Discovery and Optimization of Novel, Selective Histone

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Discovery and optimization of novel, selective histone methyltransferase SET7 inhibitors by pharmacophore- and docking-based virtual screening Fanwang Meng, Sufang Cheng, Hong Ding, Shien Liu, Yan Liu, Kongkai Zhu, Sijie Chen, Junyan Lu, Yiqian Xie, Linjuan Li, Rongfeng Liu, Zhe Shi, Yu Zhou, YuChih Liu, Mingyue Zheng, Hualiang Jiang, Wencong Lu, Hong Liu, and Cheng Luo J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01154 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 25, 2015

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Discovery and optimization of novel, selective histone methyltransferase SET7 inhibitors by pharmacophore- and docking-based virtual screening Fanwang Meng$,†,|,Sufang Cheng§,|, Hong Ding†,|, Shien Liu†, Yan Liu†, Kongkai Zhu†, Shijie Chen†, Junyan Lu†, Yiqian Xie†, Linjuan Li†,#, Rongfeng Liu¶, Zhe Shi¶, Yu Zhou§, Yu-Chih Liu¶, Mingyue Zheng†, Hualiang Jiang†,#, Wencong Lu$,*, Hong Liu§,*, and Cheng Luo†,* $

Department of Chemistry, College of Sciences, Shanghai University, Shanghai 200444, China;

†

Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute

of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; §Chinese Academy of Sciences Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China;

#

School of Life Science and

Technology, ShanghaiTech University, Shanghai 200031, China;

¶

Shanghai ChemPartner Co.,

LTD., Zhangjiang Hi-Tech Park, Shanghai 201203, China 201203 |

These authors contributed equally.

*Correspondence: Cheng Luo, E-mail: [email protected] or Hong Liu, Email: [email protected]; or Wencong Lu, Email: [email protected] Received Data (to be automatically inserted after your manuscript is accepted)

ACS Paragon Plus Environment

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ABSTRACT Histone methyltransferases are involved in various biological functions, and these methylation regulating enzymes abnormal expression or activity has been noted in several human cancers. Within this context, SET domain-containing (lysine methyltransferase) 7 (SET7, also called KMT7, SETD7, SET9) is of increasing significance due to its diverse roles in biological functions and diseases, such as diabetes, cancers, alopecia areata, atherosclerotic vascular disease, HIV and HCV. In this study, DC-S100 which was discovered by pharmacophore- and dockingbased virtual screening was identified as the hit compound of SET7 inhibitor. Structure-activity relationship (SAR) analysis was performed on analogs of DC-S100 and according to the putative binding mode of DC-S100 and structure modifications were made to improve its activity. Of note, compounds DC-S238 and DC-S239, with IC50 values of 4.88 µM and 4.59 µM, respectively, displayed selectivity for DNMT1, DOT1L, EZH2, NSD1, SETD8 and G9a. Taken together, DC-S238 and DC-S239 can serve as leads for further investigation as SET7 inhibitors and the chemical toolkits for functional biology studies of SET7.

Keywords: epigenetic; SET7 inhibitor; pharmacophore; molecular docking.


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Introduction

Histone lysine methyltransferases (HKMTs) are histone covalent modifying enzymes that transfer methyl groups to lysine or arginine on histone H3 via an S‑adenosylmethionine (SAM)dependent SN-2 mechanism. HKMTs are involved in heterochromatin formation, gene expression regulation, DNA repair, cell cycle control1 and cancer development2.Thus these epigenetic targets are vital in cancer biology. HKMTs can be divided into two categories: (i) SET domain-containing enzymes, such as SET7 (SET domain-containing lysine methyltransferase 7, also called SETD7, SET9, KMT7) and EZH2 and (ii) non-SET domain enzymes, such as DOT1L. SET7 was first characterized as a mono-methyltransferase of H3K43, 4. A series of studies later revealed that SET7 modulates various transcriptional regulators, including ARTD15, COL2A16, ERα7, FoxO38, pRb9, 10, RAR11, STAT312, SUV39H113, 14, TAF1015, p6516, Pdx117, HIF-α18. In addition to its role in transcriptional regulation16, 19, 20, SET7 also functions in cell cycle control9, 10, 21, differentiation22, DNA repair13 and DNMT1 stability23, 24, and increasing evidence suggests that SET7 is closely associated with various diseases. SET7 regulates NF-κB activity through lysine monomethylation of p65 in response to TNF-α in human kidney cells25, and knockdown of SET7 inhibits key NF-κB-dependent gene expression26. Li, Y. et al. observed increased inflammatory gene expression and SET7 recruitment in macrophages from diabetic mice, demonstrating the role of SET7 in diabetes26. Recently, it was found that epigenetic changes induced by SET7 contribute to vascular dysfunction in patients with type 2 diabetes27, indicating the targeting of SET7 as a promising option to prevent atherosclerotic vascular disease. Increased SET7 expression is also observed in peripheral blood mononuclear cells from patients with alopecia areata28. In addition, downregulation of SET7 in breast cancer cells


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attenuates estrogen-induced gene activation and that SET7 knockdown results in a concomitant decrease in the steady-state levels of estrogen receptor α (ERα) protein7, which is involved in the pathology of breast cancer, endometrial cancer and osteoporosis. Other studies also support that SET7 is linked to the pathological processes of cancers13, 29. Recently, the role of SET7 in virus infection was identified; for example, knockdown of SET7 suppresses Tat transcription of the HIV promoter30, and SET7 facilitates HCV replication by attenuating IFN signaling pathways and IFN-related effectors31. Notably, there is a controversial debate about whether the methylation of p53 by SET7 meditates p53 activity32. Some studies revealed that the SET7mediated methylation of Lys-372 at the p53 C-terminus can regulate p53 acetylation, enhancing its antitumor activity33-36. However, Lehnertz, B. et al. reported that knockdown of SET7 in mice does not affect p53-depedent transcription and tumor suppression37, results that are fully consistent with the findings of Campaner, S. et al., who reported that p53-depedent cell cycle arrest or apoptosis is not affected by SET7 deletion38. This controversial debate calls for the urgent search for a potent SET7 inhibitor that can be used as a chemical probe. As SET7 is a promising target in several diseases, including diabetes, alopecia areata, cancers and virus infection, several attempts have been made to identify SET7 inhibitors (Supporting Information Chart S1). Compounds 1 through 6 are analogs of SAM, with resulting selectivity issues39-41. Bissinger, E. M. et al. identified acyl derivatives of p-aminosulfonamides and dapsone as potent hPRMT1 inhibitors, including compound 7, yet they showed weak activity toward SET742. Using high-throughput screening, Verma, S. K. and co-workers reported that compounds 8, 9 and 10 are potent, cell-active inhibitors of EZH2, and these compounds also showed inhibitory activity toward SET743. ‘Biased-privileged’ scaffolds were used to design SET7 inhibitors, and SETin-1 (compound 11) showed inhibitory activity against both G9a and


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SET744. Compound 12, which showed weak activity toward SET7, is a coactivator-associated arginine methyltransferase 1 inhibitor with an IC50 value of 14.4 µM45. Similarly, compounds 13 to 16 are not selective SET7 inhibitors46. Following a drug repository strategy, it was found that Cyproheptadine (compound 17), an antihistaminic and antiserotonergic agent, showed inhibitory activity toward SET7 (IC50 = 20 µM)47. Recently, (R)-PFI-2 (compound 18) was reported to be a potent and selective inhibitor targeting SET7 in MCF7 cells by occupying the histone lysine binding site, as revealed by X-ray complex structure analysis48. However, most SET7 inhibitors are multi-target inhibitors with low potencies. Based on the diversity of SAM binding modes and receptor-based interactions among HKMTs, which enables the design of selective inhibitors, our research focuses on the development of potent, selective small-molecule inhibitors of SET7 targeting the co-factor binding site49, 50. With the rapid development of computational methods, many studies successfully identifying epigenetic inhibitors using this approach have been reported51-80. Our group has applied virtual screening methods to identify inhibitors of PRMT1 and DNMT1, as well as inhibitors targeting the interface of EZH2-EED and the menin–mixed lineage leukemia interface68,

81-85

; these

successes and our previous experience encouraged us to try to identify SET7 inhibitors employing virtual screening. In this study, novel, potent SAM competitive and selective inhibitors of SET7, DC-S238 (IC50 = 4.88 µM) and DC-S239 (IC50 = 4.59 µM), were identified through an integrated procedure combining pharmacophore- and docking-based virtual screening, similarity searching and hit optimization. The virtual screening identified compound DC-S100 as a hit compound possessing moderate activity toward SET7 (IC50 = 30.04 µM). Similarity-based analog searching enabled the analysis of the structure-activity relationship (SAR) of DC-S100. Based on the putative binding mode of DC-S100 and its SAR, chemical optimization was


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performed to improve its potency. Finally, compound DC-S238 (IC50 = 4.88 µM) and DC-S239 (IC50 = 4.59 µM) were obtained, displaying selectivity for DNMT1, DOT1L, EZH2, NSD1, SETD8 and G9a. In summary, this study identified DC-S238 and DC-S239 as potent and selective inhibitors against SET7, and they may serve as the lead compounds for further development and the chemical toolkits for functional biology studies of SET7.

