Synthesis and in vitro antiproliferative activity of novel 1-benzhydrylpiperazine derivatives against human cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2008.09.025

In order to explore the antiproliferative effect associated with the piperazine framework, several 1-benzhydrylpiperazine derivatives 8(a-d), 9(a-d) and 10(a-h) were synthesized. Variation in the functional group at N-terminal of the piperazine led to three sets of compounds, bearing the sulfonyl, amide and thiourea, respectively. Their chemical structures were confirmed by ¹H NMR, LCMS, IR and elemental analysis. The antiproliferative effect of the compounds were evaluated in vitro using the MTT colorimetric method against one normal cell line (NF-103 skin fibroblast cells) and four human cancer cell lines (MCF-7 breast carcinoma cell line, HepG-2 hepatocellular carcinoma cell line, HeLa cervix carcinoma cell line and HT-29 colon carcinoma cell line) for the time period of 24 h. Among the series, four compounds exhibited interesting growth inhibitory effects against all four cell lines.

Full text of DOI:10.1016/j.ejmech.2008.09.025

European Journal of Medicinal Chemistry 44 (2009) 1223–1229
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antiproliferative activity of novel 1-benzhydrylpiperazine
derivatives against human cancer cell lines
C.S. Ananda Kumar a, S.B. Benaka Prasad a, K. Vinaya a, S. Chandrappa a, N.R. Thimmegowda a,
,
Y.C. Sunil Kumar a, Sanjay Swarup b, K.S. Rangappa a
*
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India
b Department of Biological Sciences, National University of Singapore, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 April 2008
Received in revised form 29 July 2008
Accepted 4 September 2008
Available online 30 September 2008
In order to explore the antiproliferative effect associated with the piperazine framework, several 1-
benzhydrylpiperazine derivatives 8(a–d), 9(a–d) and 10(a–h) were synthesized. Variation in the func-
tional group at N-terminal of the piperazine led to three sets of compounds, bearing the sulfonyl, amide
and thiourea, respectively. Their chemical structures were confirmed by 1H NMR, LCMS, IR and elemental
analysis. The antiproliferative effect of the compounds were evaluated in vitro using the MTT colorimetric
method against one normal cell line (NF-103 skin fibroblast cells) and four human cancer cell lines (MCF-
7 breast carcinoma cell line, HepG-2 hepatocellular carcinoma cell line, HeLa cervix carcinoma cell line
and HT-29 colon carcinoma cell line) for the time period of 24 h. Among the series, four compounds
exhibited interesting growth inhibitory effects against all four cell lines.
Keywords:
1-Benzhydrylpiperazine derivatives
Sulfonyl chlorides
MTT assay
Ó 2008 Elsevier Masson SAS. All rights reserved.
Antiproliferative activity
Cell proliferation
Carcinoma cell lines
1. Introduction
either synthetic derivatives or natural products has led to the
discovery of the currently utilized anticancer drugs.
Cancer remains the leading cause of death in the World and as
a result there is a pressing need for novel and effective treatments.
One of the characteristic of cancer cells, that differs from their
Piperazines have been widely used in biological screening
resulting in numerous applications and constitute an attractive
pharmacological scaffold present in several drugs [5]. This small
and rigid heterocyclic backbone could act on various pharmaco-
logical targets. Especially, piperazine nucleus could be found in
a broad range of biological active compounds displaying anticancer
[6–13], calcium channel blockers [14–17] and histamine antago-
nists [18,19]. In the continuation of previous research on synthesis
and anticancer studies of bioactive heterocycles [20–23], herein, we
report the synthesis and antiproliferative activity of novel 1-
benzhydrylpiperazine derivatives against human cancer cell lines.
normal counterparts in
a number of biochemical processes,
particularly during the control of cell growth and division.
Despite major breakthroughs in many areas of modern medicine
over the past 100 years, the successful treatment of cancer remains
a significant challenge at the start of the 21st century. Because it is
difficult to discover novel agents that selectively kill tumor cells or
inhibit their proliferation without the general toxicity, the use of
traditional cancer chemotherapy is still very limited. In the field of
chemotherapeutic drugs, the search for new, more active, more
selective and less toxic compounds is still very intense, and new
promising anticancer approaches are being tested [1,2]. Currently,
combined anticancer therapies or multi-acting drugs are clinically
preferred to traditional cytotoxic treatment, with the aim of over-
coming resistance and toxicity drawbacks. These events often
prevent successful treatment and are responsible for reduced
survival times [3,4]. In the past 50 years, the mass screening of
2. Chemistry
1-Benzhydrylpiperazine derivatives 8(a–d), 9(a–d) and 10(a–h)
were prepared by the method summarized in Scheme 1. Initially
compound 2, benzhydrol was synthesized by Grignard reaction
under nitrogen atmosphere with benzaldehyde and phenyl
magnesium bromide, the obtained yield was found to be 60%.
