Novel small molecules as apoptosis inducers: Synthesis, preliminary structure-activity relationships, and in vitro biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2013.02.076

Inducing apoptosis is a promising therapeutic approach to overcome cancer. In this study, 30 compounds were synthesized and evaluated for their antiproliferative activity against three tumor cell lines in vitro: A875, H460 and Hela cancer cells by the MTT

Full text of DOI:10.1016/j.bmcl.2013.02.076

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2293–2297
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel small molecules as apoptosis inducers: Synthesis, preliminary
structure–activity relationships, and in vitro biological evaluation
Lifeng Zhao a, , Xiao Li a, , Lidan Zhang b, Tinghong Ye a, Yongxia Zhu a, Yuquan Wei a, Shengyong Yang a,
Luoting Yu a,
⇑
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, Sichuan, China
b College of Chemical Engineering, Sichuan University, Chengdu 610041, Sichuan, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inducing apoptosis is a promising therapeutic approach to overcome cancer. In this study, 30 compounds
were synthesized and evaluated for their antiproliferative activity against three tumor cell lines in vitro:
A875, H460 and Hela cancer cells by the MTT assay. The most potent analogue 7a, a novel compound was
first reported by our group, inhibited the proliferation of A875 cells with an IC50 value of 98 nM. Flow
cytometry analysis and morphological analysis suggested that compound 7a had potential anticancer
efficacy via G2/M cell cycle arrest, which could be attributed to its proliferation and apoptosis, and also
in a concentration-dependent manner. The SAR analysis indicated that the substituents R2 played a cru-
cial role in the antiproliferation activity.
Received 12 December 2012
Revised 13 February 2013
Accepted 15 February 2013
Available online 26 February 2013
Keywords:
Anticancer activity
Structure–activity relationships (SAR)
Apoptosis
Ó 2013 Elsevier Ltd. All rights reserved.
Chemotherapy is one of the most common treatment options
for cancer patients, especially for unresectable patients.1–3
However, conventional chemotherapy drugs have not shown
better improvement in extension and quality of patients’ life
mainly because of their poor inhibitor potency or the emergence
of drug resistance after chemotherapeutic more than six months.4,5
The discovery and development of the novel cancer inhibitors with
higher potency anticancer activity are strongly demanded at the
present time. All of the cancer cells have the same property that
they proliferate rapidly whatever they are diverse and heteroge-
neous.6 The programmed cell death, also known as apoptosis is a
complex mechanism by which cell undergo its own destruction
to control the process of cell proliferation.7,8 Apoptosis plays an
important role in tissue homoeostasis and prevention of tumor cell
study of anticancer drugs, we found a hit compound 1 (Fig. 1)
which exhibited moderate inhibition against the human melanoma
cell line A875 (IC50 = 18.6
line H460 (IC50 = 16.8 M) and the human cervical carcinoma cell
line Hela (IC50 = 25.8
M) in the MTT assay.15 The purpose here is
lM), human lung adenocarcinoma cell
l
l
to carry out a structural modification to optimize the activity of
compound 1, a series of novel compounds 6a–7r were synthe-
sized,16,17 and their anticancer activity and apoptosis ability were
tested in vitro.
Figure 1 illustrates three regions of interest for SAR evaluation
of screening hit compound 1. The synthetic strategies adopted
for the synthesis of compound 1 and other compounds 6a–7r are
depicted in Scheme 1 and 2. The starting materials 3a–h were pre-
pared from commercially available benzylamine, pyridinylamin
derivatives 2a–h, and 2-chloroacetyl chloride at room tempera-
ture. In the next step, 3a–h were condensed with 4-aminobenze-
nethiol or 5-amino-1,3,4-thiadiazole-2-thiol in THF with
refluxing, affording the amines, 4a–d and 5a, 5b, 5e–5h. Then, tar-
get compounds, 1, 6a–k and 7a–r were obtained, respectively, by
the reaction with corresponding acyl chloride in the presence of
potassium carbonate, at room temperature, in dichloromethane.18
The yield of all reactions was satisfactory. All the products were
characterized by 1H NMR, 13C NMR and ESI-MS analysis.
