Potent, Selective, and Cell Active Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor Developed by Structure-Based Virtual Screening and Hit Optimization

Article abstract of DOI:10.1021/acs.jmedchem.7b00587

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

Full text of DOI:10.1021/acs.jmedchem.7b00587

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Article
A Potent, Selective and Cell Active Protein Arginine
Methyltransferase 5 (PRMT5) Inhibitor Developed by
Structure-based Virtual Screening and Hit Optimization
Ruifeng Mao, Jingwei Shao, Kongkai Zhu, Yuanyuan Zhang, Hong Ding, Chenhua
Zhang, Zhe Shi, Hualiang Jiang, De-Qun Sun, Wenhu Duan, and Cheng Luo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00587 • Publication Date (Web): 26 Jun 2017
Downloaded from http://pubs.acs.org on June 26, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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A Potent, Selective and Cell Active Protein Arginine
Methyltransferase 5 (PRMT5) Inhibitor Developed by
Structureꢀbased Virtual Screening and Hit
Optimization
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$
,†,|
§,‡,|
#,†,|
†
†
Ruifeng Mao , Jingwei Shao , Kongkai Zhu , Yuanyuan Zhang , Hong Ding ,
¶
¶
†
$,*
§,*
Chenhua Zhang , Zhe Shi , Hualiang Jiang , Dequn Sun , Wenhu Duan and Cheng
†, *
±
Luo
$
Marine College, Shandong University, Weihai 264209, P.R. China;
†
Drug Discovery and Design Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai
201203, China;
±
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China
§
Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences (CAS), Shanghai 201203, China;
‡
University of Chinese Academy of Sciences, Beijing 100049, China;
#
School of Biological Science and Technology, University of Jinan, Jinan 250022, P.R.
China;
¶
Shanghai ChemPartner Co., LTD., Zhangjiang HiꢀTech Park, Shanghai 201203,
China
|
These authors contributed equally.
*Correspondence: Cheng Luo, Eꢀmail: cluo@simm.ac.cn or Dequn Sun, Email:
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dequn.sun@sdu.edu.cn; or Wenhu Duan, Email: whduan@simm.ac.cn
Abstract PRMT5 plays important roles in diverse cellular processes and is
upregulated in several human malignancies. Besides, PRMT5 has been validated as
antiꢀcancer target in mantle cell lymphoma. In this study, we found a potent and
selective PRMT5 inhibitor by performing structureꢀbased virtual screening and hit
optimization. The identified compound 17 (IC50 = 0.33 ꢁM) exhibited a broad
selectivity against a panel of other methyltransferases. The direct binding of 17 to
PRMT5 was validated by surface plasmon resonance experiments, with a Kd of 0.987
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ꢁM. Kinetic experiments indicated that 17 was a SAM competitive inhibitor other
than substrate. In addition, 17 showed selective antiproliferative effects against
MV4ꢀ11 cells, and further studies indicated that the mechanism of cellular antitumor
activity was due to inhibition of PRMT5 mediated SmD3 methylation. 17 may
represent a promising lead compound to understand more about PRMT5 and
potentially assist the development of treatments for leukemia indications.
Keywords:
PRMT5 inhibitor, virtual screening, anticancer target
Introduction
Acting as a bridge between the genotype and the phenotype, postꢀtranslational
modification of proteins plays a pivotal role in a variety of biological functions.
Postꢀtranslational methylation of arginine residues catalyzed by protein arginine
methyltransferases (PRMTs) is very important as it alters the activity and interactions
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of substrate proteins, with crucial influences on diverse cellular processes, including
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ꢀ3
cell growth, differentiation, proliferation and development.
Up to now, nine
mammalian PRMTs have been identified and are classified into three types (type IꢀIII)
based on the capacity of transferring one or two methyl groups to the nitrogen atoms
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of the guanidinium side chains within arginine residues using Sꢀadenosylmethionine
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(
SAM) as the methyl donor. Type I PRMT enzymes (PRMT1, ꢀ2, ꢀ3, ꢀ4, ꢀ6, and ꢀ8)
G
G
G
catalyze the formation of ωꢀN monomethyl and asymmetric ωꢀN , N ꢀdimethyl
arginine residues; Type II enzymes (PRMT5 and PRMT9) catalyze the formation of
G
G
G
ωꢀN monomethyl and ωꢀN , N’ ꢀsymmetric dimethyl arginine residues; Type III
G
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enzymes (PRMT7) catalyze the formation of ωꢀN monomethyl arginine only.
Recently, cancer epigenetic studies uncovered the dysregulation of histone
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modification as the driving force for tumorigenesis and cancer progression. Therefore,
the development of potent and selective epigenetic modulators is currently a hot topic
in the field of anticancer drug discovery.
PRMT5 is the predominant type II PRMT and in association with the
WDꢀrepeat–containing protein MEP50 (methylosome protein 50) forms large
multimolecular complexes with different binding partners. PRMT5 could specifically
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methylate a wide spectrum of substrates, including histone and nonꢀhistone
substrates (Table S1), of which the histone substrates are histones H2A residue Arg3
(H2AR3), H4 residue Arg3 (H4R3) and H3 residues Arg2 (H3R2) Arg8 (H3R8).
