Potent, Selective, and Cell Active Protein Arginine Methyltransferase 5

Article pubs.acs.org/jmc

Potent, Selective, and Cell Active Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor Developed by Structure-Based Virtual Screening and Hit Optimization Ruifeng Mao,†,‡,∇ Jingwei Shao,∥,⊥,∇ Kongkai Zhu,#,‡,∇ Yuanyuan Zhang,‡ Hong Ding,‡ Chenhua Zhang,○ Zhe Shi,○ Hualiang Jiang,‡ Dequn Sun,*,† Wenhu Duan,*,∥ and Cheng Luo*,‡,§ †

Marine College, Shandong University, Weihai 264209, P.R. China Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China § CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China ∥ Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China ⊥ University of Chinese Academy of Sciences, Beijing 100049, China # School of Biological Science and Technology, University of Jinan, Jinan 250022, P.R. China ○ Shanghai ChemPartner Co., LTD., Zhangjiang Hi-Tech Park, Shanghai 201203, China ‡

S Supporting Information *

ABSTRACT: PRMT5 plays important roles in diverse cellular processes and is upregulated in several human malignancies. Besides, PRMT5 has been validated as an anticancer target in mantle cell lymphoma. In this study, we found a potent and selective PRMT5 inhibitor by performing structure-based virtual screening and hit optimization. The identified compound 17 (IC50 = 0.33 μM) exhibited a broad selectivity against a panel of other methyltransferases. The direct binding of 17 to PRMT5 was validated by surface plasmon resonance experiments, with a Kd of 0.987 μM. Kinetic experiments indicated that 17 was a SAM competitive inhibitor other than the substrate. In addition, 17 showed selective antiproliferative effects against MV4-11 cells, and further studies indicated that the mechanism of cellular antitumor activity was due to the inhibition of PRMT5 mediated SmD3 methylation. 17 may represent a promising lead compound to understand more about PRMT5 and potentially assist the development of treatments for leukemia indications.

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INTRODUCTION Acting as a bridge between the genotype and the phenotype, post-translational modification of proteins plays a pivotal role in a variety of biological functions. Post-translational methylation of arginine residues catalyzed by protein arginine methyltransferases (PRMTs) is very important as it alters the activity and interactions of substrate proteins, with crucial influences on diverse cellular processes, including cell growth, differentiation, proliferation, and development.1−3 Up to now, nine mammalian PRMTs have been identified and are classified into three types (type I−III) based on the capacity of transferring one or two methyl groups to the nitrogen atoms of the guanidinium side chains within arginine residues using S-adenosylmethionine (SAM) as the methyl donor.4 Type I PRMT enzymes (PRMT1, -2, -3, -4, -6, and -8) catalyze the formation of ω-NG monomethyl and asymmetric ω-NG, NG-dimethyl arginine residues; type II enzymes (PRMT5 and PRMT9) catalyze the formation of ω-NG monomethyl and ω-NG, N′G-symmetric © 2017 American Chemical Society

dimethyl arginine residues; type III enzymes (PRMT7) catalyze the formation of ω-NG monomethyl arginine only.5 Recently, cancer epigenetic studies uncovered the dysregulation of histone modification as the driving force for tumorigenesis and cancer progression.6 Therefore, the development of potent and selective epigenetic modulators is currently a hot topic in the field of anticancer drug discovery. PRMT5 is the predominant type II PRMT and in association with the WD-repeat-containing protein MEP50 (methylosome protein 50) forms large multimolecular complexes with different binding partners. PRMT5 could specifically methylate a wide spectrum of substrates,7 including histone and nonhistone substrates (Table S1), of which the histone substrates are histones H2A residue Arg3 (H2AR3), H4 residue Arg3 (H4R3), and H3 residues Arg2 (H3R2) and Arg8 Received: April 18, 2017 Published: June 26, 2017 6289

DOI: 10.1021/acs.jmedchem.7b00587 J. Med. Chem. 2017, 60, 6289−6304

Journal of Medicinal Chemistry

Article

Chart 1. Structures of PRMT5 Inhibitors

Figure 1. (A) Workflow of the virtual screening strategy adopted in the present study. (B) Structures and inhibitory activities of 6 active compounds against PRMT5.

lung cancer,16 colorectal cancer,17 and glioblastoma.18 In addition, PRMT5 was validated as an anticancer target in mantle cell lymphoma (MCL) and glioblastoma.8,18 Although multiple efforts have been made to develop PRMT5 inhibitors, there are only three types of PRMT5 inhibitors reported (Chart 1). Compound 1 (EPZ015666)8 occupies the histone substrate pocket and shows dosedependent antitumor activity in multiple MCL xenograft models. Compound 2 (CMP5)19 is discovered by virtual screening methods and shows promise as a novel therapeutic approach for B-cell lymphomas. Compounds 3−5 are analogues of SAM, and all exhibit excellent PRTM5 inhibitory activity.20−23 Considering the importance of PRMT5 in tumorigenesis, it is badly in need of the identification of more potent and selective PRMT5 inhibitors to provide a powerful tool to explore and potentially further validate PRMT5 as a clinically relevant target. By performing pharmacophore- and molecule docking based virtual screening combined with an Alpha LISA assay, we found 6 PRMT5 inhibitors, which were confirmed in radioactive methylation assays resulting in the identification the new

(H3R8). Additionally, PRMT5 can methylate proteins involved in RNA splicing. Small nuclear ribonucleoproteins (snRNPs) are core integral components of the spliceosome. Sm proteins, one of which is SmD3, have a symmetric dimethyl arginine (sDMA)-modified activity, and they are necessary to assemble the snRNP in human cells. SnRNP assembly can be regulated by the interaction of methylated Sm proteins with the survival motor neuron (SMN) complex. Both PRMT5 and PRMT7 are required to catalyze cytoplasmic snRNP assembly, and both enzymes can facilitate Sm protein binding to the SMN complex. Meanwhile, knockdown of PRMT5 could decrease sDMA modification of Sm proteins and reduce snRNP assembly. Therefore, we can use the modification of SmD3 to explore the cellular biochemical activity of PRMT5. Antibodies to symmetric dimethyl arginine (sDMA) can be used to probe the cellular biochemical activity of PRMT5.8−11 PRMT5 is postulated to have a relationship with cell growth, cell cycle progression, cell death, and cell proliferation.7 Several reports stress the importance of PRMT5 in tumorigenesis, but the mechanism of PRMT5 driving tumorigenesis is still unknown. PRMT5 is upregulated in lymphomas,12−14 breast cancer,15 6290



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scaffold 6 (DC_P04) (IC50 = 24 μM). Chemical optimization of compound 6 resulted in compound 17 (IC50 = 0.33 μM), which showed good selectivity against a panel of other methyltransferases. The direct binding of 17 to PRMT5 was validated by SPR experiments, and 17 showed competitive inhibition with respect to SAM on PRMT5 and showed noncompetitive inhibition with respect to peptide on PRMT5 indicated by kinetic experiments. The antiproliferative effects of 17 against leukemia and lymphoma cells were tested, and the results indicated that 17 had selective antiproliferative effects in MV4-11 cells. Biochemical inhibition correlated well with the corresponding decreased levels of symmetrically dimethylated PRMT5 substrates and proliferation in a time- and concentration-dependent manner suggesting that the antiproliferative effects are a direct consequence of PRMT5 inhibition.

inhibition, and this result (Table 1) confirmed that compound 6 was the most active compound against PRMT5. Table 1. IC50 Values against PRMT5 of the Hits Derived from Virtual Screening Measured by Radioactive Methylation Assays compd number

IC50 (μM)

6 7 8 9

24 169 172 >200

Binding Mode Prediction of Compound 6 Provides Clues for Hit Optimization. To explore the molecular basis of inhibitory activity of compound 6 against PRMT5, as shown in Figure 2A, we used the same XP docking mode that was previously used to do the virtual screening to predict the binding mode of compound 6. The results revealed that compound 6 showed a binding mode similar to that of the SAH analogue, fitting well in the cofactor SAM binding pocket (Figure 2B). Residues involved in the interaction between compound 6 and PRMT5 were identified, and hydrogen bond and hydrophobic interaction networks are shown in Figure 2C and D, respectively. As shown in Figure 2C, the oxygen atom within the carbonyl of the ester group forms a hydrogen bond with L315 residues, so this ester group should be retained when chemistry modifications were performed. In the next step, hit optimization and SAR studies will be carried out to improve the inhibition activity of compound 6 against PRMT5. Chemical Synthesis. To discuss the SAR and improve the potency of compound 6, 31 compounds were synthesized. The PRMT5 inhibition activity was determined by a radioactive methylation assay, and compound 1 (IC50 = 0.047 μM) was used as the reference control; see Figure S2 for the IC50 results of the reference compounds. Synthetic routes for 31 compounds were outlined in Schemes 1 and 2. The synthesis started from appropriate o-nitroaniline or nitropyridine derivatives, which were commercially available or prepared according to a published procedure.36,37 Catalytic hydrogenation of the nitro group afforded diamine, which reacted with carbon disulfide in the presence of potassium hydroxide using ethanol as solvent to give the intermediates.38 Subsequent alkylation of the thio group with alkyl chlorides,39 which by reaction of substituted anilines or aminopyridine with chloroacetyl chloride in acetone, yielded the title compounds. SAR Exploration. The SAR investigation was commenced with the benzimidazole core. As shown in Table 2, substitution at the 4-position of the benzimidazole core (compounds 12− 14) led to a significant loss of PRMT5 enzyme activity. The 5position substituted 22, 15, and 17 displayed more than 100fold improvement of enzymatic potency compared with the 4position derivatives 12, 13, and 14, respectively. Encouraged by the IC50 of 0.33 μM of the 5-methoxy analogue (17), a series of 5-position substituted compounds (16, 18−21, and 23−27) was synthesized to explore the SAR, and all the assayed compounds were less potent than 17 except for 27 which showed comparable potency to that of 17. Replacement of the methoxy with isopropoxy (17 vs 20) led to 8-fold loss of potency, suggesting that it could not accommodate bulky substitution. The dimethylamino derivative 27 exhibited a more than 100-fold improvement compared with its amino analogue 25 indicating that the methyl contributed remarkably to the

