Discovery and Optimization of Novel, Selective Histone Methyltransferase SET7 Inhibitors by Pharmacophore- and Docking-Based Virtual Screening

Article abstract of DOI:10.1021/acs.jmedchem.5b01154

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 docking-based 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, structure modifications were made to improve its activity. Of note, compounds DC-S238 and DC-S239, with IC₅₀ values of 4.88 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.

Full text of DOI:10.1021/acs.jmedchem.5b01154

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Article
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, Yu-
Chih 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 pharmacophoreꢀ and dockingꢀbased virtual screening
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$
,†,|
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Fanwang Meng ,Sufang Cheng , Hong Ding , Shien Liu , Yan Liu , Kongkai Zhu , Shijie
†
†
†
†,#
¶
¶
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Chen , Junyan Lu , Yiqian Xie , Linjuan Li , Rongfeng Liu , Zhe Shi , Yu Zhou , YuꢀChih
¶
†
†,#
$,
§,
†,
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 HiꢀTech Park, Shanghai 201203, China 201203
|
These authors contributed equally.
*Correspondence: Cheng Luo, Eꢀmail: cluo@simm.ac.cn or Hong Liu, Email: hliu@simm.ac.cn;
or Wencong Lu, Email: wclu@shu.edu.cn
Received Data (to be automatically inserted after your manuscript is accepted)
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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 domainꢀcontaining (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, DCꢀS100 which was discovered by pharmacophoreꢀ and dockingꢀ
based virtual screening was identified as the hit compound of SET7 inhibitor. Structureꢀactivity
relationship (SAR) analysis was performed on analogs of DCꢀS100 and according to the putative
binding mode of DCꢀS100 and structure modifications were made to improve its activity. Of
note, compounds DCꢀS238 and DCꢀS239, with IC50 values of 4.88 ꢁM and 4.59 ꢁM,
respectively, displayed selectivity for DNMT1, DOT1L, EZH2, NSD1, SETD8 and G9a. Taken
together, DCꢀS238 and DCꢀS239 can serve as leads for further investigation as SET7 inhibitors
and the chemical toolkits for functional biology studies of SET7.
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Keywords: epigenetic; SET7 inhibitor; pharmacophore; molecular docking.
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ꢀ
Introduction
Histone lysine methyltransferases (HKMTs) are histone covalent modifying enzymes that
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transfer methyl groups to lysine or arginine on histone H3 via an S‑adenosylmethionine (SAM)ꢀ
dependent S ꢀ2 mechanism. HKMTs are involved in heterochromatin formation, gene
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expression regulation, DNA repair, cell cycle control and cancer development .Thus these
epigenetic targets are vital in cancer biology. HKMTs can be divided into two categories: (i) SET
domainꢀcontaining enzymes, such as SET7 (SET domainꢀcontaining lysine methyltransferase 7,
also called SETD7, SET9, KMT7) and EZH2 and (ii) nonꢀSET domain enzymes, such as
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DOT1L. SET7 was first characterized as a monoꢀmethyltransferase of H3K4 . A series of
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studies later revealed that SET7 modulates various transcriptional regulators, including ARTD1 ,
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COL2A1 , ERα , FoxO3 , pRb , RAR , STAT3 , SUV39H1
, TAF10 , p65 , Pdx1 ,
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HIFꢀα . In addition to its role in transcriptional regulation
, SET7 also functions in cell
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cycle control
, differentiation , DNA repair and DNMT1 stability
, and increasing
evidence suggests that SET7 is closely associated with various diseases. SET7 regulates NFꢀκB
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activity through lysine monomethylation of p65 in response to TNFꢀα in human kidney cells ,
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and knockdown of SET7 inhibits key NFꢀκBꢀdependent gene expression . Li, Y. et al. observed
increased inflammatory gene expression and SET7 recruitment in macrophages from diabetic
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mice, demonstrating the role of SET7 in diabetes . Recently, it was found that epigenetic
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changes induced by SET7 contribute to vascular dysfunction in patients with type 2 diabetes ,
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
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patients with alopecia areata . In addition, downregulation of SET7 in breast cancer cells
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attenuates estrogenꢀinduced gene activation and that SET7 knockdown results in a concomitant
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decrease in the steadyꢀstate levels of estrogen receptor α (ERα) protein , which is involved in the
pathology of breast cancer, endometrial cancer and osteoporosis. Other studies also support that
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SET7 is linked to the pathological processes of cancers
. Recently, the role of SET7 in virus
infection was identified; for example, knockdown of SET7 suppresses Tat transcription of the
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HIV promoter , and SET7 facilitates HCV replication by attenuating IFN signaling pathways
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and IFNꢀrelated effectors . Notably, there is a controversial debate about whether the
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methylation of p53 by SET7 meditates p53 activity . Some studies revealed that the SET7ꢀ
mediated methylation of Lysꢀ372 at the p53 Cꢀterminus can regulate p53 acetylation, enhancing
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its antitumor activity
. However, Lehnertz, B. et al. reported that knockdown of SET7 in mice
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does not affect p53ꢀdepedent transcription and tumor suppression , results that are fully
consistent with the findings of Campaner, S. et al., who reported that p53ꢀdepedent cell cycle
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arrest or apoptosis is not affected by SET7 deletion . 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
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issues
. Bissinger, E. M. et al. identified acyl derivatives of pꢀaminosulfonamides and
dapsone as potent hPRMT1 inhibitors, including compound 7, yet they showed weak activity
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toward SET7 . Using highꢀthroughput screening, Verma, S. K. and coꢀworkers reported that
compounds 8, 9 and 10 are potent, cellꢀactive inhibitors of EZH2, and these compounds also
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showed inhibitory activity toward SET7 . ‘Biasedꢀprivileged’ scaffolds were used to design
SET7 inhibitors, and SETinꢀ1 (compound 11) showed inhibitory activity against both G9a and
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SET7 . Compound 12, which showed weak activity toward SET7, is a coactivatorꢀassociated
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arginine methyltransferase 1 inhibitor with an IC value of 14.4 ꢁM . Similarly, compounds 13
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to 16 are not selective SET7 inhibitors . Following a drug repository strategy, it was found that
Cyproheptadine (compound 17), an antihistaminic and antiserotonergic agent, showed inhibitory
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activity toward SET7 (IC = 20 ꢁM) . Recently, (R)ꢀPFIꢀ2 (compound 18) was reported to be a
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potent and selective inhibitor targeting SET7 in MCF7 cells by occupying the histone lysine
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binding site, as revealed by Xꢀray complex structure analysis . However, most SET7 inhibitors
are multiꢀtarget inhibitors with low potencies. Based on the diversity of SAM binding modes and
receptorꢀbased interactions among HKMTs, which enables the design of selective inhibitors, our
research focuses on the development of potent, selective smallꢀmolecule inhibitors of SET7
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targeting the coꢀfactor binding site
.
