Synthesis and biological evaluation of novel synthetic chalcone derivatives as anti-tumor agents targeting Cat L and Cat K

Article abstract of DOI:10.1016/j.bmc.2017.09.019

A series of chalcone derivatives bearing benzamide or benzenesulfonamide moieties were synthesized and evaluated for their anti-tumor effect on HCT116, MCF7 and 143B cell lines in vitro. SAR analysis showed that compounds bearing a benzenesulfonamide grou

Full text of DOI:10.1016/j.bmc.2017.09.019

Accepted Manuscript
Synthesis and biological evaluation of novel synthetic chalcone derivatives as
anti-tumor agents targeting Cat L and Cat K
Yali Wang, Situ Xue, Ruolan Li, Zhihui Zheng, Hong Yi, Zhuorong Li
PII:
S0968-0896(17)31234-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.09.019
BMC 13979
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 June 2017
10 September 2017
14 September 2017
Please cite this article as: Wang, Y., Xue, S., Li, R., Zheng, Z., Yi, H., Li, Z., Synthesis and biological evaluation
of novel synthetic chalcone derivatives as anti-tumor agents targeting Cat L and Cat K, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.09.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and biological evaluation of novel synthetic chalcone
derivatives as anti-tumor agents targeting Cat L and Cat K
Yali Wang,a Situ Xue,a Ruolan Li,b Zhihui Zheng,b Hong Yia and Zhuorong Li∗a
a
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union
Medical College, Beijing 100050, China.
b
New Drug Research & Development Center, North China Pharmaceutical Group Corporation,
Shijiazhuang 050015, China.
Abstract
A series of chalcone derivatives bearing benzamide or benzenesulfonamide moieties
were synthesized and evaluated for their anti-tumor effect on HCT116, MCF7 and
143B cell lines in vitro. SAR analysis showed that compounds bearing a
benzenesulfonamide group had greater potency than those bearing a benzamide group.
It was also shown that compounds with a mono-methyl or mono-halogen group at the
3-position on the terminal phenyl ring were more effective than those with
trifluoromethyl or methoxy groups. Compound 8e exhibited the most potent
anti-tumor activities against HCT116, MCF7 and 143B cell lines, with IC50 values of
0.597, 0.886 and 0.791 µM, respectively. Molecular docking studies and enzymatic
assays demonstrated that the anti-tumor activity of compound 8e might be regulated
by Cat L and Cat K.
Key words: Anti-tumor activity, chalcone, Cat L, Cat K
1. Introduction
Cathepsins, which are involved in numerous physiological processes, can be divided
into three subgroups based on their active site amino acid; namely, cysteine (B, C, F,
H, K, L, O, S, V, W and X), aspartic (D and E), and serine (G) cathepsins.1 Cysteine
cathepsins have been reported to be important in a variety of physiological and
pathological processes, such as tumor progression and metastasis,2-5 osteoporosis,6
osteoarthritis7 and atherosclerosis.8 There is now good evidence that several cysteine
cathepsins, including cathepsin (Cat) B, F, H, L, K, S, V and X, are related to the
progression of cancer.5 Increased expression of cysteine cathepsins has been shown to
correlate with malignancy and poor prognosis of patients, indicating their potential
diagnostic and prognostic value.9, 10
The upregulation of Cat L has been reported in a wide range of human
malignancies including colon,11 breast,12 lung,13 melanoma14 and pancreatic15 cancers.
Cat L is required for cell cycle progression and knockout models exhibit reduced cell
proliferation and tumor growth.1 Cat K is overexpressed in melanoma,16 prostate
cancer,17 giant cell tumors18 and basal cell carcinoma,19 and has also been implicated
∗
Corrseponding anthor: e-mail: lizhuorong@imb.pumc.edu.cn; Tel: +86-10-83152017.

in breast cancer20 and osteosarcoma.21 Odanacatib, a Cat K inhibitor, has been shown
to have good therapeutic effects in patients with breast cancer.22 The fact that a
cysteine cathepsin inhibitor can reduce tumor growth, invasion and metastasis
suggests that targeting cysteine cathepsins could be therapeutically beneficial.23, 24
To date, there have been lots of compounds explored as cysteine cathepsin
inhibitors and the representative structure were presented in Figure 1. Several other
compounds such as quinazoline,25 chalone,26-29 pyrazoline,30 semicarbazone30 and
thiosemicarbazone,31 also have activity against cathepsins. Chalcones are an
important type of natural product and exhibit a wide range of biological effects,32
including acting as anticancer, anti-inflammatory, antiviral and antimicrobial agents.
Ramalho et al. reported on a series of chalcones that showed cytotoxic activity along
with Cat K inhibition.28 Cyclohexenyl chalcones panduratin A and nicolaioidesin C
demonstrate a high level of cytotoxicity in various human cancer cells through
inhibiting the Cat L enzyme.33 These examples highlight the growing interest in new
chalcone derivatives as cysteine cathepsin inhibitors, which prompted us to further
investigate this drug skeleton.
Figure 1. Representative inhibitors of cysteine cathepsins, including Cat K (I),34 Cat
L (II),31 Cat S (III),35 Cat B (IV)36 and Cat C (V)37.
Cysteine residues are known to be key amino acids in the active site of cysteine
cathepsins, where the reactive electrophilic warhead could interact with the thiol
moiety of cysteine.38 The electrophilic carbonyl, sulfonyl and nitrile groups act as
warheads in mediating covalent reversible inhibition. In the present study, benzamide
or benzenesulfonamide groups with carbonyl or sulfonyl pharmacophores were
introduced to the chalcone skeleton, which resulted in promising anti-cysteine
cathepsin activity. We also introduced an imidazole group to the chalcone skeleton to
enhance the anti-cancer activity, as in reported studies.39
All target compounds were evaluated for their in vitro cytotoxicity against HCT116
(human colon cancer), MCF7 (human breast cancer) and 143B (human osteosarcoma)
cell lines. Compounds possessing the greatest apparent cytotoxic activity were
evaluated for their ability to inhibit specific enzymes in vitro. Lastly, in silico
molecular docking of the most promising compound 8e were carried out.
2. Chemistry

The synthesis of target compounds 6a–o is illustrated in Scheme 1. The commercially
available 1,2-diaminobenzene and 2-hydroxypropanoic acid were refluxed in 4N
aqueous HCl solution to afford 1,40 which was precipitated from the reaction mixture
after neutralization with aqueous NH3 solution. Treatment of
1
with
2,2,6,6-tetramethylpiperidin (TEMPO), sodium hypochlorite and potassium bromide
at 10–15°C afforded intermediate 2. The aldehyde group of 3-nitrobenzaldehyde was
protected by ethylene in toluene at 120 °C to obtain 3.41 The reduction of 3 with
hydrogen and catalytic amounts of Pd/C in methanol yielded 4 at room temperature.42
Next, 4 was acylated with corresponding benzoyl chloride in the presence of pyridine
in dichloromethane, with further treatment in aqueous HCl solution mixed with
1,4-dioxane at 50°C to obtain 5a–o. Finally, target compounds 6a–o were successfully
obtained via the condensation of 5a–o with intermediate 2 in ethanol. The synthesis of
target compounds 8a–i is shown in Scheme 2. Following the procedure for 5a–o, the
intermediates 7a–i were obtained from intermediate 4 with benzene sulfonyl chloride.
Target compounds 8a–i were obtained using the same methods as for 6a–o.
Scheme 1. Synthetic route for target compounds 6a–o. Reagents and conditions: (a)
4N HCl, reflux, 5 h; (b) TEMPO, NaClO, KBr, CH3CN, 10–15 °C; (c) p-TsOH,
toluene, reflux, 8 h; (d) Pd/C, H2, CH3OH, rt; (e) (i) Py, CH2Cl2, rt; (ii) HCl,
1,4-dioxane, 50 °C; (f) NaOH, C2H5OH.

