanidine hydrochlorides with a broader variety of pharmacophoric aromatic or heteroaromatic scaffolds. We synthesized N-arylguanidines (Table S2;†8b–f, 8u, and 8x) following a reaction (Scheme 2a)53–56 of arylamines with cyanamide. Then, we prepared more structurally-varied guanidines from aromatic and heteroaromatic halides. We performed the sequential reactions (Scheme 2b)57,58 of Pd-catalyzed C–N coupling of Ar/HetAr halides with 2-amino-4,6-dimethoxy pyrimidine and subsequent acidic hydrolysis-mediated ring cleavage of 3,5-dimethoxypyrimidine motif, which produced N-Ar/HetAr-guanidine hydrochlorides (Table S3;†8g–t, 8v–w, 8y–z, 8za, and 8zb).
Synthesis of benzodithiazinedioxide-guanidines (10b–z, 10za and 10zb) and NSC-666719 analogs (11b–s).
Ar/HetAr-guanidine hydrochlorides were de-protonated by Na2CO3 in toluene and used in the in situ reaction with benzodithiazinedioxide-SMe 6 (Scheme 2c). Following the in situ methodology, benzodithiazinedioxide-guanidines were prepared (Fig. 4; 10b–z, 10za, and 10zb) in good yields. Pharmacophoric bicyclic aromatic/heteroaromatic ring motifs with anticancer importance, such as substituted aryls, morpholino phenyl, naphthyls, substituted naphthyls, naphthoyl, quinolines, substituted quinolines, and isoquinolines, substituted quinazoline, quinoxaline, indole, pyrrolopyrimidine, and pyridopyrimidinone, were assembled into the synthesized benzodithiazinedioxide-guanidine derivatives. Then, benzodithiazinedioxide-guanidine derivatives (10) were reacted with hydrazine monohydrate in ethanol (Scheme 2c; step-f) to produce the desired NSC analogs (Fig. 4, 11b–s). We considered various bicyclic aryl/heteroaryl motifs in place of naphthyl of NSC-666719 compound. The motifs are substituted and functionalized naphthyls, quinolines, and isoquinolines. Quinolines were also linked to 1,2,4-triazole amine via their different positions, such as 3, 4, 5, 6, and 8 of the ring in the synthesized compounds (Fig. 4). All the products were well characterized by spectroscopic and spectrometric techniques with 1H and 13C NMR, IR, and HRMS.
 |
| Fig. 4 Synthesized benzodithiazinedioxide-guanidine derivatives and NSC-666719 new analogs; yields of the last reaction steps are mentioned. | |
The structure of one of the synthesized compounds, i.e., 10b was determined by single X-ray crystallography (Fig. 5, CCDC no. 2269969). The molecular skeleton of the compound (10b) possesses Z-configuration of two aryls, as revealed in the X-ray crystal structure.
 |
| Fig. 5 2-Dimensional and ORTEP diagram of (Z)-2-(6-chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(4-methoxyphenyl)guanidine (10b). | |
2.2 Bio-evaluation studies
2.2.1 Antiproliferative potential of synthesized NSC-analog compounds.
The anti-proliferative activity of synthesized compounds (series I & II) was evaluated in human breast adenocarcinoma cells (MCF-7) and highly metastatic breast adenocarcinoma cells (MDA-MB-231) using MTT-assay with 48 h of treatment.59 The original NSC compound (NSC-666719; compound 11a) and an anticancer drug etoposide were used as positive control and reference to make a comparison of the potency of the synthesized NSC-analogs. It was observed that all the NSC-analog compounds showed a characteristic cytotoxicity against cancer cells and half-maximal inhibitory concentration (IC50) (Table 1). Among two series analogs, the compounds 11n, 11p, 10e, 10n, 10q, 10s, and 10w were found to be relatively more active in MCF7 and MDA-MB-231 cells with IC50 at 2.8 and 3.2 μM, 1.8 and 2.6 μM, 1 and 2.6 μM, 1.1 and 2.4 μM, 0.8 and 2.1 μM, 1 and 2.5 μM, 1.3 and 2.6 μM, respectively. These NSC-analog compounds showed antiproliferative properties with relatively higher potency compared to original NSC compound, [IC50 of NSC-666719 (11a) – 4.4, 5.5 μM] and etoposide. These selected NSC analogs were then tested in other cancer cells, HCT-116 (colon cancer), and SSC-9 (oral cancer) and were found to exhibit IC50 below 5 μM. The NSC analogs 10e and 10q were identified as most antiproliferative active with IC50 values of 3.2 and 2.8 μM in HCT-116 cells, and 0.8 and 1 μM in SCC-9 cells, respectively (Table 2). The cytotoxicity of these compounds was tested in human embryonic kidney epithelial normal cells (HEK-293) and IC50 were found to be 65–72 μM (Table 3). Interestingly, NSC analogs were selectively much higher cytotoxic to various cancer cell lines compared to normal cell line (SI > 65). All these MTT-studies indicate the important antiproliferative features of some of the NSC analogs; potent antiproliferative activity (>5-times) compared to the original NSC-666719 compound, IC50 values up to sub-micromolar concentrations, and much higher selective cytotoxicity to cancer cells compared to normal cells. Most potent analogs of NSC-666719 (compounds 10e, 10q) were selected as representative compounds for further studies of apoptosis and Polβ inhibitory activity.
Table 1 Antiproliferative activity (IC50 values) of synthesized NSC-analogs (compounds 11a–s and 10a–z) and original NSC compound (NSC-666719; compound 11a), and an anticancer drug etoposide
Table 2 Antiproliferative activity of selected more active analogs and NSC compound from series I (11) and series II (10) in various cancer cell lines
Table 3 Cytotoxicity of selected more active analogs in human embryonic kidney epithelial normal cells (HEK-293)
2.2.2 Structure–activity relationship studies (SAR).
In the 1,2,4-triazolobenzenesulphonamide compounds series (series I), the anticancer efficiency of the analogs increased when the 2-naphthyl group of the parent NSC compound (NSC-666719; compound 11a) is replaced with naphthoyl (11n), 4-bromophenyl (11f), or 4-isoquinolinyl (11p). Many scaffold-hopped analogs (series II, Table 1) showed higher antiproliferative efficiency than the NSC compound and its class of compounds (series I). In addition, the anticancer effectiveness increased when 1-naphthyl (10e), 4-morpholinophenyl (10n), 7-chloro-4-quinoline (10q), pyridopyrimidinone (10s), or 5-isoquinoline (10w) was incorporated in place of the 2-naphthyl motif. These activity profile of the series I and series II molecular skeleton-based compounds indicate that the strategy of ring-opening and ring-closing based scaffold hopping (2°) of triazolobenzenesulphonamide (series I) converting into new skeleton benzodithiazinedioxide-guanidine (series II) is important for increasing the anticancer activity.
 |
| Fig. 6 Structure–activity relationship (SAR) studies of series I and II compounds. | |
2.2.3 Compounds increase the apoptotic nuclei in cancer cells.
The apoptotic potential of compounds 10e and 10q were analysed by DAPI nuclear staining assay60 after treatment of MCF-7 cells with compound of varied concentrations for 48 h. Treatments were carried out by following concentrations of 0.5, 1.0, and 2.0 μM for 10e and 0.5, 0.8, and 2.0 μM for 10q exposure conditions with respect to control. It was observed that both the compounds with increasing concentrations showed higher shrinkage and deformation of nuclei, as compared to the control (Fig. 7A). The percentages of apoptotic and non-apoptotic nuclei are presented in bar graphs (Fig. 7B). The compounds 10e and 10q at 2.0 μM treatment caused 68% and 78% apoptotic nuclei, respectively, whereas 5% apoptotic nuclei were observed in control (untreated with compound). Results indicate that both the compounds caused a dose-dependent increase of apoptotic nuclei and a much greater number of apoptotic nuclei compared to the untreated cells.
 |
| Fig. 7 (A) DAPI-stained nuclei of MCF-7 cells after treatment with compounds 10e and 10q. (B) Apoptotic and non-apoptotic nuclei presented in bar graph. The experiment was carried out according to protocol described in the methods and material section. The results are presented as mean ± standard deviation (SD) of five individual experiments. GraphPad Prism 5.0 software, USA was used for statistical analysis. One-way ANOVA was used to analyze the data followed by Bonferroni's multiple comparison tests. The statistical significance was denoted as ‘***’ (p < 0.0001). | |
2.2.4 Compounds induces the DNA damages in cancer cells.
Gamma-H2AX (γH2AX) is a phosphorylated form of histone H2AX that acts as a highly sensitive biomarker for DNA damage. The phosphorylation of γH2AX plays an essential role in the DNA damage response (DDR) process by enabling the recruitment of DNA repair proteins to the damaged chromatin regions. As a result, an increase in γH2AX expression reflects an increase in DNA damage.61,62 Thus, to investigate the potential effect of compound in DNA damage, MCF-7 cells were treated with compounds 10e (0.5, 1.0, and 2.0 μM) and 10q (0.5, 0.8, and 2.0 μM) in a dose-dependent manner (Fig. 8A and B). Approximately, 6.9-fold and 7.3-fold (P < 0.0001) increase in γH2AX expression was observed while treated with the compounds 10e and 10q of 2.0 μM concentration, respectively, as compared to untreated control (Fig. 8B). Thus, these results suggest that the compounds 10e and 10q significantly induce DNA-damage inside cancer cells leading to apoptosis.
 |
| Fig. 8 (A) The expression of γH2AX after treatment with compounds 10e and 10q in MCF-7 cells. (B) The bar graph represents relative mean intensity in the expression of γH2AX-TRITC. The cells were grown and treated with the compounds for 48 h. The experiment was carried out according to protocol described in the methods and material section. The results are presented as mean ± standard deviation (SD) of three individual experiments. GraphPad Prism 5.0 software, USA was used for statistical analysis. One-way ANOVA was used to analyze the data followed by Bonferroni's multiple comparison tests. The statistical significance was denoted as ‘***’ (p < 0.0001). | |
2.2.5 Compounds increase the expression of caspase-3 in cancer cells.
To correlate the increased levels of γH2AX in MCF-7 cells with cell death after 10e and 10q treatments, we performed apoptosis assays. Caspase-3 activity is an important indicator or marker for apoptosis. Higher caspase-3 expression is a sign of increased apoptosis, particularly in cancer cells.61 Thus, to investigate the potential effect of 10e and 10q in inducing apoptosis in cancer cells, MCF-7 cells were treated with varied concentrations of 10e (0.5, 1.0, and 2.0 μM) and 10q (0.5, 0.8, and 2.0 μM) compounds (Fig. 9). It was observed that the expression of caspase-3 increased in a dose-dependent manner, as compared to control (Fig. 9A). Approximately, 7.8- and 8.3-folds (P < 0.0001) higher caspase-3 expression was observed in the cells treated with 2.0 μM of 10e and 10q, respectively, as compared to control (Fig. 9B). Therefore, the study indicates that the compounds show their effect of increasing caspase-3 expression in cancer cells, which ultimately leads to apoptosis.
 |
| Fig. 9 (A) The expression of caspase-3 after treatment with compounds 10e and 10q in MCF-7 cells (B). The bar graph represents a relative fold increase in the expression of caspase-3-TRITC. The detail experimental protocol was described in the methods and material section. The results were expressed as mean ± standard deviation (SD) of three independent experiments (n = 3). The statistical analysis was performed by using GraphPad Prism 5.0 software, USA. Statistical significance of differences was denoted as ‘***’ (p < 0.0001). | |
2.2.6 Compounds induce the apoptotic cell populations in cancer cells.
For determination of apoptosis, an Annexin-V-FITC/PI dual-staining assay also was performed.63 Approximately, 10.2%, 40.4% and 48.2% of apoptotic cell populations were noted in MCF-7 cells after treatment with 0.5, 1.0, and 2.0 μM, of the compound 10e. The treatment of MCF cells with the compound 10q of 0.5, 0.8, and 2.0 μM concentrations resulted in a similar apoptotic cell population, 12.6%, 38.2%, and 49.1% (Fig. 10).