With the rapid development of computational methods, many studies successfully identifying
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epigenetic inhibitors using this approach have been reported
. Our group has applied virtual
screening methods to identify inhibitors of PRMT1 and DNMT1, as well as inhibitors targeting
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the interface of EZH2ꢀEED and the menin–mixed lineage leukemia interface
; 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, DCꢀS238 (IC = 4.88 ꢁM) and DCꢀS239 (IC = 4.59 ꢁM), were identified
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through an integrated procedure combining pharmacophoreꢀ and dockingꢀbased virtual screening,
similarity searching and hit optimization. The virtual screening identified compound DCꢀS100 as
a hit compound possessing moderate activity toward SET7 (IC = 30.04 ꢁM). Similarityꢀbased
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analog searching enabled the analysis of the structureꢀactivity relationship (SAR) of DCꢀS100.
Based on the putative binding mode of DCꢀS100 and its SAR, chemical optimization was
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performed to improve its potency. Finally, compound DCꢀS238 (IC = 4.88 ꢁM) and DCꢀS239
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(
IC50 = 4.59 ꢁM) were obtained, displaying selectivity for DNMT1, DOT1L, EZH2, NSD1,
SETD8 and G9a. In summary, this study identified DCꢀS238 and DCꢀS239 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.
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ꢀ
Results and Discussion
Virtual Screening. To exploit all available structural and chemical information, an integrated
pharmacophoreꢀ and dockingꢀbased virtual screening procedure was designed to counterbalance
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their limitations . Pharmacophoreꢀbased virtual screening can be used to preꢀprocess virtual
molecule libraries to filter compounds lacking features essential for binding. The 3D
pharmacophore model which yields the lead compound DCꢀS100 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 coꢀfactor 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
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Rule of Five , and ‘panꢀassay interference compounds’ (PAINS)
were removed with
Pipeline Pilot, version 7.5 (Pipeline Pilot; Accelrys Software Inc., San Diego, CA). The
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remaining 182,014 molecules were prepared with LigPrep version 2.3 (Schrödinger, LLC: New
York, NY, 2009) to generate all stereoisomers and different protonation states by Epik version
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2.0 (Schrödinger, LLC: New York, NY, 2009). The prepared Specs library was used to
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generate an indexed multiꢀconformer 3D database in Accelrys Discovery Studio 3.0 with the
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conformation method CAESAR . Receptorꢀligand based pharmacophores, which was used to
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screen the 3D database, was generated with Accelrys Discovery Studio 3.0 . A total of 15,541
molecules matched the pharmacophore features and were subjected to two rounds of dockingꢀ
based virtual screening.
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Several docking programs were preliminarily evaluated, and Glide 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
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conformation of SAM with an RMSD value of 0.30 Å (Glide SP mode ) and 0.22 Å (Glide XP
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mode ), respectively, which agreed with our docking procedures (Supporting Information
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Figure S2). The Glide SP and XP modes, as integrated in Maestro 9.2 , were subsequently used
to dock the compound library into the SAM binding site. As lysineꢀ294 may be involved in the
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specific recognition of SAM by SET7 , we chose this residue as a hydrogen bond constraint
during the Glide XP docking procedure. The topꢀranked 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 Lysꢀ294, Alaꢀ226,
Gluꢀ228, Hisꢀ297, Gluꢀ356 and πꢀπ stacking interactions with Trpꢀ352 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 ꢁM. The enzyme solution was transferred to assay plates, and 5
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L 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 ꢁM (Figure 1B). Among those seven molecules, compound DCꢀS100 was
characterized as a structurally novel SET7 inhibitor (IC = 30.04 ꢁM).
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Similarity-Based Analog Searching and SAR analysis. To explore the structure activity
relationship (SAR) of compound DCꢀS100 and to optimize the compound, a similarityꢀbased
analog search was conducted using Pipeline Pilot V7.5 (Accelrys Inc., San Diego, CA, USA). A
total of 109 derivatives of compound DCꢀS100 were purchased for the same AlphaLISA assay
described above. Seven compounds displayed greater than 50 % inhibition of SET7 activity at
100 ꢁM (Figure 1C), and the IC values against SET7 were measured (Supporting Information
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Table S3).