Scheme 2. Synthetic route for target compounds 8a–i. Reagents and conditions: (a) (i)
Py, CH2Cl2, rt; (ii) HCl, 1,4-dioxane, 50 °C; (b) NaOH, C2H5OH.
3. Results and Discussion
3.1. In vitro anti-tumor activities and structure-activity relationships
The anti-tumor activities of the target compounds were evaluated against HCT116,
MCF7 and 143B cell lines using the MTT assay. The bioactivity data was summarized
in Tables 1 and 2, using 5-FU, paclitaxel and odanacatib as the positive controls.
Table 1 The anti-tumor activities of 6a–o against HCT116, MCF7 and 143B cell lines
in vitro
Compd.
R1
IC50 (µM) SD
HCT116
MCF7
143B
H
2-Cl
2.443 0.422
1.282 0.059
1.123 0.154
2.267 0.138
1.680 0.091
1.555 0.251
2.225 0.890
1.869 1.616
1.418 0.260
1.662 0.338
3.362 0.162
3.681 0.236
4.882 0.954
1.645 0.352
1.553 0.148
8.638 1.639
0.014 0.005
> 100
11.232 1.492
4.568 0.030
7.835 1.588
8.409 0.672
4.519 0.443
5.209 0.100
7.002 13.102
5.867 0.114
5.594 0.746
9.692 0.613
21.470 7.655
17.840 0.175
20.134 1.025
> 100
8.340 1.244
3.692 0.699
3.370 0.188
7.441 0.632
6.047 0.105
4.624 0.220
7.314 0.173
8.285 0.117
4.882 0.847
6.564 1.271
9.570 0.452
4.607 0.423
6.328 0.502
3.657 0.242
4.015 0.631
25.216 1.058
5.560 0.495
12.715 1.411
6a
6b
3-Cl
6c
4-Cl
6d
2-Br
6e
3-Br
6f
4-Br
6g
2-CH3
3-CH3
4-CH3
3-CF3
3-OCH3
4-OCH3
3,5-(Cl)2
3,5-(CH3)2
-
6h
6i
6j
6k
6l
6m
6n
24.608 6.951
32.626 2.785
1.428 0.389
45.379 3.094
6o
5-FU
Paclitaxel
Odanacatib
-
-
As illustrated in Tables 1 and 2, most of the target compounds 6a–o and 8a–i were
shown to have anti-tumor activities. With the exception of 6n, all other compounds
demonstrated greater potency than 5-FU, and all of the compounds showed greater
potency than odanacatib. Some compounds were more potent than paclitaxel against
one or more of the tumor cell lines. Compound 6a, which lacked a substituent (R1 = H)
on the terminal phenyl ring, showed moderate activity against HCT116, MCF7 and
143B cell lines, with IC50 values of 2.443, 11.232 and 8.340 µM, respectively. The
introduction of a mono-halogen (6b–g, R1 = 2-Cl, 3-Cl, 4-Cl, 2-Br, 3-Br and 4-Br,

respectively) led to an improvement in activities compared to 6a. Compounds 6h–j
with mono-methyl groups (R1 = 2-CH3, 3-CH3, and 4-CH3, respectively) also showed
greater activity than 6a. However, the introduction of a double chlorine or double
methyl group (6n, R1 = 3,5-(Cl)2; 6o, R1 = 3,5-(CH3)2) resulted in lower potency than
mono-substitution (6c and 6i). Compound 6k with a 3-CF3 substitution had lower
activity than 6a. The introduction of a methoxy group (6l, R1 = 3-OCH3; 6m, R1 =
4-OCH3) led to higher activity against the 143B cell line, but lower activity against
HCT116 and MCF7 cell lines, compared to 6a. Interestingly, the position of the R1
group was closely related to the anti-tumor activity. Compounds with a 3-substituted
phenyl group (6c, 6f and 6i) exhibited higher potency than those with a 2-substituted
(6b, 6e and 6h) or 4-substituted phenyl group (6d, 6g and 6j). Another interesting
phenomenon observed was that when introducing
a
group with strong
electron-withdrawing ability (6k, R1 = 3-CF3) or electron-donating ability (6l, R1 =
3-OCH3) into the 3-position, the resulting activity was lower than that of 6a. However,
the introduction of a chlorine (6c, R1 = 3-Cl) or methyl (6i, R1 = 3-CH3) group into the
3-position resulted in higher potency than 6a. We therefore predicted that a suitable
electron density within the terminal phenyl would be favorable to drug activity.
Preliminary structure-activity relationships (SARs) for compounds 8a-i were also
analyzed. Similar to the SARs for 6a–o, compounds with a 3-substituted phenyl group
(8b and 8e) showed higher potency than those with a 2-substituted (8a and 8d) or
4-substituted phenyl group (8c and 8f). The introduction of double chlorine or methyl
groups (8g and 8i) led to reduced activity compared to the mono-substituent (8b and
8d).
Table 2 The anti-tumor activities of 8a-i against HCT116, MCF7 and 143B cell lines
in vitro
R1
IC50 (µM) SD
MCF7
Compd.
HCT116
143B
2-Cl
3-Cl
0.987 0.034
0.737 0.068
1.956 0.255
0.955 0.153
0.597 0.166
1.989 0.126
2.844 1.038
1.224 0.294
2.230 1.451
8.638 1.639
0.014 0.005
> 100
4.284 0.653
4.159 0.113
3.822 0.573
4.132 0.204
0.886 0.131
3.216 0.108
19.995 6.169
4.566 0.297
5.859 1.084
32.626 2.785
1.428 0.389
45.379 3.094
4.789 0.140
2.506 0.055
7.445 2.198
4.063 0.617
0.791 0.078
3.220 0.243
5.951 0.162
3.330 0.927
5.048 0.035
25.216 1.058
5.560 0.495
12.715 1.411
8a
8b
4-Cl
8c
2-CH3
3-CH3
4-CH3
3,5-(Cl)2
3,4-(CH3)2
3,5-(CH3)2
-
8d
8e
8f
8g
8h
8i
5-FU
-
Paclitaxel
Odanacatib
-