Additionally, PRMT5 can methylate proteins involved in RNA splicing. Small nuclear
ribonucleoproteins (snRNPs) are core integral components of the spliceosome. Sm
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proteins, one of which is SmD3, have the symmetric dimethyl arginine
(sDMA)ꢀmodified activity. And they are necessary to assemble the snRNP in human
cells. SnRNP assembly can be regulated by interaction of methylated Sm proteins
with the survival motor neuron (SMN) complex. Both PRMT5 and PRMT7 are
required to catalyze cytoplasmic snRNP assembly. And both enzymes can facilitate
Sm proteins binding to the SMN complex, respectively. Meanwhile, knockdown of
PRMT5 could decrease sDMA modification of Sm proteins and reduce snRNP
assembly. Therefore, we can use modification of SmD3 to explore the cellular
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biochemical activity of PRMT5. Antiꢀbodies to symmetric dimethyl arginine (sDMA)
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can be used to probe the cellular bioꢀchemical activity of PRMT5.
PRMT5 is
postulated to have a relationship with cell growth, cell cycle progression, cell death
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and cell proliferation. Several reports stress the importance of PRMT5 in
tumorigenesis, but the mechanism of PRMT5 driving tumorigenesis is still unknown.
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PRMT5 is upregulated in lymphomas,
breast cancer, lung cancer, colorectal
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cancer and glioblastoma. In addition, PRMT5 was validated as anticancer target in
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mantle cell lymphoma (MCL) and glioblastoma.
Although multiple efforts have been made to develop PRMT5 inhibitors, there are
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only three types of PRMT5 inhibitors reported (Chart 1). Compound 1 (EPZ015666)
occupies histone substrate pocket and shows doseꢀdependent antitumor activity in
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multiple MCL xenograft models. Compound 2 (CMP5) is discovered by virtual
screening methods and shows promise as a novel therapeutic approach for Bꢀcell
lymphomas. Compound 3ꢀ5 are analogues of SAM and all exhibit excellent PRTM5
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inhibitory activity.
Considering the importance of PRMT5 in tumorigenesis, it is
badly in need of identifying more potent and selective PRMT5 inhibitors to provide
powerful tool in efforts to explore and potentially further validate PRMT5 as a
clinically relevant target.
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Chart 1. Structures of PRMT5 inhibitors
By performing pharmacophoreꢀ and molecule dockingꢀ based virtual screening
combined with Alpha LISA assay, we found 6 PRMT5 inhibitors, which were
confirmed in radioactive methylation assays resulting in the identification the new
scaffold 6 (DC_P04) (IC50 = 24 ꢁM). Chemical optimization of compound 6 resulted
in compound 17 (IC = 0.33 ꢁM) which showed good selectivity against a panel of
5
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other methyltransferases. The direct binding of 17 to PRMT5 was validated by SPR
experiments, and 17 showed competitive inhibition with respect to SAM on PRMT5
and showed noncompetitive inhibition with respect to peptide on PRMT5 indicated by
kinetic experiments. The antiꢀproliferative effects of 17 against leukemia and
lymphoma cells were tested and the results indicated that 17 had selective
antiꢀproliferative effects in MV4ꢀ11 cells. Biochemical inhibition correlated well with
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corresponding decreased levels of symmetrically dimethylated PRMT5 substrates and
proliferation in a timeꢀ and concentrationꢀdependent manner suggesting that the
antiꢀproliferative effects are a direct consequence of PRMT5 inhibition.
Results and Discussion
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116 candidates were selected by virtual screening.
Structureꢀbased virtual screening is an effective method in hit compound discovery
24ꢀ33
and has been widely used in the process of lead compound discovery.
In this study,
we used pharmacophoreꢀ and molecular dockingꢀ based virtual screening to screen
drugꢀlike SPECS database (http://www.specs.net), which contains about 207,342
compounds, for compounds that could inhibit PRMT5 activity (see Figure 1A for the
screening workflow). Based on the human crystal structure of PRMT5 [Protein Data
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Bank (PDB) ID code 4GQB], we created a pharmacophore model (Figure S1) using
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Discovery Studio 3.0. Then the pharmacophore model was used to screen the
SPECS 3D database which was generated by Generate Conformations with BEST
method inserted in Discovery Studio 3.0, and a total of 4,344 small molecules
matched the pharmacophore were obtained. Next, the crystal structure (4GQB) was
used as the reference structure for molecular docking based virtual screening.
Residues located within 20 Å of SAH analog (compound 5) was defined as the
binding site, then 4,344 small molecules were docked into binding site of PRMT5
with extraꢀprecision (XP) docking mode. The topꢀ866 small molecules ranked based
on the docking score were clustered into 50 clusters using Clustering Molecules
protocols integrated in Pipeline Pilot, version 7.5 (Pipeline Pilot; Accelrys Software
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Inc., San Diego, CA) for visual inspection, with the selection criteria as following: (1)
The binding poses of molecule occupied the SAM pocket and were reasonable with
no high strain energy; (2) In each clustered group, at least one molecule was selected
to retain molecular diversity; (3) For those molecules with the same scaffold, the
relatively simple structures were selected. At last, 116 candidates were selected and
purchased from SPECS.
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Figure 1. (A) Workflow of the virtual screening strategy adopted in the present study.
(B) Structures and inhibitory activities of 6 active compounds against PRMT5.
Alpha LISA and radioactive methylation assays were used to determine the
inhibition activity against PRMT5.
In order to test the inhibition activity of 116 compounds, Alpha LISA assay was
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carried out. We first tested the inhibition against PRMT5 at 100 ꢁM, and found that
among the 116 tested compounds, 15 compounds showed an inhibition larger than 50%
(Table S2), then IC50 values of these 15 compounds were determined (Table S3). The
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enzymeꢀinhibition results indicated that 6 compounds showed microꢀmolar IC lower
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than 30 ꢁM, with compound 6 showing the most active inhibition with an IC50 of 8.1
M (Figure 1B). Then 4 selected compounds were subjected to radioactive
methylation assay to further verify their enzyme (PRMT5) inhibition, and this result
Table 1) confirmed compound 6 was the most active compounds against PRMT5.