We analyzed the activity data of the analogs (see Table 1) carefully and explored the SAR of
DCꢀS100 to find clues for hit optimization. In scaffolding (I), the R group is expected to be
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aromatic rings, as opposed to other side chains, because substitutions of aromatic ring resulted in
a significant decrease in potency (DCꢀS133 and DCꢀS134), with inhibition rates of 10.64 % and
22.06 %, respectively. In contrast, when R is a derivative of furan (DCꢀS128, DCꢀS129) or
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benzene (DCꢀS130 to DCꢀS132), inhibition activity increased. The length of the amide group
and R group connecting metaꢀnitrobenzene and the aromatic ring (R ) 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 DCꢀS135 and DCꢀS136, with inhibition rates 12.14 % and
15.78 % respectively; thus, an amide group is the most appropriate linker. Moreover, the
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introduction of a bulky group into the aromatic rings (R ) is not tolerated because large groups
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often lead to a decrease in activity (DCꢀS138 to DCꢀS143). For example, when an iodine atom
was substituted in the benzene ring (DCꢀS137), 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 DCꢀS144.
Second, a larger R group also led to a dramatic decrease in potency (DCꢀS145 and DCꢀS146),
2
supporting the previous hypothesis. In summary, a metaꢀnitrobenzene 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 DCꢀS100. As depicted in
Figure 2A, the nitro group is capable of forming hydrogen bonds with Gluꢀ228 and Lysꢀ294. The
hydrogen bond lengths of Gluꢀ228 and Lysꢀ294 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 Asnꢀ296,
with a bond length of 1.9 Å. The π–π stacking interactions between indole ring of Trpꢀ352 and
the benzene ring of DCꢀS100 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. Hisꢀ297 is the key
residue forming two hydrogen bonds with SAM, but no polar interactions between DCꢀS100 and
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Hisꢀ297 exist. If the compound could form polar interactions with Gluꢀ356, disrupting polar
interactions between Gluꢀ356 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 Hisꢀ297 and Gluꢀ356, which in turn will enhance its activity against
SET7. This later proved to be a successful strategy.
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Taking feasibility and accessibility into consideration, we synthesized DCꢀS237 via the
Gewald reaction and tested its activity toward SET7. DCꢀS237 displayed activity that was
similar to DCꢀS100, with an IC value of 37.67 ꢁM (Figure 2D). In pursuit of molecular details,
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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 Gluꢀ228 disappeared, DCꢀS237
formed new polar interactions with Hisꢀ297 and Gluꢀ356. A predicted conformation alignment
revealed that both DCꢀS100 and DCꢀS237 share similar binding modes, though the direction of
the amide group in DCꢀS237 is altered (Figure 2C). The binding mode of DCꢀS237 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 DCꢀS238 and DCꢀS239 were synthesized, both of which displayed better inhibitory
activity, as expected. The IC values of DCꢀS238 and DCꢀS239 were determined to be 4.88 ꢁM
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and 4.59 ꢁM, respectively (Figure 3B).
Selectivity. We determined the selectivity of DCꢀS238 and DCꢀS239 for SET7 compared to 6
other enzymes (Table 2). The results showed that DCꢀS238 and DCꢀS239 exhibited a low
inhibition rate, below 45%, for DNMT1, DOT1L, EZH2, NSD1, SETD8 and G9a (Table 2).
Moreover, DCꢀS239 possesses a good selectivity profile over DNMT3A/3L at 100 ꢁM, with an
inhibition rate of ꢀ4.25 %. Therefore, DCꢀS238 and DCꢀS239 are selective inhibitors of SET7.
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Cell Activity. Because DCꢀS239 is a selective inhibitor of SET7, we assessed whether SET7
can show selective profiles in different cell lines. Therefore, the antiꢀproliferation activities of
compound DCꢀS239 against the MCF7, HL60, DHL4, MV4ꢀ11, and HCT116 cell lines were
determined by the MTT or alamarBlue assay. DCꢀS239 inhibited the proliferation of MCF7,
HL60 and MV4ꢀ11 cells in a doseꢀdependent manner while showing no cellular activity against
HCT116 and DHL4 cells (Figure 4). DCꢀS239 displayed IC values of 10.93 ꢁM in MCFꢀ7
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cells and 16.43 ꢁM in HL60 cells. Moreover, DCꢀS239 also displayed doseꢀdependent inhibition
activity toward MV4ꢀ11 cells. These inhibition profiles suggested that DCꢀS239 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 DCꢀS239, an analog series was synthesized, and the activities were tested against SET7
(Figure 3A). Comparing DCꢀS197, DCꢀS198, and DCꢀS199 with DCꢀS238, it can be concluded
that an amino group (R ) is essential for activity (Table 3). When the amino group is replaced
2
with another group, such as in DCꢀS197, DCꢀS198, and DCꢀS199, which are analogs from the
similarity search, no inhibitory activity was observed against SET7. Another finding is that a
bulkier terminal R group resulted in reduced activity (DCꢀS238 to DCꢀS244) from 94.71% to
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.53%. Activity gradually decreased when the R group was larger than the ethyl group; in
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particular, DCꢀS242, with the largest alkyl group tertꢀbutyl, 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
(DCꢀS249) or chlorine (DCꢀS257), a dramatic decrease in activity was observed. Other
substitutions did not contribute to activity. Thus, a nitro group at the metaꢀposition is
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indispensable. In summary, a nitro group at the metaꢀposition 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.