Figure 2. Inhibitory curves of compound 8e toward HCT116 (magenta), MCF7
(orange) and 143B (olive) cell lines.
In general, the data listed in Tables 1 and 2 showed that the activity of compounds
8a–i was higher than that of compounds 6a–o, demonstrating that introducing a
benzenesulfonamide group led to greater potency than introducing a benzamide group.
Compound 8e showed the most potent anti-tumor activity against HCT116, MCF7
and 143B cell lines, with IC50 values of 0.597, 0.886 and 0.791 µM, respectively. The
inhibitory curves (Figure 2) showed that the effects of 8e were dose-dependent.
3.2. In silico molecular docking studies and in vitro enzymatic assays
To investigate the biological mechanisms of action, molecular docking studies
combined with in vitro enzymatic assays were implemented. The crystal structures of
eight cancer-associated cysteine cathepsins were processed using the protein
preparation protocol of Sybyl-X2.0 (Tripos, USA) and the location of the ligand in the
co-crystal structure was defined as the active site. Compound 8e was docked into the
active sites of eight crystal structures, respectively. A high absolute docking score
demonstrated possible enzymatic activity. According to the results shown in Table 4,
Cat B, Cat L, Cat K and Cat S exhibited the best docking scores (< −7 kcal/mol).
These four enzymes were selected to investigate further.
Table 4 Docking scores of compound 8e with eight cancer-associated cysteine
cathepsins.
Enzyme/Pdb Code Docking score (kcal.mol-1)
Cat B/1GMY
Cat F/1M6D
Cat H/1NB3
Cat L/4AMX
Cat K/1NLJ
Cat S/4BS5
Cat V/1FHO
Cat X/1EF7
-7.46
-5.75
-5.14
-7.83
-7.96
-7.70
-6.14
-5.93
Eight compounds were screened against Cat L, Cat K, Cat B and Cat S at 5 µM.
The results summarized in Table 5 are expressed as inhibition rates (%). Against Cat L,

compounds 8e and 8f showed > 90% inhibition; compounds 6c and 6i showed 80–90%
inhibition; and compounds 8b and 8d showed 70–80% inhibition. Most of the tested
compounds demonstrated greater potency than odanacatib. Against Cat K, compounds
8d and 8e showed > 90% inhibition; compounds 8a, 8b and 8f showed 80–90%
inhibition; and compounds 6c and 6i showed 70–80% inhibition. In short, most of
them showed superior activity against Cat K. Against Cat B, all compounds resulted
in lower than 50% inhibition. Against Cat S, compound 8c showed 89.35% inhibition,
while all other compounds showed lower enzymatic activity. The enzyme activity of
these compounds was obviously improved compared with the compound without
benzenesulfonamide or benzamide group (Table S1). In general, these compounds
showed dual activity against Cat K and Cat L, whereas the effects on Cat B and Cat S
were weaker. Importantly, compound 8e, which showed > 90% inhibition of Cat L
and Cat K, also exhibited the best anti-tumor activity against HCT116, MCF7 and
143B cell lines. We speculated that compound 8e exhibited anti-tumor activity
through targeting Cat L and Cat K. Further research into the mechanisms of these
compounds was required.
Table 5 The inhibitory rate of Cat L, Cat K, Cat B and Cat S by target compounds
Compd.
Enzyme Inhibition (5 µM, %)
Cat L
Cat K
72.99 5.12
77.97 7.34
86.48 5.95
89.15 6.76
58.14 5.33
91.27 7.12
93.46 7.88
85.96 6.53
Cat B
Cat S
86.00 14.16
88.54 12.49
62.94 5.32
78.88 7.44
57.99 10.30
71.28 6.32
92.35 8.87
93.79 12.53
71.28 11.25
30.49 4.43 19.47 3.17
41.30 7.29 45.17 3.55
32.48 5.22 45.73 7.27
15.24 3.89 52.61 4.96
23.75 4.11 89.35 5.53
40.02 5.28 47.75 5.19
45.63 4.99 50.13 4.20
34.26 7.21 21.97 4.37
6c
6i
8a
8b
8c
8d
8e
8f
100
100
-
0
0
100
100
100
0
0
0
100
100
-
0
0
Odanacatib
E64
100
-
0
CA074
3.3. In silico docking analysis
In order to observe the possible binding modes of compound 8e in the Cat L and Cat
K active sites, in silico docking results were analyzed. The proposed binding mode of
compound 8e in the Cat L active site is shown in Figure 3(A). In this model, one
oxygen atom from the sulfonyl group serves as an acceptor to form H-bonds with
Cys25 and His164; the amino of the sulfamide group donates one H-bond to Gly68;
and the oxygen atom of the carbonyl group serves as an acceptor to form H-bonds
with Gln19 and His163. A suggested binding model for compound 8e in the Cat K
active site is shown in Figure 3(B). In this model, two oxygen atoms from the sulfonyl
group serve as acceptors to form H-bonds with Cys25, His162, Gln19 and Trp184;
and the amino of the imidazole ring serves as a donor to form an H-bond with Gly64.

Figure 3. (A) The virtual binding mode of compound 8e in the Cat L active site. (B)
The virtual binding mode of compound 8e in the Cat K active site. Compound 8e is
represented as stick diagrams with carbon atoms shown in green. Relevant amino acid
residues in the binding site are shown in line. Red dashed lines represent H-bonds.
Pictures were generated using Pymol.
From the docking results exhibited in Figure 3, we concluded that the binding
modes of compound 8e were different in the Cat L and Cat K active sites. Whereas,
8e could form an H-bond interaction with Cys25, which is a crucial amino acid
residue in both of the Cat L and Cat K active sites. The sulfonyl group of 8e appears
to act as a reactive electrophilic warhead that interacts with the thiol moiety of Cys25.
Furthermore, the carbonyl group introduced to the compound (6i) could also form an
H-bond interaction with Cys25 (Figure S1).
4. Conclusions
In this study, a library of new chalcone analogs possessing benzamide or
benzenesulfonamide scaffolds were synthesized and evaluated for their biological
activity. Preliminary investigation indicated that the series containing
benzenesulfonamide scaffolds demonstrate higher levels of anti-tumor activity than
those based on a benzamide scaffold. The most promising compound, 8e, had IC50
values of 0.597, 0.886 and 0.791 µM against HCT116, MCF7 and 143B cell lines,
respectively. A SAR study showed that substituting either a mono-methyl or
mono-halogen group at the 3-position on the terminal phenyl ring was beneficial in
terms of anti-tumor activity. Docking studies demonstrated that compound 8e formed
H-bond interactions with Gln19, Cys25, Gly68, His163 and Gly164 in the Cat L
active site, and Gln19, Cys25, Gly64, His162 and Trp184 in the Cat K active site.
Enzymatic assays revealed that 8e (5 µM) inhibited Cat L and Cat K by 92.35% and
93.46%, respectively. Combined results from enzymatic assays and molecular
docking analysis therefore indicated that Cat L and Cat K might be potential targets
for compound 8e.
5. Experimental section
5.1. General procedures
All melting points (m.p.) were determined using an MP90 automated melting point