proliferation,9 as
a
defense mechanism apoptosis remove
unwanted and potentially dangerous cells including tumor
cells.10,11 Its deregulation is widely believed to be involved in the
pathogenesis of cancer. Certain drugs and agents have been identi-
fied treatment of the cancers depending on triggering the apoptotic
pathway, then inducing the apoptosis process in tumor cells.12–14
Thus, development of drugs that can effectively trigger the apopto-
tic process and evaluation the apoptosis inducing ability have been
receiving considerable attention.
Our project is to develop novel small molecule anticancer drugs
that could induce apoptosis. In a previous cell-based screening
As shown in Table 1 and 29 analogs of compound 1 were syn-
thesized to survey the SAR of Region 1, Region 2 and Region 3 by
evaluating the cell growth inhibitory activity (IC50) on the three
human cell lines A875, H460 and HepG2 in the MTT assay. Most
compounds were able to increase the inhibitory activities against
the three human cell lines, some compounds were better than
⇑
Corresponding author. Tel.: +86 28 85164063; fax: +86 28 85164060.
E-mail address: yuluot@scu.edu.cn (L. Yu).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.076

2294
L. Zhao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2293–2297
replaced by chloromethyl, the groups including 5-chloro-2-pyridi-
Br
nyl (6e), 2-chloro-4-methyl-3-pyridinyl (6i), 5-methyl-2-pyridinyl
(6j), in R1 did not result in the loss of activity, these results showed
no clear SAR trends for Region 1. This observation confirmed fur-
ther that chloromethyl and 2-chloroethyl played an important role
in increasing the anticancer activity.
After identifying chloromethyl and 2-chloroethyl were the po-
tent substituent, we tried to replace Region 2 with phenyl ring.
As shown in Table 1, among 7a–7v we found the most potent anti-
cancer activity compound 7a that moderate inhibitory activity was
more than 200-fold improvement against the human melanoma
cell line A875 (IC50 = 98 nM), compared with cisplatin as a positive
O
Cl
S
S
N
N
H
NH
O
N
N
Cl
Region1
Region2
Region3
1
control (IC50 = 22.4 lM). We next turned to examining the SAR at
Region 1 and Region 3. The Region 3 was replaced with chloro-
methyl, the anticancer activity was slightly fluctuate with the
replacement of 5-chloro-2-pyridinyl (7a), 5-bromo-2-pyridinyl
(7b), 4-chloro-2-pyridinyl (7c) and decreased with the replace-
ment of 3-chlorobenzyl (7d), 4-chlorobenzyl (7e), 2-pyridinyl (7f)
in R1 group. The results suggested 2-pyridinyl ring with electron-
withdrawing chloro or bromo at the 4-position would increase
the anticancer activity. However, the potent activity was decreased
and even more loss with increasing of alky chain in R2 (7g–7k).
Introduction of 1.1-dimethylethyl (7l, 7o), trihloromethyl (7m),
dichloromethyl (7n) instead of chloromethyl in R2 group resulted
in the loss of activity, then R2 was replaced with ethenyl (7p, 7q,
Cl
O
S
N
N
H
O
Cl
N
H
7a
Figure 1. Selected regions of interest for SAR evaluation of screening hit compound
1 and the structural formula of compound 7a.
cisplatin, while six compounds had no activity against the three
human cell lines. The most potent analog 7a significantly inhibited
the proliferation of the human melanoma cell line A875 at low
nanomolar concentration (IC50 = 98 nM).
7r), IC50 values were between 14 and 24 lM against A875 cells.
This observation confirmed further that chloromethyl could play
an important role in the anticancer activities.
Through the structure–activity relationship study, we found
that chloromethyl could significantly improve the antitumor activ-
ity of this series analogs in vitro. Region 3 seemed to play a more
important role in increasing the activities than Region 1 and Region
2. This observation provided us with important information for fur-
ther molecular structure modification.
