Synthesis and biological evaluation of farnesylthiosalicylamides as potential anti-tumor agents

Article abstract of DOI:10.1016/j.bmc.2013.11.013

Fourteen hybrids of farnesylthiosalicylic acid (FTS) with various diamines were synthesized and biologically evaluated. It was found that FTS-monoamide molecules (10a-g) displayed strong anti-proliferative activity against seven human cancer cell lines, superior to FTS and FTS-bisamide compounds (11a-g). The mono-amide 10f was the most active, with IC₅₀s of 3.78-7.63 μM against all tested cancer cells, even more potent than sorafenib (9.12-22.9 μM). In addition, 10f induced SMMC-7721 cell apoptosis, down-regulated the expression of Bcl-2 and up-regulated Bax and caspase-3. Furthermore, 10f had the improved aqueous solubility relative to FTS. Finally, treatment with 10f dose-dependently inhibited the Ras-related signaling pathways in SMMC-7721 cells. Collectively, 10f could be a promising candidate for the intervention of human cancers.

Full text of DOI:10.1016/j.bmc.2013.11.013

Bioorganic & Medicinal Chemistry 22 (2014) 374–380
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of farnesylthiosalicylamides as
potential anti-tumor agents
Yong Ling a,b, Zhiqiang Wang a,b, Hongyan Zhu b, Xuemin Wang b, Wei Zhang b, Xinyang Wang b, Li Chen a,
Zhangjian Huang a,c, , Yihua Zhang a,c,
⇑
⇑
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
b Medical College, Nantong University, Nantong 226001, PR China
c Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fourteen hybrids of farnesylthiosalicylic acid (FTS) with various diamines were synthesized and biolog-
ically evaluated. It was found that FTS-monoamide molecules (10a–g) displayed strong anti-proliferative
activity against seven human cancer cell lines, superior to FTS and FTS-bisamide compounds (11a–g). The
Received 17 August 2013
Revised 17 October 2013
Accepted 8 November 2013
Available online 15 November 2013
mono-amide 10f was the most active, with IC50s of 3.78–7.63
lM against all tested cancer cells, even
more potent than sorafenib (9.12–22.9 M). In addition, 10f induced SMMC-7721 cell apoptosis,
l
down-regulated the expression of Bcl-2 and up-regulated Bax and caspase-3. Furthermore, 10f had the
improved aqueous solubility relative to FTS. Finally, treatment with 10f dose-dependently inhibited
the Ras-related signaling pathways in SMMC-7721 cells. Collectively, 10f could be a promising candidate
for the intervention of human cancers.
Keywords:
Farnesylthiosalicylic acid
Diamines
Cell apoptosis
Ras-related signaling pathway
Anti-cancer agents
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
SO2Ph
O
N
O
N
S
The Ras proteins are low molecular-weight GTP-binding
proteins, which function as biological switches playing a key role
in mediation of signal transduction between G-protein-coupled
receptors and downstream events, such as mitogen-activated
protein kinase (MAPK) and Akt.1,2 Mutations or excessive activa-
tion of Ras proteins are found in approximately 30% of human
cancers.3–5 It was reported that the inhibition of excessive acti-
vated Ras proteins may revert malignant cells to a nonmalignant
phenotype and cause tumor regression both in vitro and in vivo.6
As a result, the Ras protein has become an attractive therapeutic
target for intervention of a number of cancers.
O
N
N
O
S
COOH
FTS
1
Figure 1. The chemical structures of FTS and furoxan-based nitric oxide derivative
of FTS (compound 1).
with more potent anti-tumor activity should be of clinical
significance.
Previously, a series of furoxan-based nitric oxide donating FTS
derivatives with anti-hepatocellular carcinoma activity was
reported by our group.15 It has been observed that the amide
derivatives exhibit much stronger inhibitory effects than the corre-
sponding esters. The most active compound 1 is an amide bearing a
piperazine moiety (Fig. 1). It is known that a diamine moiety exists
in various pharmaceutical compounds16 and is often used as a
structural block in rational drug design because of its improved
water-solubility and enhanced bioavailability and metabolic sta-
bility in vivo.17 It was therefore interesting to determine whether
conjugating such diamine moiety with the carboxylic acid group
of FTS would provide the derivatives that possess enhanced
anticancer activity as well as the desired water-solubility. As part
of this ongoing program, we now describe the synthesis of
Farnesylthiosalicylic acid (FTS, Fig. 1), structurally mimicking
the carboxyl-terminal farnesylcysteine moiety of Ras proteins,
can recognize the anchorage and dislodge the active Ras protein
from the cell membrane, thereby blocking the initiation of down-
stream signaling events, inhibiting tumor cell proliferation and
promoting the tumor cell apoptosis.7–9 Although FTS was well
preclinically studied for the treatment of a wide range of malignan-
cies, including lung, breast, and pancreatic cancers,10–13 it has not
got approval for clinical trials mainly due to its limited therapeutic
efficacy.14 Therefore, development of new FTS-based Ras inhibitors
⇑
Corresponding authors. Tel./fax: +86 2583271015 (Y.Z.).
E-mail addresses: zyhtgd@hotmail.com, zyhtgd@163.com (Y. Zhang).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.013

Y. Ling et al. / Bioorg. Med. Chem. 22 (2014) 374–380
375
mono- (10a–g) and bis-amides (11a–g) of FTS, and evaluation as
potential anti-cancer agents.
gastric cancer cells), SMMC-7721 (human hepatocellular carci-
noma cells), EJ (human bladder carcinoma cells), SKOV-3 (human
ovarian cancer cells), MCF-7 (human breast cancer cells), H460
(human lung cancer cells), Panc-1 (human pancreatic carcinoma
cells) were evaluated by MTT assays in vitro using FTS and sorafe-
nib, a well-known Ras-related signal inhibitor, as positive controls.