Finally we synthesized the benzhydrol by reduction of benzophe-
none using sodium borohydride and achieved
Compound 2 was subsequently treated with thionyl chloride to
give the corresponding benzhydryl chloride (3), which was directly
a 90% yield.
* Corresponding author. Tel./fax: þ91 821 2412191.
E-mail addresses: rangappaks@gmail.com, rangappaks@chemistry.uni-mysor-
e.ac.in (K.S. Rangappa).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.025

1224
C.S.A. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 1223–1229
O
N S R
O
(iv)
N
5(a-d)
8(a-d)
O
(i)
(iii)
(ii)
(v)
N
N
N
N
R
N
O
OH
N
N H
Cl
6(a-d)
9(a-d)
1
2
4
3
S
(vi)
R
7(a-h)
10(a-h)
Scheme 1. Reagents and conditions: (i) NaBH4, methanol, r.t., 5 h. (ii) Thionyl chloride, MDC, 0–5 ꢀC, 4 h. (iii) Piperazine, K2CO3, DMF, 80 ꢀC, 8 h. (iv) Sulfonyl chlorides, MDC,
triethylamine, r.t., 5–6 h. (v) Acid chlorides, MDC, triethylamine, r.t., 5–6 h. (v) Isothiocyanates, MDC, triethylamine, r.t., 4–5 h. Compounds – 5a: 4-Chloro-2-fluorobenzene-1-
sulfonyl chloride; 6a: isoxazole-5-carbonyl chloride; 5b: camphor sulfonyl chloride; 6b: morpholine-4-carbonyl chloride; 5c: benzenesulfonyl chloride; 6c: pyrrolidine-1-carbonyl
chloride; 5d: 2,2,2-trifluoro ethanesulfonyl chloride; 6d: cyclopropanecarbonyl chloride; 7a: 2-methoxyphenylisothiocyanate; 7b: 3-methoxyphenylisothiocyanate; 7c: 4-
methoxyphenylisothiocyanate; 7d: 2-chlorophenylisothiocyanate; 7e: 3-chlorophenylisothiocyanate; 7f: 4-chlorophenylisothiocyanate; 7g: 4-fluorophenylisothiocyanate; 7h: 2,4-
dichlorophenylisothiocyanate.
treated with piperazine and anhydrous potassium carbonate using
dimethyl formamide as a solvent at 80 ꢀC to give the target key
intermediate 1-benzhydrylpiperazine (4). The nucleophilic substi-
tution reactions of 1-benzhydrylpiperazine with different sulfonyl
chlorides/acid chlorides/or isothiocyanates were carried out in the
presence of triethylamine and dichloromethane as solvent with
a good yield ranging from 65 to 88% with good purity. Compound
8c was structurally characterized by X-ray crystallographic studies
and the data of the molecule have been published [24]. The absence
of N–H proton peak in proton NMR and IR spectra confirms our
products. It is also confirmed by IR data, for sulfonamide series 8(a–
d) which showed asymmetric stretching frequency of O]S]O in
the range 1350–1370 cmꢁ1 and symmetric stretching frequency
at 1270–1290 cmꢁ1 and similarly for carboxamide series 9(a–d),
IR data showed stretching frequency of –C]O at 1630–1670 cmꢁ1
and 3250–3290 cmꢁ1 for –NH in thioureas 10(a–h). The products
obtained were purified by column chromatography using hex-
ane:ethyl acetate (8:2) as an eluent. The chemical structures
and physical data of all the synthesized compounds are given in
Table 1.
mixture was heated at 80 ꢀC for 8 h, and monitored by TLC. After
completion, the solvent was removed under reduced pressure and
residue was taken in water and extracted with ethyl acetate. Finally,
water wash was given to the organic layer and dried with anhy-
drous sodium sulphate. The solvent was evaporated and the crude
product obtained was purified by column chromatography over
silica gel (60–120 mesh) using chloroform:methanol (9:1) as an
eluent. Some of the compounds were recrystallised by using ethyl
acetate.