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RESULTS AND DISCUSSION 116 Candidates Were Selected by Virtual Screening. Structure-based virtual screening is an effective method in hit compound discovery and has been widely used in the process of lead compound discovery.24−33 In this study, we used pharmacophore- and molecular docking based virtual screening to screen the drug-like SPECS database (http://www.specs. net), which contains about 207,342 compounds, for compounds that could inhibit PRMT5 activity (see Figure 1A for the screening workflow). On the basis of the human crystal structure of PRMT5 [Protein Data Bank (PDB) ID code 4GQB],34 we created a pharmacophore model (Figure S1) using Discovery Studio 3.0.35 Then, the pharmacophore model was used to screen the SPECS 3D database, which was generated by Generate Conformations with BEST method inserted in Discovery Studio 3.0, and a total of 4,344 small molecules that matched the pharmacophore were obtained. Next, the crystal structure (4GQB) was used as the reference structure for molecular docking based virtual screening. Residues located within 20 Å of the SAH analogue (compound 5) was defined as the binding site; then, 4,344 small molecules were docked into the binding site of PRMT5 with extraprecision (XP) docking mode. The top-866 small molecules ranked based on the docking score were clustered into 50 clusters using Clustering Molecules protocols integrated in Pipeline Pilot, version 7.5 (Pipeline Pilot; Accelrys Software Inc., San Diego, CA) for visual inspection, with the selection criteria as following: (1) The binding poses of the molecule occupied the SAM pocket and were reasonable with no high strain energy; (2) in each clustered group, at least one molecule was selected to retain molecular diversity; and (3) for those molecules with the same scaffold, the relatively simple structures were selected. At last, 116 candidates were selected and purchased from SPECS. Alpha LISA and Radioactive Methylation Assays Were Used to Determine the Inhibition Activity against PRMT5. In order to test the inhibition activity of 116 compounds, an Alpha LISA assay was carried out. We first tested the inhibition against PRMT5 at 100 μM and found that among the 116 tested compounds, 15 compounds showed an inhibition larger than 50% (Table S2). Then, the IC50 values of these 15 compounds were determined (Table S3). The enzyme-inhibition results indicated that 6 compounds showed micromolar IC50 lower than 30 μM, with compound 6 showing the most active inhibition with an IC50 of 8.1 μM (Figure 1B). Then, 4 selected compounds were subjected to a radioactive methylation assay to further verify their enzyme (PRMT5) 6291



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Figure 2. Binding mode prediction of compound 6. (A) Overall view of compound 6 binding to PRMT5. PRMT5 is shown as in cartoon form, and compound 6 is shown in stick representation. (B) Compound 6 aligns with SAH analogue (compound 5), the green stick representation is compound 6, and the magenta representation is compound 5. (C) Close-up views of the hydrogen bond interactions between compound 6 and PRMT5; compound 6 and interacting residues of PRMT5 are shown in green and magenta stick representation, respectively. (D) Close-up views of the hydrogen bonds and hydrophobic interactions between compound 6 and PRMT5; compound 6 is shown in green stick representation, while the hydrogen bond interacting residues and hydrophobic interacting residues of PRMT5 are shown in magenta and yellow stick representation, respectively. 4GQB (PDB code) was used to simulate the binding mode.

17 Showed Good Selectivity against PRMT5. To test the selectivity of 17 against PRMT5, we measured the IC50 values of 17 against a panel of methyltransferases including PRMT1, PRMT3, PRMT4, PRMT6, PRMT7, PRMT8, NSD1, DNMT1, DOT1L, SET7/9, and another two epigenetic targets bromodomain protein (BRD4) and histone acetyltransferase GCN5. As shown in Table 4, IC50 values of 17 against these methyltransferases, BRD4 as well as GCN5, were all greater than 100 μM, and these results indicated that 17 had selectivity for PRMT5. Surface Plasmon Resonance (SPR) Analysis. A surface plasmon resonance (SPR) assay was employed to assess the interaction between 17 and PRMT5, for which PRMT5/ MEP50 was immobilized on CM5 sensor chips. As shown in Figure 5, compound 17 displayed extracellular binding affinity with PRMT5 with an equilibrium dissociation constant (Kd) value of 0.987 μM, and the equilibrium binding curve fit is shown in Figure S4. This result indicated the direct binding of 17 to PRMT5 at the enzyme level. Although the direct binding of 17 to PRMT5 has been proved, efforts to fully understand the detailed binding mode of 17 are urgently needed through the pursuit of a PRMT5-compound 17 cocrystal structure. Kinetic Experiments. In order to verify the mechanism of action (MOA) of compound 17, we performed a kinetic experiment. This experiment was done using varied SAM concentrations with fixed peptide substrate and varied peptide concentrations with fixed SAM concentration. Compound 1 was used as the reference compound, as it was the validated PRMT5 inhibitor and occupied the substrate pocket. The results (Figure 6) indicated that compound 17 showed competitive inhibition with respect to SAM on PRMT5 and showed noncompetitive inhibition with respect to peptide on PRMT5, while compound 1 showed uncompetitive inhibition

enzymatic activity. The PRMT5 enzymatic inhibition was enhanced as the atomic radius increased in the halogen compounds 21−23. Incorporating a nitrogen atom to the benzimidazole ring (28 and 29) led to decreased activity. These results revealed that the 5-methoxy substituted benzimidazole moiety was optimal for PRMT5 activity, and the inhibition activity curves of selected compounds are shown in Figure 3. Next, we explored the SAR of the benzene ring moiety (Table 3). In the docking analysis of our hit compound 6 and PRMT5 enzymatic domain, a hydrogen bond formed between the carbonyl oxygen in the methoxycarbonyl group and the Leu315 residue. To probe the critical H-bond, derivatives 30− 34 without an H-bond acceptor were designed and synthesized, and the significant loss of enzymatic potency of 30−34 validated our modeling study. Replacement of the methoxycarbonyl of 17 with an ethoxycarbonyl or acetyl (37 and 35) led to a 5−9-fold decrease of potency. The decreased activity of the pyridine analogue 42 revealed that the phenyl ring was essential to maintaining PRMT5 potency. As discussed above, the 5-methoxy benzimidazole moiety and 2-methoxycarbonyl phenyl segment were optimal, and compound 17 was chosen for a further evaluation. It is interesting to note that compound 17 was actually identified in our virtual screening, but according to our select criteria, we selected its analogue compound 6 for the bioassay test. In order to understand the reason why 17 showed potent PRMT5 inhibition compared with that of compound 6, molecular docking was used to explore the binding mode of 17. As shown in Figure 4, compared to 6, 17 forms an additional hydrogen bond with Y324. Besides the same hydrogen bonding network, this may be the molecular basis for the more potent PRMT5 inhibition of 17 than 6. The detailed interaction between 17 and PRMT5 is shown in Figure S3. 6292



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Scheme 1a

Reagents and conditions: (a) (i) H2, 10% Pd/C, MeOH, rt; (ii) CS2, KOH, EtOH/H2O, 70 °C; (b) 50h, K2CO3, acetone, rt; (c) for 47a, EtONa, EtOH, 80 °C; for 47b, KOH, PrOH, 60 °C; for 47c, NaOH, i-PrOH, 80 °C; for 47d, methylammonium chloride, DIPEA, NMP, 100 °C; for 47e, dimethylamine, DIPEA, NMP, 100 °C; (d) TBDMSCl, imidazole, DMF, 0 °C to rt; (e) tetrabutylammonium fluoride, THF, rt.; (f) H2, 10% Pd/C, MeOH, rt. a

with respect to SAM on PRMT5 and showed competitive inhibition with respect to peptide on PRMT5. 17 Selectively Displays Antiproliferation in MV4-11 Leukemia Cells. As 17 showed selective and potent PRMT5inhibiting activity in vitro, we further explored whether it had antiproliferation effects in PRMT5 related cancer cell lines. One leukemia cell line (MV4-11) and three lymphoma cells (KOPN8, Jeko-1, and Z138) were used to expand our analysis of the differential sensitivity to 17. The antiproliferative effects of the PRMT5 inhibitor manifested over several days, necessitating the development of a long-term proliferation assay allowing for the measurement of cell growth over 12 days. As shown in Figure 7, MV4-11 cells were the most sensitive in all of the cells; notably, 17 demonstrated potent concentrationdependent antiproliferative effects, with an EC50 value of 6.53 μM (Figure 8A). However, this compound showed much weaker growth inhibition activities against other cell lines, with EC50 values ranging from 15 to 70 μM. On the basis of the antiproliferative effects assay, MV4-11 cells displayed considerably different cytotoxicity against the compounds. Therefore, the MV4-11 cell lines were selected to explore the mechanism of cell killing in more detail.

Besides, to address the possibility of off-target effects of compound 17, we tested the cytotoxicity of 17 against two other normal cells HUVEC and RCTEC. The results (Figure S5) showed that 17 has weaker cytotoxicity against these two cells, which suggests that 17 may have no off-target effect. Characterization of Cell Methylation. More work is required for an understanding of how PRMT5 activity is upregulated in MV4-11 cell lines that are dependent on this enzyme. The representative compound 17 was assessed using Western blot analysis by an sDMA antibody, with a dose titration of 0−20 μM compound (Figure 8B). After a 6 day treatment, 17 decreased the intensity of multiple bands, including one that colocalized with the previously reported PRMT5 substrate SmD3 in a dose-dependent manner, which were comparable with (Figure S6) those of compound 1. Biochemical inhibition correlated with corresponding decreased levels of symmetrically dimethylated PRMT5 substrates and proliferation in a time- and concentration-dependent manner suggesting that the antiproliferative effects were a direct consequence of PRMT5 inhibition. 6293



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Scheme 2a

Reagents and conditions: (a) chloroacetyl chloride, K2CO3, acetone, rt; or chloroacetyl chloride, Et3N, DCM, 0 °C to rt; (b) 46g, K2CO3, acetone, rt.

a

More importantly, 17 showed weaker cytotoxicity against two normal cells HUVEC and RCTEC. We also clarified the cellular cytotoxic mechanism of representative 17 derived PRMT5 inhibitors, which was mediated through the inhibition of PRMT5 substrate SmD3 and downstream signaling proteins. We believe that 17 is a promising compound that could help us gain a better understanding of the molecular biology of PRMT5 and potentially assist in defining a therapeutic strategy to treat leukemia indications.