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To explore the molecular basis of inhibitory activity, we conducted docking studies, and the
results revealed that DCꢀS239 displays a similar binding mode as SAM, fitting closely into the
open and solventꢀaccessible binding pocket (Figure 5B). The nitro group is thought to form a
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hydrogen bond with Lysꢀ294, which may be involved in selectivity . Thus, substitution of the
nitro group disrupted hydrogen bonding with Lysꢀ294, and a loss in activity was observed. The
binding mode of DCꢀS239 (Figure 5A) suggested that the amino group is capable of forming
hydrogen bonds with Hisꢀ297 and Gluꢀ356, enhancing the binding affinity for SET7. When the
amino group was replaced with other groups, such as in compounds DCꢀS197, DCꢀS198 and
DCꢀS199, 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 Trpꢀ
352 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 DCꢀS239.
ꢀ
Conclusions
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6, 19, 20
9, 10, 21
SET7 plays a significant role in transcriptional regulation
, cell cycle control
,
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differentiation , DNA repair and DNMT1 stability
. SET7ꢀmediated methylation is also
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25, 26, 99, 100
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associated with diabetes
, cancers
, alopecia areata , atherosclerotic vascular
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disease , transcription of the HIV promoter and HCV replication . The controversial debate
regarding whether SET7 can regulate p53 highlights the need for specific and potent inhibitors.
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Notably, studies reporting the role of SET7 in the regulation of ERα , STAT3 , and NFꢀκB ,
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)ꢀPFIꢀ2 has been
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reported to be a potent selective SET7 inhibitor targeting the substrate binding site . 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
structureꢀbased virtual screening and chemical optimization. First, we employed pharmacophoreꢀ
based and dockingꢀbased virtual screening to mine potential candidate molecules. DCꢀS100 was
identified as a hit, with an IC50 value of 30.04 ꢁM. Subsequently, similarityꢀbased analog
searching was performed, and their activities were tested to explore the SAR of DCꢀS100. The
SAR of DCꢀS100 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 Hisꢀ297, Gluꢀ356 and DCꢀS100,
providing a direction for hit optimization. Considering the issues discussed above and feasibility,
DCꢀS237 was synthesized via the Gewald reaction. With respect to the binding mode, DCꢀS237
may be too large to fit well into the SAM binding pocket, which prompted us to synthesize DCꢀ
S238 and DCꢀS239 as well as their analogs. DCꢀS239 proved to be a potent SET7 inhibitor, with
an IC50 value of 4.59 ꢁM. Binding mode prediction revealed that DCꢀS239 shares a similar
binding moiety with SAM, and the key interactions are in accordance with the SAR analysis of
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DCꢀS239. Biological testing showed that DCꢀS239 displays selectivity for SET7 over DNMT1,
DNMT3A/3L, DOT1L, EZH2, NSD1, SETD8 and G9a, with inhibition ratio below 45.45%.
DCꢀS238 is also a potent SET7 inhibitor with an IC value of 4.88 ꢁM and possesses a good
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selectivity profile over DNMT1, DOT1L, EZH2, NSD1, SETD8 and G9a.
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According to the GeneCards platform
, SET7 is highly expressed in the breast cancer
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MCF7 cell line, and SET7 can modulate the stability of ERα via the methylation of K302 . ERα
is overꢀexpressed in approximately 70 % of breast cancer cases, and the blockade of ERα action
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has proved to be a successful strategy in these patients . 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 structureꢀbased drug virtual screenings to potent, selective SET7 inhibitors with novel
scaffolds using a structureꢀguided drug design approach. It also highlights the significance of
selecting a proper hit compound with low molecular weight and a structureꢀguided 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
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Lipinski’s Rule of Five , and ‘panꢀassay interference compounds’ (PAINS)
were removed
using Pipeline Pilot, version 7.5 (Pipeline Pilot; Accelrys Software Inc., San Diego, CA). The
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remaining 182,014 molecules were prepared with LigPrep to generate all stereoisomers and
different protonation states with Epik. The prepared Specs database was used to generate an
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indexed multiꢀconformer 3D database in Accelrys Discovery Studio 3.0 with the conformation
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method CAESAR .
Protein Preparation. Twentyꢀnine Xꢀray 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 domainꢀcontaining family. We obtained a maximum root meanꢀsquare derivation (RMSD)
value of 0.37, suggesting that the threeꢀdimensional 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 pharmacophoreꢀ and dockingꢀbased virtual
screening. The protein was prepared with Protein Preparation Wizard Workflow, as provided in
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Maestro , with a pH of 7.0 ± 2.0. Other parameters were set as the default.
Pharmacophore-Based Virtual Screening. The receptorꢀligand based pharmacophore model
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was generated with Accelrys Discovery Studio 3.0 using the Xꢀray structure in complexes with
SAM (PDB ID: 1N6A). The pharmacophore features were edited in combinations in Discovery
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Studio 3.0 in order to cover a border chemical space when matching the prepared multiꢀ
conformer 3D Specs database. We obtained 15,541 compounds after searching the 3D database
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with those pharmacophore models, and the 15,541 molecules were prepared using LigPrep for
further docking validation.
Docking-Based Virtual Screening. To rationalize our docking procedures, the enrichment
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factor (EF) was used to evaluate the performance of several docking programs, including
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Glide 5.5 , AutoDock Vina , LibDock in Discovery Studio and Dock 6 . 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 lysineꢀ294 may be involved in specific recognition of SAM by
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SET7 , we set lysineꢀ294 as a hydrogen bond constraint during grid generation and docking.
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Glide SP mode and Glide XP mode were subsequently used to dock the compound library
into the SAM binding site. The topꢀranked 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 DCꢀS100 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 DCꢀS100.
