Synthesis and structure-activity relationships of A novel class of dithiocarbamic acid esters as anticancer agent

Article abstract of DOI:10.1002/ardp.201000259

Based on a novel lead compound 4-methylpiperazine-1-carbodithioic acid 3-cyano-3,3-diphenylpropyl ester 1, the systematic structural modification was carried out. All the synthesized compounds were evaluated for their in-vitro anticancer activities on four to six different cell lines at three different concentrations. Most of the tested compounds could selectively inhibit the growth of HL-60 and Bel-7402 cell lines at a medium concentration. Four compounds (3f, 3g, 3n, and 5) were selected for the IC₅₀ test, and the results revealed that three compounds (3g, 3n, and 5) showed almost the same or a slightly weaker activity than compound 1 against HL-60, and three compounds (3f, 3g, and 3n) showed >2-fold higher potency than compound 1 against Bel-7402. The in-vivo efficacy of 3n · HCl was evaluated with transplanted hepatocyte carcinoma 22 as an in-vivo test model. It was found that 3n · HCl could inhibit significantly the growth of tumor, and that this effect was dose-dependent. Meanwhile, the compound 3n · HCl showed low toxicity compared with compound 1 · HCl as evidenced by the little body-weight loss. These results confirmed that compound 3n · HCl is more potent than the lead compound 1 · HCl. Preliminary structure-activity relationships indicated that: a) Both nitrile group and the cyclic amine containing at least two nitrogens were indispensable moieties to keep the activity; b) substitution of the piperazine ring is unfavorable for the improvement of activity; c) the suitable linker joining the piperazinyl dithiocarboxyl and diphenylacetonitril group should be ethylene; d) a non-coplanar arrangement of the two benzene rings appears to be essential for activity. Based on a novel lead compound 4-methyl-piperazine-1-carbodithioic acid 3-cyano-3,3-diphenyl-propyl ester 1, the systematic structural modification was carried out. Compounds 3g and 3n were found to show more potent biological activities than lead compound 1. Some useful SARs were revealed Copyright

Full text of DOI:10.1002/ardp.201000259

320
Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
Full Paper
Synthesis and Structure-Activity Relationships of A Novel
Class of Dithiocarbamic Acid Esters as Anticancer Agent
Xueling Hou1,3, Zemei Ge1, Tingmin Wang2, Wei Guo1, Jun Wu1, Jingrong Cui1, Chingsan Lai2,
and Runtao Li1
1 State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking
University, Beijing, P.R. China
2 Medinox. Inc., San Diego, USA
3 Key Laboratory of Chemistry of Plant Resources of Arid Area, Xinjiang Technical Institute of Physics and
Chemistry, Chinese Academy of Sciences, Urumqi, P.R. China
Based on a novel lead compound 4-methylpiperazine-1-carbodithioic acid 3-cyano-3,3-diphenylpropyl
ester 1, the systematic structural modification was carried out. All the synthesized compounds were
evaluated for their in-vitro anticancer activities on four to six different cell lines at three different
concentrations. Most of the tested compounds could selectively inhibit the growth of HL-60 and Bel-7402
cell lines at a medium concentration. Four compounds (3f, 3g, 3n, and 5) were selected for the IC50 test,
and the results revealed that three compounds (3g, 3n, and 5) showed almost the same or a slightly
weaker activity than compound 1 against HL-60, and three compounds (3f, 3g, and 3n) showed >2-fold
higher potency than compound 1 against Bel-7402. The in-vivo efficacy of 3n ꢀ HCl was evaluated with
transplanted hepatocyte carcinoma 22 as an in-vivo test model. It was found that 3n ꢀ HCl could inhibit
significantly the growth of tumor, and that this effect was dose-dependent. Meanwhile, the compound
3n ꢀ HCl showed low toxicity compared with compound 1 ꢀ HCl as evidenced by the little body-weight
loss. These results confirmed that compound 3n ꢀ HCl is more potent than the lead compound 1 ꢀ HCl.
Preliminary structure–activity relationships indicated that: a) Both nitrile group and the cyclic amine
containing at least two nitrogens were indispensable moieties to keep the activity; b) substitution of the
piperazine ring is unfavorable for the improvement of activity; c) the suitable linker joining the
piperazinyl dithiocarboxyl and diphenylacetonitril group should be ethylene; d) a non-coplanar
arrangement of the two benzene rings appears to be essential for activity.
Keywords: Anticancer activity / Dithiocarbamic acid ester / SARs
Received: September 8, 2010; Revised: November 1, 2010; Accepted: November 5, 2010
DOI 10.1002/ardp.201000259
Introduction
chelate heavy metals [4, 5], and to function as NO scavengers [6].
Moreover, it was recently found that some kind of dithiocarbamic
acid esters have cancer chemopreventive and anticancer action,
which have received much attention. As shown in Fig. 1, the
natural product brassinin isolated from cabbage as indoleamine
2,3-dioxygenase (IDO) inhibitor [7] and sulforamate as phase II
enzyme inducer [8] have demonstrated potential cancer chemo-
preventive action [9]. Compound RWJ-025856 [10], thalidomide
dithiocarbamates [11], chromones dithiocarbamates [12], and
quinazolinone dithiocarbamates [13] have been found showing
significant inhibitory activities against different cancer cells.
Cancer is a major killer throughout human history. Despite
major breakthroughs in many areas of modern medicine, there
still remain significant challenges because of the difficulty in
discovering novel agents to selectively kill tumor cells or
inhibit their proliferation without causing general toxicity.
Dithiocarbamic acid esters, a common class of organic mol-
ecules exhibit a variety of valuable biological effects, including
antibacterial activity [1, 2], antifungal activity [3], the ability to
Correspondence: Runtao Li, State Key Laboratory of Natural and
Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Beijing 100191, P.R. China.
E-mail: lirt@mail.bjmu.edu.cn
Fax: þ86-010-82716956
Additional correspondence: Jingrong Cui, State Key Laboratory of Natural
and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking
University, Beijing 100191, P.R. China. E-mail: jrcui@bjmu.edu.cn
Fax: þ86-010-82802467
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Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
Dithiocarbamic Acid Esters as Anticancer Agent
321
O
S
S
fication of part A has been published before [15c], here only
S
S
S
N
H
the results of the other three parts were discussed.
N
H
S
N
H
Sulforamate
Chemistry
Brassinin
O
The general synthesis of the target compounds dithiocarba-
mic acid esters is shown in Scheme 1 according to our
improved and well established method [14a]. In this one
pot reaction of amines, carbon disulfide and alkylbromide
in the presence of anhydrous potassium phosphate at room
temperature, the corresponding dithiocarbamic acid esters
were generated in high yields.
N
N
O
S
N N
S
O
O
NMe2
amine
S
Thalidomide
dithiocarbamates
RWJ-025856
S
O
N
S
O
S
NR1R2
HN
Me
S
NR1R2
The modification of segment B was outlined in Scheme 2.
Compounds with various cyclic secondary amines reacted
with carbon disulfide and 3-cyano-3,3-diphenyl-propyl bro-
mide 2 afforded the corresponding dithiocarbamic acid
esters 3a–3n, except that the preparation of compound 3e
was carried out using N,N-dimethylaminoethylamine.
Subsequently, oxidation of 3a with one or two equivalents
of m-chloroperoxybenzoic acid (m-CPBA) in acetone at 08C
afforded the corresponding compounds sulfoxide 4 or sul-
fone 5. Further treatment of 3j and 3k with iodomethane
under basic conditions gave the compounds 6 and 7, respec-
tively. Lead compound 1 reacted with iodomethane in ethyl
acetate to give the piperazinium salt 8. 2,6-Diketopiperazine
9, as an amine component for the preparation of compound
3l, was prepared from glycine via two steps according to [18].
The starting material, 3-oxo-2-phenyl-piperazine 10 for the
preparation of compound 3m was obtained according to [19].
As shown in Scheme 3, in order to test whether the dis-
tance between the two moieties (segments B and D) has effect
on the biological activities, compounds with a shorter linker
(12a), a longer linker (12b), and without the dithiocarboxylic
group (12c) were synthesized. The key intermediates, 2-cyano-
2,2-diphenyl-ethyl bromide 11a and 4-cyano-4,4-diphenyl-
butyl bromide 11b, were obtained from the C-alkylation of
diphenylacetonitrile with dibromomethane and 1,3-dibro-
mopropane under the phase transfer catalytic conditions,
respectively. The synthesis of compound 12c was carried
out by the reaction of methylpiperazine and 3-cyano-3,3-
diphenyl-propyl bromide 2 in the presence of K2CO3.
Meanwhile, compounds 14, 17, and 18 were also synthesized
to examine whether the nitrile group was the indispensable
part of the pharmacophore in lead compound 1. According to
the general procedure in Scheme 4, 3,3-diphenyl-propyl bro-
X
R
O
Quinazolinone dithiocarbamates
Chromone dithiocarbamates
Figure 1. Structures of some dithiocarbamates with anticancer
activities.
We have developed a simple and convenient one-pot method
for the synthesis of dithiocarbamic acid ester and prepared
several compounds using this method [14]. Random screen-
ing of those compounds led to the discovery of several com-
pounds with significant antitumor activities [15]. One of the
promising compounds is 4-methyl-piperazine-1-carbodithioic
acid 3-cyano-3,3-diphenyl-propyl ester 1 (Fig. 2) with 9.9 mM of
IC50 against human promyelocytic leukemic cell line HL-60
and with 27.1 mM of IC50 against human hepatocellular
carcinoma cell line Bel-7402. A further in-vivo test indicated
that 1 ꢀ HCl, which has better solubility than its free amine
form 1, inhibited significantly the growth of transplanted
hepatocyte carcinoma 22 and sarcoma 180 in mice and
implanted human gastric carcinoma in nude mice with very
low toxicity [16]. The mechanism 1 ꢀ HCl inhibits tumor
growth is related to the inhibition of the PKCa/ERK1/2
MAPK signal pathway and the deactivation of cdc2 and cyclin
B1 [17]. From the perspective of drug discovery, compound 1
as an excellent lead compound offered significant anticancer
activity and very low toxicity with a novel structure type.