Table 2. Structures and IC50 Values for 18 Synthesized Analogues of Compound 6

compds

X

Y

4

5

IC50(μM)

6 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH N CH

CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH N

H Cl Me OMe H H H H H H H H H H H H H H CH

H H H H Me OH OMe OEt OPr Oi‑Pr F Cl Br NO2 NH2 NHMe NMe2 OMe H

8.1 ± 0.85 >100 >100 >100 1.4 ± 0.07 >100 0.33 ± 0.06 0.35 ± 0.07 0.53 ± 0.08 2.8 ± 1.43 3.3 ± 4.60 0.50 ± 0.42 0.34 ± 0.11 0.83 ± 0.31 40 ± 7.07 1.6 ± 0.78 0.33 ± 0.18 8.4 ± 0.35 87 ± 2.12

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EXPERIMENTAL SECTION

Virtual Screening. Pharmacophore-Based Virtual Screening. In the crystal structure of PRMT5 (4GQB), the asymmetric unit contains a PRMT5, an MEP50, an SAH analogue (compound 5), and a peptide substrate derived from H4, so MEP50 and peptide were deleted. Then based on this modified complex structure, Discovery Studio 3.0 was used to generate the pharmacophore model. Discovery Studio 3.0 was used to perform the pharmacophore-based in silicon screening. Molecular Docking Based Virtual Screening. We employed the GLIDE 5.540,41 program to perform the molecular docking studies. The 4,344 candidate compounds that match the pharmacophore model were downloaded from the SPECS database, and then the LigPrep42 panel was used to produce multiple output structures of these compounds by generating different protonation states, stereochemistries, tautomers, and ring conformations for molecular docking. The Protein Preparation Wizard Workflow was used to prepare the protein structures. Residues located within 20 Å around compound 5 on PRMT5 were defined as binding sites in which the docking grids were created. The default settings were used. All of the 4,344 candidate compounds were docked into the defined binding site using extra precision (XP) mode without any constraint and were ranked by Glide-score. In compounds 6 and 17 against PRMT5, a similar procedure was used as described above. PRMT5 Inhibition Assays (Alpha LISA and Radioactive Methylation Assays). The PRMT5/MEP50 complex purchased from BPS, (Cat. No. 51045) was used as the enzyme to test the inhibition activities of screened compounds. The protocol of the Alpha LISA assay was as follows: (1) Prepare 1× assay buffer (modified Tris-HCl buffer); (2) transfer compounds to 384 assay plates by Echo in 100% DMSO; (3) prepare enzyme

CONCLUSION The multitude of reports implicating PRMT5 in cancer reflects the need for potent and selective inhibitors of PRMT5, so that its pathobiology can be further explored and validated. In the present study, we identified 17 as a novel inhibitor of PRMT5 which showed great selectivity and growth inhibition activities against MV4-11 cells over other leukemia and lymphoma cells. 6294



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Figure 3. IC50 values against PRMT5 of the selected compounds.

Table 3. Structures and IC50 Values for 13 Synthesized Analogues of Compound 6

compd

2′

3′

4′

Z

IC50(μM)

30 31 32 33 34 35 36 37 38 39 40 41 42

H Me Cl OMe NMe2 COMe COOH COOEt CONHMe CONMe2 H H COOMe

H H H H H H H H H H COOMe H H

H H H H H H H H H H H COOMe H

H H H H H H H H H H H H N

33 ± 33.94 >200 47 ± 0.71 19 ± 1.41 25 ± 2.83 2.9 ± 1.84 >100 1.9 ± 0.01 >200 >200 52 ± 5.66 >100 2.4 ± 0.57

Figure 4. Binding mode comparison of compounds 6 and 17. PRMT5 and the interacting residues of PRMT5 are shown as cartoon and magenta stick representation, respectively, while compounds 6 and 17 are shown in green and cyan stick representation, respectively. 4GQB (PDB code) was used to simulate the binding mode.

Table 4. Inhibitory Activities of 17 against PRMT1, PRMT3, PRMT4, PRMT6, PRMT7, PRMT8, NSD1, DNMT1, DOT1L, SET7/9, BRD4, and GCN5

solution in 1× assay buffer; (4) prepare substrate mix solution in 1× assay buffer; (5) transfer 5 μL of enzyme solution to the assay plate, or for low control, transfer 5 μL of 1× assay buffer; (6) incubate at room temperature for 15 min; (7) add 5 μL of substrate mix solution to each well to start the reaction; (8) incubate at room temperature for 60 min; (9) prepare 1× Alpha LISA buffer; (10) add 5 μL of acceptor solution, incubate for 60 min at RT, under subdued light; (11) add 10 μL of donor solution, incubate for 30 min at RT, under subdued light; (12) reading end point with Envision with Alpha mode; and (13) using GraphPad Prism V5.0 software. % inhibition = (max signal − compound signal)/(max signal − min signal) × 100. The maximum signal was obtained from the action of enzyme and substrate; the minimum signal was obtained from the substrate only. The protocol of the radioactive methylation assay was as follows: (1) Prepare 1× assay buffer (modified Tris buffer); (2) transfer compounds to the assay plate by Echo with 3-fold dilution in 100% DMSO; the final DMSO concentration is 1%; (3) prepare enzyme solution in 1× assay buffer; (4) prepare substrate solution in 1× assay

protein

IC50 (μM)

PRMT5 selectivity

PRMT1 PRMT3 PRMT4 PRMT6 PRMT7 PRMT8 NSD1 DNMT1 DOT1L SET7/9 BRD4 GCN5

>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100

>303 >303 >303 >303 >303 >303 >303 >303 >303 >303 >303 >303

buffer; (5) prepare [3H]-SAM solution in 1× assay buffer; (6) transfer 15 μL of enzyme solution to the assay plate, or for low control, transfer 15 μL of 1× assay buffer; (7) incubate at room temperature for 15 min; (8) add 5 μL of substrate solution to each well; (9) add 5 μL of 6295



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were recorded on a Bruker Avance III 126 MHz spectrometer. Chemical shifts (δ) are reported in parts per million, coupling constants (J) values are given in hertz, and peak multiplicities are expressed as follows: s, singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet; and m, multiplet. High resolution mass spectrometry was conducted using an Acquity UPLC I-Class/Xevo G2-S Q-TOF system (Waters Incorporation, Milford, MA, USA). Low-resolution mass spectra (ESI) were obtained using Agilent HPLC-MS (1260-6120B). All tested compounds were purified to ≥95% purity (Table S5) as determined by high performance liquid chromatography (HPLC). General Procedure A: Synthesis of Target Compounds 12−43. To a solution of intermediates (44a−c, 46a−d, 46f−j, or 48a−e) (0.22 mmol, 1.0 equiv) in acetone was added potassium carbonate (0.33 mmol, 1.5 equiv). After stirring for 15 min at room temperature, 2-chloroacetamide (50a−m or 52) (0.26 mmol, 1.2 equiv) was added, and the reaction mixture was stirred at room temperature for 4−12 h. The mixture was filtered, the filtrate was concentrated, and the residue was adsorbed on silica and purified by flash chromatography (dichloromethane/methanol, v/v, 99:1 to 90:10), affording the title compound. Methyl 2-(2-((4-Chloro-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (12). The title compound was prepared from 44a and 50h following general procedure A. White solid (79%). 1H NMR (300 MHz, DMSO-d6) δ 13.08 (s, 1H), 11.17 (s, 1H), 8.29 (d, J = 7.5 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.38 (s, 1H), 7.15 (m, 3H), 4.32 (s, 2H), 3.76 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.2, 166.5, 150.7, 140.4, 139.2, 136.8, 134.0, 130.5, 123.5, 122.5, 121.2, 121.1, 117.8, 116.3, 109.6, 52.3, 36.0. HRMS (ESI−) m/z calcd for C17H13N3O3ClS [M − H]−: 354.0366; found, 354.0363. Methyl 2-(2-((4-methyl-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (13). The title compound was prepared from 44b and 50h following general procedure A. White solid (82%). 1H NMR (300 MHz, DMSO-d6) δ 12.66 (s, 1H), 11.24 (s, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.21 (m, 2H), 7.00 (t, J = 7.7 Hz, 1H), 6.90 (d, J = 7.0 Hz, 1H), 4.27 (s, 2H), 3.77 (s, 3H), 2.43 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.2, 166.9, 148.7, 143.1, 139.3, 135.4, 134.0, 130.6, 126.9, 123.5,

Figure 5. Compound 17 binding to PRMT5/MEP50 was analyzed with SPR, and the dissociation constant (Kd = 0.987 μM) was calculated. [3H]-SAM solution to each well to start the reaction; (10) incubate at RT for 60 min; (11) add cold SAM in 1× assay buffer to make the stop mix; (12) stop the reaction with the addition of 5 μL per well of stop solution; (13) transfer 25 μL of volume per well to Flashplate from the assay plate; (14) incubate for 1 h minimum at room temperature; (15) wash Flashplate with dH2O + 0.1% Tween-20 three times; (16) read plate on Microbeta; and (17) fit the data in Excel to obtain inhibition values using eq 1

inh % = (max − signal)/(max − min) × 100

(1)

Fit the data in GraphPad Prism 5 to obtain IC50 values using eq 2

Y = bottom + (top − bottom)/(1 + 10((LogIC50 − X ) × Hill slope)) (2) Y is % inhibition, and X is compound concentration. Chemistry Methods. General. All chemicals and solvents were purchased from commercial sources and used without further purification unless otherwise noted. Reactions were monitored by TLC, using silica gel plates with fluorescence F254 and visualized under UV light. Purification was performed by flash chromatography using silica gel (300−400 mesh). 1H NMR spectra were recorded on a Varian Mercury Plus 300 MHz spectrometer, and 13C NMR spectra

Figure 6. Kinetic experiment results. 6296



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Figure 7. Inhibition of the proliferation of MV4-11, Jeko-1, KOPN8, and Z138 cells by compound 17 in vitro over 12 days in culture.

Figure 8. (A) Effects of the compound and 17 on the proliferation of MV4-11 cells. (B) Effects of 17 on cellular target inhibition as determined by sMDA Western blot. 122.2, 121.4, 121.0, 117.6, 114.9, 52.3, 36.0, 16.6. HRMS (ESI−) m/z calcd for C18H16N3O3S [M − H]−: 354.0912; found, 354.0910. Methyl 2-(2-((4-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (14). The title compound was prepared from 44c and 50h following general procedure A. White solid (79%). 1H NMR (300 MHz, DMSO-d6) δ 12.80 (s, 1H), 11.21 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.61 (t, J = 6.6 Hz, 1H), 7.19 (t, J = 6.5 Hz, 1H), 7.03 (s, 2H), 6.67 (s, 1H), 4.26 (s, 2H), 3.88 (s, 3H), 3.77 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.2, 166.8, 149.6, 147.0, 139.2, 137.2, 134.0, 130.6, 123.5, 122.4, 121.8, 121.0, 117.6, 110.4, 103.2, 55.5, 52.3, 36.0. HRMS (ESI−) m/z calcd for C18H16N3O4S [M − H]−: 370.0862; found, 370.0854. Methyl 2-(2-((5-Methyl-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (15). The title compound was prepared from 46a and 50h following general procedure A. White solid (56%). 1H NMR (300 MHz, CD3OD) δ 8.49 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.25 (s, 1H), 7.13 (t, J = 7.7 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 4.20 (s, 2H), 3.76 (s, 3H), 2.41 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 168.5, 168.3, 148.0, 140.5, 134.6, 132.3, 131.0, 123.9, 123.6, 120.9, 116.1, 52.6, 37.8, 21.7. HRMS (ESI−) m/z calcd for C18H16N3O3S [M − H]−: 354.0912; found, 354.0908. Synthesis of Methyl-2-(2-((5-hydroxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)-benzoate (16). Compound 16a (100 mg, 0.21 mmol, 1.0 equiv) was dissolved in 4 mL of THF, and tetrabutylammonium fluoride (1 mol/L in THF, 0.25 mL, 0.25 mmol, 1.2 equiv) was added. The mixture was stirred at room