Results and Discussion Virtual Screening. To exploit all available structural and chemical information, an integrated

pharmacophore- and docking-based virtual screening procedure was designed to counterbalance their limitations86. Pharmacophore-based virtual screening can be used to pre-process virtual molecule libraries to filter compounds lacking features essential for binding. The 3D pharmacophore model which yields the lead compound DC-S100 was shown in Supporting Information Figure S1. An assessment of the SET domain in different states suggested that the SAM binding pocket has conserved architecture (Supporting Information Figure S2 A), and the crystal structure of SET7 in complex with the bioactive co-factor SAM (PDB ID: 1N6A) was selected for the subsequent pharmacophore generation and docking studies. The Specs database (http://www.specs.net), containing more than 200,000 molecules, was filtered using Lipinski’s Rule of Five

87

, and ‘pan-assay interference compounds’ (PAINS)88-91 were removed with

Pipeline Pilot, version 7.5 (Pipeline Pilot; Accelrys Software Inc., San Diego, CA). The remaining 182,014 molecules were prepared with LigPrep version 2.392 (Schrödinger, LLC: New York, NY, 2009) to generate all stereoisomers and different protonation states by Epik version 2.093 (Schrödinger, LLC: New York, NY, 2009). The prepared Specs library was used to generate an indexed multi-conformer 3D database in Accelrys Discovery Studio 3.094 with the conformation method CAESAR95. Receptor-ligand based pharmacophores, which was used to


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screen the 3D database, was generated with Accelrys Discovery Studio 3.094. A total of 15,541 molecules matched the pharmacophore features and were subjected to two rounds of dockingbased virtual screening. Several docking programs were preliminarily evaluated, and Glide96 was chosen due to its better performance with regard to enrichment factors (Supporting Information Table S1). The redocking studies suggested that Glide is capable of reproducing the bioactive binding conformation of SAM with an RMSD value of 0.30 Å (Glide SP mode96) and 0.22 Å (Glide XP mode97), respectively, which agreed with our docking procedures (Supporting Information Figure S2). The Glide SP and XP modes, as integrated in Maestro 9.298, were subsequently used to dock the compound library into the SAM binding site. As lysine-294 may be involved in the specific recognition of SAM by SET749, we chose this residue as a hydrogen bond constraint during the Glide XP docking procedure. The top-ranked 649 molecules were clustered into 108 groups with Pipeline Pilot, version 7.5 (Pipeline Pilot; Accelrys Software Inc., San Diego, CA) using its protocol component Cluster Molecules for visual inspection. The candidate molecules were selected on the basis of the following considerations. (1) The binding poses were reasonable. Molecules with high strain energy or not occupying the SAM binding pocket were not selected for further validation. (2) Among a cluster of similar molecules, smaller ones are preferred. (3) Molecule that can form distinguishable hydrogen bonds with Lys-294, Ala-226, Glu-228, His-297, Glu-356 and π-π stacking interactions with Trp-352 are good candidates because these residues can form key interactions with SAM and the disruption of the key interactions are believed to contribute to potency. (4) In each clustered group, at least one molecule was retained to cover more chemical space, which in turn improves hit rates. Consequently, 127 compounds were purchased from Specs Company for biochemical assays.


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SET7 Enzymatic Assays. The 127 candidate molecules selected were tested for SET7 inhibition to determine their biochemical activity in vitro. We used SAH as reference compounds in the AlphaLISA assay at 100 µM. The enzyme solution was transferred to assay plates, and 5 µL of 1x assay buffer (modified Tris Buffer) for low control, both were incubated at room temperature for 15 min. Acceptor and donor beads were prepared in 1x AlphaLISA buffer and incubated for 60 min at room temperature, shielded from light. The endpoint was read using EnSpire in Alpha mode. In the assay, seven compounds demonstrated significant inhibition of SET7 activity at 100 µM (Figure 1B). Among those seven molecules, compound DC-S100 was characterized as a structurally novel SET7 inhibitor (IC50 = 30.04 µM). Similarity-Based Analog Searching and SAR analysis. To explore the structure activity relationship (SAR) of compound DC-S100 and to optimize the compound, a similarity-based analog search was conducted using Pipeline Pilot V7.5 (Accelrys Inc., San Diego, CA, USA). A total of 109 derivatives of compound DC-S100 were purchased for the same AlphaLISA assay described above. Seven compounds displayed greater than 50 % inhibition of SET7 activity at 100 µM (Figure 1C), and the IC50 values against SET7 were measured (Supporting Information Table S3). We analyzed the activity data of the analogs (see Table 1) carefully and explored the SAR of DC-S100 to find clues for hit optimization. In scaffolding (I), the R2 group is expected to be aromatic rings, as opposed to other side chains, because substitutions of aromatic ring resulted in a significant decrease in potency (DC-S133 and DC-S134), with inhibition rates of 10.64 % and 22.06 %, respectively. In contrast, when R2 is a derivative of furan (DC-S128, DC-S129) or benzene (DC-S130 to DC-S132), inhibition activity increased. The length of the amide group and R1 group connecting meta-nitrobenzene and the aromatic ring (R2) has strong effects on the


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activity. A significant decrease in activity was also observed when the linker was longer than that of the amide group, such as for DC-S135 and DC-S136, with inhibition rates 12.14 % and 15.78 % respectively; thus, an amide group is the most appropriate linker. Moreover, the introduction of a bulky group into the aromatic rings (R2) is not tolerated because large groups often lead to a decrease in activity (DC-S138 to DC-S143). For example, when an iodine atom was substituted in the benzene ring (DC-S137), the inhibition rate decreased to 1.62 %, with almost no inhibitory activity against SET7. Regarding scaffold (II), we can conclude the same results. First, a linker group that is too long is not tolerated, such as in compound DC-S144. Second, a larger R2 group also led to a dramatic decrease in potency (DC-S145 and DC-S146), supporting the previous hypothesis. In summary, a meta-nitrobenzene group and an aromatic ring linked by an amide group was used as the basic scaffold for the next stage of chemical optimization. Chemical Modification of DC-S100. To gain a better understanding of the molecular basis of the inhibitory activity, we analyzed the putative binding mode of DC-S100. As depicted in Figure 2A, the nitro group is capable of forming hydrogen bonds with Glu-228 and Lys-294. The hydrogen bond lengths of Glu-228 and Lys-294 are 2.0 Å and 2.3 Å, respectively, indicating that the hydrogen bonds are strong and essential for activity. The amide group, which is the most appropriate linker according to the SAR analysis, forms a strong hydrogen bond with Asn-296, with a bond length of 1.9 Å. The π–π stacking interactions between indole ring of Trp-352 and the benzene ring of DC-S100 are present, indicating that an aromatic ring is indispensable for activity. When the amide group is replaced by longer linkers, the position of the benzene is not favorable for π–π stacking interactions, and thus the inhibition rate decreased. His-297 is the key residue forming two hydrogen bonds with SAM, but no polar interactions between DC-S100 and


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His-297 exist. If the compound could form polar interactions with Glu-356, disrupting polar interactions between Glu-356 and SAM, its binding affinity with SET7 will be enhanced. We presumed that if we introduced an amino group to the aromatic ring, the amino group may form hydrogen bonds with His-297 and Glu-356, which in turn will enhance its activity against SET7. This later proved to be a successful strategy. Taking feasibility and accessibility into consideration, we synthesized DC-S237 via the Gewald reaction and tested its activity toward SET7. DC-S237 displayed activity that was similar to DC-S100, with an IC50 value of 37.67 µM (Figure 2D). In pursuit of molecular details, a docking simulation was conducted, and the putative binding mode is shown in Figure 2B. The binding mode suggested that, although the hydrogen bond with Glu-228 disappeared, DC-S237 formed new polar interactions with His-297 and Glu-356. A predicted conformation alignment revealed that both DC-S100 and DC-S237 share similar binding modes, though the direction of the amide group in DC-S237 is altered (Figure 2C). The binding mode of DC-S237 also suggested that the terminal isobutyl may be slightly too large to completely fit into the SAM binding pocket, which prompted us to substitute the isobutyl with smaller alkyl groups. Compound DC-S238 and DC-S239 were synthesized, both of which displayed better inhibitory activity, as expected. The IC50 values of DC-S238 and DC-S239 were determined to be 4.88 µM and 4.59 µM, respectively (Figure 3B). Selectivity. We determined the selectivity of DC-S238 and DC-S239 for SET7 compared to 6 other enzymes (Table 2). The results showed that DC-S238 and DC-S239 exhibited a low inhibition rate, below 45%, for DNMT1, DOT1L, EZH2, NSD1, SETD8 and G9a (Table 2). Moreover, DC-S239 possesses a good selectivity profile over DNMT3A/3L at 100 µM, with an inhibition rate of -4.25 %. Therefore, DC-S238 and DC-S239 are selective inhibitors of SET7.


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Cell Activity. Because DC-S239 is a selective inhibitor of SET7, we assessed whether SET7 can show selective profiles in different cell lines. Therefore, the anti-proliferation activities of compound DC-S239 against the MCF7, HL60, DHL4, MV4-11, and HCT116 cell lines were determined by the MTT or alamarBlue assay. DC-S239 inhibited the proliferation of MCF7, HL60 and MV4-11 cells in a dose-dependent manner while showing no cellular activity against HCT116 and DHL4 cells (Figure 4). DC-S239 displayed IC50 values of 10.93 µM in MCF-7 cells and 16.43 µM in HL60 cells. Moreover, DC-S239 also displayed dose-dependent inhibition activity toward MV4-11 cells. These inhibition profiles suggested that DC-S239 is more preferential toward breast cancer and leukemia cell lines. SAR Analysis of DC-S239 and Binding Mode Prediction of DC-S239. To explore the SAR of DC-S239, an analog series was synthesized, and the activities were tested against SET7 (Figure 3A). Comparing DC-S197, DC-S198, and DC-S199 with DC-S238, it can be concluded that an amino group (R2) is essential for activity (Table 3). When the amino group is replaced with another group, such as in DC-S197, DC-S198, and DC-S199, which are analogs from the similarity search, no inhibitory activity was observed against SET7. Another finding is that a bulkier terminal R3 group resulted in reduced activity (DC-S238 to DC-S244) from 94.71% to 1.53%. Activity gradually decreased when the R3 group was larger than the ethyl group; in particular, DC-S242, with the largest alkyl group tert-butyl, showed no inhibitory activity toward SET7. This may be due to steric effects hindering the molecules from fitting into the SAM binding site. Inhibitory activities also revealed that the substitution of the nitro group leads to a significant loss in potency. For example, when the nitro group was substituted with methoxy (DC-S249) or chlorine (DC-S257), a dramatic decrease in activity was observed. Other substitutions did not contribute to activity. Thus, a nitro group at the meta-position is


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indispensable. In summary, a nitro group at the meta-position of the benzene ring, a thiophene ring with an amino group at the α position, and an amide group are essential building blocks for active SET7 inhibitors. In addition, a methyl or ethyl group linked by an ester group is the most appropriate alkyl group. To explore the molecular basis of inhibitory activity, we conducted docking studies, and the results revealed that DC-S239 displays a similar binding mode as SAM, fitting closely into the open and solvent-accessible binding pocket (Figure 5B). The nitro group is thought to form a hydrogen bond with Lys-294, which may be involved in selectivity49. Thus, substitution of the nitro group disrupted hydrogen bonding with Lys-294, and a loss in activity was observed. The binding mode of DC-S239 (Figure 5A) suggested that the amino group is capable of forming hydrogen bonds with His-297 and Glu-356, enhancing the binding affinity for SET7. When the amino group was replaced with other groups, such as in compounds DC-S197, DC-S198 and DC-S199, the disruption of the hydrogen bonds resulted in a significant decrease in activity. The binding mode also suggested that π–π stacking interactions between the thiophene ring and Trp352 contributed to the activity against SET7. If the linker is longer than the amide group, the position of the aromatic rings are altered, preventing π–π stacking interactions. Therefore, the amide group is the most proper linker, and the aromatic ring is essential to activity. Additionally, the terminal alkyl group cannot be too large because large groups can result in steric effects, making the SAM pocket inaccessible. A larger terminal alkyl group leading to potency loss is in accordance with the SAR of DC-S239.