The IC50 values were summarized in Table 1. As can be seen, mono-
amides 10a–g displayed dramatically improved antiproliferative
effects compared to FTS. Among them, compounds 10c–f
2. Results and discussion
2.1. Chemistry
FTS was previously prepared by direct conjugation of thiosali-
cylic acid with farnesyl bromide in the presence of guanidine
carbonate,18 but some disadvantages limit the utility of this
approach. Firstly, hydrogen-bonding effect between the carboxyl
with the sulfhydryl of thiosalicylic acid could attenuate the reactiv-
ity of sulfhydryl with farnesyl bromide. Secondly, the carboxyl of
thiosalicylic acid could also react with farnesyl bromide, producing
some by-products, reducing the yields and leading to purification
problem. Herein, FTS was obtained using a protection strategy.
The synthetic routes of 10a–g and 11a–g including FTS are
depicted in Scheme 1. Triethylphosphonoacetate (TEPA) was trea-
ted with (E)-geranyl acetone (2) in the presence of NaH to form
(E,E)-ethyl farnesylate (3) as previously reported.19 Compound 3
was reduced by LiAlH4 giving (E,E)-farnesol (4), which was then
converted to (E,E)-farnesyl bromide (5) by treatment with PBr3.
In addition, the carboxyl group of thiosalicylic acid (6) was
protected to form methyl thiosalicylate (7). Coupling of 5 with 7
furnished methyl (E,E)-farnesylthiosalicylicate (FTM, 8), followed
by hydrolysis in aqueous solution of NaOH to provide free carbox-
ylic acid (FTS, 9). Subsequently, 9 was treated with oxalylchloride
to produce (E,E)-farnesylthiosalicyl chloride, which was treated
without further purification with several diamines including cyclic
piperazine in the presence of triethylamine, giving target
compounds 10a–g and 11a–g. The target compounds 10a–g and
11a–g were purified by column chromatography, and their
structures were characterized by IR, 1H NMR and MS.
(IC50 = 3.78–15.0
effects than sorafenib (IC50 = 9.12–22.9
most potent inhibitory activity with IC50 value range of
3.78–7.63 M against all tested cancer cells. However, compounds
l
M) showed slightly stronger antiproliferative
lM). Notably, 10f exhibited
l
11a–f, the bis-amide derivatives of FTS showed very week
inhibitory activity.
Given that FTS is a known Ras inhibitor and could block Ras-re-
lated signaling events, inducing tumor cell apoptosis, the most ac-
tive compound 10f was selected to determine its effect on SMMC-
7721 cells apoptosis. The SMMC-7721 cells were incubated with
vehicle alone or with different concentrations of 10f (3, 6 or
12 lmol/L), or FTS (12 lmol/L) for 48 h, and the percentages of
the apoptotic SMMC-7721cells were determined by FITC-Annexin
V/PI staining and flow cytometry. As shown in Figure 2, the
percentage of annexin V + apoptotic SMMC-7721 cells gradually
increased for those cells exposed to increasing concentrations of
10f (25.9% for 3
12 mol/L), demonstrating that incubation with 10f induced
SMMC-7721cell apoptosis in a dose-dependent manner. In con-
lmol/L; 48.2% for 6 lmol/L; and 84.5% for
l
trast, incubations performed using FTS (12
apoptosis of 19.4% SMMC-7721cells.
lmol/L) only induced
Next, a Western blotting analysis was conducted to check the
expression levels of Bcl-2, Bax and caspase-3 proteins. It was
observed that incubation of 10f significantly increased levels of
pro-apoptotic Bax and caspase 3, but reduced levels of anti-apop-
totic Bcl-2 (Fig. 3) in a dose-dependent manner.
2.2. Biological evaluation
Finally, in order to get insight to the mechanisms underlying the
activity of these farnesylthiosalicylamide derivatives, we examined
the inhibitory effects of the active compound 10f on the Ras-re-
The cell growth inhibitory activity of target compounds 10a–g
and 11a–g against seven human cancer cell lines SGC7901 (human
O
a
b
O
O
3
2
Br
OH
c
4
5
S
SH
SH
COOMe
e
d
COOMe
COOH
7
8
6
S
S
g
f
O
COOH
R
XH
X
9, FTS
10a-g
10-11a
X= NH, R = CH2CH2
S
X
10-11b
10-11c
10-11d
X= NH, R = CH2CH2CH2
O
X= NH, R = CH2CH2CH2CH2
X= NH, R = CH2CH2CH2CH2CH2
X
S
R
+
O
10-11e
X= NH, R = CH2CH2CH2CH2CH2CH2
X= NH, R = CH2CH2OCH2CH2
10-11f
10-11g
11a-g
N
=
X
X
R
N
Scheme 1. Reaction reagents and conditions: (a) TEPA, NaH, ether, 0 °C to rt, 12 h, 81%; (b) LiAlH4, AlCl3, ether, 0 °C to rt, 4 h, 88%; (c) PBr3, pyridine, n-hexane, ether, 0 °C to rt,
4 h, 76%; (d) SOCl2, MeOH, 0 °C-reflux, 8 h, 85%; (e) 5, K2CO3, CH3CN, 50 °C, 6 h; (f) 1 N NaOH, MeOH, 60 °C, 10 h, 82%; (g) oxalylchloride, diamines or piperazine, TEA, CH2Cl2,
0 °C to rt, 2 h.