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 384ꢀ
well 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 ꢁL of substrate solution
was added to each well to start the reaction. The substrate solution was incubated in each well for
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0 min. Acceptor and donor beads (15 ꢁL) 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 %=( MaxꢀSignal)/ (MaxꢀMin)*100
Enzymatic Selectivity Assay. A radioactive methylation inhibition assay of DNMT1 was
performed in modified Tris buffer using poly (dIꢀDC) (Sigma, USA, Product No. P4929) as a
substrate. The enzyme solution was incubated at room temperature for 15 min before 10 ꢁL of
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substrate and 10 ꢁL of [ H]ꢀ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 preꢀincubated for 15 min with 0.5 % PEI, and residual solution was
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removed by vacuum. The plates were washed 3 times with ddH O under vacuum, and the counts
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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 IDTꢀ01 Sequence 1 and
IDTꢀ01 Sequence 2 as substrates in modified Tris buffer. The enzyme solution was incubated at
room temperature for 15 min before 10 ꢁL of substrate solution was added to each well to start
the reaction. After incubation at room temperature for 4 h, cold SAM solution (10 ꢁL) was added
to each well to stop the reaction. Subsequently, 25 ꢁL 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.
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H3K27me peptide and [ H]ꢀ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 ꢁL 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 ꢁL was added per well; 25 ꢁL 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 dH O and 0.1 % Tweenꢀ20, and the radioactivity was determined by
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liquid scintillation counting (MicroBeta, PerkinElmer). GSKꢀ126 was used as the reference
compound.
A radioactive methylation inhibition assay of NSD1 was performed inꢀmodified Tris buffer.
The enzyme solution of NSD1 (10 ꢁL; Biogenie, Cat. No. M1057) was transferred to an assay
plate and incubated at room temperature for 15 min. Subsequently, 10 ꢁL of substrate solution
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was added to each well, as was 10 ꢁL of [ H]ꢀSAM solution to start the reaction. After
incubation for 4 h, 15 ꢁL of cold SAM solution was added to each well to stop the reaction. The
reaction mixture (40 ꢁL) was transferred to GF/B plate (Millipore, Cat. No. MSFBN6B50) (preꢀ
treated with 0.5 % PEI for 15 min) using Platemate, and the plate was washed 3 times with
ddH O under vacuum. The radioactivity was determined by liquid scintillation counting
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(MicroBeta, PerkinElmer). Chaetocin was used as the reference compound.
The inhibition activities of DCꢀS238 and DCꢀS239 against DOT1L were determined using
AlphaLISA protocols. In each plate well, 5 ꢁL of compound or assay buffer were added and so
was 2.5 ꢁL of DOT1L enzyme. After incubation for 15 min at room temperature, 2.5 ꢁL of
oligonucleosomes/SAM mix was added to start the reaction; the plate was covered with TopSealꢀ
A film (PerkinElmer, NO. 6050195) and incubated at room temperature for 30 min. To stop the
reaction, 5 ꢁL of highꢀsalt buffer was added. A 5 × mixture of antiꢀHistone H3 Acceptor Beads
(
PerkinElmer, NO. AL147) and biotinylated antiꢀH3K79me2 antibody (PerkinElmer, NO.
AL148) was prepared at 50 ꢁg/mL and 0.5 nM in a 25 ꢁL total assay volume. Subsequently, 5
L of 5 × acceptor beads/biotinylated antibody mix was added; the samples were covered with
TopSealꢀA film and incubated for 60 min at room temperature. A 5× Streptavidin Donor Beads
PerkinElmer, NO. 6760002) sample at 50 ꢁg/mL was prepared in 1× Detection Buffer and
shielded from light (final concentration of 10 ꢁg/mL in 25 ꢁL total assay volume). Donor beads
ꢁ
(
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(
5 ꢁL) were added, shielded from light, and the sample was covered with TopSealꢀA 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
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compounds were first transferred to assay plate by Echo in 100% DMSO. Both peptide and [ H]ꢀ
SAM in 1x assay buffer (modified Tris Buffer) were added to each well to start the reaction
before the compounds and 10 ꢁL enzyme solution were incubated at room temperature for 15
minutes. And 10 ꢁL cold SAM in 1x assay buffer was added to each well to stop the reaction
after 120 min. 25 ꢁL 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 dH O +
2
0
.1% Tweenꢀ20 three times and read on Microbeta. SAH (Sigma, Cat. No. A9384ꢀ25MG) was
used as the reference compound.
The inhibition activities of DCꢀS238 and DCꢀS239 against G9a were determined using
AlphaLISA protocols. Both DCꢀS238 and DCꢀS239 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 ꢁL of the enzyme solution was incubated in assay plate in each well at
room temperature for 15 min. The 5 ꢁL 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 ꢁL 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, MV4ꢀ11, 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 % CO atmosphere. HL60, MV4ꢀ
2
11, and DHL4 cells were cultured in 1640 medium supplemented with 10 % fetal bovine serum.
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HCT116 cells were cultured in 5A medium. DCꢀS239 was dissolved in DMSO (Sigma) and then
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stored at 4 °C. Approximately 2*10 cells were seeded in 100 µL of medium in each well of a
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6ꢀwell flatꢀbottom plate and then grown in a CO incubator overnight. The cells were incubated
2
with DCꢀS239 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 DCꢀS239 against HL60, MV4ꢀ11, and DHL4 cells was measured by
108, 109
the alamarBlue assay
. 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
1
products were characterized by their NMR and MS spectra. H NMR spectra were recorded
1
3
using a 400 MHz Varian Mercury Plus 400 instrument, and 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). Lowꢀresolution mass spectra (LRMS) were measured using a Finnigan
LCQ/DECA spectrometer. Highꢀresolution mass spectra (HRMS) were measured using a
Micromass Ultra QꢀTOF spectrometer. Melting points (uncorrected) were determined using a
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SGWXꢀ4B micro melting point apparatus. All of the microwaveꢀassisted reactions were
TM
performed using an Anton Paar Monowave 300 monomode microwave reactor.
