Synthesis and in vitro biological evaluation of farnesylthiosalicylic acid derivatives as anti-tumor carcinoma agents

Article abstract of DOI:10.1016/j.cclet.2012.08.007

Novel farnesylthiosalicylic acid (FTA) derivatives 5a-m with different substituted 1,3,4-thiadiazoles were synthesized. Compounds 5b, 5c, 5e and 5f displayed anti-tumor activities superior to FTA in most cancer cells tested. Furthermore, 5e induced tumor cell apoptosis, which was accompanied by lower Bcl-2 expression, but with higher Bax and caspase 3 expression activities in cancer cells.

Full text of DOI:10.1016/j.cclet.2012.08.007

Available online at www.sciencedirect.com
Chinese Chemical Letters 23 (2012) 1141–1144
www.elsevier.com/locate/cclet
Synthesis and in vitro biological evaluation of farnesylthiosalicylic
acid derivatives as anti-tumor carcinoma agents
Yong Ling a, You An Xiao b, Guang Tong Chen a, Dong Geng Wang a, Yu Qin Li a,
c
Xin Yang Wang a, , Heng Zheng
*
a Department of Pharmacy, School of Medical, Nantong University, Nantong 226001, China
b Medicinal College, Anhui College of Traditional Chinese Medicine, Hefei 230031, China
c Department of Bioinformatics, College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
Received 20 June 2012
Available online 21 September 2012
Abstract
Novel farnesylthiosalicylic acid (FTA) derivatives 5a–m with different substituted 1,3,4-thiadiazoles were synthesized.
Compounds 5b, 5c, 5e and 5f displayed anti-tumor activities superior to FTA in most cancer cells tested. Furthermore, 5e
induced tumor cell apoptosis, which was accompanied by lower Bcl-2 expression, but with higher Bax and caspase 3 expression
activities in cancer cells.
# 2012 Xin Yang Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Synthesis; Farnesylthiosalicylic acid; 1,3,4-Thiodiazole; Anti-tumor agents; Cytotoxic activities; Cell apoptosis
Ras proteins serve as key regulators of oncogenic processes by governing cell proliferation, migration, and survival
[1–3]. Notably, the mutations of the Ras genes usually result in the constitutive activation of Ras-GTP proteins and
overstimulation of downstream signaling, promoting the development and progression of tumors in human [4,5].
Hence, the Ras-related signaling events are the therapeutic targets for intervention of human cancers.
Farnesylthiosalicylic acid (FTA), a novel Ras inhibitor, structurally mimiced the farnesyl moiety in the carboxy
terminal of Ras proteins, and was found to induce accelerated dislodgement of Ras from the cell membrane and its
subsequent degradation [6,7]. FTA has been used for the treatment of a wide range of malignancies, including
pancreatic and lung cancers [8–10]. However, its therapeutic efficacy is limited [11]. Therefore, development of new
Ras inhibitors with potent anti-tumor activity should be of great significance. Our group has recently developed a
number of promising FTA derivatives as anticancer drug candidates [12,13].
Thiadiazoles are one of the privileged structural fragments in medicinal chemistry having broad spectrum of
pharmacological activities. Particularly, 1,3,4-thiadiazoles are much explored for their wide range of anticancer
activities [14–16]. Accordingly, we hypothesized that novel types of thiadiazoles/FTA hybrids could possess
better anticacer activities, leading to potent cytotoxicity against broad types of cancer cells. Therefore, a total of 13
target compounds 5a–m were designed and synthesized by coupling the carboxyl group of FTA with different
* Corresponding author.
E-mail address: wangxy@ntu.edu.cn (X.Y. Wang).
1001-8417/$ – see front matter # 2012 Xin Yang Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2012.08.007