 |
| Fig. 10 Annexin-V/FITC dual staining apoptosis assay using FACS. (A) Measurement of apoptosis by Annexin-V-FITC–PI dual staining assay in MCF-7 cells after treating with compounds 10e and 10q using FACS according to protocol described in methods and material section. Q1, and Q2 represent the necrotic and true apoptotic cells, respectively, and both Q3 and Q4 represent live cells. Cells were quantified as mean percent cells ± SD for each quadrant (three independent set of experiments). (B) Statistical significance was measured for every quadrant in 10e and 10q treated cells with respect to control through one-way ANOVA, where b.i–b.iii and c.i–c.iii were significantly increased (P < 0.0001) with respect to a. Similarly e.i–e.iii and f.i–f.iii were also enhanced (P < 0.0001) as compared to d. Here P < 0.0001 represented the statistical significance. | |
2.2.7 Compounds affect the expression of apoptosis-related proteins.
The expression of apoptosis-related proteins (Bcl-xl and Bax) were measured by western blot analysis64 before (control) and after the treatment of MCF-7 cells with varied concentrations of compound 10e (0.5, 1.0, and 2.0 μM) and compound 10q (0.5, 0.8, and 2.0 μM) for 48 h. After treatment with the compound 10e at its IC50 concentration (1.0 μM), we observed that the expression of Bcl-xl was downregulated up to 2-fold, whereas Bax was upregulated up to 2.7-fold, as compared to control (Fig. 11). Similarly, the compound 10q of its IC50 concentration (0.8 μM) showed a reduced expression of Bcl-xl (2.5-fold), while a 4.5-fold increased expression of Bax was noticed in comparison with control (Fig. 11). Further, the Bax/Bcl-xl ratio was elevated by 26.5- and 66-folds in MCF-7 cells treated with 2 μM of the compounds 10e and 10q, respectively, with respect to untreated control. Additionally, caspase-3 expression was also upregulated by increasing concentration of the compounds 10e and 10q. The compound 10e showed an enhanced caspase-3 expression by 2.9- and 5.1-fold at 1.0 μM and 2.0 μM treatment condition, whereas the compound 10q exposure elevated the expression by 3.2- and 5.7-fold at 0.8 μM and 2.0 μM concentration, respectively.
 |
| Fig. 11 Western blot analysis of whole cell extracts of MCF-7 cells after treating with compounds 10e and 10q. GAPDH served as loading control. Western blot was done according to protocol stated in methods and material section. The numerical values above each blot represent the relative fold change in protein expression with respect to control. | |
2.2.8 Compound 10e inhibits the Polβ activity in in vitro Polβ nucleotide insertion assay.
To evaluate potential effect of the compound on the Polβ inhibitory activity, we used a reconstituted in vitro BER assay system with purified Polβ. We investigated the effect of the parent NSC compound (NSC-666719; 11a) and the most active NSC-analog 10e on the Polβ activity using compounds of concentrations of 0–5 μM, a range covering IC50 values of both the compounds. A dose-dependent decrease in the gap-filling activity of Polβ was observed for both the compounds 11a and 10e (Fig. 12).
 |
| Fig. 12 Impact of the NSC compound and a representative most active NSC-analog (10e) on Polβ activity. (A) Schematic presentation of the insertion assay used to monitor the gap filling activity of Polβ. (B) Lane 1 is the negative enzyme control containing the reaction mixture and the gap DNA substrate but no Polβ. Lanes 2–5 are the positive control of Polβ dGTP:C insertion products in the absence of the compound. Lanes 6–9, 10–13, and 14–17 are Polβ dGTP:C insertion products in the presence of compound 10e at concentrations of 1, 3, and 5 μM, respectively, and correspond to time points of 15, 30, 45, and 60 s. (C and D) Graphs show time-dependent changes in the amount of Polβ insertion products for the compound 11a (C) and the compound 10e (D). The data represents the average from three independent experiments ± SD. | |
2.2.9 Compound 10e inhibits the Polβ activity in cancer cells in an in vivo BER assay.
Next, we performed an in vivo BER assay to confirm whether the compound 10e shows BER inhibitory activity. Plasmid-based BER assay was done to quantify BER activity.65 Approximately, 3.2-fold and 4.8-fold decreased luciferase activity was observed in U-p21p (SP-BER) and R-p21p (LP-BER) transfected cells (P < 0.0001), respectively, after treatment with 2 μM of the compound 10e (Fig. 13). Since the reduced U-p21p and R-p21p promoter activities reflect reduced BER, the results obtained here suggest that the compound 10e inhibits both SP- and LP-BER in intact cells. Thus, interestingly, these results suggest that we have identified a potent NSC-analog 10e which inhibits the BER pathway by deregulating the Polβ activity both in vitro reconstituted system and an in vivo intact cell system and enhances single strand breaks which ultimately lead to increased cytotoxicity and apoptosis in cancer cells.
 |
| Fig. 13 Bar graph represents the relative luciferase activity of U-p21p and R-p21p DNA transiently transfected in MCF-7 cells after treatment with and without compound 10e and experiment was carried out according to methods and material. One-way ANOVA was used to analyze the data followed by Bonferroni's multiple comparison tests. The statistical significance was denoted as ‘***’ (p < 0.0001), ‘**’ (p < 0.005). | |
2.2.10 Binding characteristics of Polβ and compound 10e by surface plasmon resonance (SPR) assay.
A SPR sensorgram study was done (Fig. 14A). Both the compounds 11a and 10e showed non-covalently reversible binding to Polβ. Then, concentration gradient tests were performed using both the compounds. The binding signal of the NSC-analog 10e dose-dependently increased with higher binding affinity and KD = 20.11 ± 4.412 μM (Fig. 14B and C). Meanwhile, the binding signal of the NSC compound 11a was found non-related to compound concentrations. In addition, the negative SPR signal observed for compounds 11a and 10e binding to Polβ may be due to a non-oriented functionalization of Polβ to the biosensor. It is likely that after binding with compounds 11a and 10e the Polβ protein goes through confirmational change and produces negative SPR response. In previous studies, a similar negative SPR response was observed with other proteins and their ligand interactions.66–68 Thus, the results of our studies suggest that the NSC analog 10e possess higher Polβ-binding efficiency than the original NSC compound (11a). In addition, the study provides evidence that conformational transitions of free and complexed proteins are important dynamic features involved in Polβ activity regulation.
 |
| Fig. 14 Characterization of NSC compound 11a (NSC-666719) and NSC-analog 10e binding mode using surface plasmon resonance (SPR). (A) Sensorgram of Polβ binding comparison of 40 μM NSC compound 11a and NSC-analog 10e; (B) concentration gradient testing of NSC-analog 10e; (C) KD measurement of compound 10e. | |
2.3 Computational studies of the interactions between Polβ and compounds 10e and 11a
To further confirm better binding of 10e as compared to 11a, both the compounds were docked to human DNA Polβ using PDBID: 1BPZ. Docking was performed in the same binding region as reported previously12,13 using Maestro software (13.2.128; Schrodinger). Top poses with lowest possible energy were analysed and the binding energies of the compounds are shown in Table 4. Compound 10e displayed more interaction (Fig. 15) with the protein having low binding energy as compared to compound 11a. Close inspection of the interactions of 10e revealed that the NH2 group on the linker interacts with residues Gly14 and Met18 through hydrogen bonding having bond length 1.96 Å and 2.80 Å, respectively. The terminal naphthalene ring of the molecule interacts with the residue Phe76 through π–π stacking interactions. Benzodithiazinedioxide moiety of the molecule interacts with amino acid residue of Arg89 through π–cation interaction. In contrast, compound 11a's triazole ring established hydrogen bonds with the Glu75 and Arg83 residues. However, intriguingly, the terminal aromatic rings (benzene and naphthalene) of the compound exhibited negligible interaction with the protein, thus resulting in a lower energy score in comparison to compound 10e. This difference in binding energies stems from the inherent structural differences between compounds 10e and 11a. In the case of compound 10e, its two-ring system is linked via a flexible chain, imparting a certain degree of flexibility to the overall molecule when situated within a protein structure. On the contrary, in the case of compound 11a, the connection between the two ring systems involves a triazole ring, making the compound considerably more rigid as compared to compound 10e. This increased rigidity apparently contributes to a lower energy score observed for compound 11a.
Table 4 Binding energy of compounds 11a and 10e with DNA Polβ along with interacting residues
S. no |
Code |
Binding energy ΔG (kcal mol−1) |
Physically interacting residue |
Spatial interacting residue |
1 |
11a
|
−27.75 |
Arg83, Glu75 |
Asp74, Glu71, Ilc69, Lys72, Lys84 |
2 |
10e
|
−39.59 |
Gly14, Met18, Phe76, Arg89 |
Gly80, Asp17, Lys81, Leu82, Leu85, Glu21, Glu86 |
 |
| Fig. 15 (A) and (C) 3D representations of the binding of compounds 11a (A) and 10e (C) with human DNA Polβ (PDBID: 1BPZ); (B) and (D) 2D representations of binding interactions of 11a (B) and 10e (D). | |
3 Conclusions
The present study shows importance of a medicinal chemistry strategy, pharmacophore-based molecular editing of a NSC bioactive skeleton with “cyclic-to-acyclic” and “acyclic-to-cyclic” switches on two separate motifs, which enabled discovery of potent DNA polymerase β targeting anticancer agents. NSC-666719 and its analogs were prepared by a synthetic route comprising of numerous key reaction steps, construction of benzodithiazinedioxide ring, installation of guanidine linked with ring, hydrazine-mediated cleavage of benzodithiazinedioxide and formation of triazole-sulfonamide. Total 19 analogs of series I and 26 analogs of series II with relevant substitution motifs and functionalities were synthesized and investigated. Interestingly, many compounds from both the series exhibited characteristic pronounced antiproliferative activities and several showed higher activities (sub-micromolar IC50; more than five-fold) than the original NSC-666719 (11a). Most potent compounds 10e and 10q, investigated further as representative examples, significantly induced DNA-damages in cancer cells leading to apoptosis. The compounds showed more pronounced effect in the expression of apoptotic proteins in cancer cells, such as increase in the expression of caspase-3, downregulation in the expression of Bcl-xl, upregulation of Bax and elevation in Bax/Bcl-xl ratio, and furthermore, exhibited high increase in apoptotic cell population. In vitro Polβ nucleotide insertion assays using purified reconstituted in vitro BER assay showed significant effect of compound 10e in the inhibition of Polβ-mediated dGTP:C insertion; the activity was comparable to that of the NSC-666719 (11a). Both the original NSC compound and its analog 10e exhibited similar dose-dependent decrease in the gap-filling activity of Polβ. The inhibitory effect of 10e was found to be more pronounced at higher concentrations than the IC50 values of cellular assays. Overall, there is a correlation between compound's effect on cancer cells antiproliferative activities and the inhibition of Polβ. The compound 10e displayed interesting behaviour in an in vivo BER assay; the inhibition in both SP- and LP-BER. The results indicate that the analog 10e inhibited Polβ activity in both in vitro reconstituted and in vivo intact cell systems and increases cytotoxicity and apoptosis in cellular assays. Finally, the SPR experiments showed a direct, non-covalent, reversible, and significant interaction of compound 10e to Polβ (KD = 20.11 ± 4.412 μM) that was higher binding efficiency than NSC-666719 (11a). Molecular docking studies further demonstrated Polβ-biding efficiency of NSC analog 10e as compared to parent NSC compound (NSC-666719; compound 11a). The NSC analog 10e thus makes a promising lead compound for developing clinically relevant Polβ inhibitors as cancer therapeutics and warrants further preclinical evaluation, and structural optimization as needed in the future.