Conclusions SET7 plays a significant role in transcriptional regulation16,

19, 20

, cell cycle control9,

10, 21

,

differentiation22, DNA repair13 and DNMT1 stability23, 24. SET7-mediated methylation is also


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associated with diabetes25,

26, 99, 100

, cancers7,

13, 29

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, alopecia areata28, atherosclerotic vascular

disease27, transcription of the HIV promoter30 and HCV replication31. The controversial debate regarding whether SET7 can regulate p53 highlights the need for specific and potent inhibitors. Notably, studies reporting the role of SET7 in the regulation of ERα7, STAT312, and NF-κB16, including p53, have all employed knockdown or overexpression methods, and this may obscure or exaggerate the delicate regulatory mechanism of SET7. To date, only (R)-PFI-2 has been reported to be a potent selective SET7 inhibitor targeting the substrate binding site48. Other inhibitors either lack potency or selectivity, hindering an understanding of the functional biology of SET7 as well as the exploration of its potential as a therapeutic target. Nonetheless, efforts have been made to discover potent and selective SET7 inhibitors through structure-based virtual screening and chemical optimization. First, we employed pharmacophorebased and docking-based virtual screening to mine potential candidate molecules. DC-S100 was identified as a hit, with an IC50 value of 30.04 µM. Subsequently, similarity-based analog searching was performed, and their activities were tested to explore the SAR of DC-S100. The SAR of DC-S100 suggested that the nitro group at the meta position, a linker amide group and an aromatic ring that does not cause steric effects are essential elements for activity. Docking studies revealed that there are no polar interactions between His-297, Glu-356 and DC-S100, providing a direction for hit optimization. Considering the issues discussed above and feasibility, DC-S237 was synthesized via the Gewald reaction. With respect to the binding mode, DC-S237 may be too large to fit well into the SAM binding pocket, which prompted us to synthesize DCS238 and DC-S239 as well as their analogs. DC-S239 proved to be a potent SET7 inhibitor, with an IC50 value of 4.59 µM. Binding mode prediction revealed that DC-S239 shares a similar binding moiety with SAM, and the key interactions are in accordance with the SAR analysis of


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DC-S239. Biological testing showed that DC-S239 displays selectivity for SET7 over DNMT1, DNMT3A/3L, DOT1L, EZH2, NSD1, SETD8 and G9a, with inhibition ratio below 45.45%. DC-S238 is also a potent SET7 inhibitor with an IC50 value of 4.88 µM and possesses a good selectivity profile over DNMT1, DOT1L, EZH2, NSD1, SETD8 and G9a. According to the GeneCards platform101, 102, SET7 is highly expressed in the breast cancer MCF7 cell line, and SET7 can modulate the stability of ERα via the methylation of K3027. ERα is over-expressed in approximately 70 % of breast cancer cases, and the blockade of ERα action has proved to be a successful strategy in these patients103. Thus, our small molecules have the potential to function as modulators of ERα. The study presented here details the discovery and development process of hit optimization from structure-based drug virtual screenings to potent, selective SET7 inhibitors with novel scaffolds using a structure-guided drug design approach. It also highlights the significance of selecting a proper hit compound with low molecular weight and a structure-guided drug design approach. The encouraging findings obtained in our study provide lead compounds for the further development of SET7 inhibitors and will shed light on chemical biology studies of SET7.

Experimental Section Virtual Screening. Ligand Database Preparation. The ligand database was extracted from

the Specs library, which contains approximately 200,000 molecules. The ligands were filtered by Lipinski’s Rule of Five87, and ‘pan-assay interference compounds’ (PAINS)88-91 were removed using Pipeline Pilot, version 7.5 (Pipeline Pilot; Accelrys Software Inc., San Diego, CA). The remaining 182,014 molecules were prepared with LigPrep92 to generate all stereoisomers and different protonation states with Epik. The prepared Specs database was used to generate an


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indexed multi-conformer 3D database in Accelrys Discovery Studio 3.094 with the conformation method CAESAR95. Protein Preparation. Twenty-nine X-ray crystal structures of SET7 are in the PDB database, and we aligned the SET domain of these structures for comparison, which is conserved in the SET domain-containing family. We obtained a maximum root mean-square derivation (RMSD) value of 0.37, suggesting that the three-dimensional structure of the SET domain in SET7 is conserved. Considering the structure resolution and integrality, the crystal structure of SET7 in a complex with SAM (PDB ID: 1N6A) was chosen for pharmacophore- and docking-based virtual screening. The protein was prepared with Protein Preparation Wizard Workflow, as provided in Maestro98, with a pH of 7.0 ± 2.0. Other parameters were set as the default. Pharmacophore-Based Virtual Screening. The receptor-ligand based pharmacophore model was generated with Accelrys Discovery Studio 3.094 using the X-ray structure in complexes with SAM (PDB ID: 1N6A). The pharmacophore features were edited in combinations in Discovery Studio 3.094 in order to cover a border chemical space when matching the prepared multiconformer 3D Specs database. We obtained 15,541 compounds after searching the 3D database with those pharmacophore models, and the 15,541 molecules were prepared using LigPrep92 for further docking validation. Docking-Based Virtual Screening. To rationalize our docking procedures, the enrichment factor (EF)104 was used to evaluate the performance of several docking programs, including Glide 5.5105, AutoDock Vina106, LibDock in Discovery Studio94 and Dock 6107. The results suggested that Glide 5.5 performs better than the others (Supporting Information Table S1). A redocking evaluation illustrated that Glide was capable of reproducing the active conformation, with RMSD values of 0.300 Å (Glide SP mode) and 0.218 Å (Glide XP mode), respectively


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(Supporting Information Figure S2). The grid box was set to 25 Å×25 Å×25 Å centered at the bioactive ligand SAM. Because lysine-294 may be involved in specific recognition of SAM by SET749, we set lysine-294 as a hydrogen bond constraint during grid generation and docking. Glide96 SP mode and Glide XP mode97 were subsequently used to dock the compound library into the SAM binding site. The top-ranked 649 molecules were clustered for visual inspection, and 127 compounds were selected and purchased for biological testing. Similarity-Based Analog Searching. According to the results of the biological tests, we searched the prepared Specs library for derivatives of DC-S100 using Similarity Filter from File in Pipeline Pilot, version 7.5. (Pipeline Pilot; Accelrys Software Inc., San Diego, CA). We purchased 109 compounds and tested their biological activity toward SET7 to investigate the SAR of DC-S100. SET7 Inhibition Assays. AlphaLISA was applied to determine the inhibition activity of the compounds over SET7. The purified SET7 protein was incubated in modified Tris buffer in 384well plates (Perkin Elmer, Cat. 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 endpoint was evaluated with EnSpire in Alpha mode. The experimental data were fitted in GraphPad Prism 5 to obtain inhibition values using Inhibition %=( Max-Signal)/ (Max-Min)*100 Enzymatic Selectivity Assay. 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


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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 pre-incubated for 15 min with 0.5 % PEI, and 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 radioactive methylation inhibition assay of DNMT3A/3L utilized IDT-01 Sequence 1 and IDT-01 Sequence 2 as substrates in modified Tris buffer. The enzyme solution was incubated at room temperature for 15 min before 10 µL of substrate solution was added to each well to start the reaction. After incubation at room temperature for 4 h, cold SAM solution (10 µL) was added to each well to stop the reaction. Subsequently, 25 µL of the reaction was transferred to a FlashPlate microplate (PerkinElmer Inc., USA, Cat. No. SMP410A001PK), and the radioactivity was determined by liquid scintillation counting (MicroBeta, PerkinElmer). Sinefungin (Sigma, Product No. S8559) was used as the reference compound. The radioactive methylation inhibition assay of EZH2 was performed in modified Tris buffer. H3K27me peptide and [3H]-SAM (Perkin Elmer Inc., USA, Lot. NO. 1731619) were added in 1x buffer as the substrate solution. The enzyme solution was incubated at room temperature for 15 min before 10 µL of substrate solution was added to each well to start the reaction. Cold SAM (Sigma, Cat. NO. 7007) was added in 1x buffer to prepare the stop mix (final concentration 0.5 mM), and 10 µL was added per well; 25 µL was transferred to a FlashPlate (Perkin Elmer, Cat. No. SMP410A001PK) and incubated at room temperature for a minimum of 1 h. The plate was washed three times with dH2O and 0.1 % Tween-20, and the radioactivity was determined by