With the aim of improving potency and anticancer activity,
we carried out structure–activity relationship study from lead
compound 1. For a convenient description, the compound
structure is divided into four segments (A, B, C, and D) (Fig. 2)
and a systematic study has been completed. Since the modi-
S
NC
1
H3C N
N
S
K3PO4
acetone
rt
R
+ CS
+
Br
NH
N
S
R
n
2
n
S
A
D
C
B
Figure 2. Lead compound 1 and overall strategy for its structural
Scheme 1. General method for the synthesis of the dithio-
carbamic acid esters.
modification.
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X. Hou et al.
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NC
n
b
H3C N
N
S
S
CN
CN
H
a
12a n=1
Br
Br
+
Br
n
n
12b n=3
NC
c
11a n=1
11b n=3
11c n=2
H3C N
N
12c
Scheme 3. Reaction conditions: (a) 11a or 11b, 50% NaOH,
TBAB, r.t.; (b) methylpiperazine, CS2, K3PO4, acetone, r.t.; (c)
11c, methylpiperazine, K2CO3, acetonitrile, reflux.
S
H
H
a
H3C N
N
S
H3C N NH
+ CS2 +
Br
13
14
S
EtO2C
N S
b
c
Br
COOH
Br
COOEt
H3C N
17
16
15
N
HN
S
N
N
S
S
NC
S
d
H3C N
N
H3C N
N
18
1
Scheme 4. Reaction conditions: (a) K3PO4, acetone, r.t.; (b) etha-
nol, DCC, DMAP, CH2Cl2, r.t.; (c) methylpiperazine, CS2, K3PO4,
acetone, r.t.; (d) NaN3, Et3N, toluene, reflux.
R
Br
c
a
b
CH2CN
CHCN
CHCN
20
19
21a -21e
23a R=CH3
23b R=Cl
S
NC
R
d
e
f
H3C N
N
N
S
23c R=OCH3
23d R=C(CH3)3
23e R=Br
23a -23e
S
NC
S
C(CH3)3
R
Br
HN
CN
24
22a -22e
S
NC
C(CH3)3
N
S
H3CN
25
Scheme 5. Reaction conditions: (a) Br2, 1058C; (b) substituted
benzene, AlCl3, 508C; (c) 1,2-dibromoethane, K2CO3, acetonitrile,
TBAB, reflux; (d) 1-methylpiperazine, CS2, K3PO4, acetone, r.t.; (e)
piperazine, CS2, K3PO4, acetone, r.t.; (f) 1-methylhomopiprazine,
CS2, K3PO4, acetone, r.t.
mide 13 and 4-bromo-2,2-diphenyl-butyric acid ethyl ester 16
were used as halides to afford the corresponding compounds
14 and 17. 4-Bromo-2,2-diphenyl-butyric acid ethyl ester 16
was obtained from the esterification of 4-bromo-2,2-diphenyl-
butyric acid 15. Treatment of the lead compound 1 with NaN3
gave the tetrazole derivative 18.
Scheme 2. Reagents and conditions: (a) K3PO4, acetone, r.t.;
(b) MCPBA equal molecular with 7a, acetone, 08C; (c) MCPBA
dual molecular with 3a, acetone, 08C; (d) iodomethane, K2CO3,
ethanol, 508 C; (e) iodomethane, NaH, toluene, r.t.; (f)
iodomethane, ethyl acetate, r.t.; (g) SOCl2, methanol, reflux; (h)
chloroacetamide, KHCO3, acetonitrile, reflux; (i) ethanol, Br2,
PCl3, reflux; (j) ethylenediamine, ethanol, reflux.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
Dithiocarbamic Acid Esters as Anticancer Agent
323
a
c
b
gastric carcinoma cell line (BGC-823), a human hepatocellu-
lar carcinoma cell line (Bel-7402), a human cervical carci-
noma cell line (Hela), a human prostatic carcinoma cell
line (PC 3MIE8), and a human breast carcinoma cell line
(MDA-MB-435) with at least three different concentrations.
Then, the IC50 values for the potent compounds were deter-
mined. Because most of the compounds only selectively
inhibited the growth of HL-60 and Bel-7402 cells at a medium
concentration, we herein merely listed these biological
results in Tables 1 to 5 in order to discuss the SARs.
H CHO
26
H C N OH
H
27
S
NC
e
d
H3C
N
N
S
Br
NC
H
CN
28
30
29
S
NC
f
HCl
H3C
N
N
S
31
The activity results from the compounds with the various
modification of segment B are reported in Table 1. Replacing
the N-methyl piperazine moiety of lead compound 1 with
thiomorpholine, morpholine, piperidine, 4-methylpiperidine,
and N,N-dimethylaminoethylamine resulted in a significant
reduction or complete loss of activities (Table 1, 3a–3e),
which suggested that the nitrogen atom at 4-position in
piperazine was an important structure element for the active
compounds. The ring-opened analogue 3e and ring-expanded
analogues (3f and 3g) showed obviously different activities.
The former (3e) lost the activity completely, however, the
latter (3f, 96.5% against HL-60; 95.9% against Bel-7402; 3g,
86.3% against HL-60; 90.1% against Bel-7402) exhibited high
potency. Therefore, it should be very necessary to retain the
structure of cyclic amine containing at least two nitrogen
atoms in the molecule. Furthermore, introducing the differ-
ent numbers of methyl groups (3h–3j) or oxo groups (3k–3m)
into the carbon atoms of the piperazine ring led to a dramatic
decrease or the complete loss in activity. These results
revealed that substitution of the piperazine ring is unfavor-
able for the improvement of activity, which should be the
reason that the introduction of substitutions led to the
change of conformation. However, it is interesting to find
that the sulfoxide and sulfone analogues (4 and 5) of com-
pound 3a showed quite different activities. Similar to com-
pound 3a, compound 4 showed much lower potency,
whereas compound 5 exhibited good inhibition (85.6%
against HL-60; 92.9% against Bel-7402). This finding provides
a new kind of scaffold for the further optimization of the lead
compound 1. It should be noted that compound 3n without a
substituent on 4-N of piperazine exhibited better activities
(R1 ¼ H, 98.7% against HL-60; 95.5% against Bel-7402) than
the lead compound 1 (R1 ¼ Me, 79.0% against HL-60; 75.2%
against Bel-7402). Meanwhile, the quaternary ammonium
derivatives 8 of the lead compound 1 almost lost the activity
completely. It is clear that the activity order is 8 (quaternary
amine) <1 (tertiary amine) <3n (secondary amine). The most
potent compound 3n should attribute to its capability to
form H-bonding with target molecules.
Scheme 6. Reagents and conditions: (a) NaH, DMSO, ethyl
formate, r.t.; (b) NH2OH ꢀ HCl, ethanol, r.t.; (c) SOCl2, ether, r.t.;
(d) 1,2-dibromoethane, K2CO3, acetonitrile, TBAB, reflux; (e)
methylpiperazine, CS2, K3PO4, acetone, r.t.; (f) HCl (gas), ethyl
acetate.
The modification of segment D was carried out following the
method outlined in Scheme 5. To investigate whether the
electron-donating and withdrawing groups or larger and
smaller substituents have any effect on the activities, the
phenyl-substituted analogues of the lead compound 1,
23a–23e were formed from the corresponding 4-bromo-2-sub-
stituted phenyl-2-phenylbutanenitrile (22a–22e), which were
prepared from benzeneacetonitrile 19 via bromination [20],
Friedel-Crafts arylation, and bromoethylation.
Because of the promising activity of piperazine and
4-methyl homopiperazine derivatives (3f and 3n) in the
modification of segments B, we also chose to synthesize
the compounds 24 and 25, which were obtained from the
reactions of piperazine or 4-methyl homopiperazine with
carbon disulfide and 4-bromo-2-(4-tert-butylphenyl)-2-phenyl-
butanenitrile (22d).
To demonstrate the effect of the 3D-position of the two
benzene rings on the activity, the fluorine derivative 30 was
also synthesized, as shown in Scheme 6. The compound 30
was synthesized from fluorene via several steps: Formylation
of fluorene with ethyl formate under basic conditions
afforded 26 in a quantitative yield; treatment of 26 with
hydrochloride salt of NH2OH in ethanol gave the oxime 27
[21a], which was dehydrated by the treatment with thionyl
chloride to obtain 9-cyanofluorene 28 [21b]; reaction of 28
with 1,2-dibromoethane under basic conditions gave the key
intermediate 29; by the reaction of methyl piperazine, car-
bon disulfide and 29 in presence of anhydrous potassium
phosphate in acetone at room temperature, the expected
compound 30 was prepared.
Biological results and discussion
All synthesized compounds were firstly evaluated for their in-
vitro anticancer activities on four to six different cell lines: A
human promyelocytic leukemic cell line (HL-60), a human
Table 2 shows the in-vitro anticancer activities of com-
pounds in various modification of segment C. Comparing
with the lead compound 1, the shorter linker (12a), longer
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Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
Table 1. (continued )
Table 1. In-vitro evaluation of anticancer activity as % proliferation
inhibition at concentrations of 33 mM against HL-60 and Bel-7402
cell lines of the compounds modified in segment B.