4 Experimental section
4.1 Chemistry
General information: without additional purification, all the starting materials and solvents were used as supplied from commercial sources. In some situations, anhydrous solvents were used and purified using MBRAUN's MB SPS-800 anhydrous solvent preparation apparatus. Throughout this study, all reactions were monitored using thin-layer chromatography (TLC) and visualised under UV light. For reaction monitoring, Merck Life Sciences Private Limited aluminium pre-coated TLC plates (silica gel 60 F254, 0.2 mm) were used. To isolate the products from the reaction mixture in column chromatography, silica gel 100–200 (silica gel 100–200 mesh, neutral, spherical) was used. Biotage Initiator EXP EU microwave synthesizer was utilised for carrying out reaction under microwave irradiation. On a digital melting point equipment (PERFIT INDIA), the melting points of the synthesised compounds were recorded. The ATR Microscope spectrometer (PerkinElmer FTIR) was used to record the infrared (IR) spectra of the synthesised compounds. On a Bruker Avance III-400 (400 MHz) and Jeol 500 MHz spectrometers, proton nuclear magnetic resonance spectra (1H NMR) were recorded. Chemical shifts (δ) are provided in ppm in CDCl3 or DMSO-d6 with integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dt = doublet of triplet, dd = doublet of doublet, br. = broad), and J = coupling constants (Hz) using tetramethylsilane as an internal standard. On a Bruker Avance III-400 (100 MHz) and Jeol 125 MHz spectrometers with complete proton decoupling, 13C{1H}-NMR spectra were acquired. High-resolution mass spectra (HRMS) were obtained using an Agilent technologies high-resolution LCMS/MS instrument with a “QTOF” mass analyser, and mass spectra were obtained using an MS instrument. With the LC-6AD (Shimadzu Corporation), the compounds 2–6 (Scheme 1) were synthesized following known methods.44,69
4.1.1 Procedure for the synthesis of 2-naphthylguanidine (Scheme 1, 9a).
2-Naphthylguanidine salt (1 mmol) was added to a saturated aqueous solution of sodium carbonate (Na2CO3), and the reaction mixture was stirred at room temperature under open air conditions for 6 h. After the reaction was completed as confirmed by TLC, the reaction mixture was filtered using a vacuum pump, and the filter cake was dried at reduced pressure, resulting in a pale red colour solid that was stored in a tightly packed container and used for the next reaction.
4.1.2 Procedure for the synthesis of 1-(6-chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-3-(naphthalen-2-yl) guanidine (Scheme 1, 10a).
39
Under nitrogen atmosphere, 6-chloro-7-methyl-3-(methylthio)benzo[e][1,4,2]dithiazine 1,1-dioxide (1 mmol) and 2-naphthylguanidine (1.5 mmol) were taken in an oven dried sealed tube containing a magnetic bar. The mixture was then degassed with nitrogen and was added anhydrous toluene (2 mL). The tube was capped, and the mixture was stirred at 120 °C for 48 h. The reaction mixture was diluted with ethyl acetate (30 mL). The diluted ethyl acetate solution was washed with 50 mL of 1 N NaOH solution, 50 mL of water, and 50 mL of brine solution, then dried over anhydrous sodium sulphate and concentrated under reduced pressure. The organic residue was purified using EtOAc–hexane, 70
:
30 v/v, column chromatography over silica gel, which provided the desired product in 76% yield.
4.1.3 Characterisation data of synthesized compound (Scheme 2, 10a).
4.1.3.1 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(naphthalen-2-yl)guanidine (Scheme 1, 10a).
Purification by column chromatography (EtOAc–hexane, 70
:
30 v/v) afforded 10a; white solid (327 mg, yield 76%); m.p. 257 °C; IR (KBr): νmax 3294.1, 2294.1, 1987.2, 1492.8, 1421.2, 1335.7, 1286.1, 1026.4, 929.1, 754.9 cm−1; 1H NMR (600 MHz, DMSO-d6): δ 10.18 (s, 1H), 8.50 (s, 1H), 8.07 (s, 1H), 7.93 (t, J = 4.3 Hz, 2H), 7.90–7.86 (m, 2H), 7.84 (d, J = 1.1 Hz, 1H), 7.75 (s, 1H), 7.52–7.45 (m, 2H), 7.42 (d, J = 8.6 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H}4 (151 MHz, DMSO-d6): δ 168.2, 156.8, 137.5, 136.8, 133.7, 131.5, 131.0, 130.4, 129.5, 128.9, 128.1, 127.6, 127.2, 126.7, 126.4, 123.4, 19.8; HRMS (ESI+) calcd for C19H16ClN4O2S2+ [M + H+] 431.0398, found 431.0401.
4.1.4 Procedure for the synthesis of 4-chloro-2-mercapto-5-methyl-N-(3-(naphthalen-2-ylamino)-1H-1,2,4-triazol-5-yl)benzene sulfonamide (Scheme 1, NSC-666719 or 11a).
39
Hydrazine monohydrate (1.5 mmol) was added to a solution of 1-(6-chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-3-(naphthalen-2-yl) guanidine (1 mmol) in ethanol, and the reaction mixture was stirred at room temperature under open air conditions for 48 h. After the reaction completed as confirmed by TLC, the solvent was evaporated under vacuum. The resultant mixture was subjected to column chromatography using silica gel eluting with solvent MeOH–DCM, 10
:
90 v/v, which provided the desired product in 71% yield.
4.1.5 Characterisation data of synthesized final compound (Scheme 1, NSC-666719 or 11a).
4.1.5.1 4-Chloro-2-mercapto-5-methyl-N-(3-(naphthalen-2-ylamino)-1H-1,2,4-triazol-5-yl)benzene sulfonamide (Scheme 1, NSC-666719 or 11a).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded NSC-666719; white solid (317 mg, yield 71%); m.p. 291–295 °C; IR (KBr): νmax 3295.7, 2191.6, 1618.7, 1568.9, 1440.1, 1343.9, 1295.9, 1144.7, 1068.8, 995.70, 936.4, 722.9 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.56 (s, 1H), 12.25 (s, 1H), 9.23 (s, 1H), 8.01 (d, J = 8.0 Hz, 2H), 7.81 (t, J = 8.4 Hz, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.64 (s, 1H), 7.42 (d, J = 7.9 Hz, 2H), 7.34 (t, J = 7.1 Hz, 1H), 2.31 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 148.7, 147.3, 140.1, 137.8, 137.6, 134.8, 134.2, 132.9, 131.7, 129.1, 127.9, 127.1, 127.0, 126.3, 124.2, 119.3, 111.8, 19.4; HRMS (ESI+) calcd for C19H17ClN5O2S2+ [M + H]+ 446.0507, found 446.0528.
4.1.6 Synthesis of N-Ar/HetAr-guanidine hydrochlorides from arylamines (Scheme 2, 8b–8f, 8u and 8x).
51
The reported method with some modifications was followed. Anhydrous aluminium chloride was used as catalyst instead of Sc(OTf)3 or HCl and the reaction was done following this procedure. An oven-dried sealed tube containing a magnetic bar was charged with anhydrous AlCl3 (10 mol%). Ethanol (2 mL) was slowly added to it, followed by cyanamide (2 mmol). The septum was used to close the tube, and the mixture was stirred at room temperature for 5 minutes. To the reaction mixture was then added arylamine (1 mmol). The tube was then capped, and the mixture was irradiated to microwave for 10 minutes at 130 °C. The reaction mixture was diluted with methanol. Column chromatography over silica gel, eluting with the solvent's mixture MeOH–DCM provided the desired N-arylguanidine hydrochloride. The characterization data of 8b–8f, 8u and 8x (Scheme 2, Table S2†) were in accordance with the data previously published.51,53–56
For the synthesis of N-heteroarylguanidine hydrochlorides 8e–8t, 8v, 8y–z, 8za, 8zb (Scheme 2, Table S3†) from heteroaromatic bromides, a known procedure was followed.57,58 The characterization data of 8e–8t, 8v, 8y–z, 8za, 8zb (Scheme 2, Table S3†) were mentioned in ESI.†
4.1.7 General procedure for the synthesis of benzodithiazinedioxide-guanidine derivatives (10b–z).
2-Aryl/heteroarylguanidine hydrochloride (8, 1 mmol) was added to a solution of sodium carbonate (Na2CO3, 1.5 mmol) in toluene, and the reaction mixture was stirred at room temperature under open-air conditions for 4–8 h. After the reaction completed as confirmed by TLC, 6-chloro-7-methyl-3-(methylthio)benzo[e][1,4,2]dithiazine 1,1-dioxide (6) was added to the reaction mixture in situ, and the reaction mixture was refluxed at 120 °C for 48 h. The reaction mixture was diluted with ethyl acetate (30 mL). The diluted ethyl acetate solution was washed with 50 mL of 1 N NaOH solution, 50 mL of water, and 50 mL of brine solution, then dried over anhydrous sodium sulphate and concentrated under reduced pressure. The organic residue was purified by column chromatography over silica gel, using EtOAc–hexane as solvents.
4.1.8 Characterization data of benzodithiazinedioxide-guanidine derivatives (10b–z).
4.1.8.1 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(4-methoxyphenyl)guanidine (Fig. 4, 10b).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10b; white solid (291 mg, yield 71%); m.p. 247 °C; 1H NMR (600 MHz, DMSO-d6): δ 9.90 (s, 1H), 8.42 (s, 1H), 7.91 (s, 1H), 7.79 (s, 1H), 7.73 (s, 1H), 7.19 (d, J = 7.6 Hz, 2H), 6.95 (d, J = 4.6 Hz, 2H), 3.73 (s, 3H), 2.37 (s, 3H); 13C NMR {1H} (150 MHz, DMSO-d6): δ 167.9, 137.4, 136.7, 131.1, 130.5, 127.6, 126.7, 115.1, 55.8, 19.8; HRMS (ESI+) calcd for C16H16ClN4O3S2+ [M + H+] 411.0347, found 411.0349.
4.1.8.2 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(4-chlorophenyl)guanidine (Fig. 4, 10c).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10c; white solid (232 mg, yield 54%); m.p. 218 °C; 1H NMR (600 MHz, DMSO-d6): δ 9.94 (s, 1H), 8.40 (s, 1H), 8.03 (s, 1H), 7.75 (s, 1H), 7.42 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.7 Hz, 2H), 2.38 (s, 3H); 13C NMR {1H} (150 MHz, DMSO-d6): δ 168.4, 156.42, 137.5, 136.9, 130.9, 130.2, 129.6, 127.6, 126.7, 125.7, 19.8; HRMS (ESI+) calcd for C15H13Cl2N4O2S2+ [M + H+] 414.9851, found 414.9853.
4.1.8.3 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(3,4,5-trimethoxyphenyl)guanidine (Fig. 4, 10d).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10d; white solid (272 mg, yield 58%); m.p. 261 °C; 1H NMR (600 MHz, DMSO-d6): δ 9.96 (s, 1H), 8.44 (s, 1H), 7.91 (s, 2H), 7.74 (s, 1H), 6.61 (s, 2H), 3.74 (s, 6H), 3.62 (s, 3H), 2.38 (s, 3H); 13C NMR {1H} (150 MHz, DMSO-d6): δ 167.9, 156.8, 153.6, 137.4, 136.8, 131.0, 130.5, 127.6, 126.2, 60.5, 56.4, 19.8; HRMS (ESI+) calcd for C18H20ClN4O5S2+ [M + H+] 471.0558, found 471.0559.
4.1.8.4 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(naphthalen-1-yl)guanidine (Fig. 4, 10e).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10e; white solid (198 mg, yield 46%); m.p. 251 °C; 1H NMR (600 MHz, DMSO-d6): δ 10.28 (s, 1H), 8.43 (s, 1H), 8.01–7.98 (m, 1H), 7.96–7.91 (m, 3H), 7.79 (s, 1H), 7.74 (s, 1H), 7.62–7.54 (m, 3H), 7.51 (d, J = 7.2 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H} (151 MHz, DMSO-d6): δ 168.0, 158.4, 137.4, 136.7, 134.5, 131.6, 131.1, 130.6, 129.8, 128.8, 128.6, 127.6, 127.5, 127.1, 126.7, 126.5, 125.5, 122.9, 19.8; HRMS (ESI+) calcd for C19H16ClN4O2S2+ [M + H+] 431.0398, found 431.0394.
4.1.8.5 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(quinolin-8-yl)guanidine (Fig. 4, 10f).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10f; pale red solid (130 mg, yield 48%); m.p. 235 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.35 (bs, 1H [NH]), 8.93 (d, J = 3 Hz 1H), 8.65 (bs, 1H [NH]), 8.47 (bs, 1H [NH]), 8.43 (dd, JL = 8, JS = 1, 1H), 8.29 (d, J = 5.5, 1H), 7.95 (s, 1H), 7.80 (s, 1H), 7.73 (d, J = 8 Hz, 1H), 7.64–7.59 (m, 2H), 2.39 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.7, 156.0, 149.9, 139.8, 137.6, 137.2, 137.1, 133.8, 130.9, 130.0, 128.6, 127.6, 127.2, 126.8, 125.8, 123.8, 122.8, 19.8; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0354.