General Procedure for Preparation of Acetoacetanilide Derivatives B01 – B15.
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A mixture of ethyl acetoacetate (0.06 mol), an appropriate amine (0.03 mol), and a catalytic
amount of potassium tertꢀbutoxide 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)
+
1
B01 was prepared from 3ꢀnitroaniline (A01). LRMS (ESI) m/z 223 [M+H] ; H NMR (400
MHz, DMSOꢀd ) 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
6
Hz, 1H), 3.61 (s, 2H), 2.22 (s, 3H).
3-oxo-N-phenylbutanamide (B02)
+
1
B02 was prepared from aniline (A02). LRMS (ESI) m/z 178 [M+H] ; H NMR (400 MHz,
DMSOꢀd ) 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,
6
1H), 3.54 (s, 2H), 2.20 (s, 3H).
N-(4-methoxyphenyl)-3-oxobutanamide (B03)
+
1
B03 was prepared from 4ꢀmethoxyaniline (A03). LRMS (ESI) m/z 208 [M+H] ; H NMR (400
MHz, DMSOꢀd ) δ 9.94 (s, 1H), 7.55 – 7.39 (m, 2H), 6.97 – 6.81 (m, 2H), 3.71 (s, 3H), 3.50 (s,
6
2H), 2.20 (s, 3H).
N-(4-bromophenyl)-3-oxobutanamide (B04)
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B04 was prepared from 4ꢀbromoaniline (A04). LRMS (ESI) m/z 256 [M+H] ; H NMR (400
MHz, DMSOꢀd ) δ 10.21 (s, 1H), 7.57 – 7.52 (m, 2H), 7.51 – 7.46 (m, 2H), 3.55 (s, 2H), 2.20 (s,
6
3H).
N-(2-fluorophenyl)-3-oxobutanamide (B05)
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+
1
B05 was prepared from 2ꢀfluoroaniline (A05). LRMS (ESI) m/z 196 [M+H] ; H NMR (400
MHz, DMSOꢀd ) δ 9.91 (s, 1H), 8.00 – 7.92 (m, 1H), 7.30 – 7.22 (m, 1H), 7.20 – 7.09 (m, 2H),
6
3.64 (s, 2H), 2.20 (s, 3H).
N-(3-methoxyphenyl)-3-oxobutanamide (B06)
+
1
B06 was prepared from 3ꢀmethoxyaniline (A06). LRMS (ESI) m/z 208 [M+H] ; H NMR (400
MHz, DMSOꢀd ) 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
6
(
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)
+
1
B07 was prepared from 4ꢀaminobenzamide (A07). LRMS (ESI) m/z 221 [M+H] ; H NMR (400
MHz, DMSOꢀd ) 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),
6
7.26 (s, 1H), 3.58 (s, 2H), 2.21 (s, 3H).
N-(4-chlorophenyl)-3-oxobutanamide (B08)
+
1
B08 was prepared from 4ꢀchloroaniline (A08). LRMS (ESI) m/z 212 [M+H] ; H NMR (400
MHz, DMSOꢀd ) δ 10.21 (s, 1H), 7.65 – 7.52 (m, 2H), 7.42 – 7.27 (m, 2H), 3.55 (s, 2H), 2.20 (s,
6
3H).
N-(2-chlorophenyl)-3-oxobutanamide (B09)
+
1
B09 was prepared from 2ꢀchloroaniline (A09). LRMS (ESI) m/z 212 [M+H] ; H 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 2,5ꢀdichloroaniline (A10). LRMS (ESI) m/z 246 [M+H] ; H NMR (400
MHz, DMSOꢀd ) 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 =
6
8.6, 2.5 Hz, 1H), 3.71 (s, 2H), 2.21 (s, 3H).
N-(4-chloro-2,5-dimethoxyphenyl)-3-oxobutanamide (B11)
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B11 was prepared from 4ꢀchloroꢀ2,5ꢀdimethoxyaniline (A11). LRMS (ESI) m/z 272 [M+H] ; H
NMR (400 MHz, DMSOꢀd ) δ 9.58 (s, 1H), 8.03 (s, 1H), 7.14 (s, 1H), 3.81 (s, 3H), 3.75 (s, 3H),
6
3.69 (s, 2H), 2.18 (s, 3H).
N-(2-methoxyphenyl)-3-oxobutanamide (B12)
+
1
B12 was prepared from 2ꢀmethoxyaniline (A12). LRMS (ESI) m/z 208 [M+H] ; H NMR (400
MHz, DMSOꢀd ) δ 9.47 (s, 1H), 8.06 – 7.95 (m, 1H), 7.11 – 6.99 (m, 2H), 6.94 – 6.83 (m, 1H),
6
3.83 (s, 3H), 3.66 (s, 2H), 2.18 (s, 3H).
N-(4-fluorophenyl)-3-oxobutanamide (B13)
+
1
B13 was prepared from 4ꢀfluoroaniline (A13). LRMS (ESI) m/z 196 [M+H] ; H NMR (400
MHz, DMSOꢀd ) 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
6
(s, 3H).
N-(3-chlorophenyl)-3-oxobutanamide (B14)
+
1
B14 was prepared from 3ꢀchloroaniline (A14). LRMS (ESI) m/z 212 [M+H] ; H NMR (400
MHz, DMSOꢀd ) δ 10.27 (s, 1H), 7.81 (t, J = 2.0 Hz, 1H), 7.46 – 7.24 (m, 2H), 7.12 (ddd, J =
6
7.8, 2.1, 1.2 Hz, 1H), 3.56 (s, 2H), 2.21 (s, 3H).