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Y. Ling et al. / Chinese Chemical Letters 23 (2012) 1141–1144
Scheme 1. Reagents and conditions: (a) 1. CS2, KOH, EtOH, 70–75 8C, 6 h; 2. conc.HCl, rt, 82%. (b) RCH2Br, 0.1 mol/L NaOH, 80% EtOH, rt,
24 h, 64–85%. (c) EDCI, DMAP, CH2Cl2, rt, 24 h, 60–83%.
thioether-substituted 1,3,4-thiadiazoles. Their anti-tumor activities, and apoptosis effect were evaluated in vitro.
Herein, we report the synthesis and biological evaluation of the target compounds.
The synthetic routes of 5a–m were outlined in Scheme 1. The substituted 1,3,4-thiadiazoles were prepared in a two-
step sequence. The starting material thiosemicarbazide 2 was converted to 2-amino-1,3,4-thiadiazole-5-thiol 3 by
treatment with CS2 in the presence of KOH in 82% yield. Subsequently, compound 3 was treated with corresponding
bromides in the presence of NaOH in 80% EtOH to afford intermediates 4a–m in 64–85% yields. Finally, the resulting
thiadiazoles were reacted with S-trans,trans-farnesylthiosalicylic acid (1, FTA) in CH2Cl2 in the presence of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylamino-pyridine (DMAP) to
generate the target compounds 5a–m in 61–79% yields [17].
The cytotoxicity of individual compounds against human breast adenocarcinoma cells (MDA-MB-231), human
hepatocellular carcinoma cells (Bel7402), human lung cancer cells (H520) and human glioblastoma cells (U251) was
evaluated by MTT assay using FTA as positive control. Table 1 illustrates the IC50 values of target compounds against
each tumor cell line. Most of compounds displayed inhibitory effects on the proliferation of tumor cells, which were
superior to FTA. For example, the inhibitory effects of 5b, 5c, 5e and 5f (IC50 = 15.8–23.0 mmol/L) on the
proliferation of MDA-MB-231 cells were notably stronger than that of FTA (IC50 = 59.7 mmol/L). Especially, the IC50
of 5e (13.2–20.5 mmol/L) against four tumor cells was 3-4-fold less than those of FTA (51.8–78.0 mmol/L).
Based on above results, wewere interested in determiningwhethertheobserved cytotoxicity by 5ewas dueto effect of
5eoncell apoptosis. Inordertotestthis, MDA-MB-231cellsweretreated withvehiclecontrolordifferentdosesof 5eand
the expression of the pro-apoptotic Bax and anti-apoptotic Bcl-2 was determined by western blotting analysis (Fig. 1A).
Obviously, treatment with 5e dramatically increased the relative levels of pro-apoptotic Bax expression, but reduced the
levels of anti-apoptotic Bcl-2 expression in a concentration-dependent manner. Meanwhile, given that caspases are
cysteine dependent enzymes that play an important role in the induction, transduction and amplification of intracellular
apoptotic signals, subsequent experiments to examine thelevelsof activated caspase 3 bycalorimetricassaysshowed that
incubation with 5e elevated the levels of activated caspase 3 activities in a concentration-dependent manner (Fig. 1B).
Theobtaineddatademonstratedthat theactivecompoundcouldsignificantlyinducetumorcellsapoptosisinvitro. We are
currently interested in further studies to investigate how target compounds promote tumor cell apoptosis.