instrument (Mettler Toledo, Switzerland) and were uncorrected. Mass spectra (MS)
and high resolution mass spectra (HRMS) were performed using an LCMS-2020
(Shimadzu, Japan) and an LTQ Orbitrap XL (Thermo Scientific, USA), respectively.
1H and 13C NMR spectra were performed using a Bruker BioSpin GmbH spectrometer
(Bruker Bioscience, USA). Column chromatography was performed using a 200–300
mesh silica gel from Qingdao Ocean Chemical. Unless specified, all reagents were
obtained from commercially available sources and were used without purification.
5.2. Chemical synthesis
1-(1H-benzo[d]imidazol-2-yl) ethanol (1)
A mixture of 1,2-diaminobenzene (5.41 g, 0.05 mol), 2-hydroxypropanoic acid (7.95
g, 85% in water, 0.075 mol) and 4 N aqueous HCl solution (40 mL) was refluxed for
5 h. After cooling to room temperature, the reaction mixture was neutralized with
aqueous NH3 solution. The precipitant was then filtered, washed with water and dried
to afford compound 1, a grey-white solid with 80% yield, MS (ESI) m/z: 163 (M+H)+.
The 1H NMR spectrum was in accordance with that described in the literature.40
1-(1H-benzo[d]imidazol-2-yl) ethanone (2)
To a solution of 1 (1.62 g, 0.01 mol), 2,2,6,6-tetramethylpiperidin (0.016 g, 0.1 mmol)
and potassium bromide (0.12 g, 1 mmol) in 40 mL acetonitrile/water (1:1), 8%
aqueous sodium hypochlorite (13.96 g, 0.015 mol) was added over a 15–20 minute
period, keeping the temperature of the reaction mixture between 10 and 15°C. The
mixture was stirred for 30 minutes and was extracted with ethyl acetate (50 mL × 3).
The combined extracts were washed with 60 mL of 10% aqueous sodium thiosulfate
and 60 mL of saturated sodium chloride solution. The organic phase was separated,
dried over anhydrous MgSO4 and evaporated to afford compound 2 with 85% yield,
1
MS (ESI) m/z: 161 (M+ H)+. H NMR (400 MHz, DMSO) δ 13.31 (s, 1H), 7.84 (d, J
= 8.1 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.39 (t, J = 7.1 Hz, 1H), 7.31 (d, J = 7.2 Hz,
1H), 2.72 (s, 3H).
2-(3-Nitro-phenyl)-1,3-dioxolane (3)
A solution of 3-nitrobenzaldehyde (4.53 g, 0.03 mol), ethylene glycol (2.05 g, 0.033
mol) and p-toluenesulfonic (0.02 g, 0.1 mmol) in 150 mL toluene was refluxed under
a Dean-Stark trap for 10 h. The reaction mixture was then cooled to room temperature
and washed with saturated sodium bicarbonate solution and saturated sodium chloride
solution, and dried with anhydrous MgSO4. Evaporation of the organic phase gave
1
compound 3 with 95% yield. The H NMR spectrum was in accordance with that
described in the literature.41
3-(1,3-Dioxolan-2-yl)aniline (4)
A mixture of compound 3 (1.95 g, 0.01 mol) and Pd/C (0.1 g, 10%) in 50 mL
methanol was stirred under hydrogen at room temperature for 4 h. The reaction
mixture was filtered and the filtrate was evaporated to afford compound 4, a yellow
1
oil with 97% yield, MS (ESI) m/z: 166 (M+ H)+. The H NMR spectrum was in

accordance with that described in the literature.42
General procedure for preparation of intermediates (5a–o)
To a stirred solution of compound 4 (0.50 g, 3 mmol) in 15 mL dichloromethane,
pyridine (0.24 mL, 3 mmol) and corresponding benzoyl chloride (3 mmol) were
added at 0°C. The reaction mixture was stirred for 30 minutes, after which it was
washed with 10 mL 4N aqueous HCl solution and 10 mL saturated sodium chloride
solution. The organic layer was dried with anhydrous MgSO4 and concentrated. The
residue was dissolved in 20 mL dioxane and then 15 mL of a 4N aqueous HCl
solution was added at room temperature. The reaction mixture was stirred at 50°C for
30 minutes after which it was extracted with ethyl acetate (50 mL × 3). The extract
was washed with saturated sodium chloride solution and dried with anhydrous MgSO4.
After concentration, column chromatography of the residue on silica gel (eluent
PE/EA 7:1) generated compounds 5a–o. The spectral data are summarized in the
Supplementary Information.
General procedure for preparation of target compounds (6a–o)
A mixture of an appropriate intermediates 5a–o (3 mmol), compound 2 (0.48 g, 3
mmol) and sodium hydroxide (0.12 g, 3 mmol) in ethanol (15 mL) was stirred at room
temperature for 24 h. Distilled water (5 mL) was added and the precipitate was
collected by filtration. Subsequently, it was purified by recrystallization with
methanol to afford the target compounds 6a–o.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)benzamide (6a).
Yellow solid; yield: 53%; m.p.: 233.5–235.2 °C; 1H NMR (400 MHz, DMSO) δ 10.45
(s, 1H), 8.39 (s, 1H), 8.15 (d, J = 16.1 Hz, 1H), 8.04–7.96 (m, 4H), 7.80–7.65 (m, 2H),
7.64–7.61 (m, 1H), 7.56 (m, 3H), 7.49 (t, J = 7.8 Hz, 1H), 7.39 (s, 2H); 13C NMR
(101 MHz, DMSO) δ 181.25, 166.21, 149.47, 144.50, 140.42, 135.17, 132.23, 130.03,
128.92, 128.17, 126.09, 123.41, 122.00, 119.14; HRMS calcd for C23H18N3O2
(M+H)+ 368.13935, found 368.13921.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-2-chlorobenza
1
mide (6b). Yellow solid; yield: 54%; m.p.: 275.5–277.3 °C; H NMR (400 MHz,
DMSO) δ 10.72 (s, 1H), 8.33 (s, 1H), 8.13 (d, J = 16.1 Hz, 1H), 7.96 (d, J = 16.1 Hz,
1H), 7.87 (t, J = 8.3 Hz, 2H), 7.65 (dd, J = 7.2, 1.1 Hz, 1H), 7.60 (d, J = 7.8 Hz, 3H),
13
7.56–7.48 (m, 3H), 7.41 (t, J = 7.5 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H); C NMR (101
MHz, DMSO) δ 181.20, 165.65, 149.42, 144.29, 143.51, 140.11, 137.18, 135.30,
135.25, 131.69, 130.41, 130.21, 130.15, 129.44, 127.74, 126.28, 126.03, 123.66,
122.66, 122.13, 121.64, 118.50, 113.36; HRMS calcd for C23H17ClN3O2 (M+H)+
402.10038, found 402.10029.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3-chlorobenza
1
mide (6c). Yellow solid; yield: 57%; m.p.: 205.5–206.9 °C; H NMR (400 MHz,
DMSO) δ 10.53 (s, 1H), 8.36 (s, 1H), 8.14 (d, J = 16.1 Hz, 1H), 8.07 (s, 1H),