S
NC
S
B segment
S
NC
S
B segment
No.
5
B Segment
Inhibition rate
against HL-60 (%)
Inhibition rate
against Bel-7402 (%)
No.
B Segment
Inhibition rate
against HL-60 (%)
Inhibition rate
against Bel-7402 (%)
O
85.6
92.9
S
N
O
H3C
1
79.0
7.1
0
75.2
14.2
29.2
H3C N
N
H3C N
N
6
3.0
0
CH3
3a
3b
S
N
N
N
O
O
7
8
0
14.7
5.3
H3C N
N
N
3c
0
21.3
Me
Me
+I-
3.1
N
3d
3e
0
0
25.4
5.3
H3C
N
N
N
linker (12b), and linker without dithiocarboxylic group (12c),
all led to the activity decrease significantly. Therefore, the
ethylene in lead compound 1 is a more suitable linker. This
observation could suggest that there at least existed double
binding sites between the target molecule and the active
compounds. Subsequently, replacement of the nitrile group
in the lead compound 1 with hydrogen (14), ethoxycarbonyl
group (17) or tetrazole (18), all resulted in significantly
decreased activities, although the activity of compound 14
decreased not so much. Thus, the presence of the nitrile
group can benefit the biological activity.
H
3f
H3C N
HN
N
97.6
86.3
95.9
90.1
3g
N
N
N
H3C
HN
3h
3i
54.5
37.7
0
0
H3C
HN
Table 3 shows the biological activities for those compounds
with modified segment D. Compounds 23a–23e with five
different substituents (methyl, methoxy, chloride, bromide,
H3C
H3C
HN
3j
47.5
0
N
Table 2. In-vitro evaluation of anticancer activity as % proliferation
inhibition at concentrations of 33 mM against HL-60 and Bel-7402
cell lines of the compounds modified in segment C.
CH3
O
3k
3l
11.3
16.9
0
HN
N
N
W
H3C N
N
O
HN
O
L
18.8
No.
L
W
Inhibition Inhibition
rate against rate against
HN
O
N
HL-60 (%)
Bel-7402 (%)
3m
20.6
6.8
Ph
1
12a
–CS2(CH2)2–
–CS2CH2–
CN
CN
CN
CN
H
CO2Et
79.0
26.1
13.7
50.9
67.0
ꢁ14
6.8
75.2
25.3
26.7
0
51.7
28.9
0
3n
4
98.7
13.2
95.5
HN
N
N
12b –CS2(CH2)3–
12c
14
17
18
–CH2CH2–
–CS2(CH2)2–
–CS2(CH2)2–
10.5
O S
–CS2(CH2)2– 5-(1H)-tetrazolyl
continued
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Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
Dithiocarbamic Acid Esters as Anticancer Agent
325
Table 3. In-vitro evaluation of anticancer activity as % proliferation inhibition at concentrations of 10 mM against HL-60 and Bel-7402 cell
lines of the compounds modified in segment D.
S
NC
S
S
NC
NC
N
H3C N
S
H3C N
N
S
R N
N
S
R1
R1
23 R = Me; 24 R = H
25
30
No.
R1
Inhibition rate
against HL-60 (%)
Inhibition rate
against Bel-7402 (%)
1
H
Me
Cl
MeO
t-Bu
Br
t-Bu
t-Bu
73.4
74.3
68.2
45.8
82.8
65.5
96.6
93.4
7.9
4.1
4.2
5.2
0
10.3
2.3
93.8
62.1
27.1
23a
23b
23c
23d
23e
24
25
30
and t-butyl) on the benzene ring showed similar or better
activities relative to those from the lead compound 1. The
compound 23d with the bulky group t-butyl had the best
activity (82.8% against HL-60 at the concentration of 10 mM).
Considering compounds 3f and 3n exhibited more potential
activities than the lead compound 1 in various modifications
of segments A–C, the compounds 24 and 25 were designed
and synthesized. The results of the biological activities
showed that 24 and 25 are more potent than both compound
23d and the lead compound 1. It is clear that the compounds
substituted with a t-butyl group (23d, 24, and 25) and a
methoxy group (23c) showed the highest and lowest anti-
cancer activities in these compounds, respectively. These
results hit us that a higher steric substituent on benzene
is favorable for the activity and the strong electron-donating
substituent could induce the decrease of activities.
Additionally, the fluorine derivative 30, in which two
benzene rings were fixed in one plane, showed very low
activity. Considering that both compound 1 and compound
23d showed high activity, it is further demonstrated that
the larger angle of the two benzene rings would lead to the
better activities.
On the base of the above biological activity results, we
selected four compounds with potential activity to evaluate
their IC50 against both cell lines HL-60 and Bel-7402. It was
found that compound 3n was more potent than compound 1
(Table 4).
Table 4. IC50 values of selected compounds against both cell lines
HL-60 and Bel-7402.
S
NC
R1
To evaluate the anticancer efficacy in vivo, compound 3n
was converted into its corresponding hydrochloride salt
(3n ꢀ HCl). The in-vivo efficacy of compound 3n ꢀ HCl was
compared to the lead compound 1 ꢀ HCl which was used
as the positive control. The transplanted hepatocyte carci-
noma 22 was used as in-vivo test model. As shown in Table 5,
after once-daily oral dosing for 10 days (25, 50 or 100 mg/kg
for 3n ꢀ HCl and 100 mg/kg for 1 ꢀ HCl), 3n ꢀ HCl produced a
significant reduction in tumor size, and the effect was dose-
dependent (25, 50, and 100 mg/kg; 32.3, 42.8, and 50.0%
inhibition, respectively). In comparison, 1 ꢀ HCl had low
effect on tumor reduction (32.3%) at a 100 mg/kg dosage.
Meanwhile, the significant body-weight loss was not observed
during the treatment with 3n ꢀ HCl, consistent with the
results from the treatment of 1 ꢀ HCl or no treatment.
N
S
R2
No.
1
R1R2N–
H3C N
HL-60 (IC50 mM)
Bel-7402 (IC50 mM)
9.9
27.1
N
3f
13.0
11.7
H3C N
N
3g
3n
10.3
5.3
11.4
11.5
HN
HN
N
N
O
S
5
9.6
26.5
N
O
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Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
Table 5. In-vivo efficiency of compound 3n ꢀ HCl growth inhibition of transplanted hepatocyte carcinoma 22 (H22).
Group
Dose (mg/kg)
Change of body weight (g)
(X ꢂ SD)
Tumor weight (g)
(X ꢂ SD)
Inhibition (%)
NS
1 ꢀ HCl
/
3.4 ꢂ 2.7
4.3 ꢂ 2.1
4.8 ꢂ 1.5
4.9 ꢂ 1.3
3.0 ꢂ 1.9
1.6 ꢂ 0.3
1.1 ꢂ 0.4ꢃꢃ
1.1 ꢂ 0.4
0.9 ꢂ 0.3ꢃꢃ
0.8 ꢂ 0.3ꢃꢃ
/
100
25
50
100.
32.3
32.3
43.8
50.0
3n ꢀ HCl
ꢃ
ꢃꢃ
p < 0.05, p < 0.01, compared with NS group by paired student’s t-test (n ¼ 10)
These results confirmed that compound 3n ꢀ HCl is more
potent than the lead compound 1 ꢀ HCl
General procedure for the synthesis of the target
compounds
Conclusion
Method A (Scheme 1)
A mixture of secondary amine (10 mmol), anhydrous potassium
phosphate (10 mmol), and acetone (20 mL) was stirred at room
temperature for 15 min and then carbon disulfide (12 mmol)
was added dropwise. After stirring for 30 min, bromoalkane
(10 mmol) was added. The agitation was continued until TLC
showed that the reaction was completed. The solid was filtered
off and the filtrate was concentrated. The residue was dissolved
in 20 mL ethyl acetate, and washed with water. The organic layer
was dried over anhydrous Na2SO4, the solvent was evaporated, and
the residue was purified by recrystallization or chromatography to
obtain the product.
Based on the structure of the lead compound 1, we carried
out the structural modifications in a systematic manner. A
series of new dithiocarbamic acid esters was synthesized and
evaluated for anticancer activities. Most of the tested com-
pounds could selectively inhibit the proliferation of HL-60
and Bel-7402 cell lines at medium concentration. Several
compounds exhibited higher activities than the lead com-
pound 1. The in-vivo evaluation for compound 3n ꢀ HCl
showed that 3n ꢀ HCl possessed excellent inhibition for
the growth of transplanted hepatocyte carcinoma 22 com-
pared to the control group, which is more potential than
1 ꢀ HCl. The experimental results reveal the following struc-
ture–activity relationships (SAR): a) Both the nitrile group
and the cyclic amine containing at least two nitrogen were
indispensable moieties to keep the activity; b) substitution of
the piperazine ring is unfavorable for the improvement of
activity; c) the suitable linker joining the piperazinyl dithio-
carboxyl and diphenylacetonitril group should be ethylene;
d) the two benzene rings in a vertically position is efficient for
the activity. These results provide promising information for
further development of potent inhibitors.
Thiomorpholine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3a)
Following method A, 3a was prepared from thiomorpholine in
71% yield after column chromatography (EtOAc/petroleum
ether, 1:5); mp 105–1078C; 1H-NMR (CDCl3) d 2.72 (bs, 4H,
SCH2), 2.83 (m, 2H, –CH2C(CN)Ph2), 3.39 (m, 2H, –CS2CH2–),
4.22 (bs, 2H, –CH2N–), 4.58 (bs, 2H, –CH2N–), 7.31 (t, 2H,
J ¼ 7.2 Hz, Ar–H), 7.38 (t, 4H, J ¼ 7.2 Hz, Ar–H), 7.46 (d, 4H,
J ¼ 7.2 Hz, Ar–H); MS (EI) m/z (%): 398 [Mþ] (2), 296 (4), 220
(15), 192 (34), 178 (13), 146 (100), 102 (28). Anal. calcd.: C,
63.28; H, 5.56; N, 7.03. Found: C, 63.46; H, 5.60; N, 7.06.