3.2. General procedure for synthesis of 1-benzhydrylpiperazine
derivatives 8(a–d), 9(a–d) and 10(a–h)
A
solution of 1-benzhydrylpiperazine (4) (1.0 eq) in dry
dichloromethane was taken and cooled to 0–5 ꢀC in an ice bath.
Triethylamine (3.0 eq) was added to the cold reaction mixture and
stirred for 10 min, then different sulfonyl chlorides 5(a–d)/acid
chlorides 6(a–d)/isothiocyanates 7(a–h) (1.0 eq) were added, the
reaction mixture was allowed to stir at room temperature for 4–6 h.
The progress of the reaction was monitored by TLC. Upon
completion, the solvent was removed under reduced pressure and
residue was taken in water and extracted with ethyl acetate. The
organic layer was washed with 10% ammonium chloride solution
and finally water wash was given to organic layer and dried with
anhydrous sodium sulphate. The solvent was evaporated to get
crude product which was purified by column chromatography over
silica gel (60–120 mesh) using hexane:ethyl acetate (8:2) as an
eluent.
3. Experimental
Melting points were determined using SELACO-650 hot stage
melting point apparatus and were uncorrected. Infrared (IR)
spectra were recorded using a Jasco FTIR-4100 series. Nuclear
magnetic resonance (1H NMR) spectra were recorded on Shimadzu
AMX 400-Bruker, 400 MHz spectrometer using DMSO-d6 as
a solvent and TMS as internal standard (chemical shift in
d ppm).
Spin multiplets are given as s (singlet), d (doublet), t (triplet) and m
(multiplet). Mass and purity were recorded on a LC-MSD-Trap-XCT.
Elemental (CHNS) analyses were obtained on Vario EL III Elementar.
Silica gel column chromatography was performed using Merck
7734 silica gel (60–120 mesh) and Merck-made TLC plates.
3.2.1. Synthesis of 1-benzhydryl-4-(4-chloro-2-
fluorobenzenesulfonyl)-piperazine (8a)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol),
4-chloro-2-fluorobenzene-1-sulfonyl
chloride
(0.453 g, 1.98 mmol), triethylamine (0.601 g, 5.94 mmol). The
product obtained was white crystalline solid (0.660 g). 1H NMR
3.1. General procedure for synthesis of 1-benzhydrylpiperazine (4)
(DMSO-d6, 400 MHz) d: 7.82 (1H, m, Ar–H), 7.74 (d, 1H, Ar–H), 7.55
(d, 1H, Ar–H), 7.17 (t, 2H, Ar–H), 7.25 (t, 4H, Ar–H), 7.37 (d, 4H, Ar–
H), 4.25 (s, 1H, –CH–), 3.1 (br s, 4H, –CH2–), 2.31 (br s, 4H, –CH2–).
MS (ESI, þ ion): m/z ¼ 445.2. IR (KBr, cmꢁ1): 3049, 2950, 2828,1340,
1286. Anal. calcd. for C23H22ClFN2O2S (in %): C 62.09, H 4.98, N 6.30,
S 7.21. Found C 62.05, H 4.95, N 6.35, S 7.25.
A solution of piperazine dihydrochloride (10.0 g, 62.86 mmol) in
dimethyl formamide was taken, anhydrous potassium carbonate
(43.44 g, 314.3 mmol) was added and stirred for 10 min, and then
benzhydryl chloride (11.46 g, 56.58 mmol) was added. The reaction

C.S.A. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 1223–1229
1225
Table 1
Chemical structure, physical data and purity of synthesized compounds
Table 1 (continued)
Compound
R
Yield (%)
75
M.P. (ꢀC)
213–215
Purity
Compound
R
Yield (%)
M.P. (ꢀC)
192–194
Purity
98.98
10e
97.90
8a
75
10f
80
74
78
208–210
189–191
206–208
97.62
98.60
97.80
8b
68
173–175
97.14
10g
10h
8c
8d
9a
9b
80
65
73
70
157–159
151–153
143–145
181–183
99.1
97.20
98.01
98.65
3.2.2. Synthesis of 1-(4-benzhydrylpiperazine-1-sulfonylmethyl)-
7,7-dimethyl-bicyclo [2,2,1]heptan-2-one (8b)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), (7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)metha-
nesulfonyl chloride (0.496 g, 1.98 mmol), triethylamine (0.601 g,
5.94 mmol). The product obtained was white crystalline solid
(0.627 g). 1H NMR (DMSO-d6, 400 MHz)
d: 7.18 (t, 2H, Ar–H), 7.27 (t,
4H, Ar–H), 7.44 (d, 4H, Ar–H), 4.37 (s,1H, –CH–), 3.24 (br s, 4H, –CH2–
), 2.38 (br s, 4H, –CH2–),1.38 (m, 1H, –CH–),1.9–2.9 (m,12H, –CH2–),
0.82 (s, 6H, –CH3). MS (ESI, þ ion): m/z ¼ 467.2. IR (KBr, cmꢁ1):
3053, 2959, 2838, 1345, 1280. Anal. calcd. for C27H36N2O2S (in %): C
71.64, H 8.02, N 6.19, S 7.08. Found C 71.60, H 8.05, N 6.15, S 7.05.