7.99–7.95 (m, 3H), 7.75 (s, 2H), 7.69 (d, J = 7.9 Hz, 1H), 7.62–7.57 (m, 2H), 7.49 (t, J
13
= 7.8 Hz, 1H), 7.38 (dd, J = 6.0, 3.1 Hz, 2H); C NMR (101 MHz, DMSO) δ 181.16,
164.68, 149.39, 144.40, 140.10, 137.09, 135.11, 133.73, 132.02, 130.89, 130.04,
127.91, 126.98, 126.27, 123.44, 122.04, 119.24; HRMS calcd for C23H17ClN3O2
(M+H)+ 402.10038, found 402.10037.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-4-chlorobenza
1
mide (6d). Yellow solid; yield: 62%; m.p.: 250.1–252.9 °C; H NMR (400 MHz,
DMSO) δ 10.50 (s, 1H), 8.36 (s, 1H), 8.14 (d, J = 16.1 Hz, 1H), 8.045 (d, J = 8.6 Hz,
2H), 7.99–7.95 (m, 2H), 7.75 (s, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 7.7 Hz,
1H), 7.49 (t, J = 7.8 Hz, 1H), 7.38 (dd, J = 6.2, 3.1 Hz, 2H); 13C NMR (101 MHz,
DMSO) δ 181.19, 165.09, 149.42, 144.45, 140.21, 137.09, 135.12, 133.84, 130.13,
129.00, 126.20, 123.45, 122.03, 119.24; HRMS calcd for C23H17ClN3O2 (M+H)+
402.10038, found 402.10038.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-2-bromobenza
1
mide (6e). Yellow solid; yield: 64%; m.p.: 276.2–278.1 °C; H NMR (400 MHz,
DMSO) δ 10.70 (s, 1H), 8.33 (s, 1H), 8.13 (d, J = 16.1 Hz, 1H), 7.96 (d, J = 16.1 Hz,
1H), 7.86 (d, J = 8.0 Hz, 1H), 7.75 (dd, J = 7.9, 0.9 Hz, 3H), 7.65–7.58 (m, 2H),
7.55–7.50 (m, 2H), 7.48–7.43 (m, 1H), 7.40–7.37 (m, 2H); 13C NMR (101 MHz,
DMSO) δ 181.17, 166.56, 149.40, 144.36, 140.16, 139.37, 135.26, 133.24, 131.80,
130.23, 129.38, 128.23, 126.05, 122.70, 122.14, 119.46, 118.52; HRMS calcd for
C23H17BrN3O2 (M+H)+ 446.04987, found 446.04990.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3-bromobenza
1
mide (6f). Yellow solid; yield: 45%; m.p.: 233.2–235.1 °C; H NMR (400 MHz,
DMSO) δ 10.56 (s, 1H), 8.36 (s, 1H), 8.21 (s, 1H), 8.15 (d, J = 16.0 Hz, 1H), 8.02 (d,
J = 7.7 Hz, 1H), 7.96 (t, J = 7.9 Hz, 2H), 7.81 (d, J = 7.8 Hz, 1H), 7.76 (dd, J = 5.7,
3.0 Hz, 2H), 7.59 (d, J = 7.5 Hz, 1H), 7.51 (m, 2H), 7.40 (dd, J = 6.0, 3.0 Hz, 2H);
13C NMR (101 MHz, DMSO) δ 180.77, 164.62, 149.02, 144.68, 140.13, 137.28,
135.09, 134.94, 131.15, 130.78, 127.39, 126.18, 125.26, 123.58, 122.21, 122.02,
119.54; HRMS calcd for C23H17BrN3O2 (M+H)+ 446.04987, found 446.04982.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-4-bromobenza
1
mide (6g). Yellow solid; yield: 60%; m.p.: 282.2–283.7 °C; H NMR (400 MHz,
DMSO) δ 10.50 (s, 1H), 8.35 (s, 1H), 8.14 (d, J = 16.1 Hz, 1H), 7.96 (t, J = 7.9 Hz,
4H), 7.78 (d, J = 8.4 Hz, 4H), 7.56 (d, J = 7.6 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.38
(dd, J = 5.8, 2.8 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 181.21, 165.20, 149.44,
144.42, 140.20, 135.12, 134.21, 131.94, 130.30, 130.05, 126.22, 126.04, 123.44,
122.04, 119.21; HRMS calcd for C23H17BrN3O2 (M+H)+ 446.04987, found
446.04991.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-2-methylbenza
1
mide (6h). Yellow solid; yield: 55%; m.p.: 264.1–265.9 °C; H NMR (400 MHz,

DMSO) δ 13.53 (s, 1H), 10.49 (s, 1H), 8.36 (s, 1H), 8.12 (d, J = 16.1 Hz, 1H), 7.95 (d,
J = 16.0 Hz, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 7.7
Hz, 1H), 7.53 (d, J = 7.2 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.42 (t, J = 7.3 Hz, 2H),
7.53 (m, 3H), 2.44 (s, 3H); 13C NMR (101 MHz, DMSO) δ 181.25, 168.58, 149.45,
144.49, 143.52, 140.49, 137.41, 135.82, 135.32, 135.16, 131.06, 130.27, 130.11,
127.75, 126.30, 126.14, 125.81, 123.68, 122.79, 122.03, 121.64, 118.06, 113.38,
19.85; HRMS calcd for C24H20N3O2 (M+H)+ 382.15500, found 382.15477.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3-methylbenza
1
mide (6i). Yellow solid; yield: 56%; m.p.: 225.4–227.2 °C; H NMR (400 MHz,
DMSO) δ 10.42 (s, 1H), 8.38 (s, 1H), 8.18 (d, J = 16.1 Hz, 1H), 8.01 (d, J = 7.8 Hz,
1H), 7.94 (d, J = 16.1 Hz, 1H), 7.83(t, J = 7.4 Hz, 2H), 7.74 (dd, J = 5.8, 3.1 Hz, 2H),
7.55 (d, J = 7.4 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 6.4 Hz, 2H), 7.34 (dd, J
= 6.0, 3.0 Hz, 2H), 2.42 (s, 3H); 13C NMR (101 MHz, DMSO) δ 181.58, 166.31,
150.31, 144.13, 140.46, 138.23, 135.18, 132.78, 129.98, 128.80, 128.64, 125.96,
125.35, 124.61, 123.27, 122.32, 119.08, 21.46; HRMS calcd for C24H20N3O2 (M+H)+
382.15500, found 382.15494.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-4-methylbenza
1
mide (6j). Yellow solid; yield: 52%; m.p.: 264.4–266.2 °C; H NMR (400 MHz,
DMSO) δ 10.36 (s, 1H), 8.38 (s, 1H), 8.15 (d, J = 16.1 Hz, 1H), 8.02–7.94 (m, 4H),
7.76 (s, 2H), 7.55 (d, J = 7.6 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.38 (m, 4H), 2.40 (s,
3H); 13C NMR (101 MHz, DMSO) δ 181.19, 166.01, 149.42, 144.57, 142.28, 140.50,
135.07, 132.26, 129.98, 129.44 , 128.21, 125.98, 123.41, 121.95, 119.13, 21.50;
HRMS calcd for C24H20N3O2 (M+H)+ 382.15500, found 382.15500.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-2-(trifluoromet
1
hyl)benzamide (6k). Yellow solid; yield: 51%; m.p.: 200.4–202.1 °C; H NMR (400
MHz, DMSO) δ 10.65 (s, 1H), 8.36 (d, J = 0.88 Hz, 2H), 8.33 (d, J = 8.0 Hz, 1H),
8.16 (d, J = 16.1 Hz, 1H), 8.01–7.96 (m, 3H), 7.83 (t, J = 7.8 Hz, 1H), 7.75 (s, 2H),
7.59 (d, J = 7.7 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.38 (dd, J = 6.1, 3.1 Hz, 2H); 13C
NMR (101 MHz, DMSO) δ 181.21, 164.69, 149.44, 144.37, 140.05, 136.03, 135.17,
132.35, 130.24, 130.08, 126.39, 124.79, 124.75, 124.71, 123.56, 122.10, 119.37;
HRMS calcd for C24H17F3N3O2 (M+H)+ 436.12674, found 436.12696.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-2-methoxybenz
1
amide (6l). Yellow solid; yield: 58%; m.p.: 225.9–227.4 °C; H NMR (400 MHz,
DMSO) δ 10.29 (s, 1H), 8.33 (s, 1H), 8.11 (d, J = 16.1 Hz, 1H), 7.94 (d, J = 16.1 Hz,
1H), 7.86 (d, J = 8.0 Hz, 1H), 7.75 (s, 2H), 7.66 (d, J = 7.4 Hz, 1H), 7.56–7.44 (m,
3H), 7.36 (dd, J = 5.9, 2.9 Hz, 2H), 7.17 (d, J = 8.4 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H),
3.91 (s, 3H); 13C NMR (101 MHz, DMSO) δ 181.25, 165.32, 156.96, 149.42, 144.53,
140.25, 135.18, 132.61, 130.12, 130.06, 125.56, 125.26, 122.87, 122.05, 120.94,
118.91, 112.43, 56.36; HRMS calcd for C24H20N3O3 (M+H)+ 398.14992, found
398.17975.