Morpholine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3b)
Experimental
Following method A, 3b was prepared from morpholine in 73%
yield after column chromatography (EtOAc/petroleum ether,
Chemistry
1:5); mp 142–1448C; 1H-NMR (CDCl3)
d
2.83 (m, 2H,
–CH2C(CN)Ph2), 3.40 (m, 2H, –CS2CH2–), 3.74 (bs, 4H, –O–CH2–),
3.65 (bs, 2H, –CH2N–), 4.28 (bs, 2H, –CH2N–), 7.31 (t, 2H,
J ¼ 7.2 Hz, Ar–H), 7.37 (t, 4H, J ¼ 7.5 Hz, Ar–H), 7.47 (d, 4H,
J ¼ 7.5 Hz, Ar–H); MS (ESI) m/z 383.6 (M þ 1), 405.4 (M þ Na).
Anal. calcd.: C, 65.93; H, 5.80; N, 7.32. Found: C, 65.97; H, 5.76;
N, 7.13.
Melting points were determined on X4 microscope and were
uncorrected. 1H-NMR and 13C-NMR spectra were performed on a
VXR 300 (300 MHz) instrument. Elemental analyses were per-
formed on a Vario ELIII (Germany). Mass spectra were recorded
on a ZAB-2F spectrometer or TRACE MS spectrometer (Finnigan)
at 70 eV. IR spectra were determined in KBr on a Perking-Elmer
983 spectrophotometer. Thin layer chromatography was carried
out on precoated GF254 silica gel plates. The column chroma-
tography was performed using G60 H silica gel. Anhydrous
potassium phosphate was obtained from dehydration
of K3PO4 ꢀ 7 H2O. The other reagents and solvents were of com-
mercial quality from freshly opened containers.
Piperidine-1-carbodithioic acid 3-cyano-3,3-diphenylpropyl
ester (3c)
Following method A, 3c was prepared from piperidine in 87%
yield after column chromatography (EtOAc/petroleum ether,
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Dithiocarbamic Acid Esters as Anticancer Agent
327
1
1:5); mp 117–1198C; H-NMR (CDCl3) d 1.70 (bs, 6H, –CH2–), 2.84
3-Methylpiperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3h)
(m, 2H, –CH2C(CN)Ph2), 3.38 (m, 2H, –CS2CH2–), 3.84 (bs, 2H,
–CH2N–), 4.26 (bs, 2H, –CH2N–), 7.30 (t, 2H, J ¼ 7.0 Hz, Ar–H),
7.37 (t, 4H, J ¼ 7.5 Hz, Ar–H), 7.48 (d, 4H, J ¼ 7.5 Hz, Ar–H); MS
(ESI) m/z 381.6 (M þ 1), 403.4 (M þ Na). Anal. calcd.: C, 69.43; H,
6.36; N, 7.36. Found: C, 69.19; H, 6.00; N, 7.23.
Following method A, 3h was prepared from 2-methylpiperazine
in 83% yield after column chromatography (EtOAc/petroleum
ether, 1:5); mp 114–1158C; 1H-NMR (CDCl3) d 1.11 (d, 3H, J ¼ 6.0 Hz,
CH3), 2.73 (bs, 3H, piperazine), 2.83 (m, 2H, –CH2C(CN)Ph2), 3.05
(bs, 1H, piperazine), 3.18 (bs, 1H, piperazine), 3.39 (m, 2H,
–CS2CH2–), 4.45 (bs, 2H, piperazine), 5.45 (bs, 1H, NH), 7.30
(t, 2H, J ¼ 7.5 Hz, Ar–H), 7.38 (t, 4H, J ¼ 7.5 Hz, Ar–H), 7.46
(d, 4H, J ¼ 7.5 Hz, Ar-H); MS (ESI) m/z 418.4 (M þ Na). Anal. calcd.:
C, 66.80; H, 6.37; N, 10.62. Found: C, 66.70; H, 6.06; N, 10.55.
4-Methylpiperidine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3d)
Following method A, 3d was prepared from 4-methylpiperidine
in 27% yield after column chromatography (EtOAc/petroleum
ether, 1:5); mp 62–648C; 1H-NMR (CDCl3) d 0.97 (d, 3H, J ¼ 6.0 Hz,
CH3), 1.25 (bs, 2H, piperidine ring), 1.73 (bs, 3H, piperidine ring),
2.84 (m, 2H, –CH2C(CN)Ph2), 3.06 (bs, 1H, –CH2N–), 3.14 (bs, 1H,
–CH2N–), 3.37 (m, 2H, –CS2CH2–), 4.53 (bs, 1H, –CH2N–), 5.48
(bs, 1H, –CH2N–), 7.30 (t, 2H, J ¼ 7.5 Hz, Ar–H), 7.37 (t, 4H,
J ¼ 7.5 Hz, Ar–H), 7.48 (d, 4H, J ¼ 7.5 Hz, Ar–H); MS (ESI) m/z
395.2 (M þ 1), 417.6 (M þ Na). Anal. calcd.: C, 70.01; H, 6.64;
N, 7.10. Found: C, 69.89; H, 6.49; N, 7.12.
3,5-Dimethylpiperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3i)
Following method A, 3i was prepared from 2,6-dimethylpiper-
azine in 78% yield after column chromatography (EtOAc/
petroleum ether, 1:5); mp 122–1238C; 1H-NMR (CDCl3) d 1.12
(d, 6H, J ¼ 6.0 Hz, 2CH3), 1.75 (bs, 1H, NH), 2.58 (bs, 1H, piper-
azine), 2.74 (bs, 1H, piperazine), 2.83 (m, 2H, –CH2C(CN)Ph2), 2.90
(bs, 2H, piperazine), 3.38 (bs, 2H, –CS2CH2–), 4.43 (bs, 1H, piper-
azine), 5.49 (bs, 1H, piperazine), 7.30 (t, 2H, J ¼ 7.5 Hz, Ar-H), 7.37
(t, 4H, J ¼ 7.5 Hz, Ar-H), 7.47 (d, 4H, J ¼ 7.5 Hz, Ar-H); MS (ESI) m/z
410.9 (M þ 1). Anal. calcd.: C, 67.44; H, 6.64; N, 10.26. Found:
C, 67.46; H, 6.68; N, 10.27.
N,N-Dimethylethylenediamine-1-carbodithioic acid 3-
cyano-3,3-diphenylpropyl ester (3e)
Following method A, 7e was prepared from N,N-dimethyl-ethyl-
diamine in 84% yield after column chromatography
(EtOAc/petroleum ether, 1:5); sticky oil; 1H-NMR (CDCl3)
d 2.23 (s, 6H, 2CH3), 2.50 (m, 2H, Me2NCH2–), 2.81 (m, 2H,
–CH2C(CN)Ph2), 3.29 (m, 2H, –CS2CH2–), 3.71 (t, 2H, J ¼ 6.0 Hz,
–CH2NH–), 7.27–7.48 (m, 10H, Ar–H); MS (EI) m/z (%): 383.3 [Mþ]
(1), 313 (3), 252 (9), 193 (27), 165 (13), 115 (14), 71 (62), 58 (100).
Anal. calcd.: C, 65.76; H, 6.57; N, 10.95. Found: C, 65.58; H, 6.39; N,
10.91.
2,5-Dimethylpiperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3j)
Following method A, 3j was prepared from 2,5-dimethylpiperazine
in 68% yield after column chromatography (EtOAc/petroleum
ether, 1:5); mp 90–948C; 1H-NMR (CDCl3)
d 1.18 (d, 3H,
J ¼ 6.5 Hz, CH3), 1.32 (d, 3H, J ¼ 6.5 Hz, CH3), 2.16 (s, 1H, NH),
2.62 (dd, 1H, J ¼ 2.7 Hz, 13 Hz, piperazine), 2.85 (m, 2H,
–CH2C(CN)Ph2), 3.27 (dd, 1H, J ¼ 4.0 Hz, 13 Hz, piperazine),
3.36 (m, 1H, piperazine), 3.39 (m, 2H, –CS2CH2–), 3.46 (dd, 1H,
J ¼ 4.0 Hz, 13 Hz, piperazine), 4.59 (bs, 1H, piperazine), 5.17
(bs, 1H, piperazine), 7.30 (t, 2H, J ¼ 7.0 Hz, Ar-H), 7.37 (t, 4H,
J ¼ 7.0 Hz, Ar-H), 7.48 (d, 4H, J ¼ 7.0 Hz, Ar-H); MS (ESI) m/z 410.8
(M þ 1), 432.6 (M þ Na). Anal. calcd.: C, 67.44; H, 6.64; N, 10.26.
Found: C, 67.46; H, 6.51; N, 10.12.