376
Y. Ling et al. / Bioorg. Med. Chem. 22 (2014) 374–380
Table 1
The IC50 values of 10a–g and 11a–g against seven human cancer cell lines
a
Compd
In vitro inhibition of human cancer cells proliferation (IC50 , lM)
SGC-7901
SMMC-7721
EJ
SKOV-3
MCF-7
H460
Panc-1
FTS
41.3 5.56
11.5 2.71
33.8 3.68
12.8 1.76
11.2 0.97
7.35 0.88
7.87 1.06
7.63 0.90
25.7 2.75
>50
>50
>50
>50
>50
43.8 5.25
>50
69.7 3.85
18.7 2.65
25.2 3.59
10.6 1.84
15.0 1.67
9.21 1.13
7.93 1.20
5.71 0.69
32.4 2.94
>50
>50
>50
36.2 3.75
>50
29.0 3.32
>50
47.6 3.28
22.9 3.53
>50
17.2 1.37
13.1 1.49
9.85 1.24
10.1 0.77
6.13 0.52
27.5 3.67
>50
>50
>50
39.5 4.18
43.9 4.71
27.8 3.07
>50
51.2 5.06
9.25 2.15
31.6 2.81
12.3 1.92
7.80 1.01
7.13 0.94
7.66 1.05
6.83 0.71
>50
>50
>50
>50
41.9 3.54
>50
49.1 4.66
9.12 1.80
28.7 3.42
13.7 2.01
11.2 1.33
7.83 0.68
8.01 0.93
7.29 0.93
34.1 3.01
>50
>50
45.8 3.99
>50
40.6 3.66
30.0 3.62
>50
49.2 4.47
10.8 2.36
36.0 3.83
20.4 3.36
12.5 1.62
8.84 1.10
7.52 0.89
6.89 0.85
26.3 2.64
>50
>50
>50
47.1 3.26
>50
43.5 3.27
>50
53.6 5.77
12.3 1.46
>50
11.8 1.25
10.5 1.38
7.56 1.17
8.69 1.09
3.78 0.45
22.5 2.96
>50
>50
>50
>50
>50
29.8 3.03
>50
Sorafenib
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
11e
11f
34.4 4.28
>50
11g
a
The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data are the mean values of IC50 (a dose
achieved 50% inhibition in the proliferation of cancer cells cultured) SEM from at least three independent experiments.
Figure 2. Compound 10f induced SMMC-7721 cell apoptosis in vitro. SMMC-7721 cells were incubated with the indicated concentrations of 10f and 12 lM FTS for 48 h, and
the cells were stained with FITC-Annexin V and PI, followed by flow cytometry analysis. (A) Flow cytometry analysis. (B) Quantitative analysis of apoptotic cells. Data are
expressed as means SEM of the percentages of apoptotic cells from three independent experiments. ⁄P <0.01 versus control.
lated signaling in SMMC-7721 cells using FTS as control. The cells
were incubated with vehicle alone, FTS (12 M), or with 10f (3, 6 or
12 M). The expression and phosphorylation levels of the Ras-re-
activities in the growth inhibition and apoptosis induction of the
cancer cell lines.
l
l
lated signal events, Akt, and ERK1/2 (extracellular signal-regulated
kinase 1/2) were determined using an immunoblotting assays. As
shown in Figure 4, after the treatment with 10f, the levels of phos-
pho-ERK1/2 and phospho-Akt were clearly reduced in SMMC-7721
cells in a dose-dependent manner, and at the same concentration
2.3. Aqueous solubility determination
The aqueous solubility of parent compound FTS, some selected
FTS mono-amide (10d, 10f) and bis-amide (11d) derivatives in 10%
PEG400 aqueous solution was determined using a previously
reported HPLC-based method.20 As shown in Table 2, the solubility
of mono-amides 10d, 10f were 17.97 and 23.75 mg/mL,
respectively, which were six to eightfold better than that of FTS
(3.65 mg/mL). Obviously, compound 10f with an oxygen atom
displaced in the diamine chain of 10d, got an improved solubility.
of 12 lM, treatment with 10f showed the lower levels relative to
FTS. Meanwhile the similar expression levels of ERK and Akt were
observed in groups treated with 12f and FTS, respectively. The
results demonstrated that the inhibition of Ras-related signaling
pathways by 10f may contribute to its significant antitumor

Y. Ling et al. / Bioorg. Med. Chem. 22 (2014) 374–380
377
Figure 3. Effect of 10f on the expression of apoptosis-related proteins in SMMC-7721 cells. (A) The expression of Bax, Bcl2, caspase 3 and b-actin was examined by Western
blot analysis. SMMC-7721 cells were incubated with, or without, 10f at the indicated concentrations for 48 h and the levels of protein expression were detected using specific
antibodies, and b-actin was used as the control. Data shown are representative images of each protein for three separate experiments. (B) Quantitative analysis: the relative
levels of each protein compared to control b-actin were determined by densimetric scanning. Data are expressed as means SEM from three separate experiments. ⁄P <0.05
versus control; ⁄⁄P <0.01 versus control.
Figure 4. Immunoblot analysis of the expression and phosphorylation of Akt and ERK1/2 in vitro. (A) SMMC-7721 cells were treated with vehicle (control), FTS, or different
doses of 10f were homogenized, and their lysates were subjected to immunoblot analysis using anti-ERK1/2, antiphospho-ERK1/2 (Thr202/Tyr204), anti-Akt, antiphospho-
Akt (Ser473), and anti-b-actin antibodies, respectively. b-Actin was used as the control. (B) Quantitative analysis. The relative levels of each signaling event to control b-actin
were determined by densimetric scanning. The data are expressed as means SEM of three duplicate experiments. ⁄P <0.01 versus control.
Table 2
to 10f could further improve the solubility, thus enhancing the
biological activity.
Solubilities and logP values of the selected compounds in PEG400 aqueous solution
Compound
Solubility (mg/mL)
LogP
FTS
10d
10f
11d
3.65
17.97
23.75
<0.01
6.32
5.98
4.85
3. Conclusions
In summary, a series of hybrids of FTS with various diamines
were synthesized, and their anti-proliferative activity and aqueous
solubility were evaluated. The FTS mono-amides with better aque-
ous solubility displayed stronger inhibitory activity than FTS and
bis-amides derivatives 11a–g. The mono-amide 10f was the most
14.04
In contrast, the solubility of bis-amide derivative 11d was less than
0.01 mg/mL. The logP values of these compounds were also calcu-
lated by ChembioDraw Ultra (version 2008). It was found that the
logP values of mono-amides 10d and 10f were lower than that of
FTS, and compound 11d had the highest logP value of 14.04.
active, with IC50s of 3.78–7.63 lM against all tested cancer cells,
even more potent than sorafenib, a well-known anti-cancer drug.