(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-4-methoxybenz
1
amide (6m). Yellow solid; yield: 63%; m.p.: 271.0–272.4 °C; H NMR (400 MHz,
DMSO) δ 10.27 (s, 1H), 8.36 (s, 1H), 8.13 (d, J = 16.1 Hz, 1H), 8.03–7.93 (m, 4H),
7.74 (s, 2H), 7.52 (d, J = 7.7 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.37 (dd, J = 6.1, 3.1
Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H); 13C NMR (101 MHz, DMSO) δ
181.20, 165.54, 162.50, 149.43, 144.58, 140.59, 135.03, 130.11, 129.94, 127.14,
125.86, 123.38, 121.90, 119.04, 114.11, 55.89; HRMS calcd for C24H20N3O3 (M+H)+
398.14992, found 368.14978.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3,5-dichlorobe
1
nzamide (6n). Yellow solid; yield: 36%; m.p.: 282.4–284.6 °C; H NMR (400 MHz,
DMSO) δ 10.59 (s, 1H), 8.32 (s, 1H), 8.13 (d, J = 16.1 Hz, 1H), 8.03 (s, 2H), 7.95 (t,
J = 9.7 Hz, 2H), 7.87 (s, 1H), 7.74 (s, 2H), 7.58 (d, J = 7.5 Hz, 1H), 7.49 (t, J = 7.8
Hz, 1H), 7.38 (dd, J = 5.9, 2.9 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 181.07,
163.31, 149.31, 144.38, 139.86, 138.29, 135.15, 134.82, 131.55, 130.09, 127.02,
126.47, 125.07, 123.48, 122.09, 119.34; HRMS calcd for C23H16Cl2N3O2 (M+H)+
436.06141, found 436.06139.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3,5-dimethylbe
1
nzamide (6o). Yellow solid; yield: 51%; m.p.: 220.8–222.4 °C; H NMR (400 MHz,
DMSO) δ 10.38 (s, 1H), 8.37 (s, 1H), 8.16 (d, J = 16.0 Hz, 1H), 7.98 (t, J = 16.1 Hz,
2H), 7.77 (dd, J = 6.1, 3.2 Hz, 2H), 7.63 (s, 2H), 7.57 (d, J = 7.7 Hz, 1H), 7.48 (t, J =
7.9 Hz, 1H), 7.41 (dd, J = 6.2, 3.1 Hz, 2H), 7.23 (s, 1H), 2.37 (s, 6H); 13C NMR (101
MHz, DMSO) δ 180.69, 166.40, 148.94, 144.88, 140.51, 138.06, 135.15, 135.02,
133.47, 129.97, 125.88, 125.84, 125.32, 123.47, 121.91, 119.42, 21.35; HRMS calcd
for C25H22N3O2 (M+H)+ 396.17065, found 396.17045.
General procedure for preparation of intermediates (7a–i)
According to the procedure for 5a–o, the intermediates 7a–i were obtained from
intermediate 4 with benzene sulfonyl chloride. The spectral data are summarized in
the Supplementary Information.
General procedure for preparation of target compounds (8a–i)
According to the procedure for compounds 6a–i, target compounds 8a–i were
obtained from intermediate 4 and 7a–i in the presence of NaOH in ethanol.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-2-chlorobenzen
1
esulfonamide (8a). Yellow solid; yield: 53%; m.p.: 257.3–259.1 °C; H NMR (400
MHz, DMSO) δ 13.52 (s, 1H), 10.85 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.00 (d, J =
16.1 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 16.1 Hz, 1H), 7.64 (d, J = 6.1 Hz,
2H), 7.61–7.55 (m, 3H), 7.49 (d, J = 7.7 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.35 (t, J =
7.8 Hz, 2H), 7.22 (d, J = 8.1 Hz, 1H); 13C NMR (101 MHz, DMSO) δ 181.15, 149.36,
143.75, 143.51, 138.35, 136.76, 135.68, 135.34, 132.44, 132.17, 131.23, 130.61,

128.33, 126.34, 125.34, 123.72, 122.57, 121.74, 121.70, 118.52, 113.38; HRMS calcd
for C22H17ClN3O3S (M+H)+ 438.06737, found 438.06733.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3-chlorobenzen
1
esulfonamide (8b). Yellow solid; yield: 47%; m.p.: 209.6–210.9 °C; H NMR (400
MHz, DMSO) δ 13.52 (s, 1H), 10.58 (s, 1H), 8.02 (d, J = 16.1 Hz, 1H), 7.87 (t, J =
16.0, 6.3 Hz, 2H), 7.82 (t, J = 1.7 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.74–7.71 (m,
1H), 7.62 (d, J = 7.9 Hz, 1H), 7.61–7.56 (m, 3H), 7.42 (m, 2H), 7.34 (t, J = 7.9 Hz,
1H), 7.24 (dd, J = 8.2, 1.2 Hz, 1H) ; 13C NMR (101 MHz, DMSO) δ 181.17, 149.34,
143.72, 143.50, 141.54, 138.49, 135.79, 135.31, 134.40, 133.61, 131.95, 130.72,
126.71, 126.35, 125.97, 125.93, 123.71, 123.19, 122.61, 121.68, 120.18, 113.38;
HRMS calcd for C22H17ClN3O3S (M+H)+ 438.06737, found 438.06737.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-4-chlorobenzen
1
esulfonamide (8c). Yellow solid; yield: 58%; m.p.: 261.7–263.8 °C; H NMR (400
MHz, DMSO) δ 10.58 (s, 1H), 8.02 (d, J = 16.1 Hz, 1H), 7.88–7.83 (m, 2H), 7.81 (s,
1H), 7.74 (s, 2H), 7.65 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 6.9 Hz, 2H), 7.40–7.36 (m,
3H), 7.23 (d, J = 7.5 Hz, 1H); 13C NMR (101 MHz, DMSO) δ 181.15, 149.34, 143.77,
138.65, 138.59, 138.46, 135.76, 130.67, 130.03, 129.13, 125.80, 123.02, 122.60,
119.97; HRMS calcd for C22H17ClN3O3S (M+H)+ 438.06737, found 438.06729.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-2-methylbenzen
1
esulfonamide (8d). Yellow solid; yield: 53%; m.p.: 258.4–260.0 °C; H NMR (400
MHz, DMSO) δ 13.51 (s, 1H), 10.63 (s, 1H), 8.01 (d, J = 8.6 Hz, 1H), 7.99 (s, 1H),
7.90 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 16.1 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.54 (s,
1H), 7.52 (d, J = 15.4 Hz , 1H), 7.46 (d, J = 8.2 Hz , 1H), 7.41 (t, J = 7.5 Hz, 3H),
7.34–7.38 (m, 2H), 7.20 (d, J = 8.0 Hz, 1H), 2.63 (s, 3H); 13C NMR (101 MHz,
DMSO) δ 181.15, 149.37, 143.82, 143.51, 138.87, 137.84, 137.31, 135.63, 135.32,
133.70, 133.19, 130.57, 129.93, 126.90, 126.34, 125.02, 123.71, 122.48, 121.68,
121.47, 118.07, 113.38, 20.21; HRMS calcd for C23H20N3O3S (M+H)+ 418.12199,
found 418.12181.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3-methylbenzen
1
esulfonamide (8e). Yellow solid; yield: 58%; m.p.: 244.5–256.2°C; H NMR (400
MHz, DMSO) δ 13.52 (s, 1H), 10.47 (s, 1H), 8.01 (d, J = 16.1 Hz, 1H), 7.87 (dd, J =
16.4, 8.2 Hz, 2H), 7.68 (s, 1H), 7.61 (t, J = 8.2 Hz, 2H), 7.56 (s, 1H), 7.51 (d, J = 7.7
Hz, 1H), 7.46–7.41 (m, 3H), 7.35 (t, J = 8.3 Hz, 2H), 7.24 (d, J = 8.0 Hz, 1H), 2.36 (s,
3H); 13C NMR (101 MHz, DMSO) δ 181.16, 149.36, 143.85, 143.51, 139.76, 139.57,
139.01, 135.62, 135.32, 134.19, 130.57, 129.68, 127.39, 126.33, 125.64, 124.36,
123.71, 122.74, 122.45, 121.67, 119.26, 113.38, 21.29; HRMS calcd for C23H20N3O3S
(M+H)+ 418.12199, found 418.12192.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-4-methylbenzen
1
esulfonamide (8f). Yellow solid; yield: 40%; m.p.: 283.0–284.8°C; H NMR (400