To further study the antiproliferative, 7a and 7b were selected
for evaluation of their inhibitory activities against a panel of
different types of human cancer cell lines: colon cancer cell lines
HCT-116 and HT29, lung cancer cell lines A549 and H460, liver
cancer cell lines PC-3, Bel-7402, SMMC-7721and HepG2, pancre-
atic cancer cell line BxPC-3, cervical cancer cell line Hela, mela-
noma cancer cell line A875. As shown in Table 2, compounds 7a
and 7b exhibited good anticancer activities against the different
types of human cancer cell lines in vitro, with IC50 value ranging
As illustrated in Scheme 2, 6a–6k. The Region 2 was 1,3,4-thia-
diazole, we started our SAR studies at the Region 1 and Region 3.
Introduction of a 2,6-position of the phenyl ring with the elec-
tron-withdrawing chlorine group (1) was well tolerated, whereas
the 2-methyloxyphenyl analog (6a) led to almost total loss of
activity. Meanwhile the R1 was replaced by 5-bromo-2-pyridinyl.
However, replacing the R3 with electron-donating 2-methyloxy-
phenyl resulted in the loss of activity with the introduction of
5-methyl-2-pyridinyl (6b), 5-chloro-2-pyridinyl (6c), 2-chloro-
4-methyl-3-pyridinyl (6d) in the R1 group. This suggested that
the electron-withdrawing group on the phenyl ring increased the
anticancer activity in contrast of electron-donating group.
In another variation, we replaced the substituted R2 with chlo-
romethyl, 2-chloroethyl, 3-chloropropyl and dichloromethyl
groups at R2, affording 6e–6j and 6k. As shown in Table 1, 6e–6j,
from 0.098 to 2.5 lM. This broad-spectrum antiproliferative activ-
especially 6h (IC50 = 0.43 lM) had considerably better activity than
ity is encouraging for the development of new anticancer drugs.
At the beginning of design these compounds, our project was to
develop new anticancer drugs that could induce apoptosis. There-
fore, we evaluated the apoptosis inducing ability of these
compound 1, indicating chloromethyl and 2-chloroethyl might be a
good substitution for modification compared with 3-chloropropyl
(6k) that almost loss of activity. Interestingly, when the R2 was
O
O
R1
S
b
R1
a
R1
Cl
S
N
H
NH2
2a-d
N
H
NH2
N
N
3a-d
4a-d
O
R2: cholormethyl
chloroethyl
2, 3 and 4
c
R1
S
6
S
a: R1= 5-chloro-2-pyridinyl
b: R1= 5-bromo-2-pyridinyl
c: R1= 5-methyl-2-pyridinyl
N
H
NH
O
R2
N
N
2-metyoxyphenyl
2,2-dichloromethyl
2,6-dichlorophenyl
d: R1= 2-chloro-4-methyl-3-pyridinyl
Scheme 1. Reagents and conditions: (a) 2-chloroacetyl chloride, CH2Cl2, K2CO3, rt, 4 h; (b) 5-amino-1,3,4-thiadiazole-2-thiol, THF, K2CO3, reflux, 5 h; (c) CH2Cl2, K2CO3, rt, 4 h.

L. Zhao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2293–2297
2295
O
O
R1
S
b
R1
a
R1
Cl
N
H
NH2
2a-h
N
H
NH2
3a-h
5a-h
R2: ethenyl
chloromethyl
2,3 and 5
a: R1= 5-chloro-2-pyridinyl
b: R1= 5-bromo-2-pyridinyl
e: R1= 2-pyridinyl
f: R1= 4-chloro-2-pyridinyl
g: R1= 4-chlorobenzyl
h: R1= 3-chlorobenzyl
dichloromethyl
O
c
R1
S
trichloromethyl
N
H
O
3-chloropropyl
N
H
R2
2-chloroethyl
2-metyoxyphenyl
1.1-dimethylethyl
2,6-dichlorophenyl
3-(trifluoromethyl)benzyl
7
Scheme 2. Reagents and conditions: (a) 2-chloroacetyl chloride, CH2Cl2, K2CO3, rt, 4 h; (b) 4-aminothiophenol, THF, K2CO3, reflux, 5 h; (c) CH2Cl2, K2CO3, rt, 4 h.