9c
77
66
162–164
173–175
97.36
98.21
3.2.3. Synthesis of 1-benzenesulfonyl-4-benzhydrylpiperazine (8c)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), benzenesulfonyl chloride (0.349 g, 1.98 mmol) and
triethylamine (0.601 g, 5.94 mmol). The product was a white crys-
9d
talline solid (0.622 g). 1H NMR (DMSO-d6, 400 MHz)
d: 7.48 (d, 2H,
Ar–H), 7.40 (m, 6H, Ar–H), 7.27 (t, 5H, Ar–H), 7.15 (t, 2H, Ar–H), 4.26
(s,1H, –CH–), 3.2 (br s, 4H, –CH2–), 2.50 (br s, 4H, –CH2–). MS (ESI, þ
ion): m/z ¼ 393.61. IR (KBr, cmꢁ1): 3026, 2925, 2960, 1319, 1150.
Anal. calcd. for C23H24N2O2S (in %): C 70.38, H 6.16, N 7.14, S 8.17.
Found C 70.34, H 6.11, N 7.10, S 8.15.
10a
82
158–160
97.81
3.2.4. Synthesis of 1-benzhydryl-4-(2,2,2-trifloro-ethyanesulfonyl)-
piperazine (8d)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), 2,2,2-trifluoro ethanesulfonyl chloride (0.361 g,
1.98 mmol), triethylamine (0.601 g, 5.94 mmol). The product
obtained was brown crystalline solid (0.512 g). 1H NMR (DMSO-d6,
10b
10c
10d
85
88
78
160–162
199–201
194–196
98.65
96.85
98.13
400 MHz) d: 7.18 (t, 2H, Ar–H), 7.26 (t, 4H, Ar–H), 7.45 (d, 4H, Ar–H),
4.37 (s, 1H, –CH–), 3.24 (br s, 4H, –CH2–), 2.37 (br s, 4H, –CH2–), 4.82
(s, 2H, –CH2–). MS (ESI, þ ion): m/z ¼ 399.30. IR (KBr, cmꢁ1): 3043,
2946, 2830, 1347, 1281. Anal. calcd. for C19H21F3N2O2S (in %): C
57.27, H 5.31, N 7.03, S 8.05. Found C 57.23, H 5.27, N 7.07, S 8.10.
3.2.5. Synthesis of 4-benzhydrylpiperazine-1-yl(isoxazol-5-
yl)methanone (9a)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), isoxazole-5-carbonyl chloride (0.260 g, 1.98 mmol),
triethylamine (0.601 g, 5.94 mmol). The product obtained was
off-white crystalline solid (0.501 g). 1H NMR (DMSO-d6, 400 MHz)