4.1.8.6 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(2-methoxynaphthalen-1-yl)guanidine (Fig. 4, 10g).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10g; white solid (257 mg, yield 56%); m.p. 233 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.97 (s, 1H), 8.33 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.79 (d, J = 10.2 Hz, 2H), 7.54 (dd, J = 17.0, 9.2 Hz, 3H), 7.40 (t, J = 7.3 Hz, 1H), 3.92 (s, 3H), 2.39 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 167.8, 158.3, 153.5, 137.4, 136.6, 131.5, 131.1, 130.7, 130.6, 129.2, 128.6, 128.0, 127.6, 126.6, 124.6, 121.9, 115.6, 114.9, 56.8, 19.8; HRMS (ESI+) calcd for C20H18ClN4O3S2+ [M + H+] 461.0503, found 461.0503.
4.1.8.7 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(6-hydroxynaphthalen-2-yl)guanidine (Fig. 4, 10h).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10h; white solid (272 mg, yield 61%); m.p. 207 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.07 (s, 1H), 9.79 (s, 1H), 8.44 (s, 1H), 8.00 (s, 1H), 7.92 (s, 7H), 7.81–7.59 (m, 4H), 7.27 (d, J = 8.4 Hz, 1H), 7.16–7.02 (m, 2H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.0, 157.1, 156.0, 137.4, 136.7, 133.4, 131.0, 130.5, 129.8, 128.1, 127.8, 127.6, 126.7, 124.2, 119.8, 109.1, 19.8; HRMS (ESI+) calcd for C19H16ClN4O3S2+ [M + H+] 447.0347, found 447.0344.
4.1.8.8 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(quinolin-6-yl)guanidine (Fig. 4, 10i).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10i; pale red solid (138 mg, yield 55%); m.p. 256 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.23 (s, 1H), 8.88–8.81 (m, 1H), 8.49 (s, 1H), 8.31 (d, J = 8.2 Hz, 1H), 8.18 (s, 1H), 8.10 (s, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 9.2 Hz, 2H), 7.75 (d, J = 5.5 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.52 (dd, J = 8.1, 4.2 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.4, 163.7, 156.7, 150.1, 137.5, 136.9, 136.3, 130.9, 130.5, 130.2, 128.6, 127.6, 127.0, 126.7, 126.6, 122.5, 19.8; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0350.
4.1.8.9 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(quinolin-5-yl)guanidine (Fig. 4, 10j).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10j; pale yellow solid (228 mg, yield 53%); m.p. 212 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.30 (s, 1H), 8.94 (d, J = 4.3 Hz, 1H), 8.47 (s, 1H), 8.35 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.92 (s, 2H), 7.80 (t, J = 8.0 Hz, 1H), 7.74 (d, J = 5.7 Hz, 1H), 7.65–7.53 (m, 2H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.1, 158.4, 151.5, 148.9, 137.4, 136.8, 132.0, 131.7, 131.0, 130.5, 129.9, 129.4, 129.3, 127.5, 126.6, 125.8, 125.7, 125.3, 122.5, 122.8, 19.7; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0352.
4.1.8.10 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(isoquinolin-6-yl)guanidine (Fig. 4, 10k).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10k; pale green solid (249 mg, yield 58%); m.p. 232 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.25 (s, 1H), 9.23 (s, 1H), 8.57 (s, 1H), 8.45 (d, J = 5.7 Hz, 1H), 8.26 (s, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 19.6 Hz, 2H), 7.81–7.69 (m, 2H), 7.60 (dd, J = 8.6, 1.7 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.7, 156.2, 152.3, 143.8, 138.8, 137.6, 137.0, 136.3, 130.9, 130.1, 129.4, 127.6, 126.8, 126.2, 124.3, 120.7, 118.3, 19.8; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0354.
4.1.8.11 (Z)-N-(N′-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)carbamimidoyl)-2-naphthamide (Fig. 4, 10l).
Purification by column chromatography (EtOAc–hexane, 40
:
60 v/v) afforded 10l; white solid (283 mg, yield 62%); m.p. 266 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.18 (s, 1H), 8.50 (s, 1H), 8.07 (s, 1H), 7.93 (t, J = 4.3 Hz, 2H), 7.90–7.86 (m, 2H), 7.84 (d, J = 1.1 Hz, 1H), 7.75 (s, 1H), 7.52–7.45 (m, 2H), 7.42 (d, J = 8.6 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 170.3, 169.2, 155.6, 137.9, 137.6, 135.6, 132.3, 130.4, 129.5, 129.5, 129.3, 129.1, 128.3, 128.2, 127.8, 127.0, 126.9, 124.5, 19.8; HRMS (ESI+) calcd for C20H16ClN4O3S2+ [M + H+] 459.0347, found 459.0343.
4.1.8.12 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(4-hydroxy-8-methoxynaphthalen-1-yl)guanidine (Fig. 4, 10m).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10m; white solid (218 mg, yield 46%); m.p. 201 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.24 (s, 1H), 9.91 (s, 1H), 8.44 (s, 1H), 7.97–7.84 (m, 3H), 7.74 (s, 1H), 7.48 (d, J = 8.3 Hz, 2H), 7.40–7.27 (m, 1H), 6.87 (d, J = 7.5 Hz, 1H), 3.84 (s, 3H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.1, 157.3, 154.1, 137.4, 136.8, 135.8, 131.0, 130.4, 127.8, 127.6, 126.7, 123.8, 123.7, 119.1, 116.1, 112.8, 109.1, 62.0, 19.8; HRMS (ESI+) calcd for C20H17ClN4O4S2Na+ [M + Na+] 499.0272, found 499.0267.
4.1.8.13 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(4-morpholinophenyl)guanidine (Fig. 4, 10n).
Purification by column chromatography (EtOAc–hexane, 40
:
60 v/v) afforded 10n; white solid (148 mg, yield 32%); m.p. 217 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.86 (s, 1H), 8.37 (s, 1H), 7.90 (s, 1H), 7.77 (s, 1H), 7.73 (s, 1H), 7.12 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 7.3 Hz, 2H), 3.83–3.53 (m, 4H), 3.14–2.97 (m, 4H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 167.8, 137.4, 136.7, 131.1, 130.6, 127.6, 126.7, 116.0, 115.8, 66.5, 48.8, 19.8; HRMS (ESI+) calcd for C19H21ClN5O3S2+ [M + H+] 466.0769, found 466.0767.
4.1.8.14 (Z)-1-(3-Acetyl-1-methyl-1H-indol-5-yl)-2-(6-chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)guanidine (Fig. 4, 10o).
Purification by column chromatography (EtOAc–hexane, 90
:
10 v/v) afforded 10o; red solid (152 mg, yield 32%); m.p. 289 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.04 (s, 1H), 8.40 (s, 1H), 8.35 (s, 1H), 8.04 (s, 1H), 7.91 (s, 1H), 7.77 (s, 1H), 7.72 (s, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.21 (d, J = 5.8 Hz, 1H), 3.84 (s, 3H), 2.39 (d, J = 12.1 Hz, 6H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 192.6, 167.9, 139.5, 137.4, 136.7, 131.1, 130.6, 127.6, 126.6, 126.5, 116.1, 111.9, 33.8, 27.7, 19.8; HRMS (ESI+) calcd for C20H19ClN5O3S2+ [M + H+] 476.0612, found 476.0618.
4.1.8.15 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(1-methyl-2-oxo-1,2-dihydroquinolin-6-yl)guanidine (Fig. 4, 10p).
Purification by column chromatography (EtOAc–hexane, 70
:
30 v/v) afforded 10p; white solid (96 mg, yield 21%); m.p. 296 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.27 (s, 1H), 8.35 (s, 2H), 7.90 (s, 1H), 7.85 (d, J = 9.4 Hz, 1H), 7.72 (s, 1H), 7.66 (s, 1H), 7.54 (dd, J = 21.0, 8.4 Hz, 2H), 6.60 (d, J = 9.5 Hz, 1H), 3.57 (s, 3H), 2.37 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 167.8, 161.4, 139.4, 137.4, 136.7, 131.1, 130.47, 127.6, 127.0, 126.7, 122.2, 120.8, 115.9, 29.6, 19.8; HRMS (ESI+) calcd for C19H17ClN5O3S2+ [M + H+] 462.0456, found 462.0458.
4.1.8.16 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(7-chloroquinolin-4-yl)guanidine (Fig. 4, 10q).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10q; pale yellow solid (283 mg, yield 61%); m.p. 219 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.29 (s, 1H), 8.88 (s, 1H), 8.65 (s, 1H), 8.30 (s, 1H), 8.09 (d, J = 22.1 Hz, 2H), 7.94 (s, 1H), 7.81–7.63 (m, 3H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 169.0, 156.7, 152.6, 149.6, 137.6, 137.1, 135.1, 130.8, 129.9, 128.5, 127.6, 127.6, 126.8, 125.1, 121.9, 116.6, 19.8; HRMS (ESI+) calcd for C18H14Cl2N5O2S2+ [M + H+] 465.9960, found 465.9965.
4.1.8.17 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(6,7-dimethoxyquinolin-4-yl)guanidine (Fig. 4, 10r).
Purification by column chromatography (EtOAc–hexane, 80
:
40 v/v) afforded 10r; yellow solid (240 mg, yield 49%); m.p. 268 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.33 (s, 1H), 8.63 (s, 2H), 8.23 (s, 1H), 7.93 (s, 1H), 7.74 (s, 1H), 7.39 (m, 3H), 3.92 (m, 6H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.3, 157.2, 152.9, 150.1, 148.6, 146.6, 138.9, 137.5, 136.9, 130.9, 130.4, 127.6, 126.7, 118.6, 115.0, 108.7, 100.9, 56.3, 56.3, 19.8; HRMS (ESI+) calcd for C20H19ClN5O4S2+ [M + H+] 492.0561, found 492.0562.
4.1.8.18 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)guanidine (Fig. 4, 10s).
Purification by column chromatography (MeOH–DCM, 10
:
90 v/v) afforded 10s; yellow solid (165 mg, yield 37%); m.p. 312 °C; 1H NMR (600 MHz, DMSO-d6): δ 8.98 (s, 1H), 8.39 (s, 1H), 7.96 (d, J = 14.4 Hz, 1H), 7.91 (d, J = 10.9 Hz, 2H), 7.75 (s, 1H), 7.69 (s, 1H), 7.40 (s, 1H), 7.04 (s, 2H), 2.37 (s, 3H); 13C NMR {1H} (150 MHz, DMSO-d6): δ 164.5, 145.4, 137.5, 137.0, 136.2, 131.6, 131.2, 131.0, 127.5, 126.7, 126.6, 126.6, 114.8, 19.8; HRMS (ESI+) calcd for C17H14ClN6O3S2+ [M + H+] 449.0252, found 449.0253.
4.1.8.19 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(quinolin-4-yl)guanidine (Fig. 4, 10t).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10t; yellow solid (284 mg, yield 66%); m.p. 259 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.29 (s, 1H), 8.88 (s, 1H), 8.65 (s, 1H), 8.30 (s, 1H), 8.22 (d, J = 8.5 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.94 (s, 1H), 7.81–7.63 (m, 3H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 169.0, 156.0, 152.6, 149.9, 137.4, 137.4, 135.1, 130.2, 129.3, 128.5, 127.6, 127.6, 126.4, 125.8, 121.2, 116.4, 19.3; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0351.
4.1.8.20 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(isoquinolin-4-yl)guanidine (Fig. 4, 10u).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10u; pale yellow solid (168 mg, yield 39%); m.p. 207 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.19 (s, 1H), 8.82 (d, J = 1.9 Hz, 1H), 8.49 (s, 1H), 8.28 (d, J = 2.2 Hz, 2H), 7.99 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.75 (s, 1H), 7.74–7.65 (m, 1H), 7.60 (t, J = 7.3 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.7, 156.8, 148.2, 145.7, 137.5, 137.0, 130.9, 130.1, 129.6, 129.1, 128.5, 128.1, 127.7, 127.6, 126.8, 19.8; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0356.