Y. Ling et al. / Chinese Chemical Letters 23 (2012) 1141–1144
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Table 1
IC50 values of 5a–m against four human cancer cell lines.
Compd.
In vitro cytotoxicity (IC50, mmol/L)
MDA-MB-231
Bel7402
H520
U251
FTA
5a
5b
5c
59.7
>50
23.0
19.4
>50
15.8
18.7
36.6
>50
>50
34.2
33.4
>50
78.0
33.2
25.1
21.1
>50
20.5
18.7
27.5
>50
>50
>50
41.7
>50
51.9
41.9
21.9
20.5
>50
13.2
20.4
29.3
46.1
39.8
36. 4
35.2
>50
53.7
40.6
26.5
24.8
>50
18.2
22.1
32.9
>50
>50
>50
>50
>50
5d
5e
5f
5g
5h
5i
5j
5k
5m
Fig. 1. Effect of 5e on the expression of apoptosis-related proteins in MDA-MB-231 cells. (A) The expression of Bax, Bcl-2, and b-actin was
examined by western blotting analysis. MDA-MB-231 cells were incubated with, or without, 5e at the indicated concentrations for 48 h and the
levels of protein expression were detected using specific antibodies. Data shown are representative images of each protein for three separate
experiments. (B) The activity of caspase 3 in MDA-MB-231 cells was measured using colorimetric assay and expressed as the percentage of the
activity of control cells. The values presented were mean Æ SD. *P < 0.05 vs. the vehicle control.
Acknowledgments
We gratefully acknowledge the financial support by the Natural Science Foundation of Jiangsu Province in China
(No. BK2011389) and Applied Research Projects of Nantong City (BK2012085); and also thank a project funded by
the Priority Academic Programs Development of Jiangsu Higher Education Institutions (PAPD).
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[17] Selected data of title compounds: Compound 5b: Yield 72%, white waxy solid; IR (KBr, cmÀ1): 3447, 2962, 1656, 1532, 1236, 1041; 1H NMR
(300 MHz, CDCl3): d 9.08 (m, 1H), 8.09 (m, 1H), 7.58 (m, 1H), 7.50 (m, 1H), 7.47 (m, 1H), 5.25 (m, 1H), 5.03 (m, 2H), 3.57 (d, 2H,
J = 7.8 Hz), 3.23 (m, 2H), 1.89–2.02 (m, 8H), 1.50–1.68 (m, 12H), 1.06 (m, 3H); MS (ESI) m/z = 502 [M + H]+; Anal. Calcd. for
C26H35N3OS3: C, 62.24; H, 7.03; N, 8.37. Found: C, 62.07; H, 7.20; N, 8.18. Compound 5c: Yield 75%, white waxy solid; IR (KBr,
cmÀ1): 3402, 2925, 1660, 1536, 1469, 1301, 1243, 1045; 1H NMR (300 MHz, CDCl3): d 9.15 (m, 1H), 8.13 (m, 1H), 7.54 (m, 2H), 7.43 (m,
1H), 5.25 (m, 1H), 5.04 (m, 2H), 3.55 (d, 2H, J = 7.8 Hz), 3.27 (m, 2H), 1.89–2.02 (m, 8H), 1.50–1.68 (m, 12H), 1.20 (m, 2H), 1.04 (m, 3H);
MS (ESI) m/z = 516 [M + H]+; Anal. Calcd. for C27H37N3OS3: C, 62.87; H, 7.23; N, 8.15. Found: C, 62.65; H, 7.35; N, 8.17. Compound 5e:
Yield 64%, white waxy solid; IR (KBr, cmÀ1): 3453, 2921, 1644, 1536, 1455, 1306, 1250, 1176, 1043; 1H NMR (300 MHz, CDCl3): d 9.08 (m,
1H), 8.08 (m, 1H), 7.45 (m, 2H), 7.33 (m, 1H), 7.26 (m, 4H), 5.26 (m, 1H), 5.03 (m, 2H), 4.44 (s, 2H), 3.79 (m, 5H), 1.89–2.02 (m, 8H), 1.50–
1.68 (m, 12H); MS (ESI) m/z = 594 [M + H]+; Anal. Calcd. for C32H39N3O2S3: C, 64.72; H, 6.62; N, 7.08. Found: C, 64.56; H, 6.79; N, 6.92.
Compound 5f: Yield 67%, white waxy solid; IR (KBr, cmÀ1): 3331, 3135, 2923, 1657, 1537, 1303, 1243, 1069; 1H NMR (300 MHz, CDCl3):
d 9.10 (m, 1H), 8.10 (m, 1H), 7.59 (m, 1H), 7.50 (m, 1H), 7.43 (m, 2H), 7.28 (m, 3H), 5.24 (m, 1H), 5.05 (m, 2H), 4.44 (s, 2H), 3.56 (d, 2H,
J = 7.8 Hz), 1.89–2.02 (m, 8H), 1.50–1.68 (m, 12H); MS (ESI) m/z = 643 [M + H]+; Anal. Calcd. for C31H36BrN3OS3: C, 57.93; H, 5.65; N,
6.54. Found: C, 57.75; H, 5.78; N, 5.43.