temperature for 2 h and then diluted with ethyl acetate. The solution was washed with water and brine successively, dried over anhydrous sodium sulfate, and concentrated in vacuum. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol, v/ v, 94:6) to give the title product as a white solid (50 mg, 67%). 1H NMR (300 MHz, CD3OD) δ 8.48 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 6.5 Hz, 1H), 7.54 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.14 (t, J = 7.3 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 6.70 (dd, J = 8.7, 2.0 Hz, 1H), 4.15 (s, 2H), 3.77 (s, 3H). 13C NMR (126 MHz, CD3OD) δ 169.2, 168.9, 154.9, 148.4, 141.2, 135.1, 131.9, 124.6, 122.0, 118.0, 116.1, 112.9, 99.7, 52.8, 38.1. HRMS (ESI−) m/z calcd for C17H14N3O4S [M − H]−: 356.0705; found, 356.0698. Methyl 2-(2-((5-((tert-Butyldimethylsilyl)oxy)-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (16a). The title compound was prepared from 46f and 50h following general procedure A. Yellow solid (67%). 1H NMR (300 MHz, CD3OD) δ 8.47 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.30 (d, J = 8.8 Hz, 1H), 7.09 (t, J = 7.7 Hz, 1H), 6.90 (s, 1H), 6.71 (d, J = 8.7 Hz, 1H), 4.17 (s, 2H), 3.75 (s, 3H), 0.98 (s, 9H), 0.16 (s, 6H). Methyl 2-(2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (17). The title compound was prepared from 46g and 50h following general procedure A. White solid (80%). 1H NMR (300 MHz, CD3OD) δ 8.49 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.34 (d, J = 9.0 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.81 (d, J = 9.0 Hz, 1H), 4.17 (s, 2H), 3.80 (s, 3H), 3.77 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.2, 166.9, 155.3, 147.3, 139.3, 137.9, 136.3, 134.0, 130.6, 123.4, 120.9, 117.9, 6297



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117.4, 110.7, 94.2, 55.4, 52.4, 36.0. HRMS (ESI−) m/z calcd for C18H16N3O4S [M − H]−: 370.0862; found, 370.0858. Methyl 2-(2-((5-Ethoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (18). The title compound was prepared from 48a and 50h following general procedure A. White solid (84%). 1H NMR (300 MHz, CD3OD) δ 8.47 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.93 (s, 1H), 6.78 (d, J = 8.9 Hz, 1H), 4.16 (s, 2H), 3.98 (q, J = 6.9 Hz, 2H), 3.75 (s, 3H), 1.36 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.2, 166.9, 154.5, 147.3, 139.3, 137.8, 136.2, 134.0, 130.6, 123.5, 120.9, 117.8, 117.5, 110.8, 95.0, 63.4, 52.4, 36.0, 14.7. HRMS (ESI−) m/z calcd for C19H18N3O4S [M − H]−: 384.1018; found, 384.1005. Methyl 2-(2-((5-Propoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (19). The title compound was prepared from 48b and 50h following general procedure A. White solid (66%). 1H NMR (300 MHz, CD3OD) δ 8.48 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.14 (t, J = 7.7 Hz, 1H), 6.96 (s, 1H), 6.81 (dd, J = 8.9, 2.2 Hz, 1H), 4.17 (s, 2H), 3.92 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H), 1.78 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 168.6, 168.5, 156.0, 147.2, 140.5, 134.7, 131.1, 123.7, 121.0, 116.1, 112.6, 70.3, 52.6, 38.1, 22.7, 10.7. HRMS (ESI−) m/z calcd for C20H20N3O4S [M − H]−: 398.1175; found, 398.1176. Methyl 2-(2-((5-Isopropoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (20). The title compound was prepared from 48c and 50h following general procedure A. White solid (80%). 1H NMR (300 MHz, CD3OD) δ 8.48 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 6.96 (s, 1H), 6.79 (d, J = 8.5 Hz, 1H), 4.61−4.47 (m, 1H), 4.17 (s, 2H), 3.76 (s, 3H), 1.29 (d, J = 6.0 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 168.4, 168.3, 154.4, 147.6, 140.4, 134.6, 131.0, 123.6, 120.9, 116.1, 113.9, 71.1, 52.6, 37.9, 22.2 (2C). HRMS (ESI−) m/z calcd for C20H20N3O4S [M − H]−: 398.1175; found, 398.1180. Methyl 2-(2-((5-Fluoro-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (21). The title compound was prepared from 46b and 50h following general procedure A. Yellow solid (80%). 1H NMR (300 MHz, DMSO-d6) δ 12.86 (s, 1H), 11.22 (s, 1H), 8.33 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 7.3 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.42 (s, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 6.96 (t, J = 9.0 Hz, 1H), 4.27 (s, 2H), 3.77 (s, 3H). 13C NMR (126 MHz, DMSOd6) δ 167.2 (s), 166.7 (s), 158.2 (d, J = 234.7 Hz), 139.3 (s), 134.0 (s), 130.6 (s), 123.5 (s), 121.0 (s), 117.5 (s), 109.22(d, J = 25.2 Hz), 52.4 (s), 35.9 (s). HRMS (ESI−) m/z calcd for C17H13N3O3FS [M − H]−: 358.0662; found, 358.0650. Methyl 2-(2-((5-Chloro-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (22). The title compound was prepared from 46c and 50h following general procedure A. Yellow solid (83%). 1H NMR (300 MHz, DMSO-d6) δ 11.21 (s, 1H), 8.32 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.50 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.16 (m, 2H), 4.28 (s, 2H), 3.77 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.2, 166.6, 151.1, 139.3, 134.0, 130.6, 125.9, 123.5, 121.6, 121.0, 117.5, 114.6, 52.3, 35.9. HRMS (ESI−) m/z calcd for C17H13N3O3SCl [M − H]−: 374.0366; found, 374.0366. Methyl 2-(2-((5-Bromo-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (23). The title compound was prepared from 46d and 50h following general procedure A. Yellow solid (76%). 1H NMR (300 MHz, CD3OD) δ 8.50 (d, J = 7.6 Hz, 1H), 7.97 (dd, J = 8.0, 1.5 Hz, 1H), 7.62 (s, 1H), 7.59−7.51 (m, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.28 (dd, J = 8.5, 1.8 Hz, 1H), 7.19−7.11 (m, 1H), 4.24 (s, 2H), 3.77 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 168.6, 168.3, 150.1, 140.4, 134.7, 131.1, 125.6, 123.8, 120.9, 117.7, 116.1, 115.6, 52.7, 37.6. HRMS (ESI−) m/z calcd for C17H13N3O3SBr [M − H]−: 417.9861; found, 417.9871. Methyl 2-(2-((5-Nitro-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (24). The title compound was prepared from 46h and 50h following general procedure A. Yellow solid (80%). 1H NMR (300 MHz, CDCl3) δ 11.78 (s, 1H), 8.67 (d, J = 8.4 Hz, 1H), 8.43 (s, 1H), 8.16−8.00 (m, 2H), 7.59 (t, J = 7.9 Hz, 1H), 7.51 (m, 1H), 7.17 (t, J =

7.6 Hz, 1H), 4.13 (s, 2H), 3.94 (s, 3H). 13C NMR (126 MHz, DMSOd6) δ 167.2, 166.4, 142.2, 139.3, 134.0, 130.6, 123.5, 121.0, 117.6, 117.5, 52.4, 35.9. HRMS (ESI−) m/z calcd for C17H13N4O5S [M − H]−: 385.0607; found, 385.0599. Synthesis of Methyl 2-(2-((5-Amino-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (25). Compound 24 (126 mg) was dissolved in 5 mL of methanol, and 10% palladium carbon (20 mg) was added. The mixture was stirred at room temperature under hydrogen atmosphere for 6 h. The mixture was filtered, and the filtrate was purified by silica gel chromatography(dichloromethane/methanol, v/v, 90:10) to give the title product as a yellow solid (100 mg, 86%). 1 H NMR (300 MHz, CD3OD) δ 8.47 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 6.8 Hz, 1H), 7.52 (t, J = 7.2 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 1.6 Hz, 1H), 6.67 (dd, J = 8.5, 1.9 Hz, 1H), 4.13 (s, 2H), 3.77 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.2, 167.0, 144.5, 139.3, 139.2, 136.8, 135.8, 134.0, 130.6, 123.5, 121.0, 120.8, 117.6, 110.5, 94.2, 52.4, 36.2. HRMS (ESI+) m/z calcd for C17H17N4O3S [M + H]+: 357.1019; found, 357.1016. Methyl 2-(2-((5-(Methylamino)-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (26). The title compound was prepared from 48d and 50h following general procedure A. Yellow solid (52%). 1H NMR (300 MHz, CD3OD) δ 8.48 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 7.4 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 7.13 (t, J = 7.3 Hz, 1H), 6.63 (m, 2H), 4.11 (s, 2H), 3.77 (s, 3H), 2.77 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 167.2, 167.0, 146.1, 139.3, 134.0, 130.6, 123.5, 120.9, 117.5, 109.7, 52.4, 36.2, 30.5. HRMS (ESI−) m/z calcd for C18H17N4O3S [M − H]−: 369.1021; found, 369.1012. Methyl 2-(2-((5-(Dimethylamino)-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (27). The title compound was prepared from 48e and 50h following general procedure A. Yellow solid (63%). 1 H NMR (300 MHz, CD3OD) δ 8.46 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.31 (d, J = 9.5 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.79 (d, J = 5.9 Hz, 2H), 4.12 (s, 2H), 3.75 (s, 3H), 2.86 (s, 6H). 13C NMR (126 MHz, CD3OD) δ 169.2, 169.0, 149.5, 147.6, 141.3, 135.1, 131.9 124.6, 122.0, 118.0, 116.2, 112.8, 52.8, 42.4 (2C), 38.2. HRMS (ESI−) m/z calcd for C19H19N4O3S [M − H]−: 383.1178; found, 383.1176. Methyl 2-(2-((5-Methoxy-3H-imidazo[4,5-b]pyridin-2-yl)thio)acetamido)benzoate (28). The title compound was prepared from 46i and 50h following general procedure A. Gray solid (67%). 1H NMR (300 MHz, CDCl3) δ 11.64 (s, 1H), 11.27 (s, 1H), 8.65 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.79 (s, 1H), 7.54 (t, J = 7.0 Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 6.60 (d, J = 7.9 Hz, 1H), 4.10 (s, 2H), 3.94 (s, 3H), 3.89 (s, 3H). HRMS (ESI−) m/z calcd for C17H15N4O4S [M − H]−: 371.0814; found, 371.0814. Methyl 2-(2-((3H-Imidazo[4,5-c]pyridin-2-yl)thio)acetamido)benzoate (29). The title compound was prepared from 46j and 50h following general procedure A. Yellow solid (53%). 1H NMR (300 MHz, CD3OD) δ 8.66 (s, 1H), 8.49 (d, J = 8.3 Hz, 1H), 8.11 (d, J = 6.1 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.54 (m, 2H), 7.11 (t, J = 7.7 Hz, 1H), 4.28 (s, 2H), 3.76 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.1, 167.0, 147.9, 140.6, 139.3, 136.1, 134.0, 132.9, 130.6, 123.4, 120.9, 117.5, 109.0, 52.4, 36.0. HRMS (ESI−) m/z calcd for C16H13N4O3S [M − H]−: 341.0708; found, 341.0709. 2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-phenylacetamide (30). The title compound was prepared from 46g and 50a following general procedure A. Yellow solid (83%). 1H NMR (300 MHz, DMSO-d6) δ 12.52 (s, 1H), 10.50 (s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.31 (m, 3H), 7.05 (t, J = 7.3 Hz, 1H), 6.91 (m, 1H), 6.75 (d, J = 8.5 Hz, 1H), 4.22 (s, 2H), 3.76 (s, 3H). HRMS (ESI−) m/z calcd for C16H14N3O2S [M − H]−: 312.0807; found, 312.0810. 2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-(o-tolyl)acetamide (31). The title compound was prepared from 46g and 50b following general procedure A. White solid (68%). 1H NMR (300 MHz, CD3OD) δ 7.46 (d, J = 7.4 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.11 (m, 3H), 6.99 (s, 1H), 6.84 (dd, J = 8.8, 2.3 Hz, 1H), 4.10 (s, 2H), 3.81 (s, 3H), 2.15 (s, 3H). HRMS (ESI−) m/z calcd for C17H16N3O2S [M − H]−: 326.0963; found, 326.0965. 2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-(2chlorophenyl)acetamide (32). The title compound was prepared 6298