N-(3-cyanophenyl)-3-oxobutanamide (B15)
+
1
B15 was prepared from 3ꢀaminobenzonitrile (A15). LRMS (ESI) m/z 203 [M+H] ; H NMR
400 MHz, DMSOꢀd ) δ 10.43 (s, 1H), 8.08 (d, J = 1.0 Hz, 1H), 7.79 – 7.72 (m, 1H), 7.57 – 7.49
(
6
(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 DCꢀ
S237, methyl cyanoacetate for DCꢀS238, 2ꢀcyanoacetamide for DCꢀS240, isopropyl
cyanoacetate for DCꢀS241, tertꢀbutyl cyanoacetate for DCꢀS242, cyclohexyl cyanoacetate for
DCꢀS243, and benzyl cyanoacetate for DCꢀS244) (0.005 mol) and elemental sulfur (0.005 mol)
in ethanol (isobutanol for DCꢀS237, methanol for DCꢀS238, isopropanol for DCꢀS241,
cyclohexanol for DCꢀS243, and benzyl alcohol for DCꢀS244) (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 DCꢀS249 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 DCꢀS243 and DCꢀS244, 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 DCꢀS243 (purity 93.2%) by HPLC analysis [HPLC: column, Agilent XDBꢀC18, 150
× 4.6 mm, 5 ꢁm; solvent system, MeOH‒1 ‰ DEA and water with isocratic elution (68%
organic phase for DCꢀS243, DCꢀS253, and DCꢀS254; 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)
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DC-S237 was prepared from Nꢀ(3ꢀnitrophenyl)ꢀ3ꢀoxobutanamide (B01) and isobutyl
cyanoacetate following the general procedure indicated above. Yield, 62 %; mp 190 – 191 °C;
1
H NMR (400 MHz, DMSOꢀd ) δ 10.15 (s, 1H), 8.62 (t, J = 2.1 Hz, 1H), 8.00 (dd, J = 8.2, 1.2
6
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),
13
2
.05 – 1.95 (m, 1H), 0.96 (d, J = 6.7 Hz, 6H); C NMR (125 MHz, DMSOꢀd ) δ 165.75, 165.22,
6
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 C H O N S [M+H]
17
20
5
3
378.1118, found 378.1126.
Methyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DC-S238)
DC-S238 was prepared from Nꢀ(3ꢀnitrophenyl)ꢀ3ꢀoxobutanamide (B01) and methyl cyanoacetate
1
following the general procedure indicated above. Yield, 59 %; mp 238 – 239 °C; H NMR (400
MHz, DMSOꢀd ) 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),
6
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
13
=
4.5 Hz, 3H); C NMR (125 MHz, DMSOꢀd ) δ 165.35, 165.12, 161.77, 147.89, 142.32,
6
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 C H O N S [M+H] 336.0649, found 336.0648.
1
4
14
5
3
Ethyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DC-S239)
DC-S239 was prepared from Nꢀ(3ꢀnitrophenyl)ꢀ3ꢀoxobutanamide (B01) and ethyl cyanoacetate
1
following the general procedure indicated above. Yield, 78 %; mp 202 – 204 °C; H NMR (400
MHz, DMSOꢀd ) 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),
6
7
2
1
.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,
13
H), 2.52 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); C NMR (125 MHz, DMSOꢀd ) δ 165.42, 164.87,
6
61.80, 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 C H O N S [M+H] 350.0805,
1
5
16
5
3
found 350.0811.
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-Amino-3-methyl-N -(3-nitrophenyl)thiophene-2,4-dicarboxamide (DC-S240)
DC-S240 was prepared from Nꢀ(3ꢀnitrophenyl)ꢀ3ꢀoxobutanamide (B01) and 2ꢀcyanoacetamide
1
following the general procedure indicated above. Yield, 63 %; mp 244 – 245 °C; H NMR (400
MHz, DMSOꢀd ) 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,
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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38
39
40
41
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47
48
49
50
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52
53
54
55
56
57
58
59
60
13
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); C NMR (100
MHz, DMSOꢀd ) δ 167.79, 162.41, 161.77, 148.32, 142.12, 141.08, 130.31, 126.58, 117.95,
6
+
114.64, 112.94, 110.60, 16.38; LRMS (ESI) m/z 321 [M+H] ; HRMS (ESI) m/z calcd
+
C H O N S [M+H] 321.0652, found 321.0650.
13 13
4
4
Isopropyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DC-
S241)
DC-S241 was prepared from Nꢀ(3ꢀnitrophenyl)ꢀ3ꢀoxobutanamide (B01) and isopropyl
cyanoacetate following the general procedure indicated above. Yield, 81 %; mp 197 – 199 °C;
1
H NMR (400 MHz, DMSOꢀd ) δ 10.14 (s, 1H), 8.63 (t, J = 2.1 Hz, 1H), 8.05 – 7.97 (m, 1H),
6
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),
1
3
2.51 (s, 3H), 1.29 (d, J = 6.3 Hz, 6H); C NMR (125 MHz, DMSOꢀd ) δ 165.47, 164.58, 161.82,
6
1
47.89, 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 C H O N S [M+H] 364.0962, found
16
18
5
3
364.0968.
tert-Butyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DC-
S242)
DC-S242 was prepared from Nꢀ(3ꢀnitrophenyl)ꢀ3ꢀoxobutanamide (B01) and tertꢀbutyl
cyanoacetate following the general procedure indicated above. Yield, 63 %; mp 196 – 197 °C;
1
H NMR (400 MHz, DMSOꢀd ) δ 10.12 (s, 1H), 8.63 (t, J = 2.0 Hz, 1H), 8.00 (dd, J = 8.2, 1.1
6
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
1
3
(
s, 9H); C NMR (125 MHz, DMSOꢀd ) δ 165.30, 164.71, 161.87, 147.89, 141.77, 140.41,
6
1
29.97, 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 C H O N S [M+H] 378.1118, found 378.1128.
