Structure-activity relationships of novel dithiocarbamates containing α,β-unsaturated ketone fragment as potent anticancer agents

Article abstract of DOI:10.1007/s00044-019-02356-y

Based on the structure of novel lead compound 8 discovered by our group, systematic structural modification was carried out. A series of compound 8’s derivatives were synthesized and evaluated for their activities against human non-small cell lung cancer cell line H460. Among them, twelve compounds showed significant proliferation inhibition activities with IC₅₀ values <1 μM and compound 12r was the most potent one. Further study on compound 12r revealed that it has significant inhibitory effect on the growth of eight kinds of cancer cell lines with IC₅₀ values <1 μM. Especially for cell lines A375 and HCT116, the IC₅₀ values of 12r achieved 63 nM and 66 nM, respectively. Meanwhile, our research results also revealed the following structure-activity relationships: (a) different substitutions on the benzene ring or heteroaromatic rings greatly effect on the activity; (b) the presence of α,β-unsaturated ketone fragment is favorable for the activity; (c) the receptor cavity binding with amine moiety might be a relatively small hydrophobic cavity. These results will be valuable for the further development of this novel kind of dithiocarbamates.

Full text of DOI:10.1007/s00044-019-02356-y

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02356-y
ORIGINAL RESEARCH
Structure-activity relationships of novel dithiocarbamates
containing α,β-unsaturated ketone fragment as potent
anticancer agents
1
Yuanqiang Wang1 Ridong Li1 Han Zhang1 Zhiyong Zhang1 Xin Wang1 Zemei Ge1 Runtao Li
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Received: 23 February 2019 / Accepted: 2 May 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Based on the structure of novel lead compound 8 discovered by our group, systematic structural modification was carried
out. A series of compound 8’s derivatives were synthesized and evaluated for their activities against human non-small cell
lung cancer cell line H460. Among them, twelve compounds showed significant proliferation inhibition activities with IC50
values <1 μM and compound 12r was the most potent one. Further study on compound 12r revealed that it has significant
inhibitory effect on the growth of eight kinds of cancer cell lines with IC50 values <1 μM. Especially for cell lines A375 and
HCT116, the IC50 values of 12r achieved 63 nM and 66 nM, respectively. Meanwhile, our research results also revealed the
following structure-activity relationships: (a) different substitutions on the benzene ring or heteroaromatic rings greatly effect
on the activity; (b) the presence of α,β-unsaturated ketone fragment is favorable for the activity; (c) the receptor cavity
binding with amine moiety might be a relatively small hydrophobic cavity. These results will be valuable for the further
development of this novel kind of dithiocarbamates.
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Keywords Dithiocarbamates α,β-unsaturated ketone Anticancer H460
Introduction
Ronconi et al. 2013; Bala et al. 2014; Li et al. 2015a),
especially anti-tumor activity (Gaspari et al. 2006; Huang
et al. 2009; Duan et al. 2013a; Duan et al. 2013b; Akin-
boye et al. 2015; Ding et al. 2016; Fu et al. 2016; Fu et al.
2017; Laskar et al. 2018; Liu et al. 2017; Skrott et al.
2017; Wei et al. 2018; Xie et al. 2018; Yang et al. 2018).
Some representative dithiocarbamates with anticancer
activities reported in recent were listed in Fig. 1. Dis-
ulfiram (1), an alcohol-abuse drug, was found to be
effective against diverse cancer types and targets cancer
via p97 segregase adaptor NPL4 and this result was
published in Nature (Skrott et al. 2017). Compound 2, a
novel 2-aminobenzamide containing dithiocarbamate
moiety as histone deacetylase inhibitors, displayed potent
antiproliferative activity against diverse human tumor cell
lines (Xie et al. 2018). Thieno[2,3-d]pyrimidine derivative
3 exhibited cytotoxicity in cancer cells by targeting
tubulin to activate the spindle assembly checkpoint (Yang
et al. 2018). (1S, 4S)-2,5-diazabicyclo[2.2.1]heptane-
dithiocarbamate-nitrostyrene hybrid (4) was identified as
potent antiproliferative and apoptotic inducing agent
against cervical cancer cell lines (Laskar et al. 2018).
Formononetin-dithiocarbamate hybrid (5) could inhibit
Discovery of lead compounds is one of the key steps in the
new drug development. Though the target-based drug dis-
covery (TDD) has been the dominant approach to drug
discovery in the past three decades, phenotypic drug dis-
covery (PDD) approach is still a way not to be ignored due
to their promise of delivering first in-class drugs (Swinney
and Anthony 2011).
Dithiocarbamate represents a privileged scaffold in
medicinal chemistry and this kind of compounds showed
a broad spectrum of biological activities (Buac et al. 2012;
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02356-y) contains supplementary
material, which is available to authorized users.
* Runtao Li
lirt@bjmu.edu.cn
1
State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing 100191, PR
China

Medicinal Chemistry Research
Fig. 1 Structures of some dithiocarbamates with anticancer activities
Fig. 2 The structure and the
activities of lead compound 8
growth and migration of PC-3 cells via MAPK/Wnt sig-
naling pathways (Fu et al. 2017). Our group have also
been interested in dithiocarbamates, and several novel
kinds of them were found to show excellent antitumor
activities (Hou et al. 2006; Li et al. 2011; Li et al. 2013; Li
et al. 2015b; Zhang et al. 2015; Li et al. 2018), such as
compounds 6 (Zhang et al. 2015) and 7 (Li et al. 2018) as
potent PKM2 inhibitor.
Recently, we identified compound 8 with significant
antitumor activities (Fig. 2) from a random screening of
an in-house compound library (about 1000 in total). As
far as we know, this is a new kind of dithiocarbamates
which contains α,β-unsaturated ketone fragment. Mean-
while, α,β-unsaturated ketone fragment is an important
pharmacophores (Adams et al. 2004; Chen et al. 2009;
Wu et al. 2010; Butturini et al. 2013), and it appears in
the structure of many drugs, such as Afatinib (9) (Dungo
and Keating 2013), Ibrutinib (10) (Byrd et al. 2013), and
Osimertinib (11) (Greig 2016) (Fig. 3). Thus, compound
8 is a valuable lead compound for further study. In this
paper, the structure activity relationships of compound 8
were investigated and more potent compound 12r was
identified.