ꢁ
(
Table 1. IC50 values against PRMT5 of the hits derived from virtual screening
measured by radioactive methylation assays.
Compound Number
IC (ꢁΜ)
50
6
7
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24
169
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>200
Binding mode prediction of compound 6 provides clues for hit optimization.
To explore the molecular basis of inhibitory activity of compound 6 against PRMT5,
as shown in Figure 2A, we used the same XP docking mode that was previously used
to do the virtual screening to predict the binding mode of compound 6. The results
revealed that compound 6 showed the similar binding mode with SAH analog, fitting
well in the cofactor SAM binding pocket (Figure 2B). Residues involved in the
interaction between compound 6 and PRMT5 were identified, hydrogen bond and
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hydrophobic interaction network were shown in Figure 2C and Figure 2D respectively.
As shown in Figure 2C, the oxygen atom within the carbonyl of ester group forms
hydrogen bond with L315 residues, so this ester group should be retained when
chemistry modification were performed. In the next step, hit optimization and SAR
studies will be carried out to improve the inhibition activity of compound 6 against
PRMT5.
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Figure 2. Binding mode prediction of compound 6. (A) Overall view of compound 6
binding to PRMT5, PRMT5 is shown as cartoon, and compound 6 is shown in stick
representation. (B) Compound 6 aligns with SAH analog (compound 5), the green
stick representation is compound 6 and the magenta representation is compound 5. (C)
Closeꢀup views of the hydrogen bonds interactions between compound 6 and PRMT5,
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compound 6 and interacting residues of PRMT5 are shown in green and magenta stick
representation respectively. (D) Closeꢀup views of the hydrogen bonds and
hydrophobic interactions between compound 6 and PRMT5, compound 6 is shown in
green stick representation, while the hydrogen bonds interacting residues and
hydrophobic interactions residues of PRMT5 are shown in magenta and yellow stick
representation respectively. The 4GQB (PDB code) was used to simulate the binding
mode.
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Chemical synthesis
To discuss the SAR and improve the potency of compound 6, 31 compounds were
synthesized. The PRMT5 inhibition activity was determined by radioactive
methylation assay and compound 1 (IC50 = 0.047 μM) were used as the reference
control, see Figure S2 for the IC50 results of the reference compounds. Synthetic
routes for 31 compounds were outlined in Scheme 1ꢀ2. The synthesis started from
appropriate
available or prepared according to a published procedure.
of nitro group afforded diamine, which reacted with carbon disulphide in presence of
oꢀnitroaniline or nitropyridine derivatives, which were commercially
36, 37
Catalytic hydrogenation
38
potassium hydroxide using ethanol as solvent to give the intermediates. Subsequent
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alkylation of the thio group with alkyl chlorides, which by reaction of substituted
anilines or aminopyridine with chloroacetyl chloride in acetone, yielded titled
compounds.
a
Scheme 1
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O
O
(a)
R1
R1
R1
HN
O
H
N
NO2
NH2
a
b
N
S
S
N
H
N
H
R2
R2
R2
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3a: R1 = Cl, R2 = H
3b: R1 = H, R2 = Me
3c: R1 = H, R2 = OMe
44a: R1 = Cl, R2 = H
44b: R1 = Me, R2 = H
44c: R1 = OMe, R2 = H
12: R1 = Cl, R2 = H
13: R1 = Me, R2 = H
14: R1 = OMe, R2 = H
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O
O
(
b)
HN
O
R3
R3
X
H
R3
X
NH2
NO2
N
X
N
a
b
S
S
Y
Y
Y
N
H
N
H
5a: R3 = Me, X = C, Y = C
5b: R3 = F, X = C, Y = C
5c: R3 = Cl, X = C, Y = C
3
46a: R3 = Me, X = C, Y = C
46b: R3 = F, X = C, Y = C
46c: R3 = Cl, X = C, Y = C
15: R3 = Me, X = C, Y = C
21: R3 = F, X = C, Y = C
22: R3 = Cl, X = C, Y = C
23: R3 = Br, X = C, Y = C
16a: R3 = OTBDMS, X = C, Y = C
16: R3 = OH, X = C, Y = C
4
4
4
4
5d: R = OMe, X = C, Y = C
46d: R = Br, X = C , Y = C
3
45e: R3 = OMe, X = N,Y = C
5f: R3 = H, X = C, Y = N
46e: R3 = OH, X = C,Y = C
e
d
4
46f: R3 = OTBDMS, X = C,Y = C
3
3
17: R = OMe, X = C, Y = C
4
4
4
4
6g: R = OMe, X = C, Y = C
3
3
24: R = NO2, X = C, Y = C
6h: R = NO2, X = C, Y = C
f
NH2
NO2
3
6i: R3 = OMe, X = N,Y = C
25: R = NH2, X = C,Y = C
6j: R3 = H, X = C, Y = N
28: R3
= OMe, X = N, Y = C
9: R3 = H, X = C,Y = N
R3
2
4
5g: R3 = Br
5h: R3 = OH
O
O
HN
O
4
(c)
R4
NH2
NO2
R4
H
R4
F
NH2
NO2
c
a
N
b
N
S
S
N
H
N
H
45b
7a: R4 = OEt
48a: R4 = OEt
48b: R4 = OPr
48c: R4 = O Pr
48d: R4 = NHMe
48e: R4 = NMe2
4
4
18: R = OEt
47b: R4 = OPr
19: R = OPr
4
7c: R4 = O Pr
i
i
20: R = O Pr
4
i
4
4
4
7d: R4 = NHMe
26: R4 = NHMe
7e: R4 = NMe2
27: R4 = NMe2
a
Reagents and conditions: (a) (i) H
2
, 10% Pd/C, MeOH, rt; (ii) CS
2
, KOH, EtOH/H
2
O, 70 °C; (b)
2 3
50h, K CO , acetone, rt; (c) for 47a, EtONa, EtOH, 80 °C; for 47b, KOH, PrOH, 60°C; for 47c,
NaOH, iꢀPrOH, 80 °C; for 47d, methylammonium chloride, DIPEA, NMP, 100 °C; for 47e,
dimethylamine, DIPEA, NMP, 100 °C; (d) TBDMSCl, imidazole, DMF, 0 °C to rt; (e)
2
tetrabutylammonium fluoride, THF, rt.; (f) H , 10% Pd/C, MeOH, rt.