Table 1
In vitro activities of compound 6a–7r
O
O
R1
S
S
R1
S
7
N
H
N
H
O
NH
O
R2
N
N
N
H
R2
6
R1
R2
IC50 (lM)
a
Compound
A875
H460
Hela
6a
6b
6c
6d
6e
6f
6g
6h
6i
5-Bromo-2-pyridinyl
5-Methyl-2-pyridinyl
5-Chloro-2-pyridinyl
2-Methoxyphenyl
2-Methoxyphenyl
2-Methoxyphenyl
2-Methoxyphenyl
Chloromethyl
Chloromethyl
Dichloromethyl
2-Chloroethyl
Chloromethyl
Chloromethyl
3-Chloropropyl
Chloromethyl
Chloromethyl
Chloromethyl
Chloromethyl
Chloromethyl
Chloromethyl
2-Chloroethyl
2-Chloroethyl
2-Chloroethyl
2-Chloroethyl
3-Chloropropyl
1.1-Dimethylethyl
Trichloromethyl
Dichloromethyl
1.1-Dimethylethyl
Ethenyl
>80
>80
>80
>80
8.6
>80
>80
>80
>80
5.7
>80
>80
>80
>80
3.6
3.4
15.2
5.3
70.5
10.9
>80
2.5
2.1
2.8
2-Chloro-4-methyl-3-pyridinyl
5-Chloro-2-pyridinyl
5-Bromo-2-pyridinyl
5-Bromo-2-pyridinyl
5-Bromo-2-pyridinyl
2-Chloro-4-methyl-3-pyridinyl
5-Methyl-2-pyridinyl
5-Chloro-2-pyridinyl
5-Chloro-2-pyridinyl
5-Bromo-2-pyridinyl
4-Chloro-2-pyridinyl
3-Chlorobenzyl
4-Chlorobenzyl
2-Pyridinyl
4-Chloro-2-pyridinyl
5-Bromo-2-pyridinyl
2-Pyridinyl
13.9
6.5
7.5
5.3
0.43
34.7
18.3
>80
0.098
0.29
0.85
7.8
0.56
59.6
15.4
55.2
0.42
0.53
5.6
33.4
23.1
11.9
10.9
1.6
65.4
60.8
>80
67.2
>80
47.8
35.3
19.7
76.8
74.5
6j
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
>80
>80
7.8
8.9
17.2
12.3
24.3
29.5
31.4
>80
>80
>80
56.8
>80
14.7
23.4
21.1
21.7
15.6
70.4
>80
>80
>80
>80
>80
>80
25.2
>80
>80
4-Chlorobenzyl
5-Chloro-2-pyridinyl
5-Chloro-2-pyridinyl
5-Chloro-2-pyridinyl
5-Bromo-2-pyridinyl
4-Chloro-2-pyridinyl
4-Chloro-2-pyridinyl
2-Pyridinyl
Ethenyl
Ethenyl
3-Chlorobenzyl
a
The antiproliferation activities of compounds 6a–7r in vitro were determined by the MTT assay on A875, H460, Hela cell lines, IC50 was average of three determinations
and deviation from the average was <5% of the average value.
compounds. Finally, compound 7a was selected as a lead for fur-
ther study because of its best potency against A875 cancer cells
in vitro.