1226
C.S.A. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 1223–1229
d: 7.38 (d, 4H, Ar–H), 7.24 (t, 4H, Ar–H), 7.17 (t, 2H, Ar–H), 5.5–5.6 (d,
3.2.11. Synthesis of 4-benzhydryl-N-(4-methoxyphenyl)piperazine-
2H, N–CH–CH–O–), 4.4 (s, 1H, –CH–), 3.2 (br s, 4H, –CH2–), 2.35 (br
s, 4H, –CH2–). MS (ESI, þ ion): m/z ¼ 348.2. IR (KBr, cmꢁ1): 2924,
2854, 1651. Anal. calcd. for C21H21N3O2 (in %): C 72.60, H 6.09, N
12.09. Found C 72.56, H 6.13, N 12.05.
1-carbothioamide (10c)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), 4-methoxyphenylisothiocyanate (0.354 g, 1.98 mmol)
and triethylamine (0.601 g, 5.94 mmol). The product obtained was
white fluffy solid (0.726 g). 1H NMR (DMSO-d6, 400 MHz)
d: 9.13 (s,
3.2.6. Synthesis of (4-benzhydrylpiperazin-1-
yl)(morpholino)methanone (9b)
1H, –NH), 7.46 (d, 4H, Ar–H), 7.33 (t, 4H, Ar–H), 7.22 (t, 2H, Ar–H),
7.14 (d, 2H, Ar–H), 6.86 (d, 2H, Ar–H), 4.38 (s, 1H, –CH–), 3.73 (s, 3H,
–OCH3), 3.89 (br s, 4H, –CH2–), 2.36 (br s, 4H, –CH2–). MS (ESI, þ
ion): m/z ¼ 418.1. IR (KBr, cmꢁ1): 3158, 1385. Anal. calcd. for
C25H27N3OS (in %): C 71.91, H 6.52, N 10.06, S 7.68. Found. C 71.94, H
6.54, N 10.06, S 7.70.
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), morpholine-4-carbonyl chloride (0.296 g, 1.98 mmol),
triethylamine (0.601 g, 5.94 mmol). The product obtained was
white crystalline solid (0.506 g). 1H NMR (DMSO-d6, 400 MHz)
d:
7.40 (d, 4H, Ar–H), 7.3 (t, 4H, Ar–H), 7.19 (t, 2H, Ar–H), 4.29 (s, 1H,
–CH–), 3.5 (br s, 4H, –CH2–), 3.0-3.1 (8H, –CH2–), 2.49 (br s, 4H,
–CH2–). MS (ESI, þ ion): m/z ¼ 366.0. IR (KBr, cmꢁ1): 2935, 2863,
1660. Anal. calcd. for C22H27N3O2 (in %): C 72.30, H 7.45, N 11.50.
Found C 72.26, H 7.40, N 11.46.
3.2.12. Synthesis of 4-benzhydryl-N-(2-chlorophenyl)piperazine-1-
carbothioamide (10d)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), 2-chlorophenylisothiocyanate (0.335 g, 1.98 mmol)
and triethylamine (0.601 g, 5.94 mmol). The product obtained was
off-white crystalline solid (0.651 g). 1H NMR (DMSO-d6, 400 MHz)
3.2.7. Synthesis of (4-benzhydrylpiperazin-1-yl)(pyrrolidin-1-
yl)methanone (9c)
d: 9.20 (s, 1H, –NH), 7.47 (d, 4H, Ar–H), 7.36 (t, 4H, Ar–H), 7.23–7.15
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), pyrrolidine-1-carbonyl chloride (0.264 g, 1.98 mmol),
triethylamine (0.601 g, 5.94 mmol). The product obtained was
(m, 3H, Ar–H), 7.05 (t, 2H, Ar–H), 6.70 (m, 1H, Ar–H), 4.38 (s, 1H,
–CH–), 3.35 (br s, 4H, –CH2–), 2.36 (br s, 4H, –CH2–). MS (ESI, þ ion):
m/z ¼ 422. IR (KBr, cmꢁ1): 3248, 1400. Anal. calcd. for C24H24ClN3OS
(in %): C 68.31, H 5.73, N 9.96, S 7.60. Found. C 68.33, H 5.75, N 9.99,
S 7.63.
brown crystalline solid (0.532 g). 1H NMR (DMSO-d6, 400 MHz)
d:
7.42 (d, 4H, Ar–H), 7.30 (t, 4H, Ar–H), 7.19 (t, 2H, Ar–H), 4.35 (s, 1H,
–CH–), 3.3 (br s, 4H, –CH2–), 3.1(br s, 4H, –CH2–), 2.48 (br s, 4H,
–CH2–), 1.69 (b, 4H, –CH2–). MS (ESI, þ ion): m/z ¼ 350.11. IR (KBr,
cmꢁ1): 2968, 2860, 1658. Anal. calcd. for C22H27N3O (in %): C 75.61,
H 7.79, N 12.02. Found C 75.57, H 7.75, N 12.05.