Materials and methods
All commercial reagents and solvents were used without
further purification unless otherwise specified. Column
chromatography was conducted using silica gel. Thin-layer
chromatography (TLC) was performed on silica gel GF-254
aluminium plates. Visualization was carried out using UV
(254 nm/366 nm). 1H NMR and 13C NMR spectra were
recorded on Bruker AVANCEIII 400 MHz and 101 MHz
spectrometer respectively, and NMR spectra were refer-
enced to the residual solvent shifts for 1H and 13C as
internal standards, and δ-values are given in ppm. Mass
spectrometry was performed with a Bruker Apex IV FTMS
by using the electro spray ionization (ESI) mode. Melting
points were determined on X5 microscope and were
uncorrected.
Chemistry
The synthetic routes of target compounds 8, 12a–12 s, 13
and 14a–14 h are depicted in Scheme 1. Various aryl or
hetero aryl ketones, 15 and 16a–16 s which were com-
mercially
available,
reacted
respectively
with

Medicinal Chemistry Research
Fig. 3 Chemical structures of representative drugs with α,β-unsaturated ketone
Scheme 1 Synthesis of target compounds. Reaction conditions: (a) AcOH, dimethylamine hydrochloride, (HCHO)n, reflux; (b) H2O, CS2, K2CO3;
(c) H2O, K2CO3

Medicinal Chemistry Research
paraformaldehyde and dimethylamine hydrochloride in
refluxing acetic acid to form the corresponding key inter-
mediates 17 and 18a–18s (Butturini et al. 2013) which were
used directly without further purification. Then, dimethy-
lamine hydrochloride (19) was reacted with carbon disulfide
in water using potassium carbonate as base to afford the
intermediate 20. Finally, intermediates 17 and 18a–18s
were dissolved in the aqueous potassium carbonate solution,
and intermediate 20 was added dropwise to the above
solution. The resulted mixture was stirred for 1 h to provide
the corresponding targets 8 and 12a–12s. The synthetic
route of compound 13 was similar to that of compounds 8
and 12a–12s, and the only difference is the use of propio-
phenone (21) as starting material. Compounds 14a–14h
were gained by replacing the dimethylamine moiety of
compound 12m with different secondary amines.
2-(4-Methoxybenzoyl)allyl
dimethylcarbamodithioate (12a)
Yield 17%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.80
(d, J = 8.8 Hz, 2 H, Ar-H), 6.92 (d, J = 8.8 Hz, 2 H, Ar-H),
6.20 (s, 1 H, C = CHH), 5.71 (s, 1 H, C = CHH), 4.39 (s, 2
H, C-CH2-S), 3.86 (s, 3 H, O-CH3), 3.54 (s, 3 H, N-CH3),
3.35 (s, 3 H, N-CH3); 13C NMR (101 MHz, CDCl3): δ
196.4 (C = S), 195.6 (C = O), 163.3 (Ar-C), 143.0 (C–C =
CH2), 132.1 (Ar–C), 129.9 (Ar-C), 127.1 (C = CH2), 113.5
(Ar-C), 55.5 (O-CH3), 45.5 (N-CH3), 41.5 (N-CH3), 39.90
(C-CH2-S); HRMS m/z (pos): 296.0781 C14H18NO2S2
(calcd. 296.0774).
2-(2-Methoxybenzoyl)allyl
dimethylcarbamodithioate (12b)
General method for synthesis of final compounds
Yield 21%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.39
(t, J = 7.9 Hz, 1 H, Ar-H), 7.25 (d, J = 7.4 Hz, 1 H, Ar–H),
6.95 (dd, J = 15.0, 7.8 Hz, 2 H, Ar–H), 6.32 (s, 1 H, C =
CHH), 5.76 (s, 1 H, C = CHH), 4.40 (s, 2 H, C-CH2-S),
3.77 (s, 3 H, O-CH3), 3.54 (s, 3 H, N–CH3), 3.37 (s, 3 H,
N–CH3); 13C NMR (101 MHz, CDCl3) δ 197.0 (S-C = S),
196.9 (C = O), 157.2 (Ar-C), 144.4 (C–C = CH2), 131.7
(Ar-C), 130.9 (Ar-C), 129.2 (Ar–C), 128.4 (Ar–C), 120.2
(C = CH2), 110.5 (Ar–C), 55.7 (O-CH3), 45.5 (N-CH3),
41.5 (N–CH3), 36.9 (C–CH2-S); HRMS m/z (pos):
296.0782 C14H18NO2S2 (calcd. 296.0774).
Aryl or heteroaryl ketone (15, 16a–16s or 21, 10 mmol) was
added into the mixture of paraformaldehyde (0.60 g,
20 mmol) and dimethylamine hydrochloride (1.63 g,
20 mmol) in acetic acid (25 mL). The reaction mixture was
heated under reflux for 12 h, followed by the removal of the
solvent to give the crude intermediate (17, 18a–18 s or 22).
A mixture of amine (19 or 23a–23h, 6 mmol), potassium
carbonate (1.38 g, 10 mmol) and H2O (15 mL) was stirred
for 15 min, and CS2 (0.38 g, 5 mmol) was added dropwise.
After stirring 1 h, 20 or 24a–24h was obtained. The inter-
mediate (17, 18a–18 s or 22) was dissolved in the aqueous
potassium carbonate solution (2.76 g, 20 mmol K2CO3 in
15 mL H2O), and 20 or 24a–24h was added dropwise to the
above solution. The reaction mixture was stirred at room
temperature for 1 h and then extracted with ethyl acetate
(10 mL × 3). The combined organic phase washed with
water (15 mL × 2), dried over anhydrous Na2SO4 and con-
centrated under vacuum to afford the crude product. The
crude product was purified by column chromatography to
give target compound 8, 12a–12s, 13 or 14a–14h.
2-(4-Hydroxybenzoyl)allyl
dimethylcarbamodithioate (12c)
Yield: 41%; mp 57.9–59.7 °C; 1H NMR (400 MHz, CDCl3)
δ 7.74 (d, J = 8.7 Hz, 2 H, Ar-H), 6.91 (d, J = 8.6 Hz, 2 H,
Ar-H), 6.22 (s, 1 H, C = CHH), 5.74 (s, 1 H, C = CHH),
4.40 (d, J = 8.9 Hz, 2 H, C-CH2-S), 3.55 (d, J = 11.5 Hz, 3
H, N-CH3), 3.34 (s, 3 H, N–CH3); 13C NMR (101 MHz,
CDCl3) δ 196.9 (S-C = S), 196.4 (C = O), 161.1 (Ar-C),
142.8 (C–C = CH2), 132.6 (Ar–C), 129.2 (Ar-C), 128.0 (C
= CH2), 115.4 (Ar–C), 45.6 (N–CH3), 41.6 (N–CH3), 38.9
(C–CH2-S; HRMS m/z (pos): 282.0623 C13H16NO2S2
(calcd. 282.0617).