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J = 7.5 Hz, 1H), 7.04 (s, 1H), 6.89 (d, J = 8.4 Hz, 1H), 3.95 (s, 2H), 3.88 (s, 3H), 3.85 (s, 3H). HRMS (ESI−) m/z calcd for C18H16N3O4S [M − H]−: 370.0862; found, 370.0855. Methyl 4-(2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (41). The title compound was prepared from 46g and 50m following general procedure A. White solid (78%). 1H NMR (300 MHz, CDCl3) δ 11.54 (s, 1H), 7.98 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 8.7 Hz, 1H), 7.02 (s, 1H), 6.92 (dd, J = 8.8, 2.3 Hz, 1H), 3.96 (s, 2H), 3.89 (s, 3H), 3.86 (s, 3H). HRMS (ESI−) m/z calcd for C18H16N3O4S [M − H]−: 370.0862; found, 370.0855. Methyl 3-(2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)isonicotinate (42). The title compound was prepared from 46g and 52 following general procedure A. White solid (56%). 1 H NMR (300 MHz, CDCl3) δ 11.31 (s, 1H), 9.90 (s, 1H), 8.45 (d, J = 5.0 Hz, 1H), 7.74 (d, J = 5.0 Hz, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.00 (s, 1H), 6.82 (d, J = 8.8 Hz, 1H), 4.10 (s, 2H), 3.90 (s, 3H), 3.80 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 168.1, 166.9, 156.5, 147.4, 144.8, 144.1, 135.2, 122.9, 122.7, 111.9, 55.9, 53.2, 37.3. HRMS (ESI−) m/z calcd for C17H15N4O4S [M − H]−: 371.0814; found, 371.0809. General Procedure B: Synthesis of Target Compounds 44a−c, 46a−e, 46g, 46i, 46j, and 48a−e. O-Nitroaniline or nitropyridine derivatives (43a−c, 45a−h, or 47a−e) (1.0 equiv) and 10% palladium carbon (0.1 equiv) were suspended in methanol, and the mixture was stirred in a hydrogen atmosphere for 6 h. The reaction vessel was purged with argon, and the catalyst was removed by filtration and washed with methanol. The filtrate was concentrated to dryness and dissolved in ethanol. Carbon disulfide (5.0 equiv) and potassium hydroxide (1.0 equiv, dissolved in water) were added, and the mixture was stirred at 70 °C overnight with a condensation device. After the reaction mixture was concentrated, water was added to the residue. The generated solid was collected by filtration and washed with water, affording the title compound. 4-Chloro-1H-benzo[d]imidazole-2-thiol (44a). The title compound was prepared from 43a following general procedure B. White solid (65%). 1H NMR (300 MHz, DMSO-d6) δ 13.02 (s, 1H), 12.78 (s, 1H), 7.15 (t, J = 5.8 Hz, 1H), 7.09 (m, 2H). MS (ESI) m/z 185.1 [M + H]+. 4-Methyl-1H-benzo[d]imidazole-2-thiol (44b). The title compound was prepared from 43b following general procedure B. Yellow solid (72%). 1H NMR (300 MHz, DMSO-d6) δ 12.50 (s, 2H), 7.10− 6.82 (m, 3H), 2.37 (s, 3H). MS (ESI) m/z 165.2 [M + H]+. 4-Methoxy-1H-benzo[d]imidazole-2-thiol (44c). The title compound was prepared from 43c following general procedure B. Yellow solid (81%). 1H NMR (300 MHz, DMSO-d6) δ 12.66 (s, 1H), 12.47 (s, 1H), 7.05 (t, J = 8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 2H), 3.86 (s, 3H). MS (ESI) m/z 181.1 [M + H]+. 5-Methyl-1H-benzo[d]imidazole-2-thiol (46a). The title compound was prepared from 45a following general procedure B. Beige solid (90%). 1H NMR (300 MHz, DMSO-d6) δ 12.42 (s, 2H), 7.01 (d, J = 7.9 Hz, 1H), 6.93 (d, J = 8.4 Hz, 2H), 2.33 (s, 3H). MS (ESI) m/z 165.2 [M + H]+. 5-Fluoro-1H-benzo[d]imidazole-2-thiol (46b). The title compound was prepared from 45b following general procedure B. Brown solid (67%). 1H NMR (300 MHz, DMSO-d6) δ 12.59 (s, 2H), 7.09 (dd, J = 9.0, 4.3 Hz, 1H), 6.94 (m, 2H). MS (ESI) m/z 169.0 [M + H]+. 5-Chloro-1H-benzo[d]imidazole-2-thiol (46c). The title compound was prepared from 45c following general procedure B. Gray solid (73%). 1H NMR (300 MHz, DMSO-d6) δ 12.64 (s, 2H), 7.12 (s, 3H). MS (ESI) m/z 184.9 [M + H]+. 5-Bromo-1H-benzo[d]imidazole-2-thiol (46d). The title compound was prepared from 45g following general procedure B. Brown solid (65%). 1H NMR (300 MHz, DMSO-d6) δ 12.67 (s, 1H), 12.63 (s, 1H), 7.25 (m), 7.06 (d, J = 8.9 Hz, 1H). MS (ESI) m/z 228.9, 230.9 [M + H]+. 5-Hydroxy-1H-benzo[d]imidazole-2-thiol (46e). The title compound was prepared from 45h following general procedure B. Brown solid (65%). 1H NMR (300 MHz, DMSO-d6) δ 12.23 (s, 1H), 12.19

from 46g and 50c following general procedure A. Yellow solid (61%). 1 H NMR (300 MHz, CD3OD) δ 7.97 (d, J = 8.1 Hz, 1H), 7.41−7.28 (m, 2H), 7.21 (t, J = 7.8 Hz, 1H), 7.05 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 1.8 Hz, 1H), 6.81 (dd, J = 8.8, 2.3 Hz, 1H), 4.12 (s, 2H), 3.78 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 168.7, 156.5, 149.2, 135.0, 129.5, 127.5, 125.4, 124.5, 122.9, 115.2, 111.8, 97.6, 56.0, 36.3. HRMS (ESI−) m/z calcd for C16H13N3O2SCl [M − H]−: 346.0417; found, 346.0414. 2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-(2methoxyphenyl)acetamide (33). The title compound was prepared from 46g and 50d following general procedure A. Yellow solid (63%). 1 H NMR (300 MHz, CD3OD) δ 8.06 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 8.8 Hz, 1H), 7.09−6.97 (m, 2H), 6.86 (m, 3H), 4.11 (s, 2H), 3.80 (s, 3H), 3.62 (s, 3H). HRMS (ESI−) m/z calcd for C17H16N3O3S [M − H]−: 342.0912; found, 342.0910. 2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-(2(dimethylamino)phenyl)acetamide (34). The title compound was prepared from 46g and 50e following general procedure A. Brown solid (81%). 1H NMR (300 MHz, DMSO) δ 12.60 (s, 1H), 9.90 (s, 1H), 8.05 (m, 1H), 7.39 (m, 1H), 7.01 (m, 4H), 6.77 (d, J = 8.7 Hz, 1H), 4.19 (s, 2H), 3.76 (s, 3H), 2.33 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 168.3, 156.4, 148.8, 144.0, 132.9, 124.8, 124.6, 120.6, 120.0, 111.7, 97.0, 55.9, 44.4(2C), 36.5. HRMS (ESI−) m/z calcd for C18H19N4O2S [M − H]−: 355.1229; found, 355.1232. 2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-(2acetylphenyl)acetamide (35). The title compound was prepared from 46g and 50f following general procedure A. Yellow solid (61%). 1H NMR (300 MHz, CD3OD) δ 8.53 (d, J = 8.5 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.33 (s, 1H), 7.19 (t, J = 7.7 Hz, 1H), 6.96 (s, 1H), 6.81 (d, J = 8.9 Hz, 1H), 4.16 (s, 2H), 3.80 (s, 3H), 2.55 (s, 3H). HRMS (ESI−) m/z calcd for C18H16N3O3S [M − H]−: 354.0912; found, 354.0903. 2-(2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoic Acid (36). The title compound was prepared from 46g and 50g following general procedure A. Gray solid (61%). 1H NMR (300 MHz, DMSO-d6) δ 12.70 (s, 1H), 8.47 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 7.00 (s, 1H), 6.73 (d, J = 8.1 Hz, 1H), 4.23 (s, 2H), 3.73 (s, 3H). HRMS (ESI−) m/z calcd for C17H14N3O4S [M − H]−: 356.0705; found, 356.0710. Ethyl 2-(2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (37). The title compound was prepared from 46g and 50i following general procedure A. White solid (67%). 1H NMR (300 MHz, CD3OD) δ 8.47 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.94 (s, 1H), 6.78 (d, J = 8.0 Hz, 1H), 4.26−4.08 (m, 4H), 3.76 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H). HRMS (ESI−) m/z calcd for C19H18N3O4S [M − H]−: 384.1018; found, 384.1015. 2-(2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)-Nmethylbenzamide (38). The title compound was prepared from 46g and 50j following general procedure A. White solid (71%). 1H NMR (300 MHz, CD3OD) δ 8.27 (d, J = 8.2 Hz, 1H), 7.54 (d, J = 7.4 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 6.99 (s, 1H), 6.81 (d, J = 8.9 Hz, 1H), 4.12 (s, 2H), 3.80 (s, 3H), 2.67 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 169.6, 168.0, 156.5, 147.2, 138.1, 132.4, 127.1, 124.0, 122.0, 121.9, 112.2, 55.9, 38.3, 26.9. HRMS (ESI−) m/z calcd for C18H17N4O3S [M − H]−: 369.1021; found, 369.1019. 2-(2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)N,N-dimethylbenzam-ide (39). The title compound was prepared from 46g and 50k following general procedure A. White solid (68%). 1 H NMR (300 MHz, CD3OD) δ 7.68 (d, J = 8.1 Hz, 1H), 7.50−7.34 (m, 2H), 7.34−7.17 (m, 2H), 7.02 (s, 1H), 6.83 (dd, J = 8.8, 2.2 Hz, 1H), 4.04 (s, 2H), 3.81 (s, 3H), 2.80 (s, 3H), 2.73 (s, 3H). HRMS (ESI−) m/z calcd for C19H19N4O3S [M − H]−: 383.1178; found, 383.1168. Methyl 3-(2-((5-Methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (40). The title compound was prepared from 46g and 50l following general procedure A. White solid (75%). 1H NMR (300 MHz, CDCl3) δ 11.41 (s, 1H), 8.23 (s, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.36 (t, 6299