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liquid scintillation counting (MicroBeta, PerkinElmer). GSK-126 was used as the reference compound. 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 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 GF/B plate (Millipore, Cat. No. MSFBN6B50) (pretreated with 0.5 % PEI for 15 min) using Platemate, and 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. The inhibition activities of DC-S238 and DC-S239 against DOT1L were determined using AlphaLISA protocols. In each plate well, 5 µL of compound or assay buffer were added and so 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 TopSealA 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 anti-Histone H3 Acceptor Beads (PerkinElmer, NO. AL147) and biotinylated anti-H3K79me2 antibody (PerkinElmer, NO. AL148) was 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 total assay volume). Donor beads


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(5 µL) were added, 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. The radioactive enzymatic assay of SETD8 was performed in modified Tris buffer. The compounds were first transferred to assay plate by Echo in 100% DMSO. Both peptide and [3H]SAM in 1x assay buffer (modified Tris Buffer) were added to each well to start the reaction before the compounds and 10 µL enzyme solution were incubated at room temperature for 15 minutes. And 10 µL cold SAM in 1x assay buffer was added to each well to stop the reaction after 120 min. 25 µL of volume per well was transferred to Streptavidin coated Flashplate Microplate (PerkinElmer Inc., USA, Cat. No. SMP410A001PK) from assay plate and each was incubated for 60 min minimum at room temperature. The Flashplate was washed with dH2O + 0.1% Tween-20 three times and read on Microbeta. SAH (Sigma, Cat. No. A9384-25MG) was used as the reference compound. The inhibition activities of DC-S238 and DC-S239 against G9a were determined using AlphaLISA protocols. Both DC-S238 and DC-S239 were transferred to assay plate by Echo in 100% DMSO and the enzyme solution was prepared in 1x assay buffer (modified Tris Buffer). The compounds and 5 µL of the enzyme solution was incubated in assay plate in each well at room temperature for 15 min. The 5 µL of susbstrate mix solution which was prepared in 1x assay buffer was added to each well to start the reation and was incubated at room temperature for 60 min. Then 15 µL acceptor and donor beads mix solution was added and incubated for 60 min at room temperature, subdued light. Cell Culture and Cell Viability Assay. MCF7, HL60, MV4-11, HCT116, and DHL4 cell lines were purchased from American Type Culture Collection. Fetal bovine serum was


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purchased from Life Technologies. MCF7 cells were cultured in DMEM medium supplemented with 10 % fetal bovine serum at 37 °C in an incubator with 5 % CO2 atmosphere. HL60, MV411, and DHL4 cells were cultured in 1640 medium supplemented with 10 % fetal bovine serum. HCT116 cells were cultured in 5A medium. DC-S239 was dissolved in DMSO (Sigma) and then stored at 4 °C. Approximately 2*104 cells were seeded in 100 µL of medium in each well of a 96-well flat-bottom plate and then grown in a CO2 incubator overnight. The cells were incubated with DC-S239 at different concentrations ranging from 0 to 100 µM for approximately 120 h. MCF7 and HCT116 cells were treated with 10 µL MTT solution and incubated for 2 h at 37 °C. The absorbance was measured at 490 nm using a micro plate reader (BMG Labtech) after adding 100 µL of DMSO to each well to dissolve the crystals; 630 nm was used as the reference wavelength. The activity of DC-S239 against HL60, MV4-11, and DHL4 cells was measured by the alamarBlue assay108,

109

. Fluorescence was measured using a fluorescence excitation

wavelength of 544 nm and fluorescence emission at 590 nm. To express the percent of proliferation, the absorbance and fluorescence were normalized with control wells. Chemistry. Commercially available chemicals were used without further purification. All products were characterized by their NMR and MS spectra. 1H NMR spectra were recorded using a 400 MHz Varian Mercury Plus 400 instrument, and

13

C NMR spectra were recorded

using a 400 MHz Varian Mercury Plus 400 instrument or a 500 MHz Bruker AVANCE III 500 instrument. Chemical shifts are reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m). Low-resolution mass spectra (LRMS) were measured using a Finnigan LCQ/DECA spectrometer. High-resolution mass spectra (HRMS) were measured using a Micromass Ultra Q-TOF spectrometer. Melting points (uncorrected) were determined using a


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SGWX-4B micro melting point apparatus. All of the microwave-assisted reactions were performed using an Anton Paar MonowaveTM 300 monomode microwave reactor. General Procedure for Preparation of Acetoacetanilide Derivatives B01 – B15.

A mixture of ethyl acetoacetate (0.06 mol), an appropriate amine (0.03 mol), and a catalytic amount of potassium tert-butoxide was heated in a microwave reactor at 120 °C for 2 h. The crude product was purified by flash chromatography on silica gel using dichloromethane as the eluent to generate the product as a solid in 40 % – 70 % yield. N-(3-nitrophenyl)-3-oxobutanamide (B01) B01 was prepared from 3-nitroaniline (A01). LRMS (ESI) m/z 223 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 8.61 (t, J = 2.1 Hz, 1H), 8.01 – 7.78 (m, 2H), 7.61 (t, J = 8.2 Hz, 1H), 3.61 (s, 2H), 2.22 (s, 3H). 3-oxo-N-phenylbutanamide (B02) B02 was prepared from aniline (A02). LRMS (ESI) m/z 178 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H), 7.57 (dd, J = 8.6, 1.1 Hz, 2H), 7.36 – 7.24 (m, 2H), 7.10 – 6.97 (m, 1H), 3.54 (s, 2H), 2.20 (s, 3H). N-(4-methoxyphenyl)-3-oxobutanamide (B03) B03 was prepared from 4-methoxyaniline (A03). LRMS (ESI) m/z 208 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 7.55 – 7.39 (m, 2H), 6.97 – 6.81 (m, 2H), 3.71 (s, 3H), 3.50 (s, 2H), 2.20 (s, 3H). N-(4-bromophenyl)-3-oxobutanamide (B04)


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B04 was prepared from 4-bromoaniline (A04). LRMS (ESI) m/z 256 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 7.57 – 7.52 (m, 2H), 7.51 – 7.46 (m, 2H), 3.55 (s, 2H), 2.20 (s, 3H). N-(2-fluorophenyl)-3-oxobutanamide (B05) B05 was prepared from 2-fluoroaniline (A05). LRMS (ESI) m/z 196 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.00 – 7.92 (m, 1H), 7.30 – 7.22 (m, 1H), 7.20 – 7.09 (m, 2H), 3.64 (s, 2H), 2.20 (s, 3H). N-(3-methoxyphenyl)-3-oxobutanamide (B06) B06 was prepared from 3-methoxyaniline (A06). LRMS (ESI) m/z 208 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 7.25 (t, J = 2.2 Hz, 1H), 7.19 (t, J = 8.1 Hz, 1H), 7.11 – 7.02 (m, 1H), 6.62 (ddd, J = 8.2, 2.5, 0.8 Hz, 1H), 3.71 (s, 3H), 3.52 (s, 2H), 2.19 (s, 3H). 4-(3-oxobutanamido)benzamide (B07) B07 was prepared from 4-aminobenzamide (A07). LRMS (ESI) m/z 221 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 7.87 (s, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.26 (s, 1H), 3.58 (s, 2H), 2.21 (s, 3H). N-(4-chlorophenyl)-3-oxobutanamide (B08) B08 was prepared from 4-chloroaniline (A08). LRMS (ESI) m/z 212 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 7.65 – 7.52 (m, 2H), 7.42 – 7.27 (m, 2H), 3.55 (s, 2H), 2.20 (s, 3H). N-(2-chlorophenyl)-3-oxobutanamide (B09) B09 was prepared from 2-chloroaniline (A09). LRMS (ESI) m/z 212 [M+H]+; 1H NMR (400 MHz, DMSO) δ 9.78 (s, 1H), 7.88 – 7.75 (m, 1H), 7.50 (dd, J = 8.0, 1.3 Hz, 1H), 7.39 – 7.28 (m, 1H), 7.18 (td, J = 7.9, 1.4 Hz, 1H), 3.66 (s, 2H), 2.21 (s, 3H). N-(2,5-dichlorophenyl)-3-oxobutanamide (B10)


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B10 was prepared from 2,5-dichloroaniline (A10). LRMS (ESI) m/z 246 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.25 (dd, J = 8.6, 2.5 Hz, 1H), 3.71 (s, 2H), 2.21 (s, 3H). N-(4-chloro-2,5-dimethoxyphenyl)-3-oxobutanamide (B11) B11 was prepared from 4-chloro-2,5-dimethoxyaniline (A11). LRMS (ESI) m/z 272 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H), 8.03 (s, 1H), 7.14 (s, 1H), 3.81 (s, 3H), 3.75 (s, 3H), 3.69 (s, 2H), 2.18 (s, 3H). N-(2-methoxyphenyl)-3-oxobutanamide (B12) B12 was prepared from 2-methoxyaniline (A12). LRMS (ESI) m/z 208 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.06 – 7.95 (m, 1H), 7.11 – 6.99 (m, 2H), 6.94 – 6.83 (m, 1H), 3.83 (s, 3H), 3.66 (s, 2H), 2.18 (s, 3H). N-(4-fluorophenyl)-3-oxobutanamide (B13) B13 was prepared from 4-fluoroaniline (A13). LRMS (ESI) m/z 196 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 7.64 – 7.49 (m, 2H), 7.13 (t, J = 8.9 Hz, 2H), 3.52 (s, 2H), 2.19 (s, 3H). N-(3-chlorophenyl)-3-oxobutanamide (B14) B14 was prepared from 3-chloroaniline (A14). LRMS (ESI) m/z 212 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 7.81 (t, J = 2.0 Hz, 1H), 7.46 – 7.24 (m, 2H), 7.12 (ddd, J = 7.8, 2.1, 1.2 Hz, 1H), 3.56 (s, 2H), 2.21 (s, 3H). N-(3-cyanophenyl)-3-oxobutanamide (B15) B15 was prepared from 3-aminobenzonitrile (A15). LRMS (ESI) m/z 203 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.08 (d, J = 1.0 Hz, 1H), 7.79 – 7.72 (m, 1H), 7.57 – 7.49 (m, 2H), 3.59 (s, 2H), 2.21 (s, 3H). General Procedure for Preparation of 2-Amino-4-methylthiophene Derivatives DC-S237 – DC-S258.