Additionally, 10f could induce tumor cell apoptosis, reduce the
protein levels of Bcl-2, and increase the expression of Bax and cas-
pase-3 proteins. More importantly, treatment with 10f inhibited
the Ras-related signaling pathways in a dose-dependent manner
in the cancer cells. Our findings suggest that diamine-based FTS
modification could be a useful way to enhance the anti-cancer
activity of FTS. Further studies on 10f are warranted for the treat-
ment of human cancers.
2.4. Structure–activity relationships (SARs) analysis
Analysis of SAR revealed that FTS-monoamides 10a–g displayed
stronger anti-proliferative activity than bis-amides derivatives
11a–g against seven tested cancer cells. It is likely that mono-
amide derivatives with a free amine moiety possess better aqueous
solubility and suitable logP values, which could be benificial for
them to penetrate the cancer cells and bind more effectively to
the galectin site of Ras protein, blocking Ras-related signaling
events. Among the monoamides, compounds 10d–f bearing five
or six-atom length of diamines moiety exerted the most potent ef-
fects against tumor cells growth. In addition, substitution of one
oxygen atom for methylene in the diamine chain of 10d leading
4. Experimental section
4.1. Chemistry
Infrared (IR) spectra (KBr) were recorded on a Nicolet Impact
410 instrument (KBr pellet). 1H NMR spectra were recorded with
a BrukerAvance 300 MHz spectrometer at 300 K, using TMS as an

378
Y. Ling et al. / Bioorg. Med. Chem. 22 (2014) 374–380
internal standard. MS spectra were recorded on a Mariner Mass
Spectrum (ESI). Element analysis was performed on an Eager 300
instrument. All compounds were routinely checked by TLC and
1H NMR. TLCs and preparative thin-layer chromatography were
performed on silica gel GF/UV 254, and the chromatograms were
conducted on silica gel (200–300 mesh, Merck) and visualized
under UV light at 254 and 365 nm. All solvents were reagent grade
and, when necessary, were purified and dried by standard meth-
ods. Compounds 2 and 6 were commercially available. Solutions
after reactions and extractions were concentrated using a rotary
evaporator operating at a reduced pressure of ca. 20 Torr.
4.1.5. Methyl 2-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-
yl)thio)benzoate (8)
A mixture of 7 (0.9 g, 6 mmol), K2CO3 (1.3 g, 7 mmol) and 3
(1.7 g, 6 mmol) in acetonitrile (75 mL) was refluxed for 6 h. The
solvent was evaporated in vacuum and water was added. The
solution was neutralized with 2 N HCl solution to pH 6–7, and then
extracted with CH2Cl2 (20 mL ꢀ 3). The organic layer was concen-
trated in vacuo and the residue was purified by column chroma-
tography on silica gel (petroleum ether/ethyl acetate = 30:1, v/v
as the eluent), yield 8 as a yellowish oil (1.83 g, 82%). 1H NMR
(CDCl3, 300 MHz, d ppm): 7.92–7.96 (m, H, Ar-H), 7.69–7.74 (m,
1H, Ar-H), 7.26–7.30 (m, 1H, Ar-H), 7.11–7.14 (m, 1H, Ar-H), 5.34
(bt, 1H, SCH2CH), 5.09 (bt, 2H, 2 ꢀ CH2CH@CCH3), 3.92 (s, 3H,
OCH3), 3.58 (d, 2H, J = 7.8 Hz, SCH2), 1.89–2.02 (m, 8H, 2 ꢀ CCH2-
CH2CH), 1.50–1.68 (m, 12H, 4 ꢀ CH@CCH3).22
4.1.1. (2E,6E)-Ethyl 3,7,11-trimethyldodeca-2,6,10-trienoate (3)
To a solution of NaH (60%, 3.20 g, 80 mmol) in anhydrous THF
(200 mL), triethylphosphonoacetate (TEPA) (9.00 g, 40 mmol) in
anhydrous THF (50 mL) was added dropwise at 0 °C according to
the literature.19 The obtained mixture was stirred for 2 h at the
room temperature and then was cooled to 0 °C again. Then (E)-ger-
anyl acetone (2) (4.90 g, 25 mmol) was added dropwise, and the
mixture was allowed to rise to the room temperature and stir for
4 h. The reaction mixture was poured into water (250 mL), and
extracted with diethyl ether (300 mL ꢀ 3). The combined organic
layer was washed with water and brine, dried over anhydrous Na2-
SO4, and concentrated in vacuo. The residue was purified by silica
chromatography (petroleum ether/ethyl acetate = 40:1, v/v as the
4.1.6. 2-(((2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trien-1-
yl)thio)benzoic acid (9)
To a solution of 5 (0.50 g, 1.34 mmol) in ethyl acohol (25 mL),
1 N NaOH aqueous solution (2.5 mL) was added dropwise at the
room temperature. The mixture was refluxed for 10 h, and then
cooled. The solvent was evaporated and the residue was dissolved
in 1 M HCl solution. The mixture was extracted with ethyl acetate
(30 mL ꢀ 3). The organic layer was combined, washed with brine,
dried with anhydrous Na2SO4 and concentrated in vacuo, affording
9 as a pale yellow waxy solid (4.32 g, 90%). 1H NMR (CDCl3,
300 MHz, d ppm): 8.12–8.15 (m, H, Ar-H), 7.41–7.47 (m, 2H, Ar-
H), 7.22–7.26 (m, 1H, Ar-H), 5.31 (bt, 1H, SCH2CH), 5.09 (bt, 2H,
2 ꢀ CH2CH@CCH3), 3.57 (d, 2H, J = 4.5 Hz, SCH2), 1.89–2.02 (m,
8H, 2 ꢀ CCH2CH2CH), 1.50–1.68 (m, 12H, 4 ꢀ CH@CCH3); MS (ESI)
m/z = 357 [M-1]+; Anal. Calcd for C22H30O2S: C, 73.70; H, 8.43.