2-Benzoylallyl dimethylcarbamodithioate (8)
1
Yield 25%; Yellow oil; H NMR (400 MHz, CDCl3): δ
7.73–7.75 (m, 2 H, Ar-H), 7.51–7.55 (m, 1 H, Ar-H),
7.41–7.44 (m, 2 H, Ar-H), 6.31 (s, 1 H, C = CHH), 5.78 (s,
1 H, C = CHH), 4.40 (s, 2 H, C-CH2-C = S), 3.55 (s, 3 H,
N-CH3), 3.35 (s, 3 H, N-CH3); 13C NMR (101 MHz,
CDCl3): δ 196.8 (C = S), 196.3 (C = O), 142.8 (C-C =
CH2), 137.3 (Ar-C), 132.2 (Ar-C), 129.4 (Ar-C), 129.0 (C
= CH2), 128.1 (Ar-C), 45.4 (N-CH3), 41.3 (N-CH3), 37.8
(C-CH2-S); HRMS m/z (pos): 266.0672 C13H16NO2S2
(calcd. 266.0668).
2-(4-Methylbenzoyl)allyl dimethylcarbamodithioate
(12d)
Yield: 23%; mp 51.3–52.7 °C; 1H NMR (400 MHz, CDCl3)
δ 7.65 (d, J = 8.1 Hz, 2 H, Ar–H), 7.21 (d, J = 8.0 Hz, 2 H,
Ar-H), 6.25 (s, 1 H, C = CHH), 5.74 (s, 1 H, C = CHH),
4.37 (s, 2 H, C-CH2-S), 3.52 (s, 3 H, N-CH3), 3.32 (s, 3 H,
N–CH3), 2.38 (s, 3 H, Ar-CH3); 13C NMR (101 MHz,
CDCl3) δ 196.6 (S-C = S), 196.4 (C = O), 143.2 (Ar-C),

Medicinal Chemistry Research
142.9 (C-C = CH2), 134.6 (Ar-C), 129.8 (Ar–C), 129.0 (Ar-
C), 128.4 (C = CH2), 45.4 (N–CH3), 41.5 (N–CH3), 38.2
(C–CH2–S), 21.7 (Ar–CH3); HRMS m/z (pos): 280.0831
C14H18NOS2 (calcd. 280.0824).
Ar-H), 8.09 (dd, J = 11.2, 4.5 Hz, 1 H, Ar-H), 7.67 (td, J =
8.0, 4.0 Hz, 1 H, Ar-H), 6.49–6.32 (m, 1 H, C = CHH),
5.80 (d, J = 6.3 Hz, 1 H, C = CHH), 4.40 (d, J = 14.8 Hz, 2
H, C-CH2-S), 3.57 (d, J = 10.5 Hz, 3 H, N-CH3), 3.39 (d,
J = 6.0 Hz, 3 H, N-CH3); 13C NMR (101 MHz, CDCl3) δ
195.9 (S-C = S), 194.6 (C = O), 148.1 (Ar-C), 143.0
(C-C = CH2), 135.2 (Ar-C), 130.0 (Ar-C), 129.6 (Ar-C),
129.6 (C = CH2), 126.7 (Ar-C), 124.3 (Ar-C), 45.6 (N-
CH3), 41.5 (N-CH3), 37.3 (C-CH2-S); HRMS m/z (pos):
311.0520 C13H15N2O3S2 (calcd. For 311.0519).
2-(3-Methylbenzoyl)allyl dimethylcarbamodithioate
(12e)
Yield: 29%; mp 33.5–37.0 °C; 1H NMR (400 MHz, CDCl3)
δ 7.60–7.46 (m, 2 H, Ar–H), 7.32 (dt, J = 14.9, 7.6 Hz, 2 H,
Ar–H), 6.31 (s, 1 H, C = CHH), 5.79 (s, 1 H, C = CHH),
4.41 (s, 2 H, C-CH2-S), 3.55 (s, 3 H, N-CH3), 3.36 (s, 3 H,
N-CH3), 2.39 (s, 3 H, Ar-CH3); 13C NMR (101 MHz,
CDCl3) δ 197.1 (S–C = S), 196.5 (C = O), 143.0 (C-C =
CH2), 138.1 (Ar–C), 137.4 (Ar–C), 133.2 (Ar–C), 129.9
(Ar-C), 129.2 (C = CH2), 128.1 (Ar-C), 126.9 (Ar–C), 45.6
(N-CH3), 41.5 (N–CH3), 38.0 (C–CH2-S), 21.4 (Ar–CH3);
HRMS m/z (pos): 280.0832 C14H18NOS2 (calcd. Found:
280.0824).
2-(2-Nitrobenzoyl)allyl dimethylcarbamodithioate
(12i)
Yield: 11%; mp 83.5–84.1 °C; 1H NMR (400 MHz, CDCl3)
δ 8.15 (d, J = 8.2 Hz, 1H, Ar-H), 7.73 (t, J = 7.4 Hz, 1 H,
Ar-H), 7.63 (t, J = 7.7 Hz, 1 H, Ar-H), 7.42 (d, J = 7.4 Hz,
1 H, Ar–H), 6.35 (s, 1 H, C = CHH), 5.55 (s, 1 H, C =
CHH), 4.43 (s, 2 H, C–CH2–S), 3.54 (s, 3 H, N–CH3), 3.40
(s, 3 H, N–CH3); 13C NMR (101 MHz, CDCl3) δ 196.4 (S–
C = S), 193.9 (C = O), 146.5 (Ar–C), 143.9 (C–C = CH2),
135.4 (Ar–C), 134.1 (Ar–C), 130.7 (Ar–C), 129.0 (C =
CH2), 124.5 (Ar–C), 45.7 (N–CH3), 41.6 (N–CH3), 36.2
(C–CH2–S); HRMS m/z (pos): 311.0514 C13H15N2O3S2
(calcd. 311.0519).