4.1.8.21 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(quinolin-3-yl)guanidine (Fig. 4, 10v).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10v; red solid (185 mg, yield 43%); m.p. 223 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.25 (s, 1H), 9.31 (s, 1H), 8.50 (s, 2H), 8.21 (d, J = 8.2 Hz, 1H), 8.02 (s, 1H), 7.97–7.91 (m, 2H), 7.86 (t, J = 7.6 Hz, 1H), 7.74 (t, J = 7.3 Hz, 2H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.2, 158.5, 152.5, 141.9, 137.4, 136.8, 132.5, 131.9, 131.0, 130.5, 129.3, 128.5, 127.6, 126.7, 122.0, 19.8; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0363.
4.1.8.22 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(isoquinolin-5-yl)guanidine (Fig. 4, 10w).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10w; pale green solid (185 mg, yield 43%); m.p. 214 °C; 1H NMR (500 MHz, DMSO-d6): δ 10.29 (s, 1H), 9.37 (s, 1H), 8.55 (d, J = 5.9 Hz, 1H), 8.43 (s, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.92 (s, 1H), 7.78 (d, J = 6.8 Hz, 2H), 7.74–7.68 (m, 2H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 168.1, 158.2, 153.2, 144.1, 137.4, 136.7, 132.3, 131.0, 130.5, 129.5, 128.2, 127.7, 127.6, 126.7, 115.9, 19.8; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0356.
4.1.8.23 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(quinoxalin-6-yl)guanidine (Fig. 4, 10x).
Purification by column chromatography (EtOAc–hexane, 80
:
20 v/v) afforded 10x; white solid (138 mg, yield 32%); m.p. 249 °C; 1H NMR (600 MHz, DMSO-d6): δ 10.27 (s, 1H), 8.90 (d, J = 1.5 Hz, 1H), 8.85 (d, J = 1.6 Hz, 1H), 8.54 (s, 1H), 8.26 (s, 1H), 8.15 (s, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.94 (s, 1H), 7.79 (s, 1H), 7.76 (dd, J = 9.0, 2.3 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H} (150 MHz, DMSO-d6): δ 168.9, 156.0, 146.7, 145.3, 143.1, 140.3, 138.8, 137.6, 137.1, 130.8, 130.3, 129.9, 127.7, 126.8, 126.7, 120.2, 19.8; HRMS (ESI+) calcd for C17H14ClN6O2S2+ [M + H+] 433.0303, found 432.0303.
4.1.8.24 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(quinolin-2-yl)guanidine (Fig. 4, 10y).
Purification by column chromatography (EtOAc–hexane, 60
:
40 v/v) afforded 10y; white solid (267 mg, yield 62%); m.p. 212 °C; 1H NMR (500 MHz, DMSO-d6): δ 11.41 (s, 1H), 11.24 (s, 1H), 9.38 (s, 1H), 8.35 (d, J = 8.9 Hz, 1H), 7.97 (s, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.3 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 2.40 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 169.1, 156.4, 152.5, 139.8, 137.6, 137.1, 131.1, 130.7, 130.1, 129.2, 128.3, 127.7, 127.4, 126.8, 126.2, 125.3, 124.0, 114.6, 19.8; HRMS (ESI+) calcd for C18H15ClN5O2S2+ [M + H+] 432.0350, found 432.0352.
4.1.8.25 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(2-methoxy-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)guanidine (Fig. 4, 10z).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10z; white solid (134 mg, yield 29%); yellow solid (165 mg, yield 37%); m.p. 219 °C; 1H NMR (600 MHz, DMSO-d6): δ 9.23 (s, 1H), 8.30 (s, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.94 (s, 1H), 7.47 (d, J = 3.4 Hz, 1H), 6.71 (d, J = 3.5 Hz, 1H), 3.81 (s, 3H), 2.80 (s, 3H), 2.44 (s, 3H); 13C NMR {1H} (150 MHz, DMSO-d6): δ 161.2, 151.3, 151.1, 146.4, 145.3, 138.8, 138.3, 138.1, 137.7, 133.0, 130.0, 124.4, 106.6, 102.5, 43.9, 32.1, 19.6; HRMS (ESI+) calcd for C17H17ClN7O3S2+ [M + H+] 466.0517, found 466.0520.
4.1.8.26 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(phenyl)guanidine (Fig. 4, 10za).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10za; white solid (14.0 mg, yield 38%); m.p. 209 °C; 1H NMR (600 MHz, DMSO-d6): δ 9.67 (s, 1H), 8.55 (s, 1H), 8.13 (s, 1H), 7.92 (s, 1H), 7.71 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.11 (t, J = 7.7 Hz, 2H), 6.99 (t, J = 7.2 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H} (150 MHz, DMSO-d6): δ 171.4, 158.4, 139.2, 138.5, 137.9, 133.9, 131.2, 129.6, 129.2, 128.7, 128.1, 122.7, 121.1, 19.8; HRMS (ESI+) calcd for C15H14ClN4O2S2+ [M + H+] 381.0241, found 381.0237.
4.1.8.27 (Z)-2-(6-Chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-1-(4-bromophenyl)guanidine (Fig. 4, 10zb).
Purification by column chromatography (EtOAc–hexane, 50
:
50 v/v) afforded 10zb; white solid (19.2 mg, yield 42%); m.p. 231 °C; 1H NMR (600 MHz, DMSO-d6): δ 9.92 (s, 1H), 8.21 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 7.71 (s, 1H), 7.31 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 2.37 (s, 3H); 13C NMR {1H} (150 MHz, DMSO-d6): δ 170.1, 156.2, 137.9, 136.2, 132.8, 132.2, 129.6, 125.4, 126.4, 116.1, 19.6; HRMS (ESI+) calcd for C15H13ClBrN4O2S2+ [M + H+] 458.9346, found 458.9342.
4.1.9 Procedure for the synthesis NSC-666719 analogs (Fig. 4, 11b–s).
39
Hydrazine monohydrate (1.5 mmol) was added to a solution of 1-(6-chloro-7-methyl-1,1-dioxidobenzo[e][1,4,2]dithiazin-3-yl)-3-(aryl) guanidines (1 mmol) in ethanol, and the reaction mixture was stirred at room temperature under open air conditions for 38–76 h. After the reaction completed as confirmed by TLC, the solvent was evaporated under vacuum and the reaction mixture was submitted to column chromatography with silica gel eluting with solvents mixture MeOH–DCM.
4.1.10 Characterization data for NSC-666719 (11a) and analogs (Fig. 6, 11b–s).
4.1.10.1 4-Chloro-2-mercapto-N-(3-((4-methoxyphenyl)amino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11b).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11b; white solid (138 mg, yield 75%); m.p. 243 °C; IR (KBr): νmax 3296.3, 1630.2, 1510.3, 1441.1, 1275.3, 1144.3, 1108.5, 1070.2, 1031.6, 936.9, 896.4, 831.9, 764.9, 748.3, 723.2 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.43 (s, 1H), 12.04 (s, 1H), 8.75 (s, 1H), 8.00 (s, 1H), 7.61 (s, 1H), 7.32 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 3.70 (s, 3H), 2.31 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 149.1, 148.6, 135.9, 134.6, 130.2, 127.6, 124.7, 115.1, 55.4, 19.5; HRMS (ESI+) calcd for C18H21ClN5O5S2+ [M + H]+ 426.0456, found 426.0459.
4.1.10.2 4-Chloro-2-mercapto-N-(3-((4-chlorophenyl)amino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11c).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11c; white solid (137 mg, yield 64%); m.p. 259 °C; IR (KBr): νmax 3322.3, 1630.4, 1596.7, 1562.9, 1493.5, 1441.2, 1401.8, 1341.3, 1276.1, 1143.6, 1095.4, 1070.6, 1013.3, 937.2, 902.0, 860.9, 823.2, 800.5, 748.1, 719.2 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.54 (s, 1H), 12.28 (s, 1H), 9.18 (s, 1H), 8.00 (s, 1H), 7.60 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 2.30 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 148.4, 146.4, 137.5, 136.9, 131.2, 130.0, 129.9, 128.5, 126.7, 125.4, 19.5; HRMS (ESI+) calcd for C15H14Cl2N5O2S2+ [M + H]+ 428.9888, found 428.9897.
4.1.10.3 4-Chloro-2-mercapto-N-(3-((phenyl)amino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11d).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11d; white solid (132 mg, yield 67%); m.p. 234 °C; IR (KBr): νmax 3322.6, 2191.4, 1629.8, 1597.2, 1568.5, 1440.1, 1333.0, 1282.4, 1141.4, 1101.0, 1068.9, 937.2, 896.1, 720.5 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.55 (s, 1H), 12.12 (s, 1H), 8.99 (s, 1H), 8.00 (s, 1H), 7.61 (s, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.28 (t, J = 7.7 Hz, 2H), 6.93 (t, J = 7.2 Hz, 1H), 2.32 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 148.5, 146.9, 140.7, 138.4, 136.8, 135.2, 131.9, 131.6, 129.8, 127.5, 125.9, 125.7, 19.5; HRMS (ESI+) calcd for C15H15ClN5O2S2+ [M + H]+ 396.0350, found 396.0354.
4.1.10.4 4-Chloro-2-mercapto-N-(3-((3,4,5-trimethoxyphenyl)amino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11e).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11e; white solid (85 mg, yield 35%); m.p. 267 °C; IR (KBr): νmax 3295.8, 2945.8, 1599.7, 1508.6, 1448.0, 1342.2, 1290.1, 1234.9, 1126.2, 1070.6, 1000.2, 937.0, 897.0, 827.5, 700.9, 667.5 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.39 (s, 2H), 8.95 (s, 1H), 8.03 (s, 1H), 7.62 (s, 1H), 6.79 (s, 2H), 3.73 (s, 6H), 3.59 (s, 3H), 2.26 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 148.5, 146.8, 153.6, 137.4, 136.8, 131.0, 130.5, 127.6, 126.7, 60.5, 56.4, 19.5; HRMS (ESI+) calcd for C18H21ClN5O5S2+ [M + H]+ 486.0667, found 486.0665.
4.1.10.5 4-Chloro-2-mercapto-N-(3-((4-bromophenyl)amino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11f).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11f; white solid (138 mg, yield 58%); m.p. 322 °C; IR (KBr): νmax 3308.0, 1629.9, 1595.4, 1568.5, 1440.1, 1382.8, 1342.0, 1284.6, 1144.0, 1105.9, 1068.0, 1021.2, 999.0, 934.6, 896.3, 861.0, 816.1, 733.3, 717.6, 663.0 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.46 (s, 1H), 12.24 (s, 1H), 9.11 (s, 1H), 7.95 (s, 1H), 7.56 (s, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.27 (d, J = 7.1 Hz, 2H), 2.25 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 149.0, 147.6, 140.2, 139.2, 137.8, 134.7, 132.9, 131.6, 129.1, 126.3, 124.9, 118.7, 19.4; HRMS (ESI+) calcd for C15H14BrClN5O2S2+ [M + H]+ 473.9455, found 473.9461.
4.1.10.6 4-Chloro-2-mercapto-N-(3-(naphthalen-1-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11g).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11g; white solid (149 mg, yield 67%); m.p. 298 °C; IR (KBr): νmax 3267.3, 2221.4, 1610.7, 1543.4, 1461.5, 1354.8, 1299.9, 1127.0, 1069.1, 989.2, 936.1, 723.2 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.74 (s, 1H [NH]), 11.95 (s, 1H [NH]), 9.06 (s, 1H [NH]), 8.04 (d, J = 8 Hz, 1H), 7.93 (s, 1H), 7.88 (t, J = 7 Hz, 2H), 7.60 (s, 1H), 7.56–7.52 (m, 3H), 7.41 (t, J = 8 Hz, 1H), 2.28 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 148.3, 147.5, 140.4, 137.9, 135.1, 134.8, 134.2, 132.9, 131.2, 129.0, 126.7, 126.5, 126.4, 126.3, 124.7, 122.6, 121.3, 113.7, 19.5; HRMS (ESI+) calcd for C19H17ClN5O2S2+ [M + H]+ 446.0507, found 446.0523.