Found: C, 73.77; H, 8.84.18
eluent), affording the E,E product
3 4.20 g and Z,E isomers
(2Z,6E)-ethyl 3,7,11-trimethyldodeca-2,6,10-trienoate 1.15 g, col-
orless liqid, total yield 81%.19
4.1.2. (2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trien-1-ol (4)
To a solution of LiAlH4 (1.20 g, 31 mmol) in anhydrous diethyl
ether (100 mL) was added AlCl3 (1.47 g, 11 mmol), then compound
3 (4.20 g, 17 mmol) in diethyl ether was slowly added at ꢁ5 °C. The
mixture was stirred for 1 h, and then hydrolyzed with 2 N NaOH
until the formation of white granule. The precipitation was
removed by filtration and the filtrate was extracted with ethyl
ether (100 mL ꢀ 3). The combined organic layers were dried with
anhydrous Na2SO4 and concentrated in vacuo. The crude product
was purified by silica chromatography (petroleum ether–ethyl ace-
tate = 4:1, v/v as the eluent), affording compound 4 as a colorless
liquid (3.11 g, 88%). 1H NMR (CDCl3, 300 MHz, d ppm): 5.33 (bt,
1H, SCH2CH), 5.09 (bt, 2H, 2 ꢀ CH2CH@CCH3), 4.12–4.15 (m, 2H,
OCH2), 1.90–2.02 (m, 8H, 2 ꢀ CCH2CH2CH), 1.54–1.68 (m, 12H,
4 ꢀ CH3).21
4.1.7. General procedure for the preparation of 10a–g and 11a–g
The parent compound 9 (0.36 g, 1 mmol) was dissolved in CH2-
Cl2 (6 mL), then oxalyl chloride (4 mmol) was added dropwise. The
mixture was stirred for 4 h at the room temperature. The solvent
and excess oxalyl chloride was evaporated to obtain the crude
farnesylthiosalicyl chloride. Then to a mixture of different dia-
mines (1.5 mmol) and TEA (0.45 mmol) in 10 mL CH2Cl2 was added
dropwise the CH2Cl2 solution (5 mL) of farnesylthiosalicyl chloride
(1.12 g, 0.3 mmol) at 0 °C over 1 h. After stirring for another 0.5 h
the reaction was quenched by adding 20 mL water. The mixture
was extracted with CH2Cl2 (20 mL ꢀ 3) and the organic layer was
combined, washed with brine, dried with anhydrous Na2SO4 and
concentrated in vacuo. The crude product was separated by silica
gel chromatography (petroleum ether/ethyl acetate = 1:1 v/v; then
CH2Cl2–MeOH = 4:1 as the eluent) to afford compound 10a–g and
11a–g as yellowish oil.
4.1.3. (2E,6E)-1-Bromo-3,7,11-trimethyldodeca-2,6,10-triene (5)
To a solution of 4 (2.87 g, 12.9 mmol) in diethyl ether (15 mL)
and pyridine (0.1 mL) under nitrogen atmosphere, PBr3 (1.52 g,
5.6 mmol) in n-hexane (6 mL) was added slowly at 0 °C. The rea-
tion mixture was stirred at 0 °C for 0.5 h and then was allowed
to rise to the room temperature for 2 h. The upper layer was
poured into ice water, and extracted with diethyl ether
(20 mL ꢀ 3). The organic layer was combined, washed with brine,
dried over anhydrous Na2SO4, and concentrated in vacuo to afford
5 as a colorless liquid (2.79 g, 76%).
4.1.8. N-(2-Aminoethyl)-2-(((2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl)thio)benzamide (10a)
Yellowish oil, 41.9% yield. IR (KBr, cmꢁ1): 3176, 1713, 1605,
1590, 1488; 1H NMR (CDCl3, 300 MHz, d ppm): 8.00 (br, 1H, NH),
7.75–7.78 (m, 1H, Ar-H), 7.32–7.37 (m, 2H, Ar-H), 7.18–7.20 (m,
1H, Ar-H), 5.23 (bt, 1H, SCH2CH), 5.08 (bt, 2H, 2 ꢀ CH2CH@CCH3),
3.56 (d, 2H, J = 9.0 Hz, SCH2), 2.99–3.05 (m, 4H, 2 ꢀ NCH2), 1.91–
2.05 (m, 8H, 2 ꢀ CCH2CH2CH), 1.53–1.68 (m, 12H, 4 ꢀ CH3); ESI-MS
(m/z): 401 [M+H]+; Anal. Calcd for C24H36N2OS: C, 71.95; H, 9.06; N,
6.99. Found: C, 71.86; H, 9.11; N, 7.01.
4.1.4. Methyl thiosalicylate (7)
To a methanol solution (50 mL) of 6 (5.00 g, 32.5 mmol), SOCl2
was added slowly and the mixture was stirred at 0 °C for 1 h. The
obtained mixture was then heated to reflux for 6 h. The solvent
was removed under reduced pressure. The residue was dissolved
in ethyl acetate (50 mL), and washed with saturated NaHCO3
(50 mL ꢀ 2) and brine, dried with anhydrous Na2SO4. The crude
product was purified by silica gel chromatography (petroleum
ether–ethyl acetate = 40:1 v/v as the eluent) to afford compound
7 as a colorless liquid (4.62 g, 85%).