2-(Benzo[d][1,3]dioxole-5-carbonyl)allyl
dimethylcarbamodithioate (12f)
Yield: 34%; mp 57.7–58.5 °C; 1H NMR (400 MHz, CDCl3)
δ 7.38 (dd, J = 8.1, 1.7 Hz, 1 H, Ar–H), 7.29 (d, J = 1.6 Hz,
1 H, Ar–H), 6.82 (d, J = 8.1 Hz, 1 H, Ar–H), 6.18 (s, 1 H,
C = CHH), 6.03 (s, 2 H, O-CH2-O), 5.70 (s, 1 H, C =
CHH), 4.36 (s, 2 H, C-CH2-S), 3.54 (s, 3 H, N–CH3), 3.34
(s, 3 H, N–CH3); 13C NMR (101 MHz, CDCl3) δ 196.4 (S–
C = S), 195.2 (C = O), 151.6 (Ar–C), 147.9 (Ar–C), 142.9
(C–C = CH2), 131.6 (Ar–C), 127.2 (Ar–C), 126.3 (C =
CH2), 109.5 (O-CH2-O), 107.7 (Ar–C), 101.8 (Ar–C), 45.5
(N–CH3), 41.5 (N–CH3), 38.5 (C-CH2-S); HRMS m/z (pos):
310.0565 C14H16NO3S2 (calcd. 310.0566).
2-(4-Cyanobenzoyl)allyl dimethylcarbamodithioate
(12j)
Yield: 38%; mp 64.5–66.4 °C; 1H NMR (400 MHz, CDCl3)
δ 7.84 (d, J = 8.3 Hz, 2 H, Ar–H), 7.75 (d, J = 8.4 Hz, 2 H,
Ar–H), 6.42 (s, 1 H, C = CHH), 5.77 (s, 1 H, C = CHH),
4.39 (s, 2 H, C–CH2–S), 3.57 (s, 3 H, N–CH3), 3.39 (s, 3 H,
N–CH3); 13C NMR (101 MHz, CDCl3) δ 196.1 (S–C = S),
195.5 (C = O), 143.0 (Ar-C), 141.1 (C–C = CH2), 132.1
(Ar–C), 130.3 (Ar–C), 121.9 (Ar–C), 118.0 (C = CH2),
115.6 (Ar–C), 45.6 (N–CH3), 41.5 (N-CH3), 37.3 (C–
CH2–S); HRMS m/z (pos): 291.0617 C14H15N2OS2 (calcd.
291.06203).
2-(4-Nitrobenzoyl)allyl dimethylcarbamodithioate
(12g)
Yield: 17%; mp 65.1-67.3 °C; 1H NMR (400 MHz, CDCl3)
δ 8.29 (d, J = 8.5 Hz, 2 H, Ar-H), 7.88 (d, J = 8.5 Hz, 2 H,
Ar-H), 6.44 (s, 1 H, C = CHH), 5.79 (s, 1 H, C = CHH),
4.38 (s, 2 H, C-CH2-S), 3.57 (s, 3 H, N-CH3), 3.39 (s, 3 H,
N-CH3); 13C NMR (101 MHz, CDCl3) δ 196.0 (S-C = S),
195.3 (C = O), 149.8 (Ar-C), 143.2 (C-C = CH2), 142.7
(Ar-C), 130.6 (C = CH2), 130.4 (Ar-C), 123.5 (Ar-C), 45.6
(N-CH3), 41.5 (N-CH3), 37.1 (C-CH2-S); HRMS m/z (pos):
311.0514 C13H15N2O3S2 (calcd. 311.0519).
2-(4-Fluorobenzoyl)allyl dimethylcarbamodithioate
(12k)
Yield: 25%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.79
(dd, J = 8.7, 5.5 Hz, 2 H, Ar-H), 7.10 (t, J = 8.6 Hz, 2 H,
Ar-H), 6.27 (s, 1 H, C = CHH), 5.73 (s, 1 H, C = CHH),
4.37 (s, 2 H, C-CH2-S), 3.54 (s, 3 H, N–CH3), 3.35 (s, 3 H,
N-CH3); 13C NMR (101 MHz, CDCl3) δ 196.2 (S-C = S),
195.5 (C = O), 166.3 (Ar–C, J = 253.9 Hz), 143.0 (C-C =
CH2), 133.6 (Ar–C, J = 3.0 Hz), 132.2 (Ar-C, J = 9.1 Hz),
128.5 (C = CH2), 115.4 (Ar–C, J = 21.8 Hz), 45.5
(N–CH3), 41.5 (N–CH3), 37.9 (C–CH2–S); HRMS m/z
(pos): 284.0569 C13H15FNOS2 (calcd. 284.0574).
2-(3-Nitrobenzoyl)allyl dimethylcarbamodithioate
(12h)
Yield: 14%; mp 81.4-82.7 °C; 1H NMR (400 MHz, CDCl3)
δ 8.57 (dd, J = 12.4, 1.6 Hz, 1 H, Ar-H), 8.49–8.34 (m, 1 H,

Medicinal Chemistry Research
2-(3-Fluorobenzoyl)allyl dimethylcarbamodithioate
(12l)
(N-CH3), 37.5 (C-CH2-S); HRMS m/z (pos): 333.9884
C13H14NCl2OS2 (calcd. 333.9888).
1
Yield: 21%; Yellow oil; H NMR (400 MHz, CDCl3) δ 7.49
2-(Furan-2-carbonyl)allyl dimethylcarbamodithioate
(12p)
(d, J = 7.7 Hz, 1 H, Ar-H), 7.45–7.35 (m, 2 H, Ar–H), 7.19
(td, J = 8.0, 2.2 Hz, 1 H, Ar–H), 6.29 (s, 1 H, C = CHH), 5.75
(s, 1 H, C = CHH), 4.33 (s, 2 H, C–CH2–S), 3.50 (s, 3 H,
N–CH3), 3.32 (s, 3 H, N–CH3); 13C NMR (101 MHz, CDCl3)
δ 196.0 (S–C = S), 195.5 (C = O), 162.4 (Ar–C, J =
248.1 Hz), 142.8 (C–C = CH2), 139.5 (Ar–C, J = 6.4 Hz),
130.0 (Ar–C, J = 7.7 Hz), 129.5 (C = CH2), 125.4 (Ar–C,
J = 3.0 Hz), 119.3 (Ar–C, J = 21.4 Hz), 116.2 (Ar–C, J =
22.5 Hz), 45.5 (N–CH3), 41.46 (N–CH3), 37.7 (C–CH2–S);
HRMS m/z (pos): 284.0571 C13H15FNOS2 (calcd. 284.0574).
Yield: 15%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.65
(s, 1 H, Ar–H), 7.20 (d, J = 3.4 Hz, 1 H, Ar–H), 6.54 (dd,
J = 3.3, 1.5 Hz, 1 H, Ar–H), 6.26 (s, 1 H, C = CHH), 6.22
(s, 1 H, C = CHH), 4.39 (s, 2 H, C–CH2–S), 3.54 (s, 3 H,
N–CH3), 3.35 (s, 3 H, N–CH3); 13C NMR (101 MHz,
CDCl3) δ 196.4 (S-C = S), 182.4 (C = O), 151.8 (Ar–C),
147.1 (Ar–C), 142.6 (C–C = CH2), 121.7 (Ar–C), 120.3
(C = CH2), 112.1 (Ar–C), 45.5 (N–CH3), 42.5 (N–CH3),
38.1 (C–CH2–S); HRMS m/z (pos): 256.0459 C11H14NO2S2
(calcd. 256.0461).