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(s, 1H), 9.28 (s, 1H), 6.92 (d, J = 9.1 Hz, 1H), 6.55 (m, 2H). MS (ESI) m/z 167.1 [M + H]+. 5-((tert-Butyldimethylsilyl)oxy)-1H-benzo[d]imidazole-2-thiol (46f). Compound 46e (560 mg, 5.1 mmol, 1.0 equiv) and imidazole (580 mg, 10.3 mmol, 2.0 equiv) were dissolved in DMF (15 mL). The solution was cooled to 0 °C. tert-Butyldimethylsilyl chloride (TBDMSCl) (1.60 g, 7.7 mmol, 1.5 equiv) was added, and the solution was allowed to warm to room temperature. The reaction mixture was stirred for 12 h. The solution was diluted with ethyl acetate and washed with brine. The organic layer was dried over sodium sulfate and concentrated in vacuum. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol, v/ v, 98:2) to give the title product as a beige solid (749 mg, 52%). 1H NMR (300 MHz, DMSO-d6) δ 12.36 (s, 1H), 12.30 (s, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.61 (d, J = 8.6 Hz, 1H), 6.55 (s, 1H), 0.93 (s, 9H), 0.14 (s, 6H). MS (ESI) m/z 281.3 [M + H]+. 5-Methoxy-1H-benzo[d]imidazole-2-thiol (46g). The title compound was prepared from 45d following general procedure B. Brown solid (72%). 1H NMR (300 MHz, DMSO-d6) δ 12.39 (s, 1H), 12.35 (s, 1H), 7.01 (d, J = 8.6 Hz, 1H), 6.70 (dd, J = 8.6, 2.3 Hz, 1H), 6.65 (d,J = 2.3 Hz 1H), 3.72 (s, 3H). MS (ESI) m/z 181.2 [M + H]+. 6-Methoxy-3H-imidazo[4,5-b]pyridine-2-thiol (46i). The title compound was prepared from 45e following general procedure B. Gray solid (52%). 1H NMR (300 MHz, DMSO-d6) δ 12.95 (s, 1H), 12.51 (s, 1H), 7.42 (d, J = 8.5 Hz, 1H), 6.53 (d, J = 8.5 Hz, 1H), 3.79 (s, 3H). MS (ESI) m/z 204.0 [M + Na]+. 1H-Imidazo[4,5-c]pyridine-2-thiol (46j). The title compound was prepared from 45f following general procedure B. Green solid (75%). 1 H NMR (300 MHz, DMSO-d6) δ 12.88 (s, 2H), 8.36 (s, 1H), 8.23 (d, J = 5.4 Hz, 1H), 7.16 (d, J = 5.3 Hz, 1H). MS (ESI) m/z 152.0 [M + H]+. Synthesis of 5-Ethoxy-2-nitroaniline (47a). Compound 45b (1.0 g, 6.4 mmol, 1.0 equiv) was dissolved in 15 mL of ethanol, and 2.5 mL of sodium ethoxide (20% in ethanol, 32.0 mmol, 5.0 equiv) was added. The mixture was heated to 80 °C for 15 h. Water was added, and the reaction mixture was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuum. The resulting residue was purified by silica gel chromatography (petroleum ether/ethyl acetate, v/v, 90:10) to give the desired product as a yellow solid (1.0 g, 90%. 1H NMR (300 MHz, CDCl3) δ 8.06 (d, J = 9.5 Hz, 1H), 6.27 (dd, J = 9.5, 2.5 Hz, 1H), 6.22 (s, 2H), 6.13 (d, J = 2.5 Hz, 1H), 4.04 (q, J = 7.0 Hz, 2H), 1.42 (t, J = 7.0 Hz, 3H). Synthesis of 5-Propoxy-2-nitroaniline (47b). To a solution of compound 45b (600 mg, 3.84 mmol, 1.0 equiv) in 15 mL of acetone, 2 mL of propanol was added followed by potassium hydroxide (1.1 g, 19.20 mmol, 5.0 equiv, dissolved in 3 mL water). The reaction mixture was stirred at 60 °C for 24 h. The mixture was concentrated and purified by flash chromatography (petroleum ether/ethyl acetate, v/v, 90:10), affording the title compound as a yellow solid (420 mg, 56%). 1 H NMR (300 MHz, CDCl3) δ 8.07 (d, J = 9.5 Hz, 1H), 6.28 (dd, J = 9.5, 2.4 Hz, 1H), 6.13 (d, J = 2.4 Hz, 1H), 3.93 (t, J = 6.5 Hz, 2H), 1.90−1.72 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H). Synthesis of 5-Isopropoxy-2-nitroaniline (47c). Compound 45b (936 mg, 6.0 mmol, 1.0 equiv), sodium hydroxide (360 mg, 9.0 mmol, 1.5 equiv), and i-propanol (10 mL) were mixed and heated to 80 °C for 4 h. The mixture was concentrated and purified by silica gel chromatography (petroleum ether/ethyl acetate, v/v, 85:15) to give the title product as a yellow solid (830 mg, 71%). 1H NMR (300 MHz, CDCl3) δ 8.06 (d, J = 9.5 Hz, 1H), 6.25 (dd, J = 9.5, 2.5 Hz, 1H), 6.14 (s, 2H), 6.12 (d, J = 2.5 Hz, 1H), 4.65−4.48 (m, 1H), 1.35 (d, J = 6.1 Hz, 6H). General Procedure C: Synthesis of Target Compounds 47d and 47e. A mixture of 5-fluoro-2-nitroaniline (45b) (468 mg, 3 mmol, 1.0 equiv), N,N-diisopropylethylamine (1.4 mL, 7.5 mmol, 2.5 equiv), and nucleophiles in N-methyl-2-pyrrolidone (15 mL) was heated to 100 °C for 4 h. The reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with water and brine successively, dried over anhydrous sodium sulfate, and concentrated in vacuum. The resulting residue was purified by silica gel chromatog-

raphy (dichloromethane/methanol, v/v, 97:3) to give the desired product. Synthesis of N1-Methyl-4-nitrobenzene-1,3-diamine (47d). The title compound was prepared from 45b with methylammonium chloride following general procedure C. Yellow solid (87%). 1H NMR (300 MHz, DMSO-d6) δ 7.70 (d, J = 9.5 Hz, 1H), 7.35 (s, 2H), 6.94 (m, 1H), 5.97 (dd, J = 9.5, 2.4 Hz, 1H), 5.76 (d, J = 2.4 Hz, 1H), 2.68 (d, J = 4.9 Hz, 3H). Synthesis of N1,N1-Dimethyl-4-nitrobenzene-1,3-diamine (47e). The title compound was prepared from 45b with dimethylamine following general procedure C. Yellow solid (90%). 1H NMR (300 MHz, DMSO-d6) δ 7.81 (d, J = 9.7 Hz, 1H), 7.24 (s, 2H), 6.21 (dd, J = 9.7, 2.5 Hz, 1H), 5.96 (d, J = 2.4 Hz, 1H), 3.00 (s, 6H). 5-Ethoxy-1H-benzo[d]imidazole-2-thiol (48a). The title compound was prepared from 47a following general procedure B. White solid (45%). 1H NMR (300 MHz, DMSO-d6) δ 12.35 (s, 2H), 6.99 (d, J = 8.7 Hz, 1H), 6.68 (d, J = 8.7 Hz, 1H), 6.63 (s, 1H), 3.96 (q, J = 6.9 Hz, 2H), 1.29 (t, J = 6.9 Hz, 3H). MS (ESI) m/z 195.2 [M + H]+. 5-Propoxy-1H-benzo[d]imidazole-2-thiol (48b). The title compound was prepared from 47b following general procedure B. Beige solid (75%). 1H NMR (300 MHz, DMSO-d6) δ 12.37 (s, 1H), 12.34 (s, 1H), 6.99 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 8.6 Hz, 1H), 6.64 (s, 1H), 3.87 (t, J = 6.5 Hz, 2H), 1.69 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). MS (ESI) m/z 209.2 [M + H]+. 5-Isopropoxy-1H-benzo[d]imidazole-2-thiol (48c). The title compound was prepared from 47c following general procedure B. Beige solid (79%). 1H NMR (300 MHz, DMSO-d6) δ 12.37 (s, 2H), 7.00 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 8.6 Hz, 1H), 6.64 (s, 1H), 4.51 (m,1H), 1.24 (d, J = 6.0 Hz, 6H). MS (ESI) m/z 209.2 [M + H]+. 5-(Methylamino)-1H-benzo[d]imidazole-2-thiol (48d). The title compound was prepared from 47d following general procedure B. Brown solid (56%). 1H NMR (300 MHz, DMSO-d6) δ 12.09 (s, 2H), 6.87 (d, J = 8.6 Hz, 1H), 6.38 (dd, J = 8.6, 2.0 Hz, 1H), 6.24 (d, J = 1.6 Hz, 1H), 5.58 (s, 1H), 2.64 (s, 3H). MS (ESI) m/z 180.0 [M + H]+. 5-(Dimethylamino)-1H-benzo[d]imidazole-2-thiol (48e). The title compound was prepared from 47e following general procedure B. Purple solid (62%). 1H NMR (300 MHz, DMSO-d6) δ 12.16 (s, 2H), 6.95 (d, J = 8.8 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H), 6.39 (s, 1H), 2.84 (s, 6H). MS (ESI) m/z 194.1 [M + H]+. General Procedure D: Synthesis of Target Compounds 50a−f, 50h−m, and 52. Chloroacetyl chloride (1.2 equiv) was added dropwise to a mixture of the appropriate amine (49a−f, 49h−m, or 51) (1.0 equiv) and potassium carbonate (2.0 equiv) in acetone at room temperature. After stirring for 4 h, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was washed with water and brine successively, dried over anhydrous sodium sulfate, and concentrated in vacuum. The resulting residue was purified by silica gel chromatography to give the desired product. 2-Chloro-N-phenylacetamide (50a). The title compound was prepared from 49a following general procedure D. White solid (98%). 1 H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.36 (t, J = 7.7 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 4.19 (s, 2H). MS (ESI) m/z 192.0 [M + Na]+. 2-Chloro-N-(o-tolyl)acetamide (50b). The title compound was prepared from 49b following general procedure D. Beige solid (84%). 1 H NMR (300 MHz, CDCl3) δ 8.23 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.22 (m, 2H), 7.12 (t, J = 7.4 Hz, 1H), 4.24 (s, 2H), 2.31 (s, 3H). MS (ESI) m/z 206.0 [M + Na]+. 2-Chloro-N-(2-chlorophenyl)acetamide (50c). The title compound was prepared from 49c following general procedure D. Beige solid (93%). 1H NMR (300 MHz, CDCl3) δ 8.93 (s, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 4.24 (s, 2H). MS (ESI) m/z 225.9 [M + Na]+. 2-Chloro-N-(2-methoxyphenyl)acetamide (50d). The title compound was prepared from 49d following general procedure D. White solid (90%). 1H NMR (300 MHz, CDCl3) δ 8.94 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 6.97 (t, J = 7.8 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 4.19 (s, 2H), 3.91 (s, 3H). MS (ESI) m/z 222.0 [M + Na]+. 6300