MHz, DMSO) δ 10.42 (s, 1H), 8.00 (d, J = 16.1 Hz, 1H), 7.84 (d, J = 16.1 Hz, 1H),
7.71 (d, J = 8.1 Hz, 4H), 7.56 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.6 Hz,
5H), 7.23 (d, J = 8.0 Hz, 1H), 2.31 (s, 3H); 13C NMR (101 MHz, DMSO) δ 181.16,
149.35, 143.93, 143.88, 139.09, 136.91, 135.62, 130.56, 130.26, 127.24, 125.50,
122.67, 122.46, 119.41, 21.44 ; HRMS calcd for C23H20N3O3S (M + H)+ 418.12199,
found 418.12197.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3,5-dichlorobe
1
nzenesulfonamide (8g). Yellow solid; yield: 50%; m.p.: 262.1–264.3 °C; H NMR
(400 MHz, DMSO) δ 13.52 (s, 1H), 10.66 (s, 1H), 8.04 (d, J = 16.1 Hz, 1H), 7.94 (s,
1H), 7.88 (d, J = 16.0 Hz 2H), 7.77 (s, 2H), 7.63–7.57 (m, 3H), 7.42 (dd, J = 10.0, 5.7
Hz, 2H), 7.35 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H); 13C NMR (101 MHz,
DMSO) δ 181.18, 149.34, 143.62, 142.70, 138.07, 135.91, 135.63, 133.32, 130.81,
126.25, 125.71, 123.52, 122.72, 120.73; HRMS calcd for C22H16Cl2N3O3S (M+H)+
472.02839, found 472.02839.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3,5-dimethylbe
1
nzenesulfonamide (8h). Yellow solid; yield: 41%; m.p.: 258.6–259.8 °C; H NMR
(400 MHz, DMSO) δ 10.40 (s, 1H), 8.00 (d, J = 16.1 Hz, 1H), 7.84 (d, J = 16.2 Hz,
2H), 7.63–7.53 (m, 4H), 7.49 (d, J = 7.3 Hz, 1H), 7.38–7.32 (m, 3H), 7.30 (d, J = 7.9
Hz, 1H), 7.24 (d, J = 7.6 Hz, 1H), 2.25 (s, 3H), 2.21 (s, 3H); 13C NMR (101 MHz,
DMSO) δ 181.13, 149.33, 143.87, 142.76, 139.12, 138.15, 137.10, 135.55, 130.58,
130.51, 127.77, 125.47, 124.74, 122.55, 122.37, 119.04, 19.81, 19.80; HRMS calcd
for C24H22N3O3S (M+H)+ 432.13764, found 432.13763.
(E)-N-(3-(3-(1H-benzo[d]imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-3,4-dimethylbe
nzenesulfonamide (8i). Yellow solid; yield: 53%; m.p.: 275.2–277.3 °C; 1H NMR (400
MHz, DMSO) δ 13.52 (s, 1H), 10.42 (s, 1H), 8.01 (d, J = 16.1 Hz, 1H), 7.86 (t, J =
16.2, 9.9 Hz, 2H), 7.59 (d, J = 8.1 Hz, 1H), 7.55 (s, 1H), 7.50 (d, J = 7.8 Hz, 1H),
7.47 (s, 2H), 7.42 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.36–7.33 (m, 1H),
7.25–7.22 (m, 2H), 2.32 (s, 6H); 13C NMR (101 MHz, DMSO) δ 181.15, 149.36,
143.89, 143.50, 139.76, 139.36, 139.06, 135.58, 135.32, 134.93, 130.57, 126.34,
125.69, 124.65, 123.72, 122.70, 122.38, 121.65, 118.90, 113.39, 21.19; HRMS calcd
for C24H22N3O3S (M+H)+ 432.13764, found 432.13759.
5.3. Pharmacology
5.3.1 MTT assay in vitro
The anti-tumor activities of compounds 6a–o and 8a–i were evaluated against
HCT116, MCF7 and 143B cell lines in vitro using a standard MTT assay, with
odanacatib and 5-FU as the positive controls. Compounds were tested at six
concentrations (0.001–100 µM).
The tumor cell lines were cultured in RPMI 1640 medium (Sigma, Germany),
supplemented with 10% fetal bovine serum (FBS, Sigma). Approximately 4×103 cells,