4.1.10.7 4-Chloro-2-mercapto-N-(3-(quinolin-8-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11h).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11h; white solid (129 mg, yield 58%); m.p. 325 °C; IR (KBr): νmax 3321.9, 1593.3, 1569.6, 1512.1, 1439.7, 1343.4, 1274.8, 1235.0, 1106.6, 1068.7, 988.0, 932.1, 898.2, 815.9762.5, 718.3, 697.6, 665.2 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.78 (s, 1H [NH]), 12.31 (s, 1H [NH]), 9.99 (s, 1H [NH]), 8.06 (d, J = 3 Hz, 1H), 8.35 (d, J = 8.5 Hz, 1H), 8.06 (d, J = 3 Hz, 1H), 8.18 (d, J = 7 Hz, 1H) 7.94 (s, 1H), 7.60–7.58 (m, 2H), 7.53–7.47 (m, 2H), 2.27 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 148.8, 148.3, 147.2, 140.6, 137.7, 137.0, 135.6, 134.9, 133.0, 131.3, 128.3, 127.7, 126.9, 122.7, 119.9, 113.0, 19.5; HRMS (ESI+) calcd for C18H16ClN6O2S2+ [M + H]+ 447.0459, found 447.0456.
4.1.10.8 4-Chloro-2-mercapto-N-(3-(2-methoxynaphthalen-1-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11i).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11i; pale yellow solid (74 mg, yield 31%); m.p. 279 °C; IR (KBr): νmax 3259.6, 2239.4, 1656.7, 1567.4, 1512.5, 1436.1, 1392.8, 1319.2, 1222.8, 1112.6, 1102.7, 1020.4, 965.7, 945.8, 865.7, 714.5 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 12.86 (s, 1H), 12.31 (s, 1H), 9.97 (s, 1H), 8.33 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.79 (d, J = 10.2 Hz, 1H), 7.54 (d, J = 17.0, 9.2 Hz, 2H), 7.40 (t, J = 7.3 Hz, 1H), 3.89 (s, 3H), 2.40 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 148.9, 147.7, 140.2, 138.4, 136.9, 135.8, 134.2, 133.6, 131.9, 129.3, 127.5, 126.9, 126.2, 125.3, 125.1, 123.6, 122.2, 118.1, 56.8, 19.4; HRMS (ESI+) calcd for C20H19ClN5O3S2+ [M + H]+ 476.0612, found 476.0617.
4.1.10.9 4-Chloro-2-mercapto-N-(3-(6-hydroxynaphthalen-2-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11j).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11j; pale red solid (102 mg, yield 44%); m.p. 288 °C; IR (KBr): νmax 3321.9, 3177.9, 1593.3, 1569.6, 1512.1, 1439.7, 1383.5, 1343.4, 1274.8, 1235.0, 1138.2, 1106.6, 1068.7, 1020.6, 988.0, 932.1, 898.2, 861.6, 815.9, 803.5, 762.5, 718.3, 697.6, 665.22 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.42 (s, 1H), 12.12 (s, 1H), 9.45 (s, 1H), 8.95 (s, 1H), 7.97 (s, 1H), 7.84 (s, 1H), 7.55 (dd, J = 20.3, 10.7 Hz, 3H), 7.28 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 12.6 Hz, 2H), 2.25 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 154.3, 140.3, 137.7, 135.0, 134.7, 132.9, 131.7, 130.5, 128.6, 128.6, 127.3, 126.3, 119.7, 119.5, 112.4, 109.3, 19.4; HRMS (ESI+) calcd for C19H17ClN5O3S2+ [M + H]+ 462.0456, found 462.0462.
4.1.10.10 4-Chloro-2-mercapto-N-(3-(quinolin-6-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11k).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11k; pale red solid (136 mg, yield 61%); m.p. 279 °C; IR (KBr): νmax 3274.7, 1569.0, 1519.6, 1437.2, 1385.0, 1246.0, 1142.2, 1096.5, 1069.1, 947.0, 869.9, 832.3, 797.9, 716.6 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 12.49 (s, 2H), 9.38 (s, 1H), 8.66 (d, J = 3.3 Hz, 1H), 8.10 (s, 1H), 7.97 (s, 2H), 7.87 (d, J = 9.0 Hz, 1H), 7.68–7.55 (m, 2H), 7.39 (dd, J = 8.1, 4.1 Hz, 1H), 2.22 (s, 3H); 13C NMR {1H} (100 MHz, DMSO-d6): δ 149.6, 148.3, 144.0, 140.0, 138.3, 137.9, 135.4, 134.7, 133.0, 131.9, 129.9, 129.2, 126.4, 122.9, 122.3, 111.2, 19.4; HRMS (ESI+) calcd for C18H15ClN6O2S2+ [M]+ 446.0383, found 446.0386.
4.1.10.11 4-Chloro-2-mercapto-N-(3-(quinolin-5-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11l).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11l; pale yellow solid (83 mg, yield 37%); m.p. 312 °C; IR (KBr): νmax 3267.1, 1554.6, 1540.1, 1467.5, 1390.2, 1318.9, 1254.5, 1232.4, 1176.9, 1099.0, 1076.3, 956.7, 867.7, 723.9 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 12.71 (s, 1H), 12.05 (s, 1H), 9.24 (s, 1H), 8.88 (s, 1H), 8.48 (d, J = 8.3 Hz, 1H), 7.94 (s, 1H), 7.88 (s, 1H), 7.61 (d, J = 9.9 Hz, 3H), 7.55 (s, 1H), 2.27 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 148.9, 147.2, 143.9, 142.5, 139.0, 137.3, 136.7, 135.2, 134.0, 131.7, 130.1, 129.5, 126.8, 124.0, 122.8, 116.1, 19.4; HRMS (ESI+) calcd for C18H15ClN6O2S2+ [M]+ 446.0383, found 446.0389.
4.1.10.12 4-Chloro-2-mercapto-N-(3-(isoquinolin-5-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11m).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11m; light green solid (102 mg, yield 46%); m.p. 288 °C; IR (KBr): νmax 3254.1, 1612.4, 1579.3, 1519.8, 1498.9, 1423.5, 1378.4, 1309.1, 1287.0, 1252.4, 1162.5, 1098.4, 989.6, 909.9, 894.5, 812.9, 721.0 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 12.84 (s, 1H), 12.42 (s, 1H), 9.49 (s, 1H), 8.59 (d, J = 3.3 Hz, 1H), 8.41 (s, 1H), 8.10 (s, 2H), 7.69 (d, J = 9.0 Hz, 1H), 7.45–7.39 (m, 2H), 7.21 (dd, J = 8.1, 4.1 Hz, 1H), 2.27 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 149.1, 147.9, 143.8, 141.1, 139.0, 139.2, 136.5, 135.6, 132.9, 132.5, 130.2, 129.9, 125.7, 123.8, 122.6, 116.2, 19.5; HRMS (ESI+) calcd for C18H16ClN6O2S2+ [M + H]+ 447.0459, found 447.0462.
4.1.10.13
N-(5-((4-Chloro-2-mercapto-5-methylphenyl)sulfonamido)-1H-1,2,4-triazol-3-yl)-2-naphthamide (Fig. 4, 11n).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11n; white solid (yield 26%); m.p. 278 °C; 1H NMR (500 MHz, DMSO-d6): δ 12.23 (s, 1H), 11.52 (s, 1H), 8.62 (s, 1H), 8.06–7.93 (m, 5H), 7.87 (s, 1H), 7.66–7.54 (m, 3H), 2.27 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 166.5, 161.2, 156.9, 153.1, 139.6, 137.7, 135.1, 132.8, 132.3, 130.5, 129.6, 128.8, 128.5, 128.2, 127.5, 125.0, 124.6, 19.4; HRMS (ESI+) calcd for C19H17ClN5O2S2+ [M + H]+ 474.0456, found 474.0457.
4.1.10.14 4-Chloro-2-mercapto-N-(3-(7-chloroquinolin-4-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11o).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11o; pale yellow solid (55 mg, yield 23%); m.p. 256 °C; 1H NMR (500 MHz, DMSO-d6): δ 12.57 (s, 1H), 12.12 (s, 1H), 8.85 (s, 1H), 8.12 (s, 1H), 8.02 (d, J = 22.1 Hz, 2H), 7.90 (s, 1H), 7.67–7.42 (m, 3H), 2.35 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 156.5, 154.2, 149.2, 147.2, 137.6, 137.1, 136.1, 132.6, 131.0, 128.1, 127.7, 127.0, 126.8, 125.7, 124.2, 116.6, 19.5; HRMS (ESI+) calcd for C18H14Cl2N6O2S2+ [M]+ 481.0069, found 481.0077.
4.1.10.15 4-Chloro-2-mercapto-N-(3-(isoquinolin-4-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11p).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11p; light-green solid (69 mg, yield 31%); m.p. 247 °C; IR (KBr): νmax 3273.5, 1594.9, 1579.1, 1462.4, 1444.1, 1374.8, 1348.1, 1301.3, 1250.9, 1145.6, 992.2, 954.0, 899.1, 816.9, 715.4 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 12.81 (s, 1H), 12.56 (s, 1H), 8.85 (d, J = 1.9 Hz, 1H), 8.32 (d, J = 2.2 Hz, 2H), 8.10 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.67 (s, 1H), 7.66–7.60 (m, 1H), 7.57 (t, J = 7.3 Hz, 1H), 2.38 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 151.1, 150.9, 147.2, 145.8, 138.2, 137.6, 131.3, 130.5, 129.9, 129.4, 128.9, 128.1, 127.9, 127.4, 126.6, 19.5; HRMS (ESI+) calcd for C18H16ClN6O2S2+ [M + H]+ 447.0459, found 447.0462.
4.1.10.16 4-Chloro-2-mercapto-N-(3-(isoquinolin-6-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11q).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11q; light-yellow solid (91 mg, yield 41%); m.p. 245 °C; 1H NMR (500 MHz, DMSO-d6): δ 12.67 (s, 1H), 12.44 (s, 1H), 9.12 (s, 1H), 8.42 (d, J = 5.7 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.91 (d, J = 19.6 Hz, 2H), 7.88–7.71 (m, 2H), 7.64 (dd, J = 8.6, 1.7 Hz, 1H), 2.39 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 148.7, 147.9, 144.3, 139.8, 138.4, 137.0, 136.6, 131.9, 130.1, 129.4, 128.1, 126.8, 126.2, 125.6, 121.7, 119.1, 19.4; HRMS (ESI+) calcd for C18H16ClN6O2S2+ [M + H]+ 447.0459, found 447.0460.
4.1.10.17 4-Chloro-2-mercapto-N-(3-(quinolin-3-ylamino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11r).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11r; pale pink solid (46 mg, yield 21%); m.p. 287 °C; 1H NMR (500 MHz, DMSO-d6): δ 12.71 (s, 1H), 12.05 (s, 1H), 9.32 (s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.94–7.88 (m, 2H), 7.84 (t, J = 7.6 Hz, 1H), 7.74 (t, J = 7.3 Hz, 2H), 2.37 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 148.7, 147.4, 142.9, 136.6, 136.5, 132.4, 131.9, 131.5, 130.9, 129.0, 128.8, 127.7, 126.8, 122.1, 19.5; HRMS (ESI+) calcd for C18H15ClN6O2S2+ [M]+ 446.0383, found 446.0390.
4.1.10.18 4-Chloro-2-mercapto-N-(3-((6,7-dimethoxyquinolin-4-yl)amino)-1H-1,2,4-triazol-5-yl)-5-methylbenzenesulfonamide (Fig. 4, 11s).