2-(4-Chlorobenzoyl)allyl dimethylcarbamodithioate
(12m)
2-(Thiophene-2-carbonyl)allyl
dimethylcarbamodithioate (12q)
Yield: 37%; mp 55.8–57.1 °C; 1H NMR (400 MHz, CDCl3)
δ 7.64 (d, J = 8.4 Hz, 2 H, Ar–H), 7.34 (d, J = 8.3 Hz, 2 H,
Ar–H), 6.24 (s, 1 H, C = CHH), 5.69 (s, 1 H, C = CHH),
4.30 (s, 2 H, C–CH2–S), 3.47 (s, 3 H, N–CH3), 3.29 (s, 3 H,
N–CH3); 13C NMR (101 MHz, CDCl3) δ 195.6 (S–C = S),
195.6 (C = O), 142.9 (C–C = CH2), 138.7 (Ar–C), 135.6
(Ar–C), 131.0 (Ar–C), 129.0 (C = CH2), 128.6 (Ar-C), 45.5
(N-CH3), 41.5 (N–CH3), 37.8 (C–CH2–S); HRMS m/z
(pos): 300.0278 C13H15ClNOS2 (calcd. 300.0278).
Yield: 23%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.64
(t, J = 4.3 Hz, 2 H, Ar–H), 7.09 (dd, J = 4.8, 3.9 Hz, 1 H,
Ar–H), 6.16 (s, 1 H, C = CHH), 5.95 (s, 1 H, C = CHH),
4.34 (s, 2 H, C-CH2–S), 3.50 (s, 3 H, N–CH3), 3.31 (s, 3 H,
N-CH3); 13C NMR (101 MHz, CDCl3) δ 196.1 (S-C = S),
188.2 (C = O), 143.1 (Ar–C), 143.0 (C–C = CH2), 134.3
(Ar–C), 128.0 (C = CH2), 126.7 (Ar–C), 45.5 (N–CH3),
41.5 (N–CH3), 38.3 (C–CH2–S); HRMS m/z (pos):
272.0232 C11H14NOS3 (Calcd. 272.0232).
2-(2-Chlorobenzoyl)allyl dimethylcarbamodithioate
(12n)
2-(1H-pyrrole-2-carbonyl)allyl
dimethylcarbamodithioate (12r)
Yield: 29%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.33
(dt, J = 8.1, 4.8 Hz, 2 H, Ar-H), 7.29–7.20 (m, 2 H, Ar-H),
6.41 (s, 1 H, C = CHH), 5.70 (s, 1 H, C = CHH), 4.37 (s, 2
Yield: 5%; mp 133.1-135.7 °C; 1H NMR (400 MHz,
CDCl3) δ 9.85 (s, 1 H, Ar–H), 7.05–7.06 (m, 1 H, Ar–H),
6.83–6.84 (m, 1 H, Ar–H), 6.21 (s, 1 H, Ar-H), 6.01 (s, 1 H,
C = CHH), 5.96 (s, 1 H, C = CHH), 4.33 (s, 2 H,
H, C-CH2-S), 3.49 (s, 3 H, N-CH3), 3.33 (s, 3 H, N-CH3); 13
C
NMR (101 MHz, CDCl3) δ 196.3 (S-C = S), 195.7 (C = O),
143.4 (C-C = CH2), 137.9 (Ar-C), 133.0 (Ar-C), 131.1 (Ar-
C), 131.0 (Ar-C), 130.0 (Ar-C), 129.9 (C = CH2), 126.5 (Ar-
C), 45.6 (N-CH3), 41.59 (N-CH3), 36.3 (C-CH2-S); HRMS
m/z (pos): 300.0273 C13H15ClNOS2 (calcd. 300.0278).
C–CH2–S), 3.47 (s, 3 H, N-CH3), 3.26 (s, 3 H, N-CH3); 13
C
NMR (101 MHz, CDCl3) δ 196.6 (S–C = S), 184.8 (C =
O), 142.7 (Ar–C), 130.8 (C–C = CH2), 125.7 (Ar-C), 125.6
(Ar-C), 118.9 (C = CH2), 110.8 (Ar-C), 45.5 (N-CH3), 41.4
(N–CH3), 38.7 (C–CH2–S); HRMS m/z (pos): 255.0622
C11H15N2OS2 (calcd. 255.0620).
2-(3,4-Dichlorobenzoyl)allyl
dimethylcarbamodithioate (12o)
Yield: 35%; mp 60.3–62.5 °C; 1H NMR (400 MHz, CDCl3)
δ 7.79 (s, 1 H, Ar-H), 7.55 (d, J = 8.2 Hz, 1 H, Ar-H), 7.48
(d, J = 8.1 Hz, 1 H, Ar-H), 6.30 (s, 1 H, C = CHH), 5.73 (s,
1 H, C = CHH), 4.31 (s, 2 H, C-CH2-S), 3.51 (s, 3 H, N-
CH3), 3.33 (s, 3 H, N-CH3); 13C NMR (101 MHz, CDCl3) δ
195.9 (S-C = S), 194.4 (C = O), 142.8 (C-C = CH2), 137.0
(Ar-C), 136.8 (Ar-C), 133.0 (Ar-C), 131.3 (Ar-C), 130.4
(Ar-C), 129.4 (C = CH2), 128.9 (Ar-C), 45.6 (N-CH3), 41.5
2-Nicotinoylallyl dimethylcarbamodithioate (12s)
Yield: 10%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 8.94
(d, J = 1.6 Hz, 1 H, Ar-H), 8.77 (dd, J = 4.8, 1.6 Hz, 1 H,
Ar-H), 8.05 (dt, J = 7.9, 1.9 Hz, 1 H, Ar-H), 7.40 (dd, J =
7.8, 4.9 Hz, 1 H, Ar–H), 6.42 (s, 1 H, C = CHH), 5.82 (s,
1 H, C = CHH), 4.41 (s, 2 H, C-CH2-S), 3.56 (s, 3 H,
N-CH3), 3.38 (s, 3 H, N–CH3); 13C NMR (101 MHz,

Medicinal Chemistry Research
CDCl3) δ 196.1 (S-C = S), 195.3 (C = O), 152.8 (Ar-C),
150.5 (Ar-C), 143.2 (C-C = CH2), 136.9 (Ar–C), 133.0
(Ar–C), 130.1 (Ar–C), 123.3 (C = CH2), 45.6 (N–CH3),
41.5 (N–CH3), 37.4 (C–CH2–S); HRMS m/z (pos):