suspended in RPMI 1640 medium, were plated into each well of a 96-well plate and
incubated at 37°C for 24 h in 5% CO2. The test compounds were then added to the
culture medium and the cells were incubated for a further 48 h. Fresh MTT was added
to each well at the terminal concentration (0.5 mg/mL) and the cells were incubated at
37°C for 4 h in 5% CO2. DMSO (150 µL) was then added to each well to dissolve the
formazan crystals and the absorbance was measured at 540 nm using a microplate
reader (HEALES MB-580, China). The results, expressed as IC50 values, were the
average of three determinations and were calculated using SPSS software.
5.3.2 Molecular docking
The X-ray crystal structure of Cat K (PDB code 1NLJ) was used for the docking
studies. 1NLJ was processed using the Protein Preparation protocol of Sybyl-X2.0.
Firstly, the ligand was extracted from the complex to determine the location of the
active site. Secondly, backbones and sidechains of the protein were repaired. Thirdly,
all hydrogen atoms were added and the protein was protonated at pH 7.4. Fourthly, an
Amber7 FF99 force field was applied to the biopolymer. A Surflex-Dock Control File
containing information about the protein and the active site was then defined with a
threshold value of 0.5. The maximum number of poses per ligand was set to 10. The
ligand in the crystal structure was defined as the reference molecule. Finally,
structure-based docking was carried out using the Surflex-Dock Geom mode of
Sybyl-X2.0.
The docking studies for compound 8e with other cysteine cathepsins (CatB, CatL,
CatS, CatF, CatH, CatV and CatX) were performed using the same method as for Cat
K.
5.3.3 In vitro enzymatic assays
The Cat K inhibitory activity of the compounds was determined using a previously
described method,43 with minor modifications. Compounds were screened at 5 µM to
evaluate the enzymatic inhibition.
Recombinant human Cat K (5.5 ng/mL) in reaction buffer (50 µL) containing
sodium acetate (50 mM), EDTA (2.5 mM) and DTT (1 mM) was added to a 96-well
plate containing the test compounds at pH 6.0. The plate was incubated at 37°C for 15
min and the reaction was then initiated by adding a solution of the substrate,
Z-Phe-Arg-AMC (20 µL, 10 µM). After 30 min, the fluorescence intensity was
measured (excitation at 355 nm; emission at 460 nm) using a microplate reader
(Wallac 1420 Victor2, Perkin-Elmer, USA). The inhibition rate was determined using
the fluorescence intensity.
The Cat L, Cat S and Cat B inhibitory assays were carried out using a similar
procedure to Cat K, except that the Cat S and Cat B substrates were replaced with
Ac-Lys-Gln-Lys-Leu-Arg-AM.C and Z-Arg-Arg-AMC, respectively.
Acknowledgements
The work was supported by the National Natural Science Foundation of China
(81473097), Student Fund of Innovation Project of Peking Union Medical College

(2015-1007-22), and the CAMS initiative for Innovative Medicine (2016-I2M-3-022).
Supporting information
The interactions between 6i and enzymes (Cat K and Cat L) were presented in Figure
S1. The enzyme activity of the compound without benzenesulfonamide or benzamide
group was listed in Table S1. The spectral data of intermidiates 5a–o and 7a–i are
1
summarized, followed by the H NMR spectrum, 13C NMR spectrum and high
resolution mass spectrum of target compounds 6a–o and 8a–i.
References
1. Vasiljeva O, Turk B. Biochimie. 2008; 90: 380-386.
2. Gocheva V, Joyce JA. Cell Cycle. 2007; 6: 60-64.
3. Jedeszko C, Sloane BF. Biol Chem. 2004; 385: 1017-1027.
4. Joyce JA, Hanahan D. Cell Cycle. 2004; 3: 1516-1519.
5. Mohamed MM, Sloane BF. Nat Rev Cancer. 2006; 6: 764-775.
6. Yasuda Y, Kaleta J, Bromme D. Adv Drug Deliv Rev. 2005; 57: 973-993.
7. Pozgan U, Caglic D, Rozman B, et al. Biol Chem. 2010; 391: 571-579.
8. Cheng XW, Huang Z, Kuzuya M, et al. Hypertension. 2011; 58: 978-986.
9. Kos J, Lah TT. Oncology Reports. 1998; 5: 1349-1361.
10. dos Santos DA, Deobald AM, Cornelio VE, et al. Bioorg Med Chem. 2017; 25:
4620-4627.
11. Tamhane T, Lllukkumbura R, Lu S, et al. Biochimie. 2016; 122: 208-218.
12. Lah TT, Kos J, Blejec A, et al. Pathol Oncol Res. 1997; 3: 89-99.
13. Chen Q, Fei J, Wu L, et al. Oncol Lett. 2011; 2: 693-699.
14. Frohlich E, Schlagenhauff B, Mohrle M, et al. Cancer. 2001; 91: 972-982.
15. Singh N, Das P, Datta Gupta S, et al. Cancer Invest. 2013; 31: 461-471.
16. Quintanilla-Dieck MJ, Codriansky K, Keady M, et al. J Invest Dermatol. 2008;
128: 2281-2288.
17. Herroon MK, Rajagurubandara E, Rudy DL, et al. Oncogene. 2013; 32:
1580-1593.
18. Lindeman JHN, Hanemaaijer R, Mulder A, et al. The American Journal of
Pathology. 2004; 165: 593-600.
19. Ishida M, Kojima F, Okabe H. J Eur Acad Dermatol Venereol. 2013; 27: 128-130.
20. Littlewood-Evans AJ, Bilbe G, Bowler WB, et al. Cancer Research. 1997; 57:
5386-5390.
21. Husmann K, Muff R, Bolander ME, et al. Mol Carcinog. 2008; 47: 66-73.
22. Jensen AB, Wynne C, Ramirez G, et al. Clin Breast Cancer. 2010; 10: 452-458.
23. Schurigt U, Sevenich L, Vannier C, et al. Biol Chem. 2008; 389: 1067-1074.
24. Le Gall C, Bellahcene A, Bonnelye E, et al. Cancer Research. 2007; 67:
9894-9902.
25. Raghav N, Singh M. Eur J Pharm Sci. 2014; 54: 28-39.
26. Kim SH, Lee E, Baek KH, et al. Bioorg Med Chem Lett. 2013; 23: 3320-3324.
27. Garg S, Raghav N. RSC Adv. 2015; 5: 72937-72949.
28. Ramalho SD, Bernades A, Demetrius G, et al. Chem Biodivers. 2013; 10:

1999-2006.
29. Raghav N, Singh M. Bioorg Med Chem. 2014; 22: 4233-4245.
30. Raghav N, Kaur R. Int J Biol Macromol. 2015; 80: 710-724.
31. Parker EN, Song J, Kishore Kumar GD, et al. Bioorg Med Chem. 2015; 23:
6974-6992.
32. Sahu NK, Balbhadra SS, Choudhary J, et al. Curr Med Chem. 2012; 19: 209-225.
33. Deb Majumdar I, Devanabanda A, Fox B, et al. Biochem Biophys Res Commun.
2011; 416: 397-402.
34. Gauthier JY, Chauret N, Cromlish W, et al. Bioorg Med Chem Lett. 2008; 18:
923-928.
35. Ahmad S, Siddiqi MI. J Mol Model. 2017; 23: 92-108.
36. Yamamoto A, Tomoo K, Hara T, et al. J Biochem. 2000; 127: 635-643.
37. Szcześniak P, Pieczykolan M，Stecko S. J Org Chem. 2016; 81: 1057-1074.
38. Schroder J, Klinger A, Oellien F, et al. J Med Chem. 2013; 56: 1478-1490.
39. Wu LT, Jiang Z, Shen JJ, et al. Eur J Med Chem. 2016; 114: 328-336.
40. Reddy VM, REddy KR. Chem Pharm Bull. 2010; 58: 953-956.
41. Vazquez ME, Blanco JB, Imperiali B. J Am Chem Soc. 2005; 127: 1300-1306.
42. Cai X, Qian CG, Zhai HX, US 20140018368A1.
43. Bromme D, Steinert A, Friebe S, et al. Biochem J. 1989; 264: 475-481.