Purification by column chromatography (MeOH–DCM, 1
:
10 v/v) afforded 11s; pale yellow solid (40 mg, yield 16%); m.p. 237 °C; 1H NMR (500 MHz, DMSO-d6): δ 12.58 (s, 1H), 12.15 (s, 1H), 7.94 (s, 1H), 7.77 (s, 1H), 7.38 (m, 3H), 3.94 (m, 6H), 2.37 (s, 3H); 13C NMR {1H} (125 MHz, DMSO-d6): δ 154.3, 152.4, 150.206, 148.2, 147.6, 138.1, 136.6, 136.1, 131.9, 131.3, 128.4, 127.6, 127.3, 124.4, 120.6, 112.1, 108.6, 19.4; HRMS (ESI+) calcd for C20H19ClN6O4S2+ [M]+ 506.0598, found 506.0602.
4.2 Bio-evaluation studies
4.2.1 Materials and methods.
4.2.1.1 MTT cell proliferation assay.
To check the anti-proliferative effects of synthesized compounds, MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] assay was performed as mentioned in the earlier study.59 MCF7 (# HTB-22), MDA-MB-231 (# HTB-26), HCT-116 (# CCL-247), SCC9 (# CRL-1629), and HEK-293 (# CRL-1573) cells were purchased from ATCC, USA, and cultured prior to perform cell proliferation assay. In brief, in a 96-well plate, 5000–6000 cells were added in triplicate and grown until they reached 70–80% confluence. Next, the cells were treated with the increasing concentration (up to 5 μM) of respective compounds for 48 h. After that, media was discarded followed by 100 μL of 0.05% MTT was added in each well and kept it for overnight at 37 °C. The formazan crystals formation was observed after incubation, which were solubilized in 0.2% NP-40 of 100 μL solution. Finally, the intensity of color solution was measured at 570 nm using a UV-visible spectroscopy (Berthold, Germany). Then, the data was plotted as percentage of cell viability against the concentrations of compounds.
4.2.1.2 DAPI nuclear staining assay.
To calculate the apoptotic cells percentage after treatment with respective compounds, 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining assay was carried out according to the previously describe protocol.60 In brief, 70–80% cells (MCF-7) were treated with most potent compounds such as 10e and 10q for 48 h. Next, following a 1× PBS wash, the cells were fixed for 15 min at 20 °C in the dark using acetone
:
methanol (1
:
1). Then the fixed cells were first washed with 1× PBS, then DAPI solution was added, and the cells were then incubated for 1 h at 37 °C in the dark. Finally, the stained cells were observed under a fluorescence microscope (Nikon, Japan) at a magnification of 40×.
4.2.1.3 Immunocytochemical analysis to check the expression of γH2AX and caspase-3.
The immunofluorescence technique was employed to analyze the expression of γH2AX (# E-AB-67407 from Elabscience, Texas, USA) and caspase-3 (# E-AB-66940 from Elabscience, Texas, USA) in MCF-7 breast cancer cells following treatment with two specific compounds, 10e and 10q, as described in a previously published protocol.61,70 In summary, MCF-7 cells were cultured on coverslips in a 24-well plate and treated with different concentrations of 10e and 10q, including one concentration at the IC50, one below the IC50, and one above the IC50 for 48 h. Then, the cells were collected, washed with 1× PBS, and fixed using a mixture of acetone and methanol (1
:
1) at −20 °C for 15 minutes. Subsequently, the cells were washed with 1× PBS and incubated with primary antibodies anti-γH2AX or anti-caspase-3 (diluted 1
:
300 in 1× PBS) for 4 hours at 4 °C. Unbound primary antibodies were washed away with 1× PBS, and the cells were then incubated with a secondary antibody IgG conjugated with TRITC, at room temperature for 2 hours. Following another round of washing with 1× PBS, the cells were counterstained with DAPI and observed under an inverted fluorescence microscope (Nikon, Japan) at 20× magnification. Picture were captured.
4.2.1.4 Measurement of apoptosis by Annexin-V-FITC/PI dual staining.
Annexin-V-FITC/PI dual staining flow cytometric assay was performed as mentioned in the previous protocol62,63 to examine the apoptotic populations of cells after the treatments of 10e and 10q compounds. In a 6-well plate, 1 × 105 MCF-7 cells were added in each well of plate and grown until 70–80% confluency. Next, after the treatment of above-mentioned compounds, the cells were collected, washed with 1× PBS, and then stained with Annexin-V/FITC and PI. After incubation, the cells were sorted at an event count of 10
000 cells per sample by flow cytometry (FACS CANTO II, Becton & Dickinson, CA, USA). Whereas FACS Diva software was used to analyze the data.
4.2.1.5 Quantification of protein expression by western blotting.
Western blotting assay was performed to check the expression of different proteins by using the previously described protocol.64 In brief, after the treatment of 10e and 10q compounds in MCF-7, the cells were collected followed by washed with 1× PBS. Then the modified RIPA lysis buffer was used to prepare the whole-cell lysates of cells. After that 80 g of protein samples were separated by using SDS-PAGE and then transferred to PVDF membranes and probed with specific antibody (Bax # E-AB-66518, Bcl-xL # E-AB-30640, caspase-3 # E-AB-66940, GAPDH # E-AB-40337 from Elabscience, Texas, USA). A UVP Gel Doc-It 310 Imaging system (UVP, Cambridge, UK) was used for the densitometric examination of the protein bands. The relative fold change is indicated by the numerical value above each blot.
4.2.1.6 Polβ nucleotide insertion assays.
We investigated the effect of the best active compounds 10e and 10q on Polβ activity for dGTP:C insertion using gap DNA substrate in vitro. The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 100 μg ml−1 BSA, 10% glycerol, dGTP (100 μM), and DNA substrate (500 nM), and the compounds at the final concentrations of 0, 1, 3, or 5 μM in a final volume of 10 μl. The reaction was initiated by the addition of the Polβ (10 nM) and the reaction mixtures were incubated at 37 °C for the time points as indicated in the figure legends. The reaction was quenched by mixing with an equal amount of gel loading buffer containing 95% formamide, 20 mM EDTA, 0.02% bromophenol blue, and 0.02% xylene cyanol, and separated by electrophoresis on an 18% polyacrylamide gel. The gels were finally scanned with a Typhoon PhosphorImager RGB (Amersham), and the data were analyzed using ImageQuant software as described before.71
4.2.1.7
In vivo BER assay.
To quantify the BER inhibition capability of the best potent compounds 10e and 10q in MCF-7 cells, an in vivo plasmid-based BER assay was carried out following the earlier referred protocol.65 In brief, p21 (pGL2-p21) promoter-containing closed circular DNA was deaminated by 3 M sodium bisulphite in the presence of 50 mM hydroquinone, which changes cytosine into uracil-residues (U-p21p), a substrate for SP-BER. In a subsequent step, U-p21p was treated with uracil-DNA glycosylase (UDG), and the resulting abasic site was reduced with 0.1 M sodium borohydride to yield R-p21p, a substrate for LP-BER. Using 10 μl ml−1 of lipofectamine reagent, 70–80% of confluent cells were transfected with 4 μg ml−1 of R-p21p cDNA, U-p21p, and 0.5 μg ml−1 of β-gal. The transfection efficacy was assessed using β-gal as an internal control. After 8 hours, the transfected medium was changed to fresh medium containing serum, and then cells were treated for 48 hours with various concentrations of selective compounds. The SP-BER and LP-BER activity in cellular lysates were then determined using a DLR luciferase assay apparatus (Berthold, Germany).
4.2.1.8 Surface plasmon resonance (SPR) binding.
Binding affinity measurements were conducted on a Reichert2SPR instrument (Ametek) at room temperature. Seven μg Polβ proteins were directly immobilized onto the 500
000 Da carboxymethyl dextran sensor chip at pH 5.0 using the standard amine coupling approach.72 After achieving a stable baseline, small molecule ligands were injected at a flow rate of 25 μL min−1 at different concentrations in the running buffer (25 mM HEPES pH 8.0, 150 mM NaCl, 200 mM MgCl2, 0.1% v/v Tween-20, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 1% DMSO). The association and dissociation time were 2 min and 5 min, respectively. Sensorgram data was processed using Excel and dissociation constant (KD) was calculated by GraphPad Prism9.
4.2.1.9 Docking studies.
All the procedures were achieved within the Maestro software platform (version 13.2.128; Schrödinger LLC, New York, NY, USA). The initial preparation of the human DNA Polβ protein (PDB code: 1BPZ) involved the application of the Protein Preparation Wizard. This step encompassed the removal of water molecules and the addition of bond orders and hydrogen atoms to the protein structure. The LigPrep module was then employed to construct the molecular configurations of the compounds, utilizing the OPLS3e force field. This process facilitated the exploration of various tautomeric and ionization states, ultimately yielding 3D molecular structures. For the generation of the receptor grid, specific residues T79, K81, and R83 were employed.12,13 Subsequently, the glide module was utilized to perform the docking of the compounds. The default parameters of the glide module were applied during the docking process. Prime module was used to calculate the binding free energies.
Abbreviations
NSC | Cancer Chemotherapy National Service Center number (NCI) |
BRCA2 | BReast CAncer gene 2 |
MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide |
MCF-7 | Michigan Cancer Foundation-7 |
MDA-MB-231 | M.D. Anderson-Metastasis Breast cancer-231 |
HCT-116 | Human colon cancer |
SCC-9 | Squamous cell carcinoma |
HEK-293 | Human embryonic kidney |
Annexin V-FITC | Annexin V conjugated with fluorescein isothiocyanate (FITC) |
PI | Propidium iodide |
γH2AX | Gamma-H2A histone family member X |
Conflicts of interest
The authors declare that they have no known conflicts of interest.
Acknowledgements
We gratefully acknowledge the financial support from DST-SERB, and CSIR, Government of India to S. K. G. and these studies were partially supported by the UF Health Cancer Centre A-1 Accelerator Program (P0280641) awarded to S. N. V. K. R. is thankful to NIPER for his fellowship. S. P. is thankful to DBT, and C. D. and B. D. are thankful to ICMR, Government of India, for providing their fellowship and research funding to CNK. Thanks to Panjab University for providing the X-ray crystallography facility (DST-FIST). Computations for this research were performed on the Pennsylvania State University's Institute for Computational and Data Sciences' Roar supercomputer.
References
- T. Lindahl and R. D. Wood, Science, 1999, 286, 1897–1905 CrossRef CAS PubMed.
- S. S. Lange, K.-i. Takata and R. D. Wood, Nat. Rev. Cancer, 2011, 11, 96–110 CrossRef CAS PubMed.
- T. Helleday, E. Petermann, C. Lundin, B. Hodgson and R. A. Sharma, Nat. Rev. Cancer, 2008, 8, 193–204 CrossRef CAS PubMed.
- N. S. Gavande, P. S. VanderVere-Carozza, H. D. Hinshaw, S. I. Jalal, C. R. Sears, K. S. Pawelczak and J. J. Turchi, Pharmacol. Ther., 2016, 160, 65–83 CrossRef CAS PubMed.
- W. A. Beard, J. K. Horton, R. Prasad and S. H. Wilson, Annu. Rev. Biochem., 2019, 88, 137 CrossRef CAS PubMed.
- S. H. Wilson, W. A. Beard, D. D. Shock, V. K. Batra, N. A. Cavanaugh, R. Prasad, E. W. Hou, Y. Liu, K. Asagoshi and J. K. Horton, Cell. Mol. Life Sci., 2010, 67, 3633–3647 CrossRef CAS PubMed.
- S. H. Wilson, Mutat. Res., DNA Repair Rep., 1998, 407, 203–215 CAS.
- R. K. Singhal and S. H. Wilson, J. Biol. Chem., 1993, 268, 15906–15911 CrossRef CAS PubMed.
- H.-Y. Hu, J. K. Horton, M. R. Gryk, R. Prasad, J. M. Naron, D.-A. Sun, S. M. Hecht, S. H. Wilson and G. P. Mullen, J. Biol. Chem., 2004, 279, 39736–39744 CrossRef CAS PubMed.
- S. H. Wilson, W. A. Beard, D. D. Shock, V. K. Batra, N. A. Cavanaugh, R. Prasad, E. W. Hou, Y. Liu, K. Asagoshi and J. K. Horton, Cell. Mol. Life Sci., 2010, 67, 3633–3647 CrossRef CAS PubMed.
- A. Srinivasan and B. Gold, Future Med. Chem., 2012, 4, 1093–1111 CrossRef CAS PubMed.