267.0625 C12H15N2OS2 (calcd. 267.0620).
2-(4-Chlorobenzoyl)allyl dibutylcarbamodithioate
(14c)
Yield: 22%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.73
(d, J = 8.4 Hz, 2 H, Ar-H), 7.42 (d, J = 8.4 Hz, 2 H, Ar-H),
6.29 (s, 1 H, C = CHH), 5.74 (s, 1 H, C = CHH), 4.40 (s, 2
H, C-CH2-S), 3.99–3.94 (m, 2 H, N-CH2-CH2), 3.67–3.61
(m, 2 H, N-CH2-CH2), 1.74–1.64 (m, 4 H, N-(CH2-CH2-CH2-
CH3)2), 1.39–1.32 (m, 4 H, N-(CH2-CH2-CH2-CH3)2), 0.95
(dt, J = 20.6, 7.4 Hz, 6 H, N-(CH2-CH2-CH2-CH3)2); 13C
NMR (101 MHz, CDCl3) δ 195.9 (S-C = S), 194.9 (C = O),
143.0 (C-C = CH2), 138.9 (Ar-C), 135.8 (Ar-C), 131.1 (Ar-
C), 128.6 (C = CH2), 128.6 (Ar-C), 55.2 (N-CH2-CH2), 55.5
(N-CH2-CH2), 37.4 (C-CH2-S), 29.4 (N-CH2-CH2-CH2), 28.4
(N-CH2-CH2-CH2), 20.1 (CH2-CH2-CH3), 20.1 (CH2-CH2-
CH3), 13.9 (CH2-CH3), 13.7 (CH2-CH3); HRMS m/z (pos):
384.1227 C19H27ClNOS2 (calcd. 384.1217).
2-Benzoylallyl dimethylcarbamodithioate (13)
Yield 18%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 8.00
(dd, J = 5.2, 3.3 Hz, 2 H, Ar-H), 7.60–7.54 (m, 1 H, Ar–H),
7.52–7.41 (m, 2 H, Ar-H), 4.13–3.98 (m, 1 H, C-CH-CH3),
3.67 (dd, J = 13.7, 8.2 Hz, 1 H, CH-CHH-S), 3.56 (dd, J =
13.7, 8.2 Hz, 1 H, CH-CHH-S), 3.55 (s, 3 H, N-CH3), 3.31
(s, 3 H, N-CH3), 1.33 (d, J = 7.1 Hz, 3 H, CH-CH3); 13C
NMR (101 MHz, CDCl3) δ 202.9 (S-C = S), 197.3 (C =
O), 136.1 (Ar-C), 133.2 (Ar-C), 128.7 (Ar-C), 128.6
(Ar-C), 45.4 (N-CH3), 41.5 (N-CH3), 40.5 (C-CH-CH3),
39.4 (C-CH2-S), 18.1 (C-CH3); HRMS m/z (pos): 268.0828
C13H18NOS2 (Calcd. 268.0824).
2-(4-Chlorobenzoyl)allyl pyrrolidine-1-
carbodithioate (14d)
2-(4-Chlorobenzoyl)allyl diethylcarbamodithioate
(14a)
1
Yield 36%; mp 59.8–61.5 °C; H NMR (400 MHz, CDCl3)
δ 7.73 (d, J = 8.5 Hz, 2 H, Ar-H), 7.43 (d, J = 8.5 Hz, 2 H,
Ar-H), 6.36 (s, 1 H, C = CHH), 5.77 (s, 1 H, C = CHH),
4.42 (s, 2 H, C-CH2-S), 3.96 (t, J = 6.9 Hz, 2 H, N-CH2-
CH2), 3.66 (t, J = 6.8 Hz, 2 H, N-CH2-CH2), 2.14–2.05 (m,
2 H, N-CH2-CH2), 2.04–1.95 (m, 2 H, N-CH2-CH2); 13C
NMR (101 MHz, CDCl3) δ 195.8 (S-C = S), 191.9 (C = O),
143.2 (C-C = CH2), 138.8 (Ar-C), 135.7 (Ar-C), 131.1 (Ar-
C), 129.0 (C = CH2), 128.6 (Ar-C), 55.2 (N-CH2-CH2),
50.6 (N-CH2-CH2), 36.7 (C-CH2-S), 26.1 (N-CH2-CH2),
24.3 (N-CH2-CH2); HRMS m/z (pos): 326.0435
C15H17ClNOS2 (calcd. 326.0435).
1
Yield: 24%; Yellow oil; H NMR (400 MHz, CDCl3) δ
7.72 (d, J = 8.5 Hz, 2 H, Ar–H), 7.41 (d, J = 8.5 Hz, 2 H,
Ar-H), 6.30 (s, 1 H, C = CHH), 5.74 (s, 1 H, C = CHH),
4.40 (s, 2 H, C–CH2–S), 4.04 (q, J = 6.9 Hz, 2 H,
N–CH2–CH3), 3.74 (dd, J = 13.7, 6.6 Hz, 2 H, N-CH2-
CH3), 1.27 (dd, J = 11.3, 6.7 Hz, 6 H, N–(CH2-CH3)2);
13C NMR (101 MHz, CDCl3) δ 195.9 (S–C = S), 194.5
(C = O), 143.0 (C–C = CH2), 138.8 (Ar-C), 135.7 (Ar–
C), 131.1 (Ar–C), 128.8 (C = CH2), 128.6 (Ar-C), 49.7
(CH2-CH3), 46.8 (CH2-CH3), 37.4 (C–CH2–S), 12.6
(CH2–CH3), 11.6 (CH2–CH3); HRMS m/z (pos): 328.0590
C15H19ClNOS2 (Calcd. 328.0591).
2-(4-Chlorobenzoyl)allyl piperidine-1-carbodithioate
(14e)
2-(4-Chlorobenzoyl)allyl dipropylcarbamodithioate
(14b)
Yield: 15%; mp 59.6–61.2 °C; 1H NMR (400 MHz, CDCl3) δ
7.73 (d, J = 8.6 Hz, 2 H, Ar-H), 7.44–7.39 (m, 2 H, Ar-H),
6.31 (s, 1 H, C = CHH), 5.75 (s, 1 H, C = CHH), 4.42 (s, 2 H,
C-CH2-S), 4.30 (br, 2 H, N-CH2-CH2), 3.89 (br, 2 H, N-CH2-
CH2), 1.71–1.64 (m, 6 H, CH2-(CH2)3-CH2); 13C NMR
(101 MHz, CDCl3) δ 195.9 (S-C = S), 194.6 (C = O), 143.1
(C-C = CH2), 138.8 (Ar-C), 135.7 (Ar-C), 131.1 (Ar-C), 128.8
(C = CH2), 128.6 (Ar-C), 37.4 (C-CH2-S), 24.3 (CH2-CH2-
CH2); HRMS m/z (pos): 340.0594 C16H19ClNOS2 (calcd.