4-Methylhomopiperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3f)
Following method A, 3f was prepared from 1-methyl-homopiper-
azine in 48% yield after column chromatography
(EtOAc/petroleum ether, 1:5); sticky oil; 1H-NMR (CDCl3) d 2.03
(bs, 2H, –CH2–), 2.36 (d, 3H, J ¼ 2.5 Hz, CH3), 2.55 (t, 2H,
J ¼ 5.5 Hz, homopiperazine), 2.71 (m, 2H, homopiperazine),
2.84 (m, 2H, –CH2C(CN)Ph2), 3.37 (m, 2H, –CS2CH2–), 3.89
(t, 1H, J ¼ 6.5 Hz, homopiperazine), 3.95 (t, 1H, J ¼ 5.0 Hz,
homopiperazine), 4.21 (t, 1H, J ¼ 6.0 Hz, homopiperazine),
4.35 (m, 1H, homopiperazine), 7.30 (t, 2H, J ¼ 7.0 Hz, Ar–H),
7.37 (t, 4H, J ¼ 7.5 Hz, Ar–H), 7.47 (d, 4H, J ¼ 7.5 Hz, Ar–H);
MS (ESI) m/z 410.8 (M þ 1). Anal. calcd.: C, 67.44; H, 6.64; N,
10.26. Found: C, 67.51; H, 6.46; N, 10.20.
3-Oxopiperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3k)
Following method A, 3k was prepared from 2-ketopiperazine in
84% yield after column chromatography (EtOAc/petroleum
ether, 1:5); mp 100–1028C; 1H-NMR (CDCl3) d 2.82 (m, 2H,
–CH2C(CN)Ph2), 3.40 (m, 2H, –CS2CH2–), 3.48 (m, 2H, –CH2N–),
4.50 (bs, 4H, –CH2N–), 6.79 (bs, 1H, NH), 7.31 (t, 2H, J ¼ 7.5 Hz,
Ar-H), 7.39 (t, 4H, J ¼ 7.5 Hz, Ar-H), 7.46 (d, 4H, J ¼ 7.5 Hz, Ar–H);
MS (ESI) m/z 396.8 (M þ 1), 418.3 (M þ Na). Anal. calcd.: C, 63.77;
H, 5.35; N, 10.62. Found: C, 63.73; H, 5.34; N, 10.51.
Homopiperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3g)
Following method A, 3g was prepared from homopiperazine
hydrochloride in 66% yield after column chromatography
(EtOAc/petroleum ether, 1:5). Sticky oil, 1H-NMR (CDCl3)
d 2.04–2.09 (m, 2H, –CH2–), 2.82–2.98 (m, 4H, homopiperazine,
–CH2C(CN)Ph2), 3.08–3.18 (m, 2H, homopiperazine), 3.33–3.38
(m, 2H, –CS2CH2–), 3.98–4.00 (m, 2H, homopiperazine),
4.26–4.36 (m, 2H, homopiperazine), 4.66 (s, 1H, NH), 7.26–7.48
(m, 10H, Ar–H); MS-ESI-TOFþ: m/e 396.0987 (Mþ þ 1). Anal.
calcd.: C, 66.80; H, 6.37; N, 10.62. Found: C, 66.58; H, 6.47; N,
10.52.
3,5-Dioxo-piperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3l)
a) Glycine methyl ester hydrochloride
To a suspension of glycine (1.0 g, 13.33 mmol) in methanol
(100 mL) was added thionyl chloride (0.97 mL, 13.13 mmol) at
room temperature. The mixture was refluxed for 8 h with
stirring. The solvent was evaporated under vacuum and the
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X. Hou et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
residue was washed with methylene chloride to give glycine
methyl ester hydrochloride as a white solid (1.6 g, 97%). mp
170–1758C (dec.).
removal of the solvent, the residue was chromatographed on
silica gel (EtOAc/hexane, 1:5) to give 0.34 g of compound 4 as
light yellow solid.
Yield 82%; mp 118–1208C; 1H-NMR (CDCl3) d 2.80 (bs, 4H,
–CH2N–), 2.88 (t, 2H, J ¼ 7.5 Hz, –CH2C(CN)Ph2), 3.86 (t, 2H,
J ¼ 7.5 Hz, –CS2CH2–), 4.26 (bs, 2H, –CH2SO–), 4.55 (bs, 1H,
–CH2N–), 5.50 (bs, 1H, –CH2N–), 7.31 (m, 2H, ArH), 7.38 (t, 4H,
J ¼ 7.5 Hz, ArH), 7.45 (d, 4H, J ¼ 7.5 Hz, ArH); MS (ESI) m/z 415.5
(M þ 1), 437.8 (M þ Na). Anal. calcd.: C, 60.83; H, 5.35; N, 6.76.
Found: C, 61.04; H, 5.48; N, 6.77.
b) 2,6-Dioxopiperazine (9)
To a stirred suspension of glycine methyl ester hydrochloride
(1.0 g, 7.97 mmol) and potassium bicarbonate (1.99 g,
19.93 mmol) in 60 mL acetonitrile was added 2-chloroacetamide
(0.75 g, 7.97 mmol) at room temperature. The reaction mixture
was refluxed for 8 h and then cooled to room temperature. The
mixture was filtered and the solvent was evaporated under
reduced pressure. The residue was successively washed with
chloroform and acetone to give 2,6-dioxopiperazine 9 as a
white solid.
4,4-Dioxothiomorpholine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (5)
Yield (0.30 g, 33%); mp >1508C (dec.); 1H-NMR (DMSO-d6) d, 3.07
(s, 4H, 2 CH2), 3.35 (m, 1H, NH), 10.83 (s, 1H, –CONHCO–); IR (KBr)
3298, 2986, 2715, 1695, 1272, 896, 548.
Following the preparation of 4, compound 5 was prepared with
twice the amount of m-CPBA in 75% yield after column chroma-
tography (EtOAc/petroleum ether, 1:5); mp 136–1388C; 1H-NMR
(CDCl3) d 2.50 (m, 2H, –CH2C(CN)Ph2), 2.59 (m, 2H, –CS2CH2–),
2.88 (bs, 4H, –CH2N–), 4.09 (m, 2H, –CH2SO2–), 4.57 (m, 2H,
–CH2SO2–), 7.36 (m, 6H, ArH), 7.41 (m, 4H, ArH); MS (ESI) m/z
453.3 (M þ Na). Anal. calcd.: C, 58.57; H, 5.15; N, 6.51. Found: C,
58.52; H, 5.33; N, 6.46.
c) 3,5-Dioxopiperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3l)
2,6-Dioxopiperazine 9 reacted with carbon disulfide and 2 by
method A to give compound 3l as sticky oil in 46% yield after
column chromatography (EtOAc/petroleum ether, 1:5). 1H-NMR
(CDCl3) d 2.81 (m, 2H, –CH2C(CN)Ph2), 3.41 (m, 2H, –CS2CH2–),
4.93 (s, 4H, –CH2N–), 7.29–7.45 (m, 10H, ArH), 8.17 (s, 1H, NH); MS
(EI) m/z (%): 409 [Mþ] (3), 220 (22), 217 (15), 193 (100), 165 (38), 157
(68), 99 (28), 91 (20). Anal. calcd.: C, 61.59; H, 4.68; N, 10.26.
Found: C, 61.28; H, 4.73; N, 9.99.
2,4,5-Trimethylpiperazine-1-carbodithioic acid 3-cyano-
3,3-diphenylpropyl ester (6)
To a mixture of 3j (0.62 g, 1.5 mmol) and potassium carbonate
(0.41 g, 3.0 mmol) in 20 mL ethanol was added iodomethane
(0.26 g, 1.8 mmol) at room temperature. The reaction mixture
was stirred at 508C for 6 h, and then cooled to room temperature.
After filtration and removal of the solvent, the residue was
dissolved in 20 mL ethyl acetate. The solution was washed with
water and dried over Na2SO4. After removal of the solvent, the
residue was chromatographed on silica gel (EtOAc/hexane, 1:5) to
give 0.32 g as white solid.
2-Phenyl-3-oxo-piperazine-1-carbodithioic acid 3-cyano-
3,3-diphenylpropyl ester (3m)
Following method A, compound 3m was prepared from 2-phenyl-
3-oxo-piperazine [19] in 94% yield after column chromatography
(EtOAc/petroleum ether, 1:5); mp 188–1908C; 1H-NMR (CDCl3) d
2.83 (bs, 2H, –CH2C(CN)Ph2), 3.39 (bs, 2H, –CS2CH2–), 3.55 (m, 2H,
–CH2N–), 3.82 (bs, 1H, –CH2N–), 4.39 (bs, 1H, –CH2N–), 4.95 (bs,
1H, –PhCH–), 6.67 (bs, 1H, NH), 7.29–7.47 (m, 15H, ArH); MS (EI)
m/z (%): 471 [Mþ] (9), 279 (8), 252 (37), 219 (25), 191 (18), 175 (100),
165 (27), 91 (35), 77 (13). Anal. calcd.: C, 68.76; H, 5.34; N, 8.91.
Found: C, 68.55; H, 5.21; N, 8.53.
Yield 51%; mp 100–1028C; 1H-NMR (CDCl3) d 0.95 (d, 3H,
J ¼ 6.5 Hz, CH3), 1.35 (d, 3H, J ¼ 6.5 Hz, CH3), 2.29 (s, 3H,
CH3), 2.33 (dd, 1H, J ¼ 1.7 Hz, 11.5 Hz, piperazine), 2.79
(m, 1H, piperazine), 2.83 (m, 2H, –CH2C(CN)Ph2), 3.01 (bs, 1H,
piperazine), 3.38 (m, 2H, –CS2CH2–), 3.50 (d, 1H, J ¼ 13.0 Hz,
piperazine), 4.70 (bs, 1H, piperazine), 5.25 (bs, 1H, piperazine),
7.30 (t, 2H, J ¼ 7.5 Hz, ArH), 7.37 (t, 4H, J ¼ 7.5 Hz, ArH), 7.47
(d, 4H, J ¼ 7.5 Hz, ArH); MS (ESI) m/z 424.7 (M þ 1), 446.3
(M þ Na). Anal. calcd.: C, 68.04; H, 6.90; N, 9.92. Found: C,
68.03; H, 6.57; N, 9.94.