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transfer 15 μL of 1× assay buffer; (7) incubate at room temperature for 15 min; (8) add 5 μL of substrate solution to each well; (9) add 5 μL of [3H]-SAM solution to each well to start the reaction; (10) incubate at room temperature for 60 min; (11) add 5 μL of cold SAM solution to each well to stop the reaction; (12) transfer 25 μL of the reaction system to Flashplate by Platemate; (13) read the count on MicroBeta; and (14) use GraphPad Prism V5.0 software to process the data. The inhibition activities of compound 17 against PRMT3, PRMT4, PRMT6, and PRMT8 were determined using Alpha LISA protocols. PRMT3, PRMT4, PRMT6, and PRMT8 were purchased from BPS, with catalog numbers 51043, 51047, 51049, 51052, respectively. The protocol was as follows: (1) prepare 1× assay buffer (modified Tris buffer); (2) transfer 100 nL of compound solution to each well of the assay plate; (3) prepare enzyme solution in 1× assay buffer, then transfer 5 μL of enzyme solution to the assay plate and incubate at room temperature for 15 min; (4) prepare substrate solution in 1× assay buffer and then add 5 μL of substrate solution to each well to start the reaction; (5) incubate at room temperature for 60 min; (6) prepare acceptor and donor beads solution in 1× assay buffer, then add 15 μL of acceptor and donor beads solution, and incubate for 60 min at room temperature, under subdued light; (7) read end point with EnSpire with Alpha mode; (8) fit the data in Excel to obtain inhibition values using eq 1. The inhibition activities of compound 17 against PRMT7 was determined by a radioactive methylation inhibition assay. PRMT7 was purchased from Active Motif, catalog number 31395. The protocol was as follows: (1) Prepare 1× assay buffer (modified Tris buffer); (2) compound dilution, transfer compound to assay plate by Echo in 100% DMSO; (3) prepare enzyme solution in 1× assay buffer, then transfer 10 μL of enzyme solution to the assay plate and incubate at room temperature for 15 min; (4) prepare substrate solution, add peptide and [3H]-SAM in 1× assay buffer to make the substrate solution, and then add 10 μL of substrate solution to each well to start the reaction; (5) incubate at room temperature for 240 min; (6) prepare the cold SAM in 1× assay buffer to make the stop solution and add 10 μL per well of stop solution; (7) transfer 25 μL of volume per well to Flashplate from the assay plate; (8) incubate for 1 h minimum at room temperature; (9) wash Flashplate with dH2O + 0.1% Tween-20 three times; read the plate on Microbeta; and (10) fit the data in Excel to obtain inhibition values using eq 1. A radioactive methylation inhibition assay of NSD1 was performed in modified Tris buffer. The enzyme solution of NSD1 (10 μL; Biogenie, Cat. No. M1057) was transferred to an assay plate and incubated at room temperature for 15 min. Subsequently, 10 μL of substrate (Oligonucleosome) solution was added to each well, as was 10 μL of [3H]-SAM solution to start the reaction. After incubation for 4 h, 15 μL of cold SAM solution was added to each well to stop the reaction. The reaction mixture (40 μL) was transferred to a GF/B plate (Millipore, Cat. No. MSFBN6B50) (pretreated with 0.5% PEI for 15 min) using Platemate, and then the plate was washed 3 times with ddH2O under vacuum. The radioactivity was determined by liquid scintillation counting (MicroBeta, PerkinElmer). Chaetocin was used as the reference compound. A radioactive methylation inhibition assay of DNMT1 was performed in modified Tris buffer using poly(dI-DC) (Sigma, USA, Product No. P4929) as a substrate. The enzyme solution was incubated at room temperature for 15 min before 10 μL of substrate and 10 μL of [3H]-SAM (PerkinElmer Inc., USA, Lot. No. 1790854) were added to start the reaction. After incubation at 37 °C for 120 min, the reactions were transferred to filter plates; the plates were preincubated for 15 min with 0.5% PEI, and the residual solution was removed by vacuum. The plates were washed 3 times with ddH2O under vacuum and the counts measured using a MicroBeta (PerkinElmer). The maximum signal was obtained with the addition of the enzyme and substrate, and the minimum signal was obtained with the substrate only. SAH (Sigma, Product No. A9384) was used as the reference compound. The inhibition activities of compound 17 against DOT1L were determined using Alpha LISA protocols. In each plate well, 5 μL of

2-Chloro-N-(2-(dimethylamino)phenyl)acetamide (50e). The title compound was prepared from 49e following general procedure D. White solid (45%). 1H NMR (300 MHz, CDCl3) δ 9.64 (s, 1H), 8.34 (d, J = 7.6, 1H), 7.24−7.04 (m, 3H), 4.21 (s, 2H), 2.68 (s, 6H). N-(2-Acetylphenyl)-2-chloroacetamide (50f). The title compound was prepared from 49f following general procedure D. Brown solid (97%). 1H NMR (300 MHz, CDCl3) δ 12.49 (s, 1H), 8.71 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.19 (t, J = 7.7 Hz, 1H), 4.19 (s, 2H), 2.67 (s, 3H). MS (ESI) m/z 234.0 [M + Na]+. 2-(2-Chloroacetamido)benzoic Acid (50g). Compound 49g (500 mg, 3.64 mmol, 1.0 equiv) was dissolved in dichloromethane. Triethylamine (1.0 mL, 7.28 mmol, 2 equiv) was added, and the reaction mixture was cooled to 0 °C. Chloroacetyl chloride (0.35 mL, 4.37 mmol, 1.2 equiv) was added while maintaining the temperature at 0 °C. The reaction was then stirred at room temperature for 4 h. The reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuum. The resulting residue was purified by silica gel chromatography to give the desired product as a beige solid (622 mg, 80%). 1H NMR (300 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.53 (d, J = 8.3 Hz, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.63 (t, J = 7.9 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 4.45 (s, 2H). MS (ESI) m/z 235.9 [M + Na]+. Methyl 2-(2-Chloroacetamido)benzoate (50h). The title compound was prepared from 49h following general procedure D. White solid (90%). 1H NMR (300 MHz, CDCl3) δ 11.89 (s, 1H), 8.70 (d, J = 8.5 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.15 (t, J = 7.7 Hz, 1H), 4.21 (s, 2H), 3.96 (s, 3H). MS (ESI) m/z 250.0 [M + Na]+. Ethyl 2-(2-Chloroacetamido)benzoate (50i). The title compound was prepared from 49i following general procedure D. White solid (87%). 1H NMR (300 MHz, CDCl3) δ 11.94 (s, 1H), 8.70 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.16 (t, J = 7.7 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 4.21 (s, 2H), 1.42 (t, J = 7.1 Hz, 3H). MS (ESI) m/z 264.0 [M + Na]+. 2-(2-Chloroacetamido)-N-methylbenzamide (50j). The title compound was prepared from 49j following general procedure D. Beige solid (77%). 1H NMR (300 MHz, CDCl3) δ 11.91 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H), 7.47 (m, 2H), 7.14 (d, J = 7.2 Hz, 1H), 6.33 (s, 1H), 4.17 (s, 2H), 3.02 (d, J = 4.8 Hz, 3H). MS (ESI) m/z 249.0 [M + Na]+. 2-(2-Chloroacetamido)-N,N-dimethylbenzamide.(50k). The title compound was prepared from 49k following general procedure D. Brown solid (96%). 1H NMR (300 MHz, CDCl3) δ 9.80 (s, 1H), 8.10 (d, J = 8.3 Hz, 1H), 7.40 (dd, J = 11.5, 4.2 Hz, 1H), 7.27 (dd, J = 7.6, 0.9 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 4.15 (s, 2H), 3.12 (s, 3H), 3.01 (s, 3H). MS (ESI) m/z 241.0 [M + H]+. Methyl 3-(2-Chloroacetamido)benzoate (50l). The title compound was prepared from 49l following general procedure D. White solid (92%). 1H NMR (300 MHz, CDCl3) δ 8.35 (s, 1H), 8.07 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 4.22 (s, 2H), 3.92 (s, 3H). MS (ESI) m/z 249.9 [M + Na]+. Methyl 4-(2-Chloroacetamido)benzoate (50m). The title compound was prepared from 49m following general procedure D. White solid (95%). 1H NMR (300 MHz, CDCl3) δ 8.38 (s, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 4.21 (s, 2H), 3.91 (s, 3H). MS (ESI) m/z 250.0 [M + Na]+. Methyl 3-(2-Chloroacetamido)isonicotinate (52). The title compound was prepared from 51 following general procedure D. Yellow solid (52%). 1H NMR (300 MHz, CDCl3) δ 11.51 (s, 1H), 10.01 (s, 1H), 8.50 (d, J = 4.9 Hz, 1H), 7.83 (d, J = 5.0 Hz, 1H), 4.24 (s, 2H), 4.00 (s, 3H). MS (ESI) m/z 229.0 [M + H]+. Enzymatic Selectivity Assay. A radioactive methylation inhibition assay of PRMT1 was performed as follows: (1) Prepare 1× assay buffer (modified Tris buffer); (2) transfer compounds to the assay plate by Echo; (3) prepare enzyme (PRMT1, purchased from BPS, Cat. No. 51041) solution in 1× assay buffer; (4) prepare substrate (peptide, purchased from GL China) solution in 1× assay buffer; (5) prepare [3H]-SAM (PerkinElmer Inc.) solution in 1× assay buffer; (6) transfer 15 μL of enzyme solution to the assay plate, or for low control, 6301