17 20 5 3
Cyclohexyl 2-amino-4-methyl-5-((3-nitrophenyl)carbamoyl)thiophene-3-carboxylate (DC-
S243)
26
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Journal of Medicinal Chemistry
Page 28 of 65
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DC-S243 was prepared from Nꢀ(3ꢀnitrophenyl)ꢀ3ꢀoxobutanamide (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.
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Yield, 52 %; mp 189 – 191 °C; H NMR (400 MHz, DMSOꢀd ) δ 10.12 (s, 1H), 8.61 (s, 1H),
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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
1
3
Hz, 3H), 1.44 – 1.28 (m, 3H); C NMR (125 MHz, DMSOꢀd ) δ 165.62, 164.57, 161.82, 147.90,
6
141.65, 140.38, 129.98, 125.97, 117.73, 114.07, 111.85, 105.93, 71.25, 65.00, 44.23, 31.13,
+
2
4.97, 23.12, 16.93; LRMS (ESI) m/z 404 [M+H] ; HRMS (ESI) m/z calcd C H O N S
1
9
22
5
3
+
[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 Nꢀ(3ꢀnitrophenyl)ꢀ3ꢀoxobutanamide (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.
1
Yield, 33 %; mp 205 – 207 °C; H NMR (400 MHz, DMSOꢀd ) δ 10.12 (s, 1H), 9.11 – 6.78 (m,
6
13
1
1H), 5.29 (s, 2H), 2.50 (s, 3H); C NMR (125 MHz, DMSOꢀd ) δ 165.86, 164.64, 161.76,
6
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 C H O N S [M+H]
20
18
5
3
412.0962, found 412.0953.
Ethyl 2-amino-4-methyl-5-(phenylcarbamoyl)thiophene-3-carboxylate (DC-S245)
DC-S245 was prepared from 3ꢀoxoꢀNꢀphenylbutanamide (B02) and ethyl cyanoacetate following
1
the general procedure indicated above. Yield, 65 %; mp 178 – 179 °C; H NMR (400 MHz,
DMSOꢀd ) 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 =
6
1
3
7
.4 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 2.49 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); C NMR (125 MHz,
DMSOꢀd ) δ 165.11, 164.95, 161.42, 140.29, 139.14, 128.59, 123.32, 120.05, 112.96, 105.40,
6
+
59.31, 16.58, 14.33; LRMS (ESI) m/z 305 [M+H] ; HRMS (ESI) m/z calcd C H O N S
15
17
3
2
+
[
M+H] 305.0954, found 305.0948.
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Page 29 of 65
Journal of Medicinal Chemistry
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Ethyl 2-amino-5-((4-methoxyphenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DC-
S246)
DC-S246 was prepared from Nꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀoxobutanamide (B03) and ethyl
cyanoacetate following the general procedure indicated above. Yield, 49 %; mp 160 – 161 °C;
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H NMR (400 MHz, DMSOꢀd ) δ 9.55 (s, 1H), 7.72 (s, 2H), 7.56 – 7.46 (m, 2H), 6.91 – 6.84 (m,
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1
3
2
H), 4.22 (q, J = 7.1 Hz, 2H), 3.72 (s, 3H), 2.48 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); C NMR (125
MHz, DMSOꢀd ) δ 164.97, 161.11, 155.37, 139.82, 132.19, 121.74, 113.71, 113.08, 105.33,
6
+
5
9.29, 55.17, 16.55, 14.33; LRMS (ESI) m/z 335 [M+H] ; HRMS (ESI) m/z calcd C H O N S
16 19
4
2
+
[
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 Nꢀ(4ꢀbromophenyl)ꢀ3ꢀoxobutanamide (B04) and ethyl cyanoacetate
1
following the general procedure indicated above. Yield, 73 %; mp 170 – 171 °C; H NMR (400
MHz, DMSOꢀd ) δ 9.81 (s, 1H), 7.77 (s, 2H), 7.63 – 7.54 (m, 2H), 7.52 – 7.44 (m, 2H), 4.23 (q,
6
1
3
J = 7.1 Hz, 2H), 2.49 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H); C NMR (125 MHz, DMSOꢀd ) δ 165.21,
6
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 C H O N BrS [M+H] 383.0060, found
15 16
3
2
3
83.0063.
Ethyl 2-amino-5-((2-fluorophenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DC-S248)
DC-S248 was prepared from Nꢀ(2ꢀfluorophenyl)ꢀ3ꢀoxobutanamide (B05) and ethyl cyanoacetate
1
following the general procedure indicated above. Yield, 55 %; mp 140 – 141 °C; H NMR (400
MHz, DMSOꢀd ) 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),
6
1
3
4
.23 (q, J = 7.1 Hz, 2H), 2.54 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); C NMR (125 MHz, DMSOꢀd )
6
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,
26.03, 124.26 (d, J = 3.3 Hz), 115.66 (d, J = 19.8 Hz), 111.99, 105.68, 59.36, 16.42, 14.31;
1
+
+
LRMS (ESI) m/z 323 [M+H] ; HRMS (ESI) m/z calcd C H O N FS [M+H] 323.0860, found
1
5
16
3
2
3
23.0865.
Ethyl 2-amino-5-((3-methoxyphenyl)carbamoyl)-4-methylthiophene-3-carboxylate (DC-
S249)
28
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