4.1.9. N,N0-(Ethane-1,2-diyl)bis(2-(((2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trien-1-yl)thio)benzamide) (11a)
Yellowish oil, 36.2% yield. IR (KBr, cmꢁ1): 3132, 1708, 1611,
1583, 1471; 1H NMR (CDCl3, 300 MHz, d ppm): 7.65 (d, 2H,
J = 7.5 Hz, Ar-H), 7.41–7.45 (m, 2H, Ar-H), 7.25–7.33 (m, 4H,

Y. Ling et al. / Bioorg. Med. Chem. 22 (2014) 374–380
379
Ar-H), 6.86–6.89 (m, 2H, NH), 5.29–5.33 (m, 2H, SCH2CH), 5.07–
4.1.15. N,N0-(Pentane-1,5-diyl)bis(2-(((2E,6E)-3,7,11-
5.10 (m, 4H, CH2CH@), 3.53–3.57 (d, 4H, J = 5.7 Hz, SCH2), 3.19–
3.23 (m, 4H, 2 ꢀ NCH2), 1.96–2.00 (m, 16H, 4 ꢀ CCH2CH2CH),
1.53–1.63 (m, 24H, 8 ꢀ CH3); MS (ESI) m/z = 742 [M+H]+; Anal.
Calcd for C46H64N2O2S2: C, 74.55; H, 8.70; N, 3.78. Found: C,
74.62; H, 8.61; N, 3.73.
trimethyldodeca-2,6,10-trien-1-yl)thio)benzamide) (11d)
Yellowish oil, 30.4% yield. IR (KBr, cmꢁ1): 3127, 1710, 1603,
1574, 1448; 1H NMR (CDCl3, 300 MHz, d ppm): 7.68 (d, 2H,
J = 7.5 Hz, Ar-H), 7.41–7.43 (m, 2H, Ar-H), 7.21–7.33 (m, 2H, Ar-
H), 6.86–6.87 (m, 2H, NH), 5.24–5.27 (m, 2H, 2 ꢀ SCH2CH),
5.03ꢂ5.07 (m, 4H, 4 ꢀ C@CH), 3.49–3.52 (m, 8H, 2 ꢀ SCH2,
2 ꢀ NCH2), 1.95–2.04 (m, 16H, 4 ꢀ CCH2CH2CH), 1.39–1.71 (m,
30H, 8 ꢀ CH@CCH3, 2 ꢀ NCH2CH2, NCH2CH2CH2); MS (ESI) m/
z = 784 [M+1]+; Anal. Calcd for C49H70N2O2S2: C, 75.14; H, 9.01;
N, 3.58. Found: C, 75.23; H, 9.12; N, 3.46.
4.1.10. N-(3-Aminopropyl)-2-(((2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl)thio)benzamide (10b)
Yellowish oil, 45.3% yield. IR (KBr, cmꢁ1): 3144, 1718, 1615,
1580, 1446; 1H NMR (DMSO-d6, 300 MHz, d ppm): 7.79–7.93 (m,
2H, Ar-H), 7.26–7.32 (m, 2H, Ar-H), 5.21 (br, 1H, SCH2CH), 5.12
(br, 2H, 2 ꢀ CH2CH@), 3.51 (d, 2H, J = 9.0 Hz, SCH2), 3.05 (m, 4H,
2 ꢀ NCH2), 1.90–1.99 (m, 8H, 2 ꢀ CCH2CH2CH), 1.54–1.69 (m,
14H, 4 ꢀ CH3, CH2CH2CH2); ESI-MS (m/z): 415 [M+H]+; Anal. Calcd
for C25H38N2OS0: C, 72.42; H, 9.24; N, 6.76. Found: C, 72.29; H, 9.18;
N, 6.82.
4.1.16. N-(6-Aminohexyl)-2-(((2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl)thio)benzamide (10e)
Yellowish oil, 60.2% yield. IR (KBr, cmꢁ1): 3184, 1723, 1625,
1588, 1468; 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.20 (br, 1H,
NH), 7.75–7.81 (m, 1H, Ar-H), 7.30–7.36 (m, 2H, Ar-H), 7.20 (m,
1H, Ar-H), 5.22 (bt, 1H, SCH2CH), 5.06 (bt, 2H, 2 ꢀ C@CH), 3.55
(d, 2H, J = 9.0 Hz, SCH2), 3.15–3.21 (m, 4H, 2 ꢀ NCH2), 1.95–2.05
(m, 8H, 2 ꢀ CCH2CH2CH), 1.47–1.63 (m, 16H, 2 ꢀ NCH2CH2,
4 ꢀ CH3), 1.31–1.36 (m, 2H, NCH2CH2CH2); ESI-MS (m/z): 457
[M+H]+; Anal. Calcd for C28H44N2OS: C, 73.63; H, 9.71; N, 6.13.
Found: C, 73.48; H, 9.76; N, 6.25.
4.1.11. N,N0-(Propane-1,3-diy)bis(2-(((2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trien-1-yl)thio)benzamide) (11b)
Yellowish oil, 31.5% yield. IR (KBr, cmꢁ1): 3206, 1720, 1619,
1583, 1458; 1H NMR (CDCl3, 300 MHz, d ppm): 7.66–7.82 (m, 2H,
Ar-H), 7.43–7.47 (m, 2H, Ar-H), 7.22–7.30 (m, 4H, Ar-H), 6.85–
6.88 (m, 2H, NH), 5.30–5.33 (m, 2H, SCH2CH), 5.08–5.12 (m, 4H,
CH2CH@), 3.53–3.57 (d, 4H, J = 9.0 Hz, SCH2), 3.19–3.23 (m, 4H,
2 ꢀ NCH2), 1.96–2.00 (m, 16H, 4 ꢀ CCH2CH2CH), 1.53–1.63 (m,
26H, 8 ꢀ CH3, NCH2CH2); MS (ESI) m/z = 756 [M+H]+; Anal. Calcd
for C47H66N2O2S2: C, 74.75; H, 8.81; N, 3.71. Found: C, 74.78; H,
8.76; N, 3.85.