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These compounds were prepared via the Gewald reaction. To a stirred equimolecular suspension of acetoacetanilide derivative (0.005 mol), ethyl cyanoacetate (isobutyl cyanoacetate for DCS237, methyl cyanoacetate for DC-S238, 2-cyanoacetamide for DC-S240, isopropyl cyanoacetate for DC-S241, tert-butyl cyanoacetate for DC-S242, cyclohexyl cyanoacetate for DC-S243, and benzyl cyanoacetate for DC-S244) (0.005 mol) and elemental sulfur (0.005 mol) in ethanol (isobutanol for DC-S237, methanol for DC-S238, isopropanol for DC-S241, cyclohexanol for DC-S243, and benzyl alcohol for DC-S244) (25 mL) were added simultaneously in quintuple, with an excess amount of morpholine. The mixture was stirred at 80 °C for 1 – 2 h. The precipitate formed after cooling was filtered off and washed with ethanol to afford the desired compound. As DC-S249 has good solubility in ethanol, no precipitate was formed after cooling, and it was obtained via purification of the crude product by flash chromatography on silica gel. For preparation of DC-S243 and DC-S244, the reaction mixture was diluted with ethanol after cooling to room temperature and stirred, and the precipitate then formed. The purity of the synthesized compounds were determined to be greater than 95% except for DC-S243 (purity 93.2%) by HPLC analysis [HPLC: column, Agilent XDB-C18, 150 × 4.6 mm, 5 µm; solvent system, MeOH‒1 ‰ DEA and water with isocratic elution (68% organic phase for DC-S243, DC-S253, and DC-S254; and 80% for the rest compounds); flow rate, 1.0 mL/min; UV detection, 254 nm; oven, 20 ℃. Agilent 1260, Agilent Technologies,


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California, USA]. Detailed HPLC analysis data of the synthesized compounds were displayed in Table S4 (Supporting Information). Isobutyl

2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate

(DC-

S237) DC-S237 was prepared from N-(3-nitrophenyl)-3-oxobutanamide (B01) and isobutyl cyanoacetate following the general procedure indicated above. Yield, 62 %; mp 190 – 191 °C; 1

H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.62 (t, J = 2.1 Hz, 1H), 8.00 (dd, J = 8.2, 1.2

Hz, 1H), 7.95 – 7.83 (m, 3H), 7.60 (t, J = 8.2 Hz, 1H), 4.00 (d, J = 6.4 Hz, 2H), 2.54 (s, 3H), 2.05 – 1.95 (m, 1H), 0.96 (d, J = 6.7 Hz, 6H); 13C NMR (125 MHz, DMSO-d6) δ 165.75, 165.22, 161.81, 147.89, 141.63, 140.36, 129.98, 126.01, 117.75, 114.10, 111.78, 105.62, 69.54, 27.35, 19.17, 16.80; LRMS (ESI) m/z 378 [M+H]+; HRMS (ESI) m/z calcd C17H20O5N3S [M+H]+ 378.1118, found 378.1126. Methyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DC-S238) DC-S238 was prepared from N-(3-nitrophenyl)-3-oxobutanamide (B01) and methyl cyanoacetate following the general procedure indicated above. Yield, 59 %; mp 238 – 239 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.62 (t, J = 2.1 Hz, 1H), 8.00 (ddd, J = 8.2, 2.0, 0.8 Hz, 1H), 7.91 (ddd, J = 8.2, 2.3, 0.8 Hz, 1H), 7.84 (s, 2H), 7.60 (t, J = 8.2 Hz, 1H), 3.75 (s, 3H), 2.51 (d, J = 4.5 Hz, 3H);

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C NMR (125 MHz, DMSO-d6) δ 165.35, 165.12, 161.77, 147.89, 142.32,

140.38, 129.95, 126.04, 117.73, 114.13, 111.58, 105.61, 50.81, 16.48; LRMS (ESI) m/z 336 [M+H]+; HRMS (ESI) m/z calcd C14H14O5N3S [M+H]+ 336.0649, found 336.0648. Ethyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DC-S239) DC-S239 was prepared from N-(3-nitrophenyl)-3-oxobutanamide (B01) and ethyl cyanoacetate following the general procedure indicated above. Yield, 78 %; mp 202 – 204 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.63 (t, J = 2.1 Hz, 1H), 8.00 (ddd, J = 8.2, 1.9, 0.7 Hz, 1H), 7.91 (ddd, J = 8.2, 2.3, 0.8 Hz, 1H), 7.84 (s, 2H), 7.60 (t, J = 8.2 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 2.52 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H);

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C NMR (125 MHz, DMSO-d6) δ 165.42, 164.87,

161.80, 147.89, 142.12, 140.38, 129.97, 126.01, 117.73, 114.10, 111.66, 105.73, 59.41, 16.62, 14.31; LRMS (ESI) m/z 350 [M+H]+; HRMS (ESI) m/z calcd C15H16O5N3S [M+H]+ 350.0805, found 350.0811.


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5-Amino-3-methyl-N2-(3-nitrophenyl)thiophene-2,4-dicarboxamide (DC-S240) DC-S240 was prepared from N-(3-nitrophenyl)-3-oxobutanamide (B01) and 2-cyanoacetamide following the general procedure indicated above. Yield, 63 %; mp 244 – 245 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.63 (t, J = 2.1 Hz, 1H), 8.04 – 7.97 (m, 1H), 7.89 (ddd, J = 8.2, 2.3, 0.8 Hz, 1H), 7.59 (t, J = 8.2 Hz, 1H), 7.37 (s, 2H), 7.05 (s, 2H), 2.51 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 167.79, 162.41, 161.77, 148.32, 142.12, 141.08, 130.31, 126.58, 117.95, 114.64, 112.94, 110.60, 16.38; LRMS (ESI) m/z 321 [M+H]+; HRMS (ESI) m/z calcd C13H13O4N4S [M+H]+ 321.0652, found 321.0650. Isopropyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DCS241) DC-S241 was prepared from N-(3-nitrophenyl)-3-oxobutanamide (B01) and isopropyl cyanoacetate following the general procedure indicated above. Yield, 81 %; mp 197 – 199 °C; 1

H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.63 (t, J = 2.1 Hz, 1H), 8.05 – 7.97 (m, 1H),

7.91 (ddd, J = 8.2, 2.2, 0.7 Hz, 1H), 7.84 (s, 2H), 7.60 (t, J = 8.2 Hz, 1H), 5.13 – 5.04 (m, 1H), 2.51 (s, 3H), 1.29 (d, J = 6.3 Hz, 6H); 13C NMR (125 MHz, DMSO-d6) δ 165.47, 164.58, 161.82, 147.89, 141.86, 140.39, 129.97, 125.96, 117.72, 114.06, 111.79, 105.93, 66.83, 21.83, 16.79; LRMS (ESI) m/z 364 [M+H]+; HRMS (ESI) m/z calcd C16H18O5N3S [M+H]+ 364.0962, found 364.0968. tert-Butyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DCS242) DC-S242 was prepared from N-(3-nitrophenyl)-3-oxobutanamide (B01) and tert-butyl cyanoacetate following the general procedure indicated above. Yield, 63 %; mp 196 – 197 °C; 1

H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.63 (t, J = 2.0 Hz, 1H), 8.00 (dd, J = 8.2, 1.1

Hz, 1H), 7.90 (dd, J = 8.2, 1.5 Hz, 1H), 7.81 (s, 2H), 7.59 (t, J = 8.2 Hz, 1H), 2.49 (s, 3H), 1.52 (s, 9H);

13

C NMR (125 MHz, DMSO-d6) δ 165.30, 164.71, 161.87, 147.89, 141.77, 140.41,

129.97, 125.93, 117.69, 114.02, 111.57, 106.89, 80.20, 28.13, 16.98; LRMS (ESI) m/z 378 [M+H]+; HRMS (ESI) m/z calcd C17H20O5N3S [M+H]+ 378.1118, found 378.1128. Cyclohexyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DCS243)


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DC-S243 was prepared from N-(3-nitrophenyl)-3-oxobutanamide (B01) and cyclohexyl cyanoacetate following the general procedure indicated above. After cooling to room temperature, the reaction mixture was diluted with ethanol and stirred, and the precipitate then formed. Yield, 52 %; mp 189 – 191 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.61 (s, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.89 (dd, J = 8.1, 1.4 Hz, 1H), 7.84 (s, 2H), 7.59 (t, J = 8.2 Hz, 1H), 4.97 – 4.79 (m, 1H), 2.53 (s, 3H), 1.83 (d, J = 3.7 Hz, 2H), 1.66 (s, 2H), 1.53 (dd, J = 19.3, 10.0 Hz, 3H), 1.44 – 1.28 (m, 3H); 13C NMR (125 MHz, DMSO-d6) δ 165.62, 164.57, 161.82, 147.90, 141.65, 140.38, 129.98, 125.97, 117.73, 114.07, 111.85, 105.93, 71.25, 65.00, 44.23, 31.13, 24.97, 23.12, 16.93; LRMS (ESI) m/z 404 [M+H]+; HRMS (ESI) m/z calcd C19H22O5N3S [M+H]+ 404.1275, found 404.1270. Benzyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DC-S244) DC-S244 was prepared from N-(3-nitrophenyl)-3-oxobutanamide (B01) and benzyl cyanoacetate following the general procedure indicated above. After cooling to room temperature, the reaction mixture was diluted with ethanol and stirred, and the precipitate then formed. Yield, 33 %; mp 205 – 207 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.11 – 6.78 (m, 11H), 5.29 (s, 2H), 2.50 (s, 3H);

13

C NMR (125 MHz, DMSO-d6) δ 165.86, 164.64, 161.76,

147.90, 141.76, 140.34, 136.67, 129.98, 128.51, 127.96, 126.01, 117.76, 114.10, 111.95, 105.37, 64.96, 16.78; LRMS (ESI) m/z 412 [M+H]+; HRMS (ESI) m/z calcd C20H18O5N3S [M+H]+ 412.0962, found 412.0953. Ethyl 2-amino-4-methyl-5-(phenylcarbamoyl)thiophene-3-carboxylate (DC-S245) DC-S245 was prepared from 3-oxo-N-phenylbutanamide (B02) and ethyl cyanoacetate following the general procedure indicated above. Yield, 65 %; mp 178 – 179 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 7.75 (s, 2H), 7.60 (d, J = 7.6 Hz, 2H), 7.37 – 7.23 (m, 2H), 7.05 (t, J = 7.4 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 2.49 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 165.11, 164.95, 161.42, 140.29, 139.14, 128.59, 123.32, 120.05, 112.96, 105.40, 59.31, 16.58, 14.33; LRMS (ESI) m/z 305 [M+H]+; HRMS (ESI) m/z calcd C15H17O3N2S [M+H]+ 305.0954, found 305.0948.