340.0591).
Yield: 24%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.73
(d, J = 8.5 Hz, 2 H, Ar-H), 7.43 (d, J = 8.5 Hz, 2 H, Ar-H),
6.30 (s, 1 H, C = CHH), 5.75 (s, 1 H, C = CHH), 4.40 (s, 2
H, C-CH2-S), 3.97–3.87 (m, 2 H, N-CH2-CH2), 3.66–3.58
(m, 2 H, N-CH2-CH2), 1.84–1.66 (m, 4 H, N-(CH2-CH2-
CH3)2), 0.95 (q, J = 7.1 Hz, 6 H, N-(CH2-CH2-CH3)2); 13C
NMR (101 MHz, CDCl3) δ 193.0 (S-C = S), 195.1 (C = O),
143.0 (C-C = CH2), 138.9 (Ar-C), 135.8 (Ar-C), 131.1 (Ar–
C), 128.8 (C = CH2), 128.6 (Ar–C), 57.0 (N–CH2–CH2),
54.4 (N-CH2-CH2), 37.4 (C-CH2-S), 20.7 (CH2–
CH2–CH3), 19.6 (CH2–CH2–CH3), 11.2 (CH2–CH3);
HRMS m/z (pos): 356.0901 C17H23ClNOS2 (calcd.
356.0904).
2-(4-Chlorobenzoyl)allyl morpholine-4-
carbodithioate (14f)
Yield: 23%; mp 75.1-76.3 °C; 1H NMR (400 MHz, CDCl3)
δ 7.72 (d, J = 8.5 Hz, 2 H, Ar-H), 7.43 (d, J = 8.5 Hz, 2 H,

Medicinal Chemistry Research
Ar-H)), 6.33 (s, 1 H, C = CHH), 5.79 (s, 1 H, C = CHH),
4.44 (s, 2 H, C-CH2-S), 4.15 (m, 4 H, N-(CH2-CH2)2), 3.77
(br, 4 H, O-(CH2-CH2)2); 13C NMR (101 MHz, CDCl3) δ
196.7 (S-C = S), 195.7 (C = O), 142.7 (C-C = CH2), 139.0
(Ar-C), 135.6 (Ar-C), 131.1 (Ar-C), 129.3 (C = CH2),
128.6 (Ar-C), 66.3 (O-(CH2-CH2)2), 52.1-50.2 (N-(CH2-
CH2)2), 37.2 (C-CH2-S); HRMS m/z (pos): 342.0380
C15H17ClNO2S2 (calcd. 342.0383).
and MDA-MB-231, and the antiproliferative activity of
the target compounds was determined by MTT assay on
cell line H460. The antiproliferative activity of compound
12r was determined by MTT assay on the following cell
lines: H460, A549, MDA-MB-231, MCF-7, A375,
HCT116, PC3 and HepG2. All cells used in the research
were prepared at 3.5 × 103cells/mL concentration and each
100 μL cell suspension was seeded onto 96-well microtiter
plates for 24 h (37 °C, 5% CO2). Then various appropriate
dilutions of tested compounds were added and incubated
for 72 h. For the control group, equivalent concentration
of DMSO (final concentration 0.5%) was added. MTT (3-
[4,5-dimethylthiazol-2yl]-diphenyl tetrazolium bromide)
method was used to measure the number of surviving cells
and recorded the OD value at 492/620 nm. The inhibition
rate of the compounds was calculated, and the IC50 values
were calculated using Prism Graphpad software of the
triplicate experiment.
(S)-Methyl 1-(((2-(4-chlorobenzoyl)allyl)thio)
carbonothioyl) pyrrolidine-2-carbo- xylate (14g)
Yield: 17%; Yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.71 -
7.69 (m, 2 H, Ar-H), 7.42 (d, J = 8.4 Hz, 2 H, Ar-H), 6.29 (d,
J = 9.6 Hz, 1 H, C = CHH), 5.74 (d, J = 9.5 Hz, 1 H, C =
CHH), 4.92 (ddd, J = 10.3, 8.7, 2.2 Hz, 1 H, N-CH-
CO2CH3), 4.41–4.32 (m, 2 H, C-CH2-S), 4.20–3.68 (m, 2 H,
N-CH2-CH2), 3.71 (d, J = 35.3 Hz, 3 H), 2.40–2.01 (m, 4 H,
CH2-(CH2)2-CH); 13C NMR (101 MHz, CDCl3) δ 195.8
(C = S), 195.7 (C = S), 194.3 (C = O), 193.4 (C = O), 171.0
(C = O), 170.6 (C = O), 142.6 (C-C = CH2), 138.8 (Ar-C),
135.7 (Ar-C), 131.1 (Ar-C), 129.2 (C = CH2), 128.9 (C =
CH2), 128.6 (Ar-C), 128.6 (Ar-C), 66.4 (N-CH-CO2CH3),
62.6 (N-CH-CO2CH3), 55.5 (O-CH3), 52.4 (O-CH3), 52.7 (N-
CH2-CH2), 50.9 (N-CH2-CH2), 37.0 (C-CH2-S), 36.9 (C-
CH2-S), 31.5 (CH-CH2-CH2), 29.2 (CH-CH2-CH2), 24.6
(CH2-CH2-CH2), 22.3 (CH2-CH2-CH2); HRMS m/z (pos):