4.1.17. N,N0-(Hexane-1,6-diyl)bis(2-(((2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trien-1-yl)thio)benzamide) (11e)
Yellowish oil, 25.8% yield. IR (KBr, cmꢁ1): 3166, 1708, 1603,
1576, 1452; 1H NMR (CDCl3, 300 MHz, d ppm): 7.67 (d, 2H,
J = 7.5 Hz, Ar-H), 7.41–7.47 (m, 2H, Ar-H), 7.24–7.33 (m, 4H, Ar-
H), 6.86–6.87 (br, 2H, NH), 5.24–5.27 (m, 2H, 2 ꢀ SCH2CH), 5.04–
5.09 (m, 4H, 4 ꢀ C@CH), 3.45–3.54 (m, 8H, 2 ꢀ SCH2, 2 ꢀ NCH2),
1.95–2.09 (m, 16H, 4 ꢀ CCH2CH2CH), 1.48–1.70 (m, 32H, 8 ꢀ CH3,
2 ꢀ NCH2CH2, 2 ꢀ NCH2CH2CH2); MS (ESI) m/z = 798 [M+1]+; Anal.
Calcd for C50H72N2O2S2: C, 75.33; H, 9.10; N, 3.51. Found: C, 75.42;
H, 9.06; N, 3.39.
4.1.12. N-(4-Aminobutyl)-2-(((2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl)thio)benzamide (10c)
Yellowish oil, 46.9% yield. IR (KBr, cmꢁ1): 3184, 1715, 1610,
1578, 1457; 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.19 (br, 1H,
NH), 7.78–7.84 (m, 1H, Ar-H), 7.36–7.41 (m, 2H, Ar-H), 7.20–
7.23 (m, 1H, Ar-H), 5.23 (bt, 1H, SCH2CH), 5.07 (bt, 2H,
2 ꢀ C@CH), 3.55 (d, 2H, J = 9.0 Hz, SCH2), 3.12–3.27 (m, 4H,
2 ꢀ NCH2), 1.91–2.03 (m, 8H, 2 ꢀ CCH2CH2CH), 1.53–1.64 (m,
12H, 4 ꢀ CH3, 2 ꢀ NCH2CH2); ESI-MS (m/z): 429 [M+H]+; Anal.
Calcd for C26H40N2OS: C, 72.85; H, 9.41; N, 6.53. Found: C,
72.79; H, 9.42; N, 6.63.
4.1.18. N-(2-(2-Aminoethoxy)ethyl)-2-(((2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trien-1-yl)thio)benzamide (10f)
Yellowish oil, 52.6% yield. IR (KBr, cmꢁ1): 3181, 1719, 1616,
1585, 1466; 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.18 (br, 1H,
NH), 7.41–7.46 (m, 3H, Ar-H), 7.22–7.26 (m, 1H, Ar-H), 5.28–5.32
(m, 1H, SCH2CH), 5.07–5.09 (m, 2H, 2 ꢀ CH2CH@CCH3), 3.49–3.82
(m, 10H, SCH2, 2 ꢀ NCH2CH2O), 1.91–2.01 (m, 8H, 2 ꢀ CCH2CH2-
CH), 1.53–1.68 (m, 12H, 4 ꢀ CH3); MS (ESI) m/z = 445 [M+1]+; Anal.
Calcd for C26H40N2O2S: C, 70.23; H, 9.07; N, 6.30. Found: C, 70.17;
H, 9.01; N, 6.35.
4.1.13. N,N0-(Butane-1,4-diyl)bis(2-(((2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trien-1-yl)thio)benzamide) (11c)
Yellowish oil, 33.0% yield. IR (KBr, cmꢁ1): 3145, 1716, 1612,
1580, 1460; 1H NMR (CDCl3, 300 MHz, d ppm): 7.66 (d, 2H,
J = 7.5 Hz, Ar-H), 7.40–7.45 (m, 2H, Ar-H), 7.23–7.36 (m, 4H,
Ar-H), 6.88–6.93 (br, 2H, NH), 5.23–5.27 (d, 2H, J = 5.7 Hz,
2 ꢀ SCH2CH), 5.03–5.08 (m, 4H, 4 ꢀ C@CH), 3.53–3.52 (m, 8H,
2 ꢀ SCH2, 2 ꢀ NCH2), 1.97–2.01 (m, 16H, 4 ꢀ CCH2CH2CH), 1.50–
1.77 (m, 28H, 8 ꢀ CH3, 2 ꢀ NCH2CH2); MS (ESI) m/z = 770
[M+H]ꢁ; Anal. Calcd for C48H68N2O2S2: C, 74.95; H, 8.91; N, 3.64.
Found: C, 75.10; H, 8.87; N, 3.52.
4.1.19. N,N0-(Oxybis(ethane-2,1-diyl)bis(2-(((2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trien-1-yl)thio)benzamide) (11f)
Yellowish oil, 29.2% yield. IR (KBr, cmꢁ1): 3169, 1717, 1618,
1581, 1463; 1H NMR (CDCl3, 300 MHz, d ppm): 7.68–7.72 (m, 2H,
Ar-H), 7.42–7.76 (m, 2H, Ar-H), 7.23–7.32 (m, 2H, Ar-H), 6.65–
6.68 (br, 2H, NH), 5.31 (m, 2H, 2 ꢀ SCH2CH), 5.08 (m, 4H, 4 ꢀ CH2-
CH@CCH3), 3.47–3.79 (m, 12H, 2 ꢀ SCH2, 2 ꢀ NCH2CH2O), 1.89–
2.03 (m, 16H, 4 ꢀ CCH2CH2CH), 1.53–1.69 (m, 24H, 8 ꢀ CH3); MS
(ESI) m/z = 786 [M+1]+; Anal. Calcd for C48H68N2O3S2: C, 73.42; H,
8.73; N, 3.57. Found: C, 73.56; H, 8.82; N, 3.43.