To further evaluate the antiproliferative mechanism of com-
pound 7a, flow cytometric analysis was used to quantitatively as-
sess and measure the apoptotic cells (sub-G1 cells) and the cell

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L. Zhao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2293–2297
cycle after PI-stained.19 As shown in Figure 2A and B, The percent-
age of apoptotic cells were 3.67%, 25.45%, 42.88% for 24 h and
11.17%, 41.59%, 61.59% for 48 h, respectively, after treatment with
accumulation of cells in the G2/M and S phase, accompanied by a
decrease in G0/G1 phase was observed after treatment with
compound 7a in A875 cancer cells, (Fig. 2C). The percentage of cells
in the G0/G1 phase was 78.18% for the control, and 60.01%, 48.94%
and 30.52% for 48 h, respectively, after treatment with 0.38, 0.75
0.38, 0.75 and 1.5
lM 7a. The results suggested that 7a inhibited
the proliferation of A875 cancer cells by inducing apoptosis in a
concentration-dependent manner. In addition,
a
significant
and 1.5
lM compound 7a. The results suggested that compound
Table 2
The antiproliferation activities of compounds 7a, 7b and cisplatin against various cancer cell lines
a
Compound
IC50
(
lM)
HT29
Hela
Bel-7402
SMMC-7721
HepG2
A549
HCT-116
A875
H460
BxPC-3
7a
7b
Cisplatin
8.7
13.7
28.9
2.5
2.1
19.8
0.45
0.67
17.2
0.35
0.43
12.6
3.6
5.8
23.1
0.59
6.2
14.5
0.61
1.2
15.6
0.098
0.29
22.4
0.42
0.53
8.7
0.19
0.28
13.2
a
Values were average of three determinations and deviation from the average is <5% of the average value.
Figure 2. Effect of 7a on the induction of apoptosis and cell cycle distribution: (A) DNA fluorescence histograms of PI-stained A875 cells. Cells were treated with 0.38
0.75 M, 1.5 M for 24 h; (B) DNA fluorescence histograms of PI-stained A875 cells. Cells were treated with 0.38 M, 0.75 M, 1.5 M for 48 h. The cells in the sub-G1 phase
were considered as apoptotic cells; (C) effect of cell cycle distribution with 0.38 M, 0.75 M, 1.5 M in A875 cells for 48 h.
lM,
l
l
l
l
l
l
l
l
Figure 3. Effect of 7a on cell morphology: (A) Bright-field microscopy images and (B) fluorescence microscopic appearance of Hoechst 33342 staining nuclei of A875 cancer
cells, after incubation with 7a for 36 h at varying concentrations: 0.38 lM, 0.75 lM, 1.5 lM. Apoptosis cells were observed in treated cells containing condensed and
fragmented fluorescent nuclei.

L. Zhao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2293–2297
2297
7a could remarkably arrested G2/M and S phase and induced apop-
References and notes
tosis at low micromolar concentrations in vitro.
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Furthermore, fluorescence microscopic examination of Hoechst
33342-stained cell confirmed the inducing apoptosis effect of 7a.20
The apoptotic cells are often characterized by its morphological,
including cell shrinkage, membrane blabbing, chromatin conden-
sation and DNA fragmentation.21 When treated with 7a for 36 h,
A875 cancer cells shrank and rounded up, these phenomena be-
came more significant with the 7a concentration increasing. In
contrast, the untreated A875 cells retained their normal size and
shape, kept proliferating with time and became over-crowded by
36 h (Fig. 3A). Morphological changes were also characteristic of
apoptosis after Hoechst 33342 staining. Apoptosis cells, bright blue
fluorescent and condensed nuclei, were observed in a large number
of treated cells in A875, and the change was concentration-depen-
dent, whereas there was no significant apoptosis in untreated A875
cells that showed blue, diffusely stained intact nuclei (Fig. 3B).
These results indicated that 7a induced apoptosis in A875 cancer
cells.