a
Scheme 2
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1
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2
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2
2
2
2
2
2
2
3
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3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
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5
6
7
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9
0
1
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5
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7
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9
0
1
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9
0
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3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
2 3
Reagents and conditions: (a) chloroacetyl chloride, K CO , acetone, rt; or chloroacetyl chloride,
3 2 3
Et N, DCM, 0 °C to rt; (b) 46g, K CO , acetone, rt.
SAR exploration
The SAR investigation was commenced with the benzimidazole core. As shown in
Table 2, substitution at the 4ꢀposition of benzimidazole core (compound 12ꢀ14) led to
a significant loss of PRMT5 enzyme activity. The 5ꢀposition substituted 22, 15 and 17
displayed more than 100ꢀfold improvement of enzymatic potency compared with the
4
ꢀposition derivatives 12, 13, and 14, respectively. Encouraged by IC50 of 0.33 µM of
the 5ꢀMethoxy analogue (17), a series of 5ꢀposition substituted compound (16, 18ꢀ21,
3ꢀ27) was synthesized to explore the SAR, and all the assayed compounds were less
2
potent than 17 except for 27 which showed comparable potency to 17. Replacement
of the methoxy with isopropoxy (17 vs 20) led to 8ꢀfold loss of potency, suggesting
that it could not accommodate bulky substitution. The dimethylamino derivative 27
exhibited a more than 100ꢀfold improvement compared with its amino analogue 25
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indicating that the methyl contributed remarkably to the enzymatic activity. The
PRMT5 enzymatic inhibition was enhanced as the atomic radius increased in the
halogen compounds 21ꢀ23. Incorporating a nitrogen atom to the benzimidazole ring
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
(
28 and 29) led to decreased activity. These results revealed that the 5ꢀmethoxy
substituted benzimidazole moiety was optimal for PRMT5 activity, and the inhibition
activity curves of selected compounds were shown in Figure 3.
Table 2. Structures and IC values for 18 synthesized analogues of compound 6.
50
Compounds
6
X
Y
4
H
5
H
IC50(ꢁM)
8.1 0.85
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
±
12
13
14
15
16
Cl
Me
OMe
H
H
>100
>100
>100
H
H
Me
OH
OMe
OEt
OPr
1.4
±
0.07
H
>100
17
18
19
H
0.33
±
±
±
0.06
H
0.35
0.53
0.07
0.08
H
iꢀ
20
21
22
23
24
H
O Pr
2.8
3.3
±
1.43
4.60
H
F
±
H
Cl
Br
0.50
0.34
0.83
±
0.42
0.11
0.31
H
±
±
H
NO
2
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3
4
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4
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
2
2
5
6
7
8
CH
CH
CH
N
CH
CH
CH
CH
N
H
H
NH
NHMe
NMe
2
40±7.07
1.6±0.78
0.33±0.18
8.4±0.35
87±2.12
H
2
H
OMe
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29
CH
CH
Figure 3. IC50 values against PRMT5 of the selected compounds.
Next we explored the SAR of the benzene ring moiety (Table 3). In the docking
analysis of our hit compound 6 and PRMT5 enzymatic domain, a hydrogen bond
formed between the carbonyl oxygen in methoxycarbonyl group and the Leu315
residue. To probe the critical Hꢀbond, derivatives 30ꢀ34 without Hꢀbond acceptor
were designed and synthesized, and the significant loss of enzymatic potency of 30ꢀ34
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validated our modeling study. Replacement of the methoxycarbonyl of 17 with
ethoxycarbonyl or acetyl (37 and 35) led to 5ꢀ9 fold decrease of potency. The
decreased activity of the pyridine analogue 42 revealed that the phenyl ring was
essential to maintain PRMT5 potency. As discussed above, the 5ꢀmethoxy
benzimidazole moiety and 2ꢀmethoxycarbonyl phenyl segment were optimal and
compound 17 was chosen for a further evaluation. It is interesting to note that
compound 17 was actually identified in our virtual screening, but according to our
select criteria, we selected its analog compound 6 for bioassay test.
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
Table 3. Structures and IC50 values for 13 synthesized analogues of compound 6.
Compound
2’
3’
4’
Z
IC50(ꢁM)
3
3
3
3
3
3
3
3
3
0
1
2
3
4
5
6
7
8
H
Me
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
33±33.94
>200
47±0.71
19±1.41
25±2.83
2.9±1.84
>100
OMe
NMe
2
COMe
COOH
COOEt
CONHMe
1.9±0.01
>200
39
CONMe
2
>200
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1
1
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2
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2
2
2
2
2
2
2
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3
3
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3
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5
5
5
5
5
6
4
4
4
0
1
2
H
H
COOMe
H
COOMe
H
H
H
N
52±5.66
>100
H
H
COOMe
2.4±0.57
0
1
2
3
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6
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9
0
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5
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9
0
1
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In order to understand the reason why 17 showed potent PRMT5 inhibition compared
with compound 6, molecular docking was used to explore the binding mode of 17. As
shown in Figure 4, compared to 6, 17 forms an additional hydrogen bond with Y324,
besides the same hydrogen bonding network, this may be the molecular basis for the
more potent PRMT5 inhibition of 17 than 6. The detailed interaction between 17 and
PRMT5 is shown in Figure S3.