4.1.14. N-(5-Aminopentyl)-2-(((2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl)thio)benzamide (10d)
Yellowish oil, 48.1% yield. IR (KBr, cmꢁ1): 3181, 1716, 1611,
1582, 1461; 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.18 (br, 1H,
NH), 7.78–7.83 (m, 1H, Ar-H), 7.35–7.42 (m, 2H, Ar-H), 7.18–7.22
(m, 1H, Ar-H), 5.23 (bt, 1H, SCH2CH), 5.02–5.03 (m, 2H, 2 ꢀ C@CH),
3.56 (d, 2H, J = 9.0 Hz, SCH2), 3.17–3.33 (m, 4H, 2 ꢀ NCH2), 1.92–
2.06 (m, 8H, 2 ꢀ CCH2CH2CH), 1.49–1.63 (m, 16H, 4 ꢀ CH3,
2 ꢀ NCH2CH2), 1.31–1.35 (m, 2H, NCH2CH2CH2); ESI-MS (m/z):
443 [M+H]+; Anal. Calcd for C27H42N2OS: C, 73.25; H, 9.56; N,
6.33. Found: C, 73.12; H, 9.61; N, 6.48.
4.1.20. Piperazin-1-yl(2-(((2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl)thio)phenyl)methanone (10g)
Yellowish oil, 44.7% yield. IR (KBr, cmꢁ1): 3170, 1715, 1608,
1581, 1461; 1H NMR (DMSO-d6, 300 MHz, d ppm): 7.41–7.47 (m,
3H, Ar-H), 7.23–7.26 (m, 1H, Ar-H), 5.31 (bt, 1H, SCH2CH), 5.09
(bt, 2H, 2 ꢀ CH2CH@CCH3), 3.57 (d, 2H, J = 4.5 Hz, SCH2), 3.06–
3.23 (m, 8H, 4 ꢀ NCH2), 1.89–2.02 (m, 8H, 2 ꢀ CCH2CH2CH),
1.50–1.68 (m, 12H, 4 ꢀ CH3); MS (ESI) m/z = 427 [M+1]+; Anal.

380
Y. Ling et al. / Bioorg. Med. Chem. 22 (2014) 374–380
Calcd for C26H38N2OS: C, 73.19; H, 8.98; N, 6.57. Found: C, 73.03; H,
8.91; N, 6.74.
relative levels of each signaling event to control b-actin were
determined by densimetric scanning.
4.1.21. Piperazine-1,4-diylbis((2-(((2E,6E)-3,7,11-
4.3. Aqueous solubility
trimethyldodeca-2,6,10-trien-1-yl)thio)phenyl)methanone)
(11g)
Each test compound (about 20.0 mg) was placed in a 25 mL
ground flask. Then 5 mL 10% PEG400 water solution was added
and exposed to ultrasonic oscillation for 10 min. The mixture was
allowed to stir for 24 h in a water bath at 25 °C. The obtained water
suspension solution was centrifuged for 10 min (10,000 rpm), and
the clear supernatant was filtered out through a microporous
membrane and injected to the HPLC system for concentration
determination using a standard curve.
Yellowish oil, 36.2% yield. IR (KBr, cmꢁ1): 3147, 1708, 1606,
1583, 1459; 1H NMR (CDCl3, 300 MHz, d ppm): 7.42–7.57 (m, 4H,
Ar-H), 7.32–7.39 (m, 2H, Ar-H), 7.17–7.21 (m, 2H, Ar-H), 5.31 (bt,
2H, 2 ꢀ SCH2CH), 5.04–5.09 (m, 2H, 4 ꢀ CH2CH@), 3.57 (d, 4H,
J = 4.5 Hz, 2 ꢀ SCH2), 3.12–3.21 (m, 8H, NCH2), 1.89–2.02 (m,
16H, 4 ꢀ CCH2CH2CH), 1.50–1.68 (m, 24H, 8 ꢀ CH3); MS (ESI) m/
z = 768 [M+1]+; Anal. Calcd for C48H66N2O2S2: C, 75.15; H, 8.67;
N, 3.65. Found: C, 75.32; H, 8.76; N, 3.49.
Acknowledgments
4.2. Biological evaluation
We gratefully acknowledge the financial support by the Natural
Science Foundation of China (Grant Nos. 81302628 and 81202467)
and Jiangsu Province (Grant Nos. BK2011389 and BK2012232), the
Natural Science Research Project of Universities in Jiangsu Province
of China (11KJB350004), 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).
4.2.1. MTT assay
SGC7901 (human gastric cancer cells), SMMC-7721 (human
hepatocellular carcinoma cells), EJ (human bladder carcinoma
cells), SKOV-3 (human ovarian cancer cells), MCF-7 (human breast
cancer cells), H460 (human lung cancer cells), or Panc-1 (human
pancreatic carcinoma cells) at 104 cells per well were cultured in
10% FBS DMEM in 96-well flat-bottom microplates overnight.
The cells were incubated in triplicate with, or without, different
concentrations of each test compound for 48 h. During the last
References and notes
4 h incubation, 30
(5 mg mLꢁ1) was added to each well. The resulting MTT-formazan
crystals were dissolved in 150 L DMSO, and absorbance was
measured spectrophotometrically at 570 nm using an ELISA plate
reader. The inhibition induced by each test compound at the
indicated concentrations was expressed as a percentage. The
concentration required for 50% inhibition (IC50) was calculated
usingthe software (GraphPadPrism Version 4.03).
l
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and transferred onto nitrocellulose membranes. After blocked with
5% fat-free milk, the target proteins were probed with anti-Bcl-2,
anti-Baxanti-caspase-3, anti-Akt, anti-phospho-Akt (Ser473),
anti-ERK, anti-phospho- ERK (Thr202/Tyr204), and anti-b-actin
antibodies (Cell Signaling, Boston), respectively. The bound anti-
bodies were detected by HRP-conjugated second antibodies and
visualized using the enhanced chemiluminescent reagent. The