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compound or assay buffer was added as was 2.5 μL of DOT1L enzyme. After incubation for 15 min at room temperature, 2.5 μL of oligonucleosomes/SAM mix was added to start the reaction; the plate was covered with TopSeal-A film (PerkinElmer, No. 6050195) and incubated at room temperature for 30 min. To stop the reaction, 5 μL of high-salt buffer was added. A 5× mixture of antihistone H3 Acceptor Beads (PerkinElmer, No. AL147) and biotinylated antiH3K79me2 antibody (PerkinElmer, NO. AL148) were prepared at 50 μg/mL and 0.5 nM in a 25 μL total assay volume. Subsequently, 5 μL of 5× acceptor beads/biotinylated antibody mix was added; the samples were covered with TopSeal-A film and incubated for 60 min at room temperature. A 5× Streptavidin Donor Beads (PerkinElmer, NO. 6760002) sample at 50 μg/mL was prepared in 1× detection buffer and shielded from light (final concentration of 10 μg/mL in 25 μL of total assay volume). Donor beads (5 μL) were added and shielded from light, and the sample was covered with TopSeal-A film and incubated shielded from light for 30 min at room temperature. The signal was measured in Alpha mode with Envision. EPZ004777 (Selleckchem, NO.S7353) was used as the reference compound. Alpha LISA was applied to determine the inhibition activity of the compound 17 against SET7/9. The purified SET7/9 protein was incubated in modified Tris buffer in 384-well plates (PerkinElmer, catalog NO. 6007299) at room temperature for 15 min. The compounds were transferred to the assay plate using Echo in 100% DMSO, and 5 μL of substrate solution was added to each well to start the reaction. The substrate solution was incubated in each well for 60 min. Acceptor and donor beads (15 μL) were added and incubated for 60 min at room temperature, shielded from light. The end point was evaluated with EnSpire in Alpha mode. The experimental data were fitted in Excel to obtain inhibition values using eq 1. The inhibition activities of compound 17 against BRD4 were determined using Alpha LISA protocols. Purified proteins were incubated with compound 17 on 384-well plates (OptiPlate-384, PerkinElmer) for 10 min at room temperature with the assay buffer (0.1% Triton X-100, 1 mM DTT, and 0.1% bovine serum albumin(w/ v)) before 25 nM biotinylated peptide was added to every plate hole. The substrate peptide was synthesized by Shanghai ChinaPeptide Corporation with the sequence SGRG-K(Ac)-GG-K(Ac)-GLG-K(Ac)-GGA-K(Ac)-RHRKVGG-K(Biotin) and was incubated at room temperature for 10 min. After the Ni2+ chelate acceptor beads (PerkinElmer) were added, the mixture was incubated for 30 min. The compound JQ-1 (Sigma) was used as the positive control. Finally, streptavidin coated donor beads (PerkinElmer) were added, and the mixtures were incubated in subdued light for 30 min at room temperature. The signals for the 384-well plates were read in alpha mode using an EnVision reader (PerkinElmer). Alpha LISA was applied to determine the inhibition activity of compound 17 against GCN5. Purified GCN5 protein was incubated with the compound at 100 μM concentration for 10 min. Then, the cofactor Ac-coa and substrate H3 were transferred to the assay plate (white opaque OptiPlate-384, PerkinElmer) to start the reaction at room temperature. After the reaction lasted for 30 min, the acceptor beads/anti-H3K14-ac antibody mixture were added to the whole system and incubated for another 30 min. Finally, streptavidin donor beads were added, and the final signals were collected using EnVision readers (PerkinElmer). Kinetic Experiment. (1) Prepare 1× assay buffer (modified Tris buffer); (2) compound serial dilution: transfer serially diluted compound DMSO solutions to assay plate by Echo; DMSO’s final fraction is 1%; (3) prepare enzyme solution in 1× assay buffer; (4) prepare peptide solution in 1× assay buffer; for the MOA study with peptide, prepare peptide solution at 7 concentrations; (5) prepare [3H]-SAM solution in 1× assay buffer; for the MOA study with SAM, prepare [3H]-SAM solution at 7 concentrations; (6) transfer 15 μL of enzyme solution to the assay plate, or for low control transfer, 15 μL of 1× assay buffer; (7) incubate at room temperature for 15 min; (8) add 5 μL of peptide solution to each well; (9) add 5 μL of [3H]-SAM solution to each well to start the reaction; (10) incubate at room temperature for 60 min; (11) add cold SAM in 1× assay buffer to make the stop solution, and stop the reaction with the addition of 5 μL

per well of stop solution; (12) transfer 25 μL of volume per well to Flashplate from the assay plate; (13) incubate for 1 h minimum at room temperature; (14) wash Flashplate with dH2O + 0.1% Tween-20 three times; (15) read plate on Microbeta; (16) fit the data in Excel to obtain inhibition values using eq 1; fit the data in GraphPad Prism 5 to obtain IC50 values using eq 2. Cell Lines. Cell lines used in these experiments were obtained from various sources and were cultured according to the conditions specified by the respective cell banks. The following cell lines were purchased from ATCC: Z-138 (CRL-3001), MV4-11 (CRL-9591), and HUVEC (PCS-100−010). KOPN8, Jeko-1, and RCTEC cells were obtained from Professor of Yongjun Dang, and all of the cells were grown in RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin under 5% CO2 at 37 °C. In Vitro Compound Treatment. Cultured cells in linear/logphase growth were split to a seeding density of 2 × 105 cells/mL in 1− 20 mL of media, depending on the yield required at the end of the growth period. Compound was diluted in DMSO and added to each culture vessel with a final DMSO concentration of 0.2%. Cells were allowed to grow for 96 h undisturbed. At the conclusion of each treatment period, cells were harvested by centrifugation (5 min, 1,000 rpm), and cell pellets were rinsed once with PBS before undergoing further processing. In Vitro Proliferation Assay. For assessment of the effect of 17 treatment on the cell lines, they were plated in 24-well plates at a density of 0.5−1 × 105 cell/well in 1 mL of media. After 6 h of incubation, the increasing concentrations of 17 (0−100 μM) were added to the medium. Cells were counted and replated at equal cell numbers in fresh media with fresh compound every 4 days. All assays about the cancer cell lines were carried out for 12 days, while the two normal cells were carried out for 8 days. Viable cell number was determined using the Cell Titer-Glo Luminescent Cell Viability assays (Promega), and luminescence was recorded using an EnVision Multilabel Plate Readers (PerkinElmer) according to the manufacturer’s protocol. For each cell line, the concentration-dependence curves were determined at each time point using GraphPad Prism software. Western Blot Assays. The primary antibodies against SDMA and GAPDH were obtained from Cell Signaling Technology. Cells were incubated with different concentrations of compounds. After 6 days of treatment, whole-cell lysates were collected and boiled for 10 min in 2× SDS sample buffer and subjected to 4−12% SDS−PAGE. The proteins were transferred to nitrocellulose membranes (Millpore) and blocked in 5% nonfat dry milk in TBST for 0.5 h at room temperature. Antibodies were incubated with the membranes at 4 °C overnight. The membranes were washed with TBST at room temperature for 15 min three times. HRP-conjugated secondary antibodies (Millipore) were added for 1 h at room temperature, and then, the membranes were washed three times with TBST for 15 min. ECL substrate (Millipore) reagent was added to develop the signal on the membranes in Amersham Imager 600.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00587.

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Molecular formula strings (CSV) Additional figures and tables illustrating the inhibition data and HPLC analysis of all target compounds (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(D.S.) E-mail: [email protected]. *(W.D.) E-mail: [email protected]. *(C.L.) E-mail: [email protected]. 6302



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ORCID

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Cheng Luo: 0000-0003-3864-8382 Author Contributions ∇

R.M., J.S., and K.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Yongjun Dang for providing KOPN8, Jeko1, and RCTEC cell lines. The computation resources were supported by Computer Network Information Center, the fund from Chinese Academy of Sciences (XDA12020304 to C.L.) and Tianjin Supercomputing Center. We are extremely grateful to National Centre for Protein Science Shanghai (Shanghai Science Research Center, Protein Expression and Purification system) for their instrument support and technical assistance. We gratefully acknowledge financial support from the Ministry of Science and Technology of China (2015CB910304 to Y.Z.); the National Natural Science Foundation of China (21472208, 81625022, and 81430084 to C.L., and 21210003 and 81230076 to H.J.), the Fund of State Key Laboratory of Toxicology and Medical Countermeasures, Academy of Military Medical Science (TMC201505 to C.L.), Shandong Talents Team Cultivation Plan of University Preponderant Discipline (10027 to K.Z.); and China Postdoctoral Science Foundation (2016M601676 to Y.Z.), sponsored by Shanghai Sailing Program (17YF1423100 to Y.Z.)

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ABBREVIATIONS USED PRMT5, protein arginine methyltransferase 5; SAM, Sadenosylmethionine; NSD1, nuclear receptor binding SET domain protein 1; DNMT1, DNA (cytosine-5)-methyltransferase 1; DOT1L, DOT1-like (disruptor of telomeric silencing 1like) methyltransferase; SET7/9, SET domain-containing lysine methyltransferase 7; BRD4, bromodomain-containing protein 4; GCN5, histone acetyltransferase GCN5; DIPEA, N,Ndiisopropylethylamine; DMF, N,N-dimethylformamide; NMP, N-methyl- 2-pyrrolidone; TBDMSCl, tert-butyldimethylsilyl chloride

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