Figure 4. Binding mode comparison of compound 6 and 17, PRMT5 and the
interacting residues of PRMT5 is shown as cartoon and magenta stick representation
respectively, while compound 6 and 17 is shown in green and cyan stick
representation, respectively. The 4GQB (PDB code) was used to simulate the binding
mode.
17 showed good selectivity against PRMT5.
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To test the selectivity of 17 against PRMT5, we measured the IC50 values of 17
against a panel of methyltransferases including PRMT1, PRMT3, PRMT4, PRMT6,
PRMT7, PRMT8, NSD1, DNMT1, DOT1L, SET7/9, and another two epigenetic
targets bromodomain protein (BRD4) and histone acetyltransferase GCN5. As shown
in Table 4, IC50 values of 17 against these methyltransferases, BRD4 as well as GCN5
were all greater than 100 ꢁΜ, these results indicated that 17 had selectivity for
PRMT5.
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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
Table 4. Inhibitory activities of 17 against PRMT1, PRMT3, PRMT4, PRMT6,
PRMT7, PRMT8, NSD1, DNMT1, DOT1L, SET7/9, BRD4 and GCN5.
Protein
PRMT1
PRMT3
PRMT4
PRMT6
PRMT7
PRMT8
NSD1
IC50 (ꢁΜ)
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
PRMT5 selectivity
> 303
> 303
> 303
> 303
> 303
> 303
> 303
DNMT1
DOT1L
SET7/9
BRD4
> 303
> 303
> 303
> 303
GCN5
> 303
Surface plasmon resonance (SPR) analysis.
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1
2
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2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A surface plasmon resonance (SPR) assay was employed to assess the interaction
between 17 and PRMT5, for which PRMT5/MEP50 was immobilized on CM5 sensor
chips. As shown in Figure 5, compound 17 displayed extracellular binding affinity
with PRMT5 with an equilibrium dissociation constant (Kd) value of 0.987 ꢁM, the
equilibrium binding curve fit is shown in Figure S4. This result indicated the direct
binding of 17 to PRMT5 at enzyme level. Although the direct binding of 17 to
PRMT5 has been proved, efforts to fully understand the detailed binding mode of 17
are urgently needed through the pursuit of a PRMT5ꢀcompound 17 cocrystal
structure.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Compound 17 binding to PRMT5/MEP50 was analyzed with SPR, and the
dissociation constant (Kd = 0.987 ꢁM) was calculated.
Kinetic experiments
In order to verify the Mechanism of Action (MOA) of compound 17, we performed
the kinetic experiment. This experiment was done using varied SAM concentrations
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with fixed peptide substrate and varied peptide concentrations with fixed SAM
concentration, compound 1 was used as the reference compound, as it was the
validated PRMT5 inhibitor and occupied the substrate pocket. The results (Figure 6)
indicated that compound 17 showed competitive inhibition with respect to SAM on
PRMT5 and shows noncompetitive inhibition with respect to peptide on PRMT5.
While compound 1 showed uncompetitive inhibition with respect to SAM on PRMT5
and showed competitive inhibition with respect to peptide on PRMT5.
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 6. The kinetic experiments results.
17 displays selectively antiꢀproliferation in MV4ꢀ11 leukemia cells.
As 17 showed selective and potent PRMT5ꢀinhibiting activity in vitro, we further
explored whether it had antiꢀproliferation effects in PRMT5 related cancer cell lines.
One leukemia cell line (MV4ꢀ11) and three lymphoma cells (KOPN8, Jekoꢀ1 and
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Z138) were used to expand our analysis of the differential sensitivity to 17. The
antiꢀproliferative effects of the PRMT5 inhibitor manifested over several days,
necessitating the development of a longꢀterm proliferation assay allowing for the
measurement of cell growth over 12 days. As shown in Figure 7, MV4ꢀ11 cells were
the most sensitive in all of the cells notably 17 demonstrated potent
concentrationꢀdependent antiꢀproliferative effects, with EC50 value of 6.53 ꢁM
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figure 8A). However this compound showed much weaker growth inhibition
activities against other cell lines, with EC50 values ranging from 15 to 70 ꢁM. Based
on the antiꢀproliferative effects assay, MV4ꢀ11 cells displayed considerably different
cytotoxicity against the compounds. Therefore, the MV4ꢀ11 cell lines were selected to
explore the mechanism of cell killing in more detail.
Besides, to address the possibility of offꢀtarget effects of compound 17, we tested the
cytotoxicity of 17 against another two normal cells HUVEC and RCTEC. The results
(Figure S5) showed that 17 has weaker cytotoxicity against these two cells, which
suggests that 17 may have no offꢀtarget effect.
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Figure 7. Inhibition of proliferation of MV4ꢀ11, Jekoꢀ1, KOPN8 and Z138 cells by
compound 17 in vitro over 12 days in culture.
Figure 8. (A) Effects of the compound and 17 on the proliferation of MV4ꢀ11 cells.
(B) Effects of 17 on cellular target inhibition as determined by sMDA western blot.
Characterization of cell methylation.