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Ethyl

2-amino-5-((4-methoxyphenyl)carbamoyl)-4-methylthiophene-3-carboxylate

(DC-

S246) DC-S246 was prepared from N-(4-methoxyphenyl)-3-oxobutanamide (B03) and ethyl cyanoacetate following the general procedure indicated above. Yield, 49 %; mp 160 – 161 °C; 1

H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 7.72 (s, 2H), 7.56 – 7.46 (m, 2H), 6.91 – 6.84 (m,

2H), 4.22 (q, J = 7.1 Hz, 2H), 3.72 (s, 3H), 2.48 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 164.97, 161.11, 155.37, 139.82, 132.19, 121.74, 113.71, 113.08, 105.33, 59.29, 55.17, 16.55, 14.33; LRMS (ESI) m/z 335 [M+H]+; HRMS (ESI) m/z calcd C16H19O4N2S [M+H]+ 335.1060, found 335.1057. Ethyl 2-amino-5-((4-bromophenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DC-S247) DC-S247 was prepared from N-(4-bromophenyl)-3-oxobutanamide (B04) and ethyl cyanoacetate following the general procedure indicated above. Yield, 73 %; mp 170 – 171 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.81 (s, 1H), 7.77 (s, 2H), 7.63 – 7.54 (m, 2H), 7.52 – 7.44 (m, 2H), 4.23 (q, J = 7.1 Hz, 2H), 2.49 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 165.21, 164.91, 161.45, 140.94, 138.55, 131.39, 121.93, 114.92, 112.46, 105.51, 59.35, 16.59, 14.32; LRMS (ESI) m/z 383 [M+H]+; HRMS (ESI) m/z calcd C15H16O3N2BrS [M+H]+ 383.0060, found 383.0063. Ethyl 2-amino-5-((2-fluorophenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DC-S248) DC-S248 was prepared from N-(2-fluorophenyl)-3-oxobutanamide (B05) and ethyl cyanoacetate following the general procedure indicated above. Yield, 55 %; mp 140 – 141 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.35 (s, 1H), 7.78 (s, 2H), 7.59 (td, J = 7.8, 1.9 Hz, 1H), 7.29 – 7.11 (m, 3H), 4.23 (q, J = 7.1 Hz, 2H), 2.54 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 165.32, 164.93, 161.39, 155.30 (d, J = 2476.4 Hz), 141.55, 126.28 (d, J = 7.5 Hz), 126.11, 126.03, 124.26 (d, J = 3.3 Hz), 115.66 (d, J = 19.8 Hz), 111.99, 105.68, 59.36, 16.42, 14.31; LRMS (ESI) m/z 323 [M+H]+; HRMS (ESI) m/z calcd C15H16O3N2FS [M+H]+ 323.0860, found 323.0865. Ethyl

2-amino-5-((3-methoxyphenyl)carbamoyl)-4-methylthiophene-3-carboxylate

(DC-

S249)


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DC-S249 was prepared from N-(3-methoxyphenyl)-3-oxobutanamide (B06) and ethyl cyanoacetate following the general procedure indicated above. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed with H2O and then brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel. Chromatographic eluent: 20 % EtOAc in petroleum ether. Yield, 38 %; mp 124 – 125 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 7.76 (s, 2H), 7.34 – 7.27 (m, 1H), 7.25 – 7.08 (m, 2H), 6.63 (dt, J = 7.2, 2.4 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.72 (s, 3H), 2.48 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H);

13

C NMR (125 MHz, DMSO-d6) δ 165.14, 164.95, 161.42, 159.43, 140.35,

140.34, 129.35, 112.96, 112.26, 108.80, 105.70, 105.42, 59.32, 54.98, 16.59, 14.32; LRMS (ESI) m/z 335 [M+H]+; HRMS (ESI) m/z calcd C16H19O4N2S [M+H]+ 335.1060, found 335.1057. Ethyl 2-amino-5-((4-carbamoylphenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DCS250) DC-S250 was prepared from 4-(3-oxobutanamido)benzamide (B07) and ethyl cyanoacetate following the general procedure indicated above. Yield, 47 %; mp 270 – 272 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H), 7.90 – 7.76 (m, 5H), 7.67 (d, J = 8.8 Hz, 2H), 7.26 (s, 1H), 4.23 (q, J = 7.1 Hz, 2H), 2.50 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H);

13

C NMR (125 MHz, DMSO-d6) δ

167.42, 165.29, 164.92, 161.56, 141.85, 141.09, 128.75, 128.23, 118.98, 112.55, 105.56, 59.37, 16.62, 14.32; LRMS (ESI) m/z 348 [M+H]+; HRMS (ESI) m/z calcd C16H18O4N3S [M+H]+ 348.1013, found 348.1013. Ethyl

2-amino-5-((4-chlorophenyl)carbamoyl)-4-methylthiophene-3-carboxylate

(DC-

S251) DC-S251 was prepared from N-(4-chlorophenyl)-3-oxobutanamide (B08) and ethyl cyanoacetate following the general procedure indicated above. Yield, 53 %; mp 168 – 169 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.78 (s, 2H), 7.69 – 7.56 (m, 2H), 7.46 – 7.23 (m, 2H), 4.23 (q, J = 7.1 Hz, 2H), 2.49 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 165.65, 165.37, 161.90, 141.35, 138.58, 128.92, 127.34, 122.02, 112.92, 105.97, 59.79, 17.02, 14.76; LRMS (ESI) m/z 339 [M+H]+; HRMS (ESI) m/z calcd C15H16O3N2ClS [M+H]+ 339.0565, found 339.0573.


29

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Ethyl

2-amino-5-((2-chlorophenyl)carbamoyl)-4-methylthiophene-3-carboxylate

(DC-

S252) DC-S252 was prepared from N-(2-chlorophenyl)-3-oxobutanamide (B09) and ethyl cyanoacetate following the general procedure indicated above. Yield, 44 %; mp 154 – 156 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 7.80 (s, 2H), 7.70 (dd, J = 8.0, 1.5 Hz, 1H), 7.51 (dd, J = 8.0, 1.4 Hz, 1H), 7.34 (td, J = 7.8, 1.4 Hz, 1H), 7.22 (td, J = 7.8, 1.6 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 2.59 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H);

13

C NMR (100 MHz, DMSO-d6) δ 165.84, 165.36,

161.70, 141.93, 135.73, 129.89, 128.32, 127.92, 127.05, 127.01, 112.63, 106.24, 59.83, 16.85, 14.76; LRMS (ESI) m/z 339 [M+H]+; HRMS (ESI) m/z calcd C15H16O3N2ClS [M+H]+ 339.0565, found 339.0563. Ethyl 2-amino-5-((2,5-dichlorophenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DCS253) DC-S253 was prepared from N-(2,5-dichlorophenyl)-3-oxobutanamide (B10) and ethyl cyanoacetate following the general procedure indicated above. Yield, 64 %; mp 173 – 175 °C; 1

H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 7.88 (d, J = 2.6 Hz, 1H), 7.85 (s, 2H), 7.56 (d, J

= 8.6 Hz, 1H), 7.29 (dd, J = 8.6, 2.6 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 2.60 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 166.06, 165.30, 161.67, 142.70, 137.02, 131.97, 131.21, 126.40, 125.75, 112.19, 106.39, 59.88, 16.87, 14.75; LRMS (ESI) m/z 373 [M+H]+; HRMS (ESI) m/z calcd C15H15O3N2Cl2S [M+H]+ 373.0175, found 373.0177. Ethyl

2-amino-5-((4-chloro-2,5-dimethoxyphenyl)carbamoyl)-4-methylthiophene-3-

carboxylate (DC-S254) DC-S254 was prepared from N-(4-chloro-2,5-dimethoxyphenyl)-3-oxobutanamide (B11) and ethyl cyanoacetate following the general procedure indicated above. Yield, 67 %; mp 190 – 191 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H), 7.92 (s, 1H), 7.83 (s, 2H), 7.18 (s, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.83 (s, 3H), 3.78 (s, 3H), 2.59 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H);

13

C

NMR (125 MHz, DMSO-d6) δ 165.50, 164.86, 160.70, 148.18, 143.56, 141.10, 127.13, 115.06, 113.11, 112.70, 106.29, 105.97, 59.44, 56.88, 56.36, 16.07, 14.31; LRMS (ESI) m/z 399 [M+H]+; HRMS (ESI) m/z calcd C17H20O5N2ClS [M+H]+ 399.0776, found 399.0780.