Piperazine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (3n)
Following method A, compound 3n was prepared from pipera-
zine in 59% yield after column chromatography (EtOAc/
petroleum ether, 1:5); mp 124–1278C; 1H-NMR (CDCl3) d 1.72
(bs, 1H, NH), 2.83 (m, 2H, –CH2C(CN)Ph2), 2.93 (bs, 4H,
–CH2N–), 3.39 (m, 2H, –CS2CH2–), 3.87 (bs, 2H, –CH2N–), 4.29
(bs, 2H, –CH2N–), 7.30 (t, 2H, ArH), 7.38 (dd, 4H, J ¼ 5.5,
1.9 Hz, ArH), 7.46 (d, 4H, J ¼ 8.0 Hz, ArH); MS (ESI) m/z 382.4
(M þ 1). Anal. calcd.: C, 66.10; H, 6.08; N, 11.01. Found: C, 66.25;
H, 6.15; N, 10.79.
3-Oxo-4-methylpiperazine-1-carbodithioic acid 3-cyano-
3,3-diphenylpropyl ester (7)
To a mixture of 3k (0.99 g, 2.5 mmol) and sodium hydride
(0.07 g, 3.0 mmol) in 20 mL toluene was added iodomethane
(0.43 g, 3.0 mmol) at room temperature. The reaction mixture
was stirred at room temperature for 24 h and quenched with
water. The organic phase was separated, washed with water,
and dried over anhydrous Na2SO4. After the solvent was
removed, the residue was chromatographed on silica gel
(EtOAc/hexane, 1:5) to give 0.07 g of compound 7 as light yellow
solid.
4-Oxothiomorpholine-1-carbodithioic acid 3-cyano-3,3-
diphenylpropyl ester (4)
To a solution of 3a (0.4 g, 1.0 mmol) in 20 mL acetone was added
3-chloroperoxybenzoic acid (m-CPBA, 0.27 g, 1.2 mmol) at 08C.
The reaction mixture was stirred at 08C for 0.5 h, and Na2SO3
(0.15 g, 1.2 mmol) was added. The solvent was evaporated and
the residue was dissolved in 25 mL ethyl acetate. The solution
was washed with water and dried over anhydrous Na2SO4. After
Yield 7%; mp 125–1268C; 1H-NMR (CDCl3) d 2.82 (m, 2H,
–CH2C(CN)Ph2), 3.03 (s, 3H, CH3), 3.39 (m, 2H, –CS2CH2–), 3.45
(t, 2H, J ¼ 5.0 Hz, –CH2N–), 4.47 (bs, 4H, –CH2N–), 7.31 (t, 2H,
J ¼ 7.0 Hz, ArH), 7.38 (t, 4H, J ¼ 7.0 Hz, ArH), 7.46 (d, 4H,
J ¼ 7.0 Hz, ArH); MS (ESI) m/z 410.6 (M þ 1), 432.4 (M þ Na).
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Dithiocarbamic Acid Esters as Anticancer Agent
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Anal. calcd.: C, 64.52; H, 5.66; N, 10.26. Found: C, 64.42; H, 5.51; N,
10.14.
Yield 38%; mp 99–1018C; 1H-NMR (DMSO-d6) d 2.11 (s, 3H,
CH3), 2.25 (m, 2H, –CH2C(CN)Ph2), 2.30 (bs, 8H, piperazine),
2.65 (m, 2H, –CH2N–), 7.32 (m, 2H, ArH), 7.40 (m, 4H, ArH),
7.43 (m, 4H, ArH); MS (ESI) m/z 320.5 (M þ 1), 342.4 (M þ Na).
Anal. calcd.: C, 78.96; H, 7.89; N, 13.15. Found: C, 78.84; H, 7.92;
N, 13.47.
4,4-Dimethylpiperazinium-1-carbodithioic acid 3-cyano-
3,3-diphenylpropyl ester iodide (8)
The lead compound 1 (1.35 g, 3.4 mmol) was treated with MeI
(0.63 g, 5.0 mmol) in ethyl acetate (10 mL) at room temperature
over night. The precipitate was filtered and washed with ethyl
acetate to give compound 8 as a white solid.
Yield 82%; mp 229–2318C; 1H-NMR (DMSO-d6) d 2.85 (m, 2H,
–CH2C(CN)Ph2), 3.21 (s, 3H, CH3), 3.25 (m, 2H, –CS2CH2–), 3.33
(s, 3H, CH3), 3.55 (t, 4H, J ¼ 5.2 Hz, –CH2N–), 4.26 (bs, 2H,
–CH2N–), 4.53 (bs, 2H, –CH2N–), 7.37 (m, 2H, ArH), 7.44 (m, 8H,
ArH); 13C-NMR (DMSO-d6) d 32.63, 36.97, 50.58, 51.14, 59.50,
121.50 (ArC), 126.46 (ArC), 128.19 (ArC), 129.16 (ArC), 139.12
(ArC), 196.45 (CO); MS (ESI) m/z 411.0 (M ꢁ I). Anal. calcd.: C,
51.39; H, 5.25; N, 7.82. Found: C, 51.17; H, 5.31; N, 7.78.
4-Methylpiperazine-1-carbodithioic acid 3,3-
diphenylpropyl ester (14)
Following method A, compound 14 was prepared from
compound 13 in 63% yield after column chromatography
(EtOAc/petroleum ether, 1:5); mp 107–1088C; 1H-NMR (CDCl3) d
2.33 (s, 3H, CH3), 2.45 (bs, 4H, piperazine), 2.48 (m, 2H,
Ph2CHCH2–), 3.26 (m, 2H, –CS2CH2–), 3.96 (bs, 2H, piperazine),
4.09 (t, 1H, J ¼ 7.5 Hz, –CH–), 4.35 (bs, 2H, piperazine), 7.18
(m, 2H, ArH), 7.28 (m, 8H, ArH); MS (ESI) m/z 371.5 (M þ 1).
Anal. calcd.: C, 68.06; H, 7.07; N, 7.56. Found: C, 67.89; H,
7.23; N, 7.52.
4-Methylpiperazine-1-carbodithioic acid 4-cyano-4,4-
diphenylethyl ester (12a)
4-Methylpiperazine-1-carbodithioic acid 3,3-diphenyl-3-
ethoxycarbonylpropyl ester (17)
a) 2,2-Diphenyl-3-bromopropylnitrile (11a)
To a solution of diphenylacetonitrile (5.80 g, 30.0 mmol) in
dibromomethane (10 mL) was added TBAB (0.80 g, 3.0 mmol)
and 50% NaOH (15 mL). The mixture was stirred at 408C until
the reaction was completed. The organic layer was separated,
and dried over anhydrous Na2SO4. The solvent was removed
under reduced pressure to obtain the crude product, which
was used in the next step without purification.
a) Compound 16 was obtained from esterification of 2,2-
diphenyl-4-bromo-butyl acid (0.64 g, 2 mmol) with ethanol
(2 mL) using CH2Cl2 (20 mL) as solvent in presence of DCC
(0.8 g, 4 mmol) and DMAP (80 mg, 0.4 mmol) at room
temperature for 3 days in 40% yield.
b) Following method A, compound 17 was prepared from 16 in
23% yield after column chromatography (EtOAc/petroleum
ether, 1:5). 1H-NMR (CDCl3) d 1.69 (t, 3H, J ¼ 7.0 Hz, CH3), 2.30
(s, 3H, -NCH3), 2.45 (m, 4H, piperazine), 2.78 (m, 2H,
–CH2C(COOEt)Ph2), 3.10 (m, 2H, –CS2CH2–), 3.90 (bs, 2H, piper-
azine), 4.18 (q, 2H, J ¼ 7.0 Hz, –COOCH2–), 4.31 (bs, 2H, piper-
azine), 7.26 (m, 2H, ArH), 7.33 (m, 8H, ArH); 13C-NMR (CDCl3)
d 14.36, 33.31, 37.67, 45.77, 49.92, 51.03, 54.52, 60.53, 61.51,
127.15, 128.14, 129.15, 142.25, 173.97, 197.02; MS (ESI) m/z
443.6 (M þ 1). Anal. calcd.: C, 65.12; H, 6.83; N, 6.33. Found: C,
65.31; H, 7.04; N, 6.57.
b) 4-Methylpiperazine-1-carbodithioic acid 4-cyano-4,4-
diphenylethyl ester (12a)
Following method A, 12a was obtained from 11a in 16% yield
after column chromatography (EtOAc/petroleum ether, 1:5); mp
117–1188C; 1H-NMR (DMSO-d6) d 2.18 (s, 3H, CH3), 2.35 (bs, 4H,
–CH2N–), 3.85 (bs, 2H, –CH2N–), 4.23 (bs, 2H, –CH2N–), 4.71 (s, 2H,
–CS2CH2–), 7.38 (t, 2H, J ¼ 6.5 Hz, ArH), 7.47 (m, 8H, ArH); MS
(ESI) m/z 382.7 (M þ 1), 404.2 (M þ Na). Anal. calcd.: C, 66.12; H,
6.03; N, 11.02. Found: C, 65.95; H, 6.01; N, 10.91.