In conclusion, we have described the optimization of the induc-
ing apoptosis compound 1, 29 analogs were synthesized and eval-
uated the antiproliferative activity in vitro. The SAR analysis
indicated that the substituents in R2 played a crucial role in the
antiproliferation activity, especially chloromethyl that could signif-
icantly increase antiproliferation ability. The most potent analog
7a displayed very good inhibitory activity against a panel of differ-
ent types of human cancer cell lines with IC50 values in the low
micromolar range and exhibited an IC50 value of 98 nM compared
14. Wang, Y.; Xiao, J.; Zhou, H.; Yang, S.; Wu, X.; Jiang, C.; Zhao, Y.; Liang, D.; Li, X.;
Liang, G. J. Med. Chem. 2011, 54, 3768.
15. Briefly, cells (3 Â 103/well) were seeded in 96-well plates and cultured for 24 h,
followed by compounds treatment for 48 h. A volume of 20 ll of 5 mg/ml MTT
was added per well and incubated for another 2 h at 37 °C, then the
supernatant fluid was removed and DMSO was added 150 l/well for 15–
l
20 min. The absorptions (OD) were measured at 570 nm with SpectraMAX M5
microplate spectrophotometer (Molecular Devices). The effect of compounds
on tumor cells viability was expressed by IC50 of each cell line.
16. Baraldi, P. G.; Preti, D.; Tabrizi, M. A.; Fruttarolo, F.; Saponaro, G.; Baraldi, S.;
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18. Typical procedure for the synthesis of 1a–1e and 1g–1r. Compound 7a was
prepared as follows: To
a stirred solution of 5a (15 mmol) in 50 mL
with cisplatin as a positive control, with an IC50 value of 22.4 lM
tetrahydrofuran was added potassium carbonate (30 mmol) and stirred at
room temperature for 5 min and then cooled to 0–5 °C. 2-Chloroacetyl chloride
(22.5 mmol) was added dropwise at 0–5 °C and after addition the reaction
mixture was allowed to warm to room temperature and then stirred for 4 h
until TLC analysis indicated the complete disappearance of starting material.
The reaction was quenched by addition of 20 mL distilled water, addition
dichloromethane was added and the dichloromethane layer after
extraction(50 mL Â 2) was separated, dried (Na2SO4) and concentrated in
vacuo to afford crud product. The crud product was purified by column
chromatography on silica gel using petroleum ether/ethyl acetate (1/4) as
eluent, the yield was 86%.
against A875 cells. The results of flow cytometry analysis and mor-
phological analysis indicated that compound 7a have potential
anticancer efficacy via G2/M of A875 cell cycle arrest, which could
be attributed to its proliferation and apoptosis, and also in a con-
centration-dependent manner.
Our results will provide useful information for the design of no-
vel compounds with better potency as antitumor agents. Further
study on the antitumor activities in vivo and exact biological
mechanism is underway
19. Briefly, cells were seeded in 6-well plates at the density of (1 Â 105/well) and
cultured for 24 h, followed by 7a treatment for another 24 h. At the indicated
intervals, both attached cells and floating cells were harvested, sediments were
resuspended in 1 mL hypotonic fluorochrome solution containing 50 lg/mL PI
Acknowledgments
in 0.1% sodium citrate plus 0.1% Triton X-100, and then analyzed by flow
cytometer (ESP Elite, Beckman-Coulter, Miami, FL). Apoptotic cells appeared in
the cell cycle distribution as cells with a DNA content of less than that of G1
cells and were estimated with Listmode software.
This work was supported by National Science and Technology
Major Project of China (2009ZX09103-132).
20. Briefly, cells (1 Â 105/well) were seeded in 6-well plates and cultured for 24 h,
followed by 7a treatment for another 24 h. After rinsing with PBS, the cells
were fixed using 75% of ethanol. The morphological change of cell was
examined by inverted microscope, then the cells were stained with Hoechst
Supplementary data
33342 (2 lg/mL, in PBS) and analyzed under fluorescence microscope (Zeiss,
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.076.
Axiovert 200, Germany) to identify the apoptotic cells.
21. Fan, C. D.; Zhao, B. X.; Wei, F.; Zhang, G. H.; Dong, W. L.; Miao, J. L. Bioorg. Med.
Chem. Lett. 2008, 18, 3860.