30


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Ethyl 2-amino-5-((2-methoxyphenyl)carbamoyl)-4-methylthiophene-3-carboxylate

Page 32 of 65

(DC-

S255) DC-S255 was prepared from N-(2-methoxyphenyl)-3-oxobutanamide (B12) and ethyl cyanoacetate following the general procedure indicated above. Yield, 48 %; mp 142 – 143 °C; 1

H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 7.95 (dd, J = 7.9, 1.4 Hz, 1H), 7.79 (s, 2H), 7.20

– 7.00 (m, 2H), 6.99 – 6.82 (m, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.85 (s, 3H), 2.59 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H);

13

C NMR (125 MHz, DMSO-d6) δ 165.33, 164.91, 160.65, 149.55, 140.46,

127.37, 124.44, 121.37, 120.41, 113.10, 111.08, 105.84, 59.40, 55.98, 16.06, 14.32; LRMS (ESI) m/z 335 [M+H]+; HRMS (ESI) m/z calcd C16H19O4N2S [M+H]+ 335.1060, found 335.1058. Ethyl 2-amino-5-((4-fluorophenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DC-S256) DC-S256 was prepared from N-(4-fluorophenyl)-3-oxobutanamide (B13) and ethyl cyanoacetate following the general procedure indicated above. Yield, 51 %; mp 151 – 153 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 7.75 (s, 2H), 7.67 – 7.54 (m, 2H), 7.14 (t, J = 8.9 Hz, 2H), 4.22 (q, J = 7.1 Hz, 2H), 2.48 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H);

13

C NMR (125 MHz, DMSO-d6) δ

165.11, 164.94, 161.37, 158.12 (d, J = 239.8 Hz), 140.53, 135.48, 121.91 (d, J = 7.7 Hz), 115.14 (d, J = 22.1 Hz), 112.61, 105.44, 59.32, 16.56, 14.32; LRMS (ESI) m/z 323 [M+H]+; HRMS (ESI) m/z calcd C15H16O3N2FS [M+H]+ 323.0860, found 323.0856. Ethyl 2-amino-5-((3-chlorophenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DC-S257) DC-S257 was prepared from N-(3-chlorophenyl)-3-oxobutanamide (B14) and ethyl cyanoacetate following the general procedure indicated above. Yield, 58 %; mp 154 – 156 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.79 (s, 2H), 7.78 (t, J = 2.0 Hz, 1H), 7.53 (ddd, J = 8.3, 1.9, 0.9 Hz, 1H), 7.32 (t, J = 8.1 Hz, 1H), 7.10 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H), 4.22 (q, J = 7.1 Hz, 2H), 2.49 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 165.29, 164.90, 161.58, 141.26, 140.66, 132.92, 130.27, 122.95, 119.41, 118.35, 112.27, 105.58, 59.37, 16.59, 14.31; LRMS (ESI) m/z 339 [M+H]+; HRMS (ESI) m/z calcd C15H16O3N2ClS [M+H]+ 339.0565, found 339.0555. Ethyl 2-amino-5-((3-cyanophenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DC-S258) DC-S258 was prepared from N-(3-cyanophenyl)-3-oxobutanamide (B15) and ethyl cyanoacetate following the general procedure indicated above. Yield, 66 %; mp 209 – 210 °C; 1H NMR (400


31

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MHz, DMSO-d6) δ 9.98 (s, 1H), 8.19 – 8.00 (m, 1H), 7.91 – 7.84 (m, 1H), 7.82 (s, 2H), 7.60 – 7.41 (m, 2H), 4.22 (q, J = 7.1 Hz, 2H), 2.50 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H);

13

C NMR (125

MHz, DMSO-d6) δ 165.38, 164.88, 161.72, 141.87, 140.01, 130.06, 126.74, 124.61, 122.76, 118.77, 111.81, 111.42, 105.70, 59.40, 16.60, 14.31; LRMS (ESI) m/z 330 [M+H]+; HRMS (ESI) m/z calcd C16H16O3N3S [M+H]+ 330.0907, found 330.0905.


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Associated Content

S Supporting Information ○

Additional figures and tables illustrate the inhibition data, assessment of docking programs, validation of docking procedure, and 2D interaction analysis of DC-S100, DC-S237 and DCS239. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author *Cheng Luo, telephone: 86-21-50806600. E-mail: [email protected]. or Hong Liu, telephone: 86-21-50806600, Email: [email protected] or Wencong Lu, telphone: 86-21-66132406, Email: [email protected]

Author Contributions |Fanwang Meng, Sufang Cheng, Hong Ding contributed equally to this work. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS

This work was supported by the Hi-Tech Research and Development Program of China (2012AA020302 to C.L. and 2012AA01A305 to H.J.), the Ministry of Science and Technology of China (2015CB910304 to H.L.), the National Natural Science Foundation of China (21210003 and 81230076 to H.J., 91229204 to H.L., 21472208 and 8143000629 to C.L.), the National


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Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” (2014ZX09507002 to M.Z.).

ABBREVIATIONS

HIV, human immunodeficiency virus; HCV, hepatitis C virus; structure-activity relationship, SAR; IC50, half-maximum inhibitory concentration; HKMTs, histone lysine methyltransferases; SET7, SET domain containing lysine methyltransferase 7; SAM, S‑adenosylmethionine; SAH, S-adenosyl-L-homocysteine, PAINS, pan-assay interference compounds; RMSD, root-mean square deviation; PDB, Protein Data Bank; XP, extra precision; SP, standard precision; ARTD1, ADP-ribosyltransferase diphtheria toxin-like 1; COL2A1, collagen alpha-1(II) chain; ERα, estrogen receptor α; FoxO3, forkhead box protein O3; pRb, retinoblastoma protein; STAT3, signal transducer and activator of transcription 3; SUV39H1, suppressor of variegation 3-9 homolog 1; TAF10, transcription initiation factor TFIID subunit 10; p65, Transcription factor p65; p53, cellular tumor antigen p53; Pdx1, pancreatic duodenal homeobox 1; HIFα, hypoxiainducible factor; DNMT1, DNA (cytosine-5)-methyltransferase 1; DNMT3A/3L, DNA (cytosine-5)-methyltransferase 3A; DOT1L, DOT1-like histone H3K79 methyltransferase; EZH2, enhancer of zeste homolog 2; NSD1, nuclear receptor binding SET domain protein 1; SETD8, SET domain containing lysine methyltransferase 8; G9a, Euchromatic histone-lysine Nmethyltransferase 2 (EHMT2); MTT, 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.


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85. Xie, Y.; Zhou, R.; Lian, F.; Liu, Y.; Chen, L.; Shi, Z.; Zhang, N.; Zheng, M.; Shen, B.; Jiang, H.; Liang, Z.; Luo, C. Virtual screening and biological evaluation of novel small molecular inhibitors against protein arginine methyltransferase 1 (PRMT1). Org. Biomol. Chem. 2014, 12, 9665-9673. 86. McInnes, C. Virtual screening strategies in drug discovery. Curr. Opin. Chem. Biol. 2007, 11, 494-502. 87. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 2001, 46, 3-26. 88. Baell, J. B. Observations on screening-based research and some concerning trends in the literature. Future Med. Chem. 2010, 2, 1529-1546. 89. Baell, J. B.; Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53, 2719-2740. 90. Whitty, A. Growing PAINS in academic drug discovery. Future Med. Chem. 2011, 3, 797-801. 91. Baell, J.; Walters, M. A. Chemistry: Chemical con artists foil drug discovery. Nature 2014, 513, 481. 92. LigPrep, version 2.3; Schrödinger, LLC: New York, NY, 2009.


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93. Shelley, J.; Cholleti, A.; Frye, L.; Greenwood, J.; Timlin, M.; Uchimaya, M. Epik: a software program for pK a prediction and protonation state generation for drug-like molecules. J. Comput.-Aided Mol. Des. 2007, 21, 681-691. 94. Accelrys Discovery Studio 3.0, Accelrys: San Diego, CA, 2010. 95. Li, J.; Ehlers, T.; Sutter, J.; Varma-O'Brien, S.; Kirchmair, J. CAESAR: A New Conformer Generation Algorithm Based on Recursive Buildup and Local Rotational Symmetry Consideration. J. Chem. Inf. Model. 2007, 47, 1923-1932. 96. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J. Med. Chem. 2004, 47, 1739-1749. 97. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein−Ligand Complexes. J. Med. Chem. 2006, 49, 6177-6196. 98. Maestro, version 9.0, Schrödinger, LLC, New York, NY, 2009. 99. Siebel, A. L.; Fernandez, A. Z.; El-Osta, A. Glycemic memory associated epigenetic changes. Biochem. Pharmacol. 2010, 80, 1853-9. 100. Yang, X.-D.; Huang, B.; Li, M.; Lamb, A.; Kelleher, N. L.; Chen, L.-F. Negative regulation of NF-kappa B action by Set9-mediated lysine methylation of the RelA subunit. EMBO J. 2009, 28, 1055-1066.


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101. Stelzer, G.; Dalah, I.; Stein, T. I.; Satanower, Y.; Rosen, N.; Nativ, N.; Oz-Levi, D.; Olender, T.; Belinky, F.; Bahir, I.; Krug, H.; Perco, P.; Mayer, B.; Kolker, E.; Safran, M.; Lancet, D. In-silico human genomics with GeneCards. Hum. Genomics 2011, 5, 709-717. 102. Belinky, F.; Bahir, I.; Stelzer, G.; Zimmerman, S.; Rosen, N.; Nativ, N.; Dalah, I.; Iny Stein, T.; Rappaport, N.; Mituyama, T.; Safran, M.; Lancet, D. Non-redundant compendium of human ncRNA genes in GeneCards. Bioinformatics 2013, 29, 255-61. 103. Oesterreich, S.; Davidson, N. E. The search for ESR1 mutations in breast cancer. Nat. Genet. 2013, 45, 1415-6. 104. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J. Med. Chem. 2004, 47, 1750-1759. 105. Glide, 5.5; Schrödinger, Inc., New York, 2009. 106. Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455-61. 107. Allen, W. J.; Balius, T. E.; Mukherjee, S.; Brozell, S. R.; Moustakas, D. T.; Lang, P. T.; Case, D. A.; Kuntz, I. D.; Rizzo, R. C. DOCK 6: Impact of new features and current docking performance. J. Comput. Chem. 2015, 36, 1132-56. 108. Hamid, R.; Rotshteyn, Y.; Rabadi, L.; Parikh, R.; Bullock, P. Comparison of alamar blue and MTT assays for high through-put screening. Toxicol. In Vitro 2004, 18, 703-10.