4-Methylpiperazine-1-carbodithioic acid 4-cyano-4,4-
diphenylbutyl ester (12b)
4-Methylpiperazine-1-carbodithioic acid 3-(5-tetrazolyl)-
3,3-diphenylpropyl ester (18)
Following the method of 12a, compound 12b was prepared from
compound 11b in 45% yield after column chromatography
(EtOAc/petroleum ether, 1:5); mp 92–988C; 1H-NMR (CDCl3)
d 1.86 (m, 2H, –CH2–), 2.32 (s, 3H, CH3), 2.47 (bs, 4H, –CH2N–),
2.52 (m, 2H, –CH2C(CN)Ph2), 3.37 (t, 2H, J ¼ 7.2 Hz, –CS2CH2–),
3.93 (bs, 2H, –CH2N–), 4.33 (bs, 2H, –CH2N–), 7.20 (m, 2H, ArH),
7.30 (m, 2H, ArH), 7.38 (m, 6H, ArH); MS (ESI) m/z 410.7 (M þ 1).
Anal. calcd.: C, 67.44; H, 6.64; N, 10.26. Found: C, 67.39; H, 6.42; N,
10.01.
A mixture of the lead compound 1 (792 mg, 2.0 mmol), sodium
azide (390 mg, 6.0 mmol) and triethylamine hydrochloride
(828 mg, 6.0 mmol) in toluene (10 mL) was refluxed for 48 h.
After cooling, the product was extracted with water (2 ꢄ 10 mL).
The aqueous layer was neutralized to pH 7 with 5% HCl solution,
and then extracted with ethyl acetate (4 ꢄ 10 mL). The organic
phase was dried over anhydrous Na2SO4 and the solvent was
evaporated. The residue was recrystallized from ethyl acetate to
give product 18 in 17% yield as yellow solid; mp 234–2368C;
1H-NMR (DMSO-d6) d 2.25 (s, 3H, CH3), 2.45 (m, 4H, piperazine),
2.50 (bs, 1H, NH), 3.03 (m, 4H, –CS2CH2CH2–), 3.88 (bs, 2H,
piperazine), 4.17 (bs, 2H, piperazine), 7.22 (d, 4H, J ¼ 7.8 Hz,
ArH), 7.28 (t, 2H, J ¼ 7.5 Hz, ArH), 7.34 (t, 4H, J ¼ 7.5 Hz, ArH);
13C-NMR (DMSO-d6) d 32.56, 38.04, 44.74, 49.21, 50.06, 51.26,
53.66, 127.00, 128.22, 128.44, 143.67, 161.90, 194.88; MS (ESI)
m/z 439.8 (M þ 1). Anal. calcd.: C, 60.24; H, 5.97; N, 19.16.
Found: C, 59.99; H, 5.78; N, 18.87.
4-(4-Methylpiperazin-1-yl)-2,2-diphenylbutanenitrile (12c)
A mixture of methylpiperazine (0.25 g, 2.5 mmol), 2 (0.75 g,
2.5 mmol), and potassium carbonate (0.69 g, 5.0 mmol) in aceto-
nitrile (20 mL) was refluxed for 15 h. The solid was filtered off
and the filtrate was concentrated under reduced pressure. The
residue was chromatographed on silica gel (EtOAc/CH3OH, 1:5) to
give 12c as white solid.
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MS (EI) m/z (%): 425 [Mþ] (1), 284 (1), 222 (6), 190 (3), 175 (20),
143 (61), 99 (7), 83 (26), 70 (100). Anal. calcd.: C, 64.91; H, 6.39; N,
9.87. Found: C, 64.72; H, 6.52; N, 9.59.
4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-phenyl-
3-(4-methylphenyl)propyl ester (23a)
a) Phenyl(p-tolyl)acetonitrile (21a)
The bromine (17 mL, 0.33 mol) was added dropwise to benzyl
cyanide 19 (35 g, 0.30 mol) under stirring at 105–1108C during
30 min. The reaction was continued for another 30 min under the
same conditions to get the crude bromobenzyl cyanide 20. To a
solution of aluminum chloride (42 g, 0.32 mol) in toluene
(150 mL) was added the crude bromobenzyl cyanide 20 within
20–30 min at room temperature. The reaction mixture was
heated to 60–658C for 1 h, then cooled to room temperature
and poured into the solution of hydrochloric acid (20 mL) in
ice water (800 mL). The organic phase was separated and the
water phase was extracted with toluene (3 ꢄ 100 mL). The
combined organic phase was dried over anhydrous Na2SO4 and
the solvent was removed. The residue was recrystallized from
ethanol to give phenyl-(p-tolyl)acetonitrile 21a as white solid in
65% yield; mp 58–608C.
4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-phenyl-
3-(4-t-butylphenyl)propyl ester (23d)
Following the procedure of 23a, compound 23d was obtained
from 4-bromo-2-phenyl-2-(4-t-butylphenyl)-butyronitrile 22d as
sticky oil in 25% yield (two steps from 21d) after column chroma-
tography (EtOAc/petroleum ether, 1:5). 1H-NMR (CDCl3) d 1.26
(s, 9H, –C(CH3)3), 2.32 (s, 3H, CH3), 2.48 (bs, 4H, piperazine), 2.81
(m, 2H, –CH2C(CN)Ph2), 3.38 (m, 2H, –CS2CH2–), 3.92 (bs, 2H,
piperazine), 4.32 (bs, 2H, piperazine), 7.30–7.50 (m, 9H, ArH).
MS (EI) m/z (%): 451 [Mþ] (1), 310 (1), 248 (2), 203 (4), 175 (20),
143 (59), 99 (9), 83 (27), 70 (100). Anal. calcd.: C, 69.14; H, 7.36; N,
9.30. Found: C, 69.35; H, 7.55; N, 9.03.
4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-phenyl-
3-(4-bromo-phenyl)-propyl ester (23e)
b) 4-Bromo-2-phenyl-2-p-tolylbutyronitrile (22a)
Following the procedure of 23a, compound 23e was obtained from
4-bromo-2-phenyl-2-(4-bromo-phenyl)-butyronitrile 22e as sticky oil
in 61% yield (two steps from 21e) after column chromatography
A mixture of 21a (1.0 g, 4.8 mmol), potassium carbonate (1.32 g,
9.6 mmol), TBAB (0.08 g, 0.24 mmol), and 1,2-dibromoethane
(1.8 g, 9.6 mmol) in acetonitrile (20 mL) was refluxed for 6 h.
The solid was filtered off and the filtrate was concentrated under
reduced pressure. The residue was chromatographed on silica gel
(EtOAc/hexane, 1:10) to give 22a as sticky oil in 79% yield. 1H-NMR
(CDCl3) d, 2.31 (s, 3H, CH3), 3.02 (m, 2H, –CH2–), 3.40 (m, 2H,
BrCH2–), 7.23–7.49 (m, 9H, ArH).
1
(EtOAc/petroleum ether, 1:5). H-NMR (CDCl3) d 2.32 (s, 3H, CH3),
2.48 (bs, 4H, piperazine), 2.80 (m, 2H, –CH2C(CN)Ph2), 3.36 (m, 2H,
–CS2CH2–), 3.91 (bs, 2H, piperazine), 4.32 (bs, 2H, piperazine), 7.32–
7.53 (m, 9H, ArH). MS (EI) m/z (%): 473 [Mþ] (1), 271 (1), 190 (12), 175
(32), 143 (60), 83 (24), 70 (100). Anal. calcd.: C, 55.69; H, 5.10; N, 8.86.
Found: C, 55.78; H, 5.26; N, 8.71.
c) 4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-phenyl-
3-(4-methylphenyl)propyl ester (23a)
Piperazine-1-carbodithioic acid 3-cyano-3-phenyl-3-(4-t-
butylphenyl)propyl ester (24)
Following method A, 23a was obtained from 22a in 34% yield (two
steps from 21a) as sticky oil after column chromatography (EtOAc/
petroleum ether, 1:5). 1H-NMR (CDCl3) d 2.31 (s, 3H, CH3), 2.32 (s, 3H,
CH3), 2.46 (bs, 4H, piperazine), 2.80 (m, 2H, –CH2C(CN)Ph2), 3.37 (m,
2H, –CS2CH2–), 3.90 (bs, 2H, piperazine), 4.32 (bs, 2H, piperazine),
7.15–7.47 (m, 9H, ArH). MS (EI) m/z (%): 409 [Mþ] (4), 268 (1), 206 (4),
190 (16), 175 (19), 143 (60), 99 (14), 83 (23), 70 (100). Anal. calcd.: C,
67.44; H, 6.64; N, 10.26. Found: C, 67.25; H, 6.84; N, 9.80.
Following method A, 24 was prepared from 4-bromo-2-phenyl-2-(4-
t-butylphenyl)butyronitrile 22d as sticky oil in 35% yield after
column chromatography (EtOAc/petroleum ether, 1:5). 1H-NMR
(CDCl3) d 1.29 (s, 9H, –C(CH3)3), 2.82 (m, 2H, –CH2C(CN)Ph2), 2.97
(t, 4H, J ¼ 5.1 Hz, piperazine), 3.39 (m, 2H, –CS2CH2–), 4.12 (bs, 4H,
piperazine), 7.28–7.50 (m, 9H, ArH). MS (EI) m/z (%): 437 [Mþ] (3), 369
(4), 310 (14), 249 (4), 189 (12), 161 (11), 129 (96), 69 (100). Anal.
calcd.: C, 68.61; H, 7.14; N, 9.60. Found: C, 68.43; H, 7.20; N, 9.42.