- A. S. Jaiswal, S. Banerjee, H. Panda, C. D. Bulkin, T. Izumi, F. H. Sarkar, D. A. Ostrov and S. Narayan, Mol. Cancer Res., 2009, 7, 1973–1983 CrossRef CAS PubMed.
- A. S. Jaiswal, S. Banerjee, R. Aneja, F. H. Sarkar, D. A. Ostrov and S. Narayan, PLoS One, 2011, 6, e16691 CrossRef CAS PubMed.
- D. Starcevic, S. Dalal and J. B. Sweasy, Cell Cycle, 2004, 3, 996–999 CrossRef PubMed.
- A. Matsukage, M. Yamaguchi, K. R. Utsumi, Y. Hayashi, R. Ueda and M. C. Yoshida, Jpn. J. Cancer Res., 1986, 77, 330–333 CAS.
- A. J. Reed, R. Vyas, A. T. Raper and Z. Suo, J. Am. Chem. Soc., 2017, 139, 465–471 CrossRef CAS PubMed.
- W. A. Beard, D. D. Shock, X.-P. Yang, S. F. DeLauder and S. H. Wilson, J. Biol. Chem., 2002, 277, 8235–8242 CrossRef CAS PubMed.
- S. Narayan and R. Sharma, Life Sci., 2015, 139, 145–152 CrossRef CAS PubMed.
- I. I. Dianova, K. M. Sleeth, S. L. Allinson, J. L. Parsons, C. Breslin, K. W. Caldecott and G. L. Dianov, Nucleic Acids Res., 2004, 32, 2550–2555 CrossRef CAS PubMed.
- M. Sukhanova, S. Khodyreva and O. Lavrik, Mutat. Res., Fundam. Mol. Mech. Mutagen., 2010, 685, 80–89 CrossRef CAS PubMed.
- Y. Liu, W. A. Beard, D. D. Shock, R. Prasad, E. W. Hou and S. H. Wilson, J. Biol. Chem., 2005, 280, 3665–3674 CrossRef CAS PubMed.
- R. A. Bennett, D. M. Wilson, D. Wong and B. Demple, Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 7166–7169 CrossRef CAS PubMed.
- P. S. Kedar, S.-J. Kim, A. Robertson, E. Hou, R. Prasad, J. K. Horton and S. H. Wilson, J. Biol. Chem., 2002, 277, 31115–31123 CrossRef CAS PubMed.
- A. Balliano, F. Hao, C. Njeri, L. Balakrishnan and J. J. Hayes, Biochemistry, 2017, 56, 647–656 CrossRef CAS PubMed.
- W. A. Beard and S. H. Wilson, Chem. Rev., 2006, 106, 361–382 CrossRef CAS PubMed.
- M. R. Albertella, A. Lau and M. J. O'Connor, DNA Repair, 2005, 4, 583–593 CrossRef CAS PubMed.
- T. Louat, L. Servant, M.-P. Rols, A. Bieth, J. Teissie, J.-S. Hoffmann and C. Cazaux, Mol. Pharmacol., 2001, 60, 553–558 CAS.
- V. Bergoglio, Y. Canitrot, L. Hogarth, L. Minto, S. B. Howell, C. Cazaux and J.-S. Hoffmann, Oncogene, 2001, 20, 6181–6187 CrossRef CAS PubMed.
- X.-H. Tan, M. Zhao, K.-F. Pan, Y. Dong, B. Dong, G.-J. Feng, G. Jia and Y.-Y. Lu, Cancer Lett., 2005, 220, 101–114 CrossRef CAS PubMed.
- K.-H. Chen, F. M. Yakes, D. K. Srivastava, R. K. Singhal, R. W. Sobol, J. K. Horton, B. Van Houten and S. H. Wilson, Nucleic Acids Res., 1998, 26, 2001–2007 CrossRef CAS PubMed.
- Y. Canitrot, C. Cazaux, M. FREchet, K. Bouayadi, C. Lesca, B. Salles and J.-S. Hoffmann, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 12586–12590 CrossRef CAS PubMed.
- J. L. Illuzzi and D. M. Wilson III, Curr. Med. Chem., 2012, 19, 3922–3936 CrossRef CAS PubMed.
- N. H. Nicolay, T. Helleday and R. A. Sharma, Curr. Mol. Pharmacol., 2012, 5, 54–67 CrossRef CAS PubMed.
- D. Fu, J. A. Calvo and L. D. Samson, Nat. Rev. Cancer, 2012, 12, 104–120 CrossRef CAS PubMed.
- A. S. Jaiswal, H. Panda, B. K. Law, J. Sharma, J. Jani, R. Hromas and S. Narayan, PLoS One, 2015, 10, e0123808 CrossRef PubMed.
- T. Strittmatter, A. Brockmann, M. Pott, A. Hantusch, T. Brunner and A. Marx, ACS Chem. Biol., 2014, 9, 282–290 CrossRef CAS PubMed.
- D. Arian, M. Hedayati, H. Zhou, Z. Bilis, K. Chen, T. L. DeWeese and M. M. Greenberg, J. Am. Chem. Soc., 2014, 136, 3176–3183 CrossRef CAS PubMed.
- S. C. Yuhas, D. J. Laverty, H. Lee, A. Majumdar and M. M. Greenberg, J. Am. Chem. Soc., 2021, 143, 8099–8107 CrossRef CAS PubMed.
- Z. Brzozowski, Acta Pol. Pharm., 1995, 52, 91–101 CAS.
- R. Ali, A. Alblihy, I. M. Miligy, M. L. Alabdullah, M. Alsaleem, M. S. Toss, M. Algethami, T. Abdel-Fatah, P. Moseley and S. Chan, Oncogene, 2021, 40, 2496–2508 CrossRef CAS PubMed.
- C. Ballatore, D. M. Huryn and A. B. Smith III, ChemMedChem, 2013, 8, 385–395 CrossRef CAS PubMed.
- A. Giraudo, J. Krall, B. Nielsen, T. E. Sørensen, K. T. Kongstad, B. Rolando, D. Boschi, B. Frølund and M. L. Lolli, Eur. J. Med. Chem., 2018, 158, 311–321 CrossRef CAS PubMed.
- M. M. Attwood, D. Fabbro, A. V. Sokolov, S. Knapp and H. B. Schiöth, Nat. Rev. Drug Discovery, 2021, 20, 839–861 CrossRef CAS PubMed.
- Z. Brzozowski, F. S
czewski and M. Gdaniec, Bioorg. Med. Chem., 2003, 11, 3673–3681 CrossRef CAS PubMed.
- Z. Brzozowski, F. Sączewski and M. Gdaniec, Eur. J. Med. Chem., 2003, 38, 991–999 CrossRef CAS PubMed.
- Z. Brzozowski, F. Sączewski and N. Neamati, Bioorg. Med. Chem., 2006, 14, 2985–2993 CrossRef CAS PubMed.
- Z. Brzozowski, F. Sączewski, J. Sławiński, P. J. Bednarski, R. Grünert and M. Gdaniec, Bioorg. Med. Chem., 2007, 15, 2560–2572 CrossRef CAS PubMed.
- Z. Brzozowski and F. S
czewski, J. Med. Chem., 2002, 45, 430–437 CrossRef CAS PubMed.
- Z. Brzozowski, B. Żołnowska and J. Sławiński, Monatsh. Chem., 2013, 144, 1397–1405 CrossRef CAS PubMed.
- Z. Hosseinzadeh, A. Ramazani and N. Razzaghi-Asl, Curr. Org. Chem., 2018, 22, 2256–2279 CrossRef CAS.
- K. Tsubokura, T. Iwata, M. Taichi, A. Kurbangalieva, K. Fukase, Y. Nakao and K. Tanaka, Synlett, 2014, 25, 1302–1306 CrossRef.
- A. Pogorzelska, J. Sławiński, K. Brożewicz, S. Ulenberg and T. Bączek, Molecules, 2015, 20, 21960–21970 CrossRef CAS PubMed.
- J. Shao, W. Chen, M. A. Giulianotti, R. A. Houghten and Y. Yu, Org. Lett., 2012, 14, 5452–5455 CrossRef CAS PubMed.
- M. Q. Tran, L. Ermolenko, P. Retailleau, T. B. Nguyen and A. Al-Mourabit, Org. Lett., 2014, 16, 920–923 CrossRef CAS PubMed.
- H. Huang, W. Guo, W. Wu, C.-J. Li and H. Jiang, Org. Lett., 2015, 17, 2894–2897 CrossRef CAS PubMed.
- H.-H. Ha, J. S. Kim and B. M. Kim, Bioorg. Med. Chem. Lett., 2008, 18, 653–656 CrossRef CAS PubMed.
- J. W. Shaw, D. H. Grayson and I. Rozas, Eur. J. Org. Chem., 2014, 2014, 3565–3569 CrossRef CAS.
- J. W. Shaw, L. Barbance, D. H. Grayson and I. Rozas, Tetrahedron Lett., 2015, 56, 4990–4992 CrossRef CAS.
- B. Das, C. Sethy, S. Chatterjee, S. R. Dash, S. Sinha, S. Paul, K. Goutam and C. N. Kundu, J. Cell Commun. Signaling, 2023, 1–18 Search PubMed.
- S. Sisodiya, S. Paul, H. Chaudhary, P. Grewal, G. Kumar, D. P. Daniel, B. Das, D. Nayak, S. K. Guchhait and C. N. Kundu, Bioorg. Med. Chem. Lett., 2021, 49, 128274 CrossRef CAS PubMed.
- S. R. Dash, S. Chatterjee, S. Sinha, B. Das, S. Paul, R. Pradhan, C. Sethy, R. Panda, J. Tripathy and C. N. Kundu, Nanomed.: Nanotechnol., Biol. Med., 2022, 40, 102502 CrossRef CAS PubMed.
- S. Paul, S. Chatterjee, S. Sinha, S. R. Dash, R. Pradhan, B. Das, K. Goutam and C. N. Kundu, Expert Opin. Ther. Targets, 2023,(10), 999–1015 CrossRef CAS PubMed.
- S. Sinha, S. Chatterjee, S. Paul, B. Das, S. R. Dash, C. Das and C. N. Kundu, Exp. Cell Res., 2022, 420, 113338 CrossRef CAS PubMed.
- S. R. Dash, B. Das, C. Das, S. Sinha, S. Paul, R. Pradhan and C. N. Kundu, Nanomedicine, 2023, 18, 19–33 CrossRef CAS PubMed.
- S. Chatterjee, A. K. Dhal, S. Paul, S. Sinha, B. Das, S. R. Dash and C. N. Kundu, J. Cancer Res. Clin. Oncol., 2022, 148, 3521–3535 CrossRef CAS PubMed.
- H. Bonnet, L. Coche-Guérente, E. Defrancq, N. Spinelli, A. Van der Heyden and J. Dejeu, Anal. Chem., 2021, 93, 4134–4140 CrossRef CAS PubMed.
- C. Crauste, N. Willand, B. Villemagne, M. Flipo, E. Willery, X. Carette, M. M. Dimala, A.-S. Drucbert, P.-M. Danze and B. Deprez, Anal. Biochem., 2014, 452, 54–66 CrossRef CAS PubMed.
- D. Dobrovodský and C. Di Primo, Biosens. Bioelectron., 2023, 232, 115296 CrossRef PubMed.
- E. H. Huntress and F. H. Carten, J. Am. Chem. Soc., 1940, 62, 511–514 CrossRef CAS.
- C. Das, S. R. Dash, S. Sinha, S. Paul, B. Das, S. Bhal, C. Sethy and C. N. Kundu, Med. Oncol., 2023, 40(12), 351 CrossRef CAS PubMed.
- M. Çağlayan, Nucleic Acids Res., 2020, 48, 3708–3721 CrossRef PubMed.
- Q. Fang, J. Andrews, N. Sharma, A. Wilk, J. Clark, J. Slyskova, C. A. Koczor, H. Lans, A. Prakash and R. W. Sobol, Nucleic Acids Res., 2019, 47, 6269–6286 CrossRef CAS PubMed.
|
This journal is © The Royal Society of Chemistry 2024 |
Click here to see how this site uses Cookies. View our privacy policy here.