More work is required for an understanding of how PRMT5 activity is upregulated in
MV4ꢀ11 cell lines that are dependent on this enzyme. The representative compound
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
1
7 was assessed using Western blot analysis by sDMA antibody, with a dose titration
of 0ꢀ20 ꢁM compound (Figure 8B). After a 6 days treatment, 17 decreased the
intensity of multiple bands, including one that colocalized with the previously
reported PRMT5 substrate SmD3, in a doseꢀdependent manner, which were
comparable with (Figure S6) compound 1. Biochemical inhibition correlated with
corresponding decreased levels of symmetrically dimethylated PRMT5 substrates and
proliferation in a timeꢀ and concentrationꢀdependent manner suggesting that the
antiꢀproliferative effects were a direct consequence of PRMT5 inhibition.
Conclusion
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The multitude of reports implicating PRMT5 in cancer reflects the need for potent and
selective inhibitors of PRMT5, so that its pathobiology can be further explored and
validated. In the present study, we identified 17 as a novel inhibitor of PRMT5 which
showed great selectivity and growth inhibition activities against MV4−11 cells over
other leukemia and lymphoma cells. More importantly, 17 showed weaker
cytotoxicity against two normal cells HUVEC and RCTEC. We also clarified the
cellular cytotoxic mechanism of representative 17 derived PRMT5 inhibitors, which
was mediated through inhibition of PRMT5 substrate SmD3 and downstream
signaling proteins. We believe 17 is a promising compound that could help us gain a
better understanding of the molecular biology of PRMT5 and potentially assist in
defining a therapeutic strategy to treat leukemia indications.
Experimental Section
Virtual screening.
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Pharmacophoreꢀbased virtual screening.
In the crystal structure of PRMT5 (4GQB), the asymmetric unit contains a PRMT5, a
MEP50, a SAH analog (compound 5) and a peptide substrate derived from H4, so
MEP50 and peptide were deleted, then based on this modified complex structure
Discovery Studio 3.0 was used to generate the pharmacophore model. Then
Discovery Studio 3.0 was used to perform the pharmacophoreꢀbased in silicon
screening.
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
Molecular docking based virtual screening.
4
0, 41
We employed GLIDE 5.5
program to perform the molecular docking studies. The
4
,344 candidate compounds that match the pharmacophore model were downloaded
4
2
from SPECS database, and then the LigPrep panel was used to produce multiple
output structures of these compounds by generating different protonation states,
stereochemistry, tautomers, and ring conformations for molecular docking. The
Protein Preparation Wizard Workflow was used to prepare the protein structures.
Residues located within 20 Å around compound 5 on PRMT5 were defined as binding
sites in which the docking grids were created. The default settings were used. All the
4,344 candidate compounds were docked into the defined binding site using extra
precision (XP) mode without any constraint and were ranked by Glideꢀscore. In the
compound 6 and 17 against PRMT5, a similar procedure was used as above.
PRMT5 inhibition assays (Alpha LISA and radioactive methylation assays).
The PRMT5/MEP50 complex purchased from BPS, (Cat. No. 51045) was used as
enzyme to test the inhibition activities of screened compounds.
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6
The protocol of Alpha LISA assay was as following: 1. Prepare 1x assay buffer
(modified TrisꢀHCl Buffer); 2. Transfer compounds to 384 assay plate by Echo in 100%
DMSO; 3. Prepare enzyme solution in 1x assay buffer; 4. Prepare substrate mix
solution in 1x assay buffer; 5. Transfer 5 ꢁL of enzyme solution to assay plate or for
low control transfer 5 ꢁL of 1x assay buffer; 6. Incubate at room temperature for 15
minutes; 7. Add 5 ꢁL of substrate mix solution to each well to start reaction; 8.
Incubate at room temperature for 60 min; 9. Prepare 1x Alphalisa buffer; 10. Add 5
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ꢁ
L acceptor solution, incubate for 60 min at RT, subdued light; 11. Add 10 ꢁL donor
solution, incubate for 30 min at RT, subdued light; 12. Reading endpoint with
Envision with Alpha mode; 13. Using GraphPad Prism V5.0 software. %Inhibition =
(Max signal ꢀ Compound signal) / (Max signal ꢀ Min signal) ×100. Max signal was
obtained from the action of Enzyme and Substrate; Min signal was obtained from the
Substrate only.
The protocol of radioactive methylation assay was as following: 1. Prepare 1x assay
buffer (modified Tris Buffer); 2. Transfer compounds to assay plate by Echo with
3
ꢀfold dilution in 100% DMSO. Final DMSO concentration is 1%; 3. Prepare enzyme
solution in 1x assay buffer; 4. Prepare substrate solution in 1x assay buffer; 5. Prepare
3
[
H]ꢀSAM solution in 1x assay buffer; 6. Transfer 15 ꢁL of enzyme solution to assay
plate or for low control transfer 15 ꢁL of 1x assay buffer; 7. Incubate at room
temperature for 15 minutes; 8. Add 5 ꢁL of substrate solution to each well; 9. Add 5
3
ꢁL of [ H]ꢀSAM solution to each well to start reaction; 10. Incubate at RT for 60 mins;
11. Add cold SAM in 1x assay buffer to make the stop mix; 12. Stop reaction with
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addition of 5 µL per well of stop solution; 13. Transfer 25 µL of volume per well to
Flashplate from assay plate; 14. Incubate for 1 h minimum at room temperature; 15.