384.0499 C17H19ClNOS2 (calcd. 384.0489).
Results and Discussion
Modification of lead compound 8
Optimization of aromatic rings
Our optimization effort started with the modification of
the aromatic rings of lead compound 8 (Table 1). A
variety of substituted aromatic rings and heteroaromatic
rings were selected to replace the benzene ring in lead
compound 8. All target compounds were synthesized and
evaluated for their anti-proliferative activities against
(R)-Methyl 1-(((2-(4-chlorobenzoyl)allyl)thio)
carbonothioyl) pyrrolidine-2-carbo- xylate (14h)
1
Yield: 20%; Yellow oil; H NMR (400 MHz, CDCl3) δ
H460
cell
based
on
our
previous
results
7.71–7.69 (m, 2 H, Ar-H), 7.42 (d, J = 8.4 Hz, 2 H, Ar-H),
6.29 (d, J = 9.7 Hz, 1 H, C = CHH), 5.74 (d, J = 9.5 Hz,
1 H, C = CHH), 4.92 (ddd, J = 10.3, 8.7, 2.1 Hz, 1 H, N-
CH-CO2CH3), 4.41–4.32 (m, 2 H, C-CH2-S), 4.20 - 3.69
(m, 2 H, N-CH2-CH2), 3.71 (d, J = 35.5 Hz, 3 H),
2.43–2.01 (m, 4 H, CH2-(CH2)2-CH); 13C NMR (101 MHz,
CDCl3) δ 195.8 (C = S), 195.7 (C = S), 194.3 (C = O),
1935 (C = O), 171.0 (C = O), 170.6 (C = O), 142.6 (C =
CH2), 138.8 (Ar-C), 135.7 (Ar-C), 131.1 (Ar-C), 129.2
(C = CH2), 128.9 (C = CH2), 128.6 (Ar-C), 66.4 (N-CH-
CO2CH3), 62.8 (N-CH-CO2CH3), 55.5 (O-CH3), 52.4 (O-
CH3), 52.7 (N-CH2-CH2), 50.9 (N-CH2-CH2), 37.0 (C-CH2-
S), 36.9 (C-CH2-S), 31.5 (CH-CH2-CH2), 29.2 (CH-CH2-
CH2), 24.6 (CH2-CH2-CH2), 22.4 (CH2-CH2-CH2); HRMS
m/z (pos): 384.0491 C17H19ClNOS2 (calcd. 384.0489).
(Li et al. 2015b).
The results showed in Table 1 indicated that most of the
compounds exhibited better activity than lead compound 8.
The position of substituents on the benzene ring had a
remarkable impact on the activity. Electron donating group
on the para position (12c, 4-OH, IC50 = 0.61 μM; 12d, 4-
Me, IC50 = 1.09 μM) was beneficial for the activity. On the
contrary, ortho substituted electron donating group, such as
12b (2-OMe-, IC50 = 6.51 μM), led to a 6-fold decrease in
potency. Electron withdrawing group was suitable in all
positions (12g–12j). However, halogen atoms had different
influence on the activity. For example, the activity of 4-
chlorine substituted compound 12m (IC50 = 0.57 μM) was
six times more potent than that of 2-chlorine substituted
compound 12n (IC50 = 3.61 μM). It was noteworthy that
disubstituted derivatives (12f and 12o) also showed excel-
lent activity. Moreover, the modification with five-
Antiproliferative activity assays
membered heteroaromatic rings (12p, furan-2-yl-, IC50
=
The inhibition rate of compound 8 was determined by
MTT assay on cell line H460, BGC823, Hela, HCT116
0.52 μM; 12q, thiophene-2-yl-, IC50 = 1.23 μM; 12r, pyr-
role-2-yl-, IC50 = 0.46 μM) also gave a satisfied results. In

Medicinal Chemistry Research
Table 1 Structures and activities of compounds 12a–12s against H460 cell linea
aH460 cell line: human non-small cell lung cancer
bDose-response curves were determined at five concentrations
The IC50 values are the concentrations needed to inhibit cell growth by 50% as determined from these curves

Medicinal Chemistry Research
comparison, the activity was slightly decreased by mod-
ification with six-membered heteroaromatic rings (12 s,
pyridin-3-yl-, IC50 = 2.10 μM).
In order to confirm the function of the α,β-unsaturated
ketone fragment, compound 13, a double bond reduced
derivatives of compound 8, was also designed and synthe-
sized. As expected, compound 13 (IC50 = 4.88 μM) was less
potent 3 times than compound 8 (IC50 = 1.59 μM). Thus, α,
β-unsaturated ketone fragment plays a critical role in the
anti-cancer activity.
Table 2 The anti-proliferative activity of compound 14a–14h against H460a
aH460 cell line: human non-small cell lung cancer.
bDose-response curves were determined at five concentrations. The IC50 values are the concentrations needed to inhibit cell growth by 50% as
determined from these curves.

Medicinal Chemistry Research
Optimization of amine moiety
Conclusion
Based on the optimization results of the aromatic ring
moiety, we chose compound 12m to further optimize the
amine moiety. Replacing dimethylamine with various sec-
ondary amines, including common cyclic and acyclic
amines frequently occurring in drug structure, led to the
compounds 14a–14 h. As shown in Table 2, it was clear
that the activity decreased dramatically with the increase of
amine size. The IC50 values of compounds 12 m (Me2N-),
14a (Et2N-), 14b (n-Pr2N-) and 14c (n-Bu2N-) were 0.57,
3.04, >50 and >50 μM, respectively. The activities of deri-
vatives (14d and 14e) with pyrrolidine and piperidine as
amine moiety were similar to that of 14a. These results
suggested that the receptor cavity binding with amine
moiety might be relatively small. Changing the piperidyl
group in compound 14e to morpholinyl group resulted in
compound 14f, which was less potent about two times than
14e. Thus, the receptor cavity binding with amine moiety
should be a hydrophobic cavity. Meanwhile, we also pre-
pared 14g and 14h by using methyl L-prolinate and methyl
D-prolinate as amine moiety, respectively. Both compound
14g and 14h had similar activities, which proved that
antitumor activity was not affected by the chiral amine
moiety. Therefore, small size hydrophobic amino groups
were more beneficial for the activity.
A systematic structural modification was carried out based
on the structure of novel lead compound 8. A series of
compound 8’s derivatives were synthesized and evaluated
for their in vitro antitumor activities. The compound 12r
was identified as the most potent one which had significant
inhibitory effect on the growth of eight kinds of cancer cell
lines with IC50 values <1 μM. Especially for cell lines A375
and HCT116, the IC50 values achieved 63 nM and 66 nM,
respectively. Meanwhile, some valuable structure-activity
relationships were revealed as follows: (a) different sub-
stitution on the benzene ring or heteroaromatic rings greatly
affected on the activity; (b) the presence of α,β-unsaturated
ketone fragment was favorable for the activity; (c) the
receptor cavity binding with amine moiety might be a
relatively small hydrophobic cavity. The further studies of
12r are currently in progress.
Acknowledgements This work was supported by the National Natural
Science Foundation of China (No. 21372019).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
The antitumor spectrum of 12r
Based on the above optimized results, 12r was identified as
the most potent antitumor agent. We next assessed the
antiproliferative effect of 12r against eight kinds of tumor
cell lines, including H460, A549, MDA-MB-231, MCF-7,
A375, HCT116, PC3 and HepG2. As shown in Table 3, 12r
could inhibit the growth of eight cell lines with IC50 values
<1 μM. Especially for cell lines A375 and HCT116, the IC50
values achieved 63 nM and 66 nM, respectively.
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