4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-phenyl-
3-(4-chlorophenyl)propyl ester (23b)
4-Methylhomopiperazine-1-carbodithioic acid 3-cyano-3-
phenyl-3-(4-t-butylphenyl)propyl ester (25)
Following the procedure of 23a, compound 23b was obtained
from 4-bromo-2-phenyl-2-(4-chlorophenyl)-butyronitrile 22b as
sticky oil in 62% yield (two steps from 21b) after column chroma-
tography (EtOAc/petroleum ether, 1:5). 1H-NMR (CDCl3) d 2.32
(s, 3H, CH3), 2.47 (bs, 4H, piperazine), 2.80 (m, 2H, –CH2C(CN)Ph2),
3.36 (m, 2H, –CS2CH2–), 3.91 (bs, 2H, piperazine), 4.33
(bs, 2H, piperazine), 7.29–7.46 (m, 9H, ArH). MS (EI) m/z (%): 429
[Mþ] (1), 227 (2), 190 (9), 175 (27), 143 (54), 83 (17), 70 (100). Anal.
calcd.: C, 61.45; H, 5.63; N, 9.77. Found: C, 61.37; H, 5.78; N, 9.58.
Following method A, 25 was prepared from 4-bromo-2-phenyl-2-(4-
t-butylphenyl)butyronitrile 22d as sticky oil in 43% yield after
column chromatography (EtOAc/petroleum ether, 1:5). 1H-NMR
(CDCl3) d 1.30 (s, 9H, –C(CH3)3), 2.05 (m, 2H, –CH2–), 2.38 (d, 3H,
J ¼ 3.0 Hz, CH3), 2.56 (m, 2H, –CH2C(CN)Ph2), 2.79 (m, 4H, homo-
piperazine), 3.37 (m, 2H, –CS2CH2–), 3.90 (t, 1H, J ¼ 6.3 Hz, homo-
piperazine), 3.97 (t, 1H, J ¼ 4.8 Hz, homopiperazine), 4.22 (t, 1H,
J ¼ 6.3 Hz, homopiperazine), 4.36 (bs, 1H, homopiperazine), 7.30–
7.50 (m, 9H, ArH). MS (EI) m/z (%): 465 [Mþ] (1), 365, 310, 249, 217,
189, 157 (100), 128, 113, 96, 70. Anal. calcd.: C, 69.63; H, 7.57; N,
9.02. Found: C, 69.50; H, 7.28; N, 8.81.
4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-phenyl-
3-(4-methoxyphenyl)propyl ester (23c)
Following the procedure of 23a, compound 23c was obtained
from 4-bromo-2-phenyl-2-(4-methoxyphenyl)-butyronitrile 22c as
sticky oil in 39% yield (two steps from 21c) after column chroma-
tography (EtOAc/petroleum ether, 1:5). 1H-NMR (CDCl3) d 2.15
(s, 3H, –NCH3), 2.30 (s, 3H, –OCH3), 2.47 (bs, 4H, piperazine), 2.77
(m, 2H, –CH2C(CN)Ph2), 3.35 (m, 2H, –CS2CH2–), 3.89 (bs, 2H,
piperazine), 4.30 (bs, 2H, piperazine), 7.24–7.43 (m, 9H, ArH).
4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-
(9-fluorenyl)propyl ester (30)
a) 9-Cyanofluorene (28)
A mixture of 0.5 g (3.0 mmol) fluorene and 0.8 g (33 mmol) NaH
in 10 mL anhydrous DMSO was stirred at room temperature for
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Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
Dithiocarbamic Acid Esters as Anticancer Agent
331
10 min. 1.5 mL (18.6 mmol) ethyl formate were added drop wise
under ice cooling. After the reaction was finished, the reaction
mixture was adjusted by hydrochloric acid to acid and extracted
by ethyl ether (3 ꢄ 5 mL). The organic phase was separated
and dried over anhydrous Na2SO4. After removing the solvent,
the crude product of 26 was mixed with 0.31 g (4.5 mmol)
hydroxylamine hydrochloride and 0.5 g (6.0 mmol) sodium
bicarbonate in 15 mL alcohol and stirred at room
temperature for 50 min. The reaction mixture was filtered
and the filtrate was concentrated under reduced pressure to
get the crude product of 27 which was used in next step
without purification. The mixture of oxime 27 and 0.2 mL
(2.7 mmol) SOCl2 in 10 mL diethyl ether was stirred at room
temperature for 1 h. The solid was filtered off and the filtrate was
washed with water (3 ꢄ 5 mL). The organic phase was dried over
anhydrous Na2SO4 and removed the solvent. The residue was
recrystallized from ethanol to give 0.52 g of compound 28 as
yellow solid.
carcinoma cell line (Bel-7402), human cervical carcinoma cell
line (Hela), human prostatic carcinoma cell line (PC 3MIE8),
and human breast carcinoma cell line (MDA-MB-435) were
grown and maintained in RPMI-1640 medium supplemented
with 10% fetal bovine serum, penicillin (100 U/mL), strepto-
mycin (100 mg/mL) at 378C in a humidified incubators in an
atmosphere of 5% CO2. All of experiments were performed on
exponentially growing cancer cells.
Assay of cancer cells proliferation
Numbers of viable cell of cancer were determined by MTT and
SRB assays [22]. Briefly, cancer cells (1–2.5 ꢄ 104 cells mLꢁ1
)
were inoculated in 96-well culture plates (180 mL/well). After
24 h, 20 mL of culture medium containing testing com-
pounds of various concentrations were added to the wells
and RPMI-1640 medium in control cells; then, the cells were
incubated for 48 h. HL-60 cells were assayed by MTT, and the
Bel-7402 cells were assayed by SRB. The absorbance of each
well was measured using a microculture plate reader at
570 nm (MTT) and 540 nm (SRB). The IC50 values (concen-
trations to inhibit 50% cancer cells proliferation) were deter-
mined by replicate of 4 wells for each point and three
independent experiments.
Yield 91% (three steps); mp 151–1528C; 1H-NMR (CDCl3)
d 4.93 (s, 1H, CH), 7.39–7.53 (m, 4H, ArH), 7.71–7.80 (m, 4H,
ArH).
b) 4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-(9-
fluorenyl)propyl ester (30)
The intermediate 29 was synthesized by the method of
compound 22. Following method A, the target compound
30 was prepared as white solid by column chromatography
(EtOAc/petroleum ether, 1:5).
Yield 64% (two steps); mp 108–1098C; 1H-NMR (CDCl3) d 2.45
(s, 3H, CH3), 2.69 (bs, 4H, piperazine), 2.72 (m, 2H, fluorenyl
–CH2–), 2.87 (m, 2H, SCH2–), 3.84 (bs, 2H, piperazine), 4.28 (bs,
2H, piperazine), 7.41–7.52 (m, 4H, ArH), 7.12–7.77 (m, 4H, ArH).
Anal. calcd.: C, 67.14; H, 5.89; N, 10.68. Found: C, 66.92; H, 5.83; N,
10.46.
Animals and tumors
Male ICR mice (body weight, 18–22 g) were obtained from the
Department of Laboratory Animal Science of Peking
University Health Science Center (Beijing, China) and kept
at four mice/cage ad libitum at 22 ꢂ 28C on a 12 h light/dark
cycle with food and water available freely. All animals were
treated in accordance with the guidelines established by the
Institutional Animal Care and Use Committee of Peking
University. The murine H22 ascites tumor cells (supplied by
the cell bank of the pharmacology group of National Key
Laboratory of Natural and Biomimetic Drugs, Peking
University) were diluted with 0.9% normal saline solution
to 1 ꢄ 106 cells/mL and transplanted s.c. via trocar into the
left armpits by using an aseptic manipulation, 0.2 mL/mouse.
1 ꢀ HCl and 3n ꢀ HCl were treated intragastically at various
doses. Each mouse was weighed three times a week and at the
end of the study; tumor was weighted by electron scales and
tumor inhibition rate was calculated according to the follow-
ing formula: (mean tumor weight of negative group – mean
tumor weight of treated group)/mean tumor weight of nega-
tive group ꢄ 100%.
4-Methylpiperazine-1-carbodithioic acid 3-cyano-3-
(9-fluorene)propyl ester hydrochloride (30 ꢀ HCl)
1.0 g (2.5 mmol) 30 was dissolved in 10 mL ethyl acetate and the
solution was saturated with anhydrous hydrochloric acid. The
target compound 30 ꢀ HCl was got as crystals.
Yield 83%; mp 2608C (dec.); 1H-NMR (DMSO-d6) d 2.69–2.89 (m, 7H,
CH3 and SCH2CH2–), 3.40 (bs, 2H, piperazine), 3.61 (bs, 2H, piper-
azine), 4.91 (bs, 2H, piperazine), 5.23 (bs, 2H, piperazine), 7.48–7.61
(m, 4H, ArH), 7.85–7.87 (d, 2H, J ¼ 7.2 Hz, ArH), 7.99–8.02 (d, 2H,
J ¼ 7.5 Hz, ArH); MS (EI) 393.2 (M ꢁ HCl). Anal. calcd.: C, 61.45; H,
5.63; N, 9.77. Found: C, 61.31; H, 5.66; N, 9.67.
Biology
Chemicals
Chemicals were synthesized by our group. RPMI-1640
medium was produced from GIBCO. Sulforhodamine B
(SRB), tetrazolium salt and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased from
Microplate reader (FLUOsta OPTIMA, Germany).
This research was supported by the Natural Science Foundation of China
(NSFC 20672009). Biological activities were completed by National Research
Laboratory of Natural and Biomimetic Drugs, Beijing, and National
Center For Drug Screening, Shanghai Institute of Medica, Chinese
Academy of Sciences.
Cell lines
Human promyelocytic leukemic cell line (HL-60), human
gastric carcinoma cell line (BGC-823), human hepatocellular
The authors have declared no conflict of interest.
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332
X. Hou et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 320–332
[11] M. A. H. Zahran, T. A. R. Salemb, R. M. Samaka, H. S. Agwa,
A. R. Awad, Bioorg. Med. Chem. 2008, 16, 9708–9718.
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