2
Wash Flashplate with dH O + 0.1% Tweenꢀ20 three times; 16. Read plate on
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Microbeta; 17. Fit the data in Excel to obtain inhibition values using equation (1)
Equation (1): inh % = ( MaxꢀSignal)/ (MaxꢀMin)*100
Fit the data in GraphPad Prism 5 to obtain IC50 values using equation (2)
Equation (2): Y = Bottom + (TopꢀBottom) / (1+10^ ((LogIC50ꢀX)*Hill Slope))
Y is %inhibition and X is compound concentration.
Chemistry methods
General. All chemicals and solvents were purchased from commercial sources
and used without further purification unless otherwise noted. Reactions were
monitored by TLC, using silica gel plates with fluorescence F254 and visualized
under UV light. Purification was performed by flash chromatography using silica gel
1
(
300–400 mesh). H NMR spectra were recorded on Varian Mercury Plus 300 MHz
13
spectrometer, and C NMR spectra were recorded on Bruker Avance III 126 MHz
spectrometer. Chemicals shifts (δ) are reported in parts per million, coupling constants
(J) values are given in hertz, and peak multiplicities are expressed as follows: s,
singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet; m, multiplet. High
resolution mass spectrometry was conducted using an ACQUITY UPLC IꢀClass/Xevo
G2ꢀS QꢀTOF system (Waters Incorporation, Milford, MA, USA). Lowꢀresolution
mass spectra (ESI) was obtained using Agilent HPLCꢀMS (1260ꢀ 6120B). All tested
compounds were purified to
≥95% purity (Table S5) as determined by high
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performance liquid chromatography (HPLC).
General Procedure A: Synthesis of Target Compounds 12
-
43. To a solution of
intermediates (44aꢀc, 46aꢀd, 46fꢀj or 48aꢀe) (0.22 mmol, 1.0 equiv) in acetone was
added potassium carbonate (0.33 mmol, 1.5 equiv). After stirring for 15 min at room
temperature, 2ꢀchloroacetamide (50aꢀm or 52) (0.26 mmol, 1.2 equiv) was added and
the reaction mixture was stirred at room temperature for 4ꢀ12 h. The mixture was
filtered, and the filtrate was concentrated and the residue was adsorbed on silica and
purified by flash chromatography (dichloromethane/methanol, v/v, 99:1 to 90:10),
affording the title compound.
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Methyl 2-(2-((4-chloro-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (12).
The title compound was prepared from 44a and 50h following general procedure A.
1
White solid (79%). H NMR (300 MHz, DMSOꢀd
6
) δ 13.08 (s, 1H), 11.17 (s, 1H),
8.29 (d, J = 7.5 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.38 (s,
13
1
H), 7.15 (m, 3H), 4.32 (s, 2H), 3.76 (s, 3H). C NMR (126 MHz, DMSOꢀd ) δ
6
167.2, 166.5, 150.7, 140.4, 139.2, 136.8, 134.0, 130.5, 123.5, 122.5, 121.2, 121.1,
ꢀ
ꢀ
1
17.8, 116.3, 109.6, 52.3, 36.0. HRMS (ESI ) m/z calcd for C17
13 3 3
H N O ClS [MꢀH] :
354.0366; found 354.0363.
Methyl 2-(2-((4-methyl-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (13).
The title compound was prepared from 44b and 50h following general procedure A.
1
White solid (82%). H NMR (300 MHz, DMSOꢀd
6
) δ 12.66 (s, 1H), 11.24 (s, 1H),
8.31 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.21 (m,
2
H), 7.00 (t, J = 7.7 Hz, 1H), 6.90 (d, J = 7.0 Hz, 1H), 4.27 (s, 2H), 3.77 (s, 3H), 2.43
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1
3
(
s, 3H). C NMR (126 MHz, DMSOꢀd
6
) δ 167.2, 166.9, 148.7, 143.1, 139.3, 135.4,
1
34.0, 130.6, 126.9, 123.5, 122.2, 121.4, 121.0, 117.6, 114.9, 52.3, 36.0, 16.6. HRMS
ꢀ
ꢀ
(
ESI ) m/z calcd for C18
16 3 3
H N O S [MꢀH] : 354.0912; found 354.0910.
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Methyl 2-(2-((4-methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (14).
The title compound was prepared from 44c and 50h following general procedure A.
1
White solid (79%). H NMR (300 MHz, DMSOꢀd
6
) δ 12.80 (s, 1H), 11.21 (s, 1H),
8
.32 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.61 (t, J = 6.6 Hz, 1H), 7.19 (t, J =
13
6
.5 Hz, 1H), 7.03 (s, 2H), 6.67 (s, 1H), 4.26 (s, 2H), 3.88 (s, 3H), 3.77 (s, 3H). C
NMR (126 MHz, DMSOꢀd ) δ 167.2, 166.8, 149.6, 147.0, 139.2, 137.2, 134.0, 130.6,
6
ꢀ
1
23.5, 122.4, 121.8, 121.0, 117.6, 110.4, 103.2, 55.5, 52.3, 36.0. HRMS (ESI ) m/z
ꢀ
calcd for C18
H
16
N
3
O
4
S [MꢀH] : 370.0862; found 370.0854.
Methyl 2-(2-((5-methyl-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (15).
The title compound was prepared from 46a and 50h following general procedure A.
1
White solid (56%). H NMR (300 MHz, CD OD) δ 8.49 (d, J = 8.5 Hz, 1H), 7.95 (d,
3
J = 7.8 Hz, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.25 (s, 1H), 7.13 (t,
13
J = 7.7 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 4.20 (s, 2H), 3.76 (s, 3H), 2.41 (s, 3H). C
NMR (126 MHz, CDCl
3
) δ 168.5, 168.3, 148.0, 140.5, 134.6, 132.3, 131.0, 123.9,
ꢀ
1
23.6, 120.9, 116.1, 52.6, 37.8, 21.7. HRMS (ESI ) m/z calcd for C18
H
16
N
3
O
3
S
ꢀ
[MꢀH] : 354.0912; found 354.0908.