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109. Al-Nasiry, S.; Geusens, N.; Hanssens, M.; Luyten, C.; Pijnenborg, R. The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Hum. Reprod. 2007, 22, 1304-9. 110. Schrödinger, LLC. The PyMOL Molecular Graphics System, Version 1.3r1. 2010.


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Figure Legends Figure 1. Virtual screening procedures and activity assays for SET7 in vitro. (A) An integrated virtual screening procedure combining pharmacophore- and docking-based virtual screening. Numerals denote the number of molecules in each stage. (B) Inhibitory activity of the 127 candidate molecules based on virtual screening at 100 µM. The red columnar bar represents the activity of DC-S100, and the blue bar denotes the reference compound SAH. (C) Inhibitory activity of the 109 analogous compounds of DC-S100 selected from the similarity search, in which the inhibition rate was determined at 100 µM. The blue columnar bar represents the activity of the reference compound SAH. Figure 2. Putative binding modes and activities of DC-S100 and DC-S237. (A) Binding mode of DC-S100 (PDB ID: 1N6A). Carbon atoms of DC-S100 are displayed in yellow; nitrogen atoms are in blue and oxygen atoms in red. Key residues are shown as sticks, and carbon is in grey, nitrogen in blue and oxygen in red. Key hydrogen bonds are shown as a red dashed line. All of the residues and compounds are labeled. (B) Binding mode of DC-S237 (PDB ID: 1N6A). DCS237 is shown as yellow sticks, and key interacting residues are shown as grey sticks. All of the oxygen atoms are in red, and nitrogen atoms are in blue. Hydrogen bonds between DC-S237 and SET7 are represented as a red dashed line. (C) Superimposition of the binding modes of DCS100 and DC-S237 (PDB ID: 1N6A). Both DC-S100 and DC-S237 share a similar binding conformation anchored in the cofactor binding pocket. DC-S237 is shown as blue sticks, and DC-S100 is depicted in cyan, with the surface of SET7 depicted in vacuum electrostatics. Key residue Lys-294 is highlighted. All of the binding mode figures were generated with PyMOL, version 1.3r1110. (D) Inhibitory activities of DC-S100 and DC-S237 against SET7.


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Figure 3. Activities of synthesized compounds (A) Inhibitory activity of synthesized compounds. Activity data of reference compound SAH is represented as a blue columnar bar. (B) IC50 curve of DC-S238 and DC-S239. The green sigmoidal curve graph depicts the activity profile of DC-S238 (IC50 = 4.88 µM), and the pink sigmoidal curve graph displays that of DCS239 (IC50 = 4.59 µM). Figure 4. Cellular activities of DC-S239 against SET7 in different cell lines. (A) IC50 curve of DC-S239 toward the MCF7 cell line. (B) IC50 curve of DC-S239 toward the HL60 cell line. (C) Dose-response relationship of DC-S239 toward the MV4-11 cell line. (D) Activity of DC-S239 toward the HCT116 cell line. (E) Activity of DC-S239 toward the DHL4 cell. The antiproliferation activity of DC-S239 is determined by the MTT (MCF7, MV4-11) or alamarBlue assay (HL60, MV4-11, DHL4). The experimental data were analyzed with GraphPad Prism 5.0. Figure 5. Predicted binding mode of DC-S239 (PDB ID: 1N6A). (A) Key interactions involved in stabilizing DC-S239 at the co-factor binding site. DC-S239 is depicted as yellow sticks, and key residues are labeled. Hydrogen bonds are shown as red lines, and the π-π stacking interaction involving residue Trp-352 is also highlighted. (B) Superimposition of the binding modes of DCS239 and SAM. The structure of SET7 is displayed in vacuum electrostatics. DC-S239 is shown as yellow sticks, and SAM is displayed as grey sticks. Key residue Lys-294 is highlighted.


53

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Abstract Figure


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Table 1. Structure-Activity Relationship (SAR) of DC-S100

NO.

Scaffold

R1

DC-S128

-

DC-S129

-

R2

Inhibition Ratio at 100 µM /% (IC50/µM)

35.53

53.32 (89.18) 62.10 DC-S130

(189.70)

DC-S131

O

DC-S132

O2N

N H

R1

R2

36.07

58.26 (61.75)

(I) DC-S133

-

10.64

DC-S134

-

22.06

DC-S135

12.14

DC-S136

15.78


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DC-S137

-

1.62

DC-S138

-

8.52

Cl

DC-S139

Cl

-

9.10

N H3C

O

DC-S140

-

13.07

DC-S141

-

18.60

DC-S142

-

10.62

DC-S143

-

18.32

DC-S100

-

50.31 (30.04)

DC-S144

11.73 (II)

DC-S145

-


4.62

56


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DC-S146

-


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6.55

57

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Table 2. Selectivity of DC-S238 and DC-S239 over Other Epigenetic Proteins Compound NO.

DC-S238

DC-S239

Target

Inhibition Ratio at 100 µM/%

SET7

94.71

DNMT1

9.82

DOT1L

-28.88

EZH2

19.61

NSD1

25.05

SETD8

45.45

G9a

29.86

SET7

90.51

DNMT1

16.07

DNMT3A/3L

-4.25

DOT1L

-5.42

EZH2

19.61

NSD1

27.16

SETD8

40.89

G9a

-0.71


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Table 3. Structure-Activity Relationship (SAR) of DC-S239

NO.

R1

R2

R3

Inhibition Ratio at 100 µM/%

IC50 (µM)

DC-S197

-26.80

DC-S198

-16.20

DC-S199

-25.36

DC-S238 DC-S239

94.71 90.51

4.88 4.59

DC-S237

61.34

37.67

DC-S240

54.36

49.52

DC-S241

48.03

DC-S242

-22.24

DC-S243

1.53

DC-S244

11.50


59

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DC-S245

-16.46

DC-S246

-25.76

DC-S247

19.07

DC-S248

-21.13

DC-S249

-13.28

DC-S250

-9.98

DC-S251

-12.38

DC-S252

-15.31

DC-S253

-4.70

DC-S254

-7.88

DC-S255

-6.45

DC-S256

-5.57

DC-S257

-2.55

DC-S258

70.44


61.55

60


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

Figure 1. Virtual screening procedures and activity assays for SET7 in vitro. (A) An integrated virtual screening procedure combining pharmacophore- and docking-based virtual screening. Numerals denote the number of molecules in each stage. (B) Inhibitory activity of the 127 candidate molecules based on virtual screening at 100 µM. The red columnar bar represents the activity of DC-S100, and the blue bar denotes the reference compound SAH. (C) Inhibitory activity of the 109 analogous compounds of DC-S100 selected from the similarity search, in which the inhibition rate was determined at 100 µM. The blue columnar bar represents the activity of the reference compound SAH. 199x96mm (300 x 300 DPI)


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Figure 2. Putative binding modes and activities of DC-S100 and DC-S237. (A) Binding mode of DC-S100 (PDB ID: 1N6A). Carbon atoms of DC-S100 are displayed in yellow; nitrogen atoms are in blue and oxygen atoms in red. Key residues are shown as sticks, and carbon is in grey, nitrogen in blue and oxygen in red. Key hydrogen bonds are shown as a red dashed line. All of the residues and compounds are labeled. (B) Binding mode of DC-S237 (PDB ID: 1N6A). DC-S237 is shown as yellow sticks, and key interacting residues are shown as grey sticks. All of the oxygen atoms are in red, and nitrogen atoms are in blue. Hydrogen bonds between DC-S237 and SET7 are represented as a red dashed line. (C) Superimposition of the binding modes of DC-S100 and DC-S237 (PDB ID: 1N6A). Both DC-S100 and DC-S237 share a similar binding conformation anchored in the cofactor binding pocket. DC-S237 is shown as blue sticks, and DC-S100 is depicted in cyan, with the surface of SET7 depicted in vacuum electrostatics. Key residue Lys-294 is highlighted. All of the binding mode figures were generated with PyMOL, version 1.3r1110. (D) Inhibitory activities of DC-S100 and DC-S237 against SET7. 199x180mm (300 x 300 DPI)



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

Figure 3. Activities of synthesized compounds (A) Inhibitory activity of synthesized compounds. Activity data of reference compound SAH is represented as a blue columnar bar. (B) IC50 curve of DC-S238 and DCS239. The green sigmoidal curve graph depicts the activity profile of DC-S238 (IC50 = 4.88 µM), and the pink sigmoidal curve graph displays that of DC-S239 (IC50 = 4.59 µM). 199x83mm (300 x 300 DPI)


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Figure 4. Cellular activities of DC-S239 against SET7 in different cell lines. (A) IC50 curve of DC-S239 toward the MCF7 cell line. (B) IC50 curve of DC-S239 toward the HL60 cell line. (C) Dose-response relationship of DC-S239 toward the MV4-11 cell line. (D) Activity of DC-S239 toward the HCT116 cell line. (E) Activity of DC-S239 toward the DHL4 cell. The anti-proliferation activity of DC-S239 is determined by the MTT (MCF7, MV4-11) or alamarBlue assay (HL60, MV4-11, DHL4). The experimental data were analyzed with GraphPad Prism 5.0. 199x90mm (300 x 300 DPI)



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

Figure 5. Predicted binding mode of DC-S239 (PDB ID: 1N6A). (A) Key interactions involved in stabilizing DC-S239 at the co-factor binding site. DC-S239 is depicted as yellow sticks, and key residues are labeled. Hydrogen bonds are shown as red lines, and the π-π stacking interaction involving residue Trp-352 is also highlighted. (B) Superimposition of the binding modes of DC-S239 and SAM. The structure of SET7 is displayed in vacuum electrostatics. DC-S239 is shown as yellow sticks, and SAM is displayed as grey sticks. Key residue Lys-294 is highlighted. 199x90mm (300 x 300 DPI)


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Discovery and Optimization of Novel, Selective Histone

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