Synthesis of methyl-2-(2-((5-hydroxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)-
benzoate (16). Compound 16a (100 mg, 0.21 mmol, 1.0 equiv) was dissolve in 4 mL
THF, and tetrabutylammonium fluoride (1 mol/L in THF, 0.25 mL, 0.25 mmol, 1.2
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equiv) was added. The mixture was stirred at room temperature for 2 h and then
diluted with ethyl acetate. The solution was washed with water and brine successively,
dried over anhydrous sodium sulfate, and concentrated in vacuum. The resulting
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residue was purified by silica gel chromatography (dichloromethane/methanol, v/v,
1
9
4:6) to give the title product as white solid (50 mg, 67%). H NMR (300 MHz,
3
CD OD) δ 8.48 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 6.5 Hz, 1H), 7.54 (t, J = 7.9 Hz, 1H),
7
.27 (d, J = 8.7 Hz, 1H), 7.14 (t, J = 7.3 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 6.70 (dd, J
13
=
8.7, 2.0 Hz, 1H), 4.15 (s, 2H), 3.77 (s, 3H). C NMR (126 MHz, CD
3
OD) δ 169.2,
1
3
68.9, 154.9, 148.4, 141.2, 135.1, 131.9, 124.6, 122.0, 118.0, 116.1, 112.9, 99.7, 52.8,
ꢀ
ꢀ
8.1. HRMS (ESI ) m/z calcd for C17
14 3 4
H N O S [MꢀH] : 356.0705; found 356.0698.
Methyl 2-(2-((5-((tert-butyldimethylsilyl)oxy)-1H-benzo[d]imidazol-2-yl)thio)ace-
tamido)benzoate (16a). The title compound was prepared from 46f and 50h following
1
general procedure A. Yellow solid (67%). H NMR (300 MHz, CD
3
OD) δ 8.47 (d, J =
8
.5 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.30 (d, J = 8.8 Hz, 1H),
7.09 (t, J = 7.7 Hz, 1H), 6.90 (s, 1H), 6.71 (d, J = 8.7 Hz, 1H), 4.17 (s, 2H), 3.75 (s,
3
H), 0.98 (s, 9H), 0.16 (s, 6H).
Methyl 2-(2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (17).
The title compound was prepared from 46g and 50h following general procedure A.
1
White solid (80%). H NMR (300 MHz, CD OD) δ 8.49 (d, J = 8.5 Hz, 1H), 7.96 (d,
3
J = 7.8 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.34 (d, J = 9.0 Hz, 1H), 7.14 (t, J = 7.6 Hz,
1
3
1H), 6.97 (s, 1H), 6.81 (d, J = 9.0 Hz, 1H), 4.17 (s, 2H), 3.80 (s, 3H), 3.77 (s, 3H). C
NMR (126 MHz, DMSOꢀd ) δ 167.2, 166.9, 155.3, 147.3, 139.3, 137.9, 136.3, 134.0,
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ꢀ
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30.6, 123.4, 120.9, 117.9, 117.4, 110.7, 94.2, 55.4, 52.4, 36.0. HRMS (ESI ) m/z
ꢀ
calcd for C18
H
16
N
3
O
4
S [MꢀH] : 370.0862; found 370.0858.
Methyl 2-(2-((5-ethoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (18).
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The title compound was prepared from 48a and 50h following general procedure A.
1
White solid (84%). H NMR (300 MHz, CD
3
OD) δ 8.47 (d, J = 8.3 Hz, 1H), 7.91 (d,
J = 7.9 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.09 (t, J = 7.4 Hz,
1
H), 6.93 (s, 1H), 6.78 (d, J = 8.9 Hz, 1H), 4.16 (s, 2H), 3.98 (q, J = 6.9 Hz, 2H), 3.75
13
(
s, 3H), 1.36 (t, J = 6.9 Hz, 3H). C NMR (126 MHz, DMSOꢀd
6
) δ 167.2, 166.9,
1
54.5, 147.3, 139.3, 137.8, 136.2, 134.0, 130.6, 123.5, 120.9, 117.8, 117.5, 110.8,
ꢀ
ꢀ
9
18 3 4
5.0, 63.4, 52.4, 36.0, 14.7. HRMS (ESI ) m/z calcd for C19H N O S [MꢀH] :
384.1018; found 384.1005.
Methyl 2-(2-((5-propoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate (19).
The title compound was prepared from 48b and 50h following general procedure A.
1
White solid (66%). H NMR (300 MHz, CD OD) δ 8.48 (d, J = 8.4 Hz, 1H), 7.96 (d,
3
J = 8.0 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.14 (t, J = 7.7 Hz,
1
H), 6.96 (s, 1H), 6.81 (dd, J = 8.9, 2.2 Hz, 1H), 4.17 (s, 2H), 3.92 (t, J = 6.4 Hz, 2H),
1
3
3.77 (s, 3H), 1.78 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl
3
) δ
1
68.6, 168.5, 156.0, 147.2, 140.5, 134.7, 131.1, 123.7, 121.0, 116.1, 112.6, 70.3, 52.6,
ꢀ
ꢀ
38.1, 22.7, 10.7. HRMS (ESI ) m/z calcd for C H N O S [MꢀH] : 398.1175; found
20
20
3
4
3
98.1176.
Methyl
2-(2-((5-isopropoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate
(20). The title compound was prepared from 48c and 50h following general procedure
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