3.2.13. Synthesis of 4-benzhydryl-N-(3-chlorophenyl)piperazine-1-
carbothioamide (10e)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), 3-chlorophenylisothiocyanate (0.335 g, 1.98 mmol)
and triethylamine (0.601 g, 5.94 mmol). The product obtained was
3.2.8. Synthesis of (4-benzhydrylpiperazin-1-
yl)(cyclopropyl)methanone (9d)
white fluffy solid (0.626 g). 1H NMR (DMSO-d6, 400 MHz)
d: 9.23 (s,
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), cyclopropanecarbonyl chloride (0.207 g, 1.98 mmol),
triethylamine (0.601 g, 5.94 mmol). The product obtained was off-
1H, –NH), 7.45 (d, 4H, Ar–H), 7.39 (t, 4H, Ar–H), 7.27 (t, 4H, Ar–H),
6.89 (t, 2H, Ar–H), 6.67 (m, 1H, Ar–H), 4.39 (s, 1H, –CH–), 3.37 (br s,
4H, –CH2–), 2.36 (br s, 4H, –CH2–). MS (ESI, þ ion): m/z ¼ 422.4. IR
(KBr, cmꢁ1): 3275, 1321. Anal. calcd. for C24H24ClN3OS (in %): C
68.31, H 5.73, N 9.96, S 7.60. Found. C 68.32, H 5.76, N 9.97, S 7.64.
white crystalline solid (0.418 g). 1H NMR (DMSO-d6, 400 MHz)
d: 7.45
(d, 4H, Ar–H), 7.28 (t, 4H, Ar–H), 7.21 (t, 2H, Ar–H), 4.32 (s,1H, –CH–),
3.4 (br s, 2H, –CH2–), 3.28 (br s, 2H, –CH2–), 2.5 (br s, 2H, –CH2–), 2.32
(br s, 2H, –CH2–), 0.6–0.7 (m, 4H, –CH–). MS (ESI, þ ion): m/z ¼ 321.2.
IR (KBr, cmꢁ1): 2960, 2856, 1650. Anal. calcd. for C21H24N2O (in %): C
78.72, H 7.55, N 8.74. Found C 78.70, H 7.52, N 8.70.
3.2.14. Synthesis of 4-benzhydryl-N-(4-chlorophenyl)piperazine-1-
carbothioamide (10f)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), 4-chlorophenylisothiocyanate (0.335 g, 1.98 mmol) and
triethylamine (0.601 g, 5.94 mmol). The product obtained was off-
3.2.9. Synthesis of 4-benzhydryl-N-(2-methoxyphenyl)piperazine-
1-carbothioamide (10a)
white crystalline solid (0.668 g). 1H NMR (DMSO-d6, 400 MHz)
d:
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), 2-methoxyphenylisothiocyanate (0.354 g, 1.98 mmol)
and triethylamine (0.601 g, 5.94 mmol). The product obtained was
9.14 (s, 1H, –NH), 7.48 (d, 4H, Ar–H), 7.33 (t, 4H, Ar–H), 7.25 (t, 2H,
Ar–H), 7.14 (d, 2H, Ar–H), 6.90 (d, 2H, Ar–H), 4.39 (s, 1H, –CH–), 3.87
(br s, 4H, –CH2–), 2.38 (br s, 4H, –CH2–). MS (ESI, þ ion): m/z ¼ 422.1.
IR (KBr, cmꢁ1): 3286, 1346. Anal. calcd. for C24H24ClN3OS (in %): C
68.31, H 5.73, N 9.96, S 7.60. Found. C 68.35, H 5.75, N 9.97, S 7.62.
white crystalline solid (0.677 g). 1H NMR (DMSO-d6, 400 MHz)
d:
9.18 (s, 1H, –NH), 7.44 (d, 5H, Ar–H), 7.34 (t, 5H, Ar–H), 7.25 (t, 2H,
Ar–H), 7.20 (m, 2H, Ar–H), 4.34 (s, 1H, –CH–), 3.35 (br s, 4H, –CH2–),
2.39 (br s, 4H, –CH2–). MS (ESI, þ ion): m/z ¼ 418.1. IR (KBr, cmꢁ1):
3289, 1334. Anal. calcd. for C25H27N3OS (in %): C 71.91, H 6.52, N
10.06, S 7.68. Found. C 71.94, H 6.55, N 10.10, S 7.70.
3.2.15. Synthesis of 4-benzhydryl-N-(4-fluorophenyl)piperazine-1-
carbothioamide (10g)
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), 4-fluorophenylisothiocyanate (0.303 g, 1.98 mmol) and
triethylamine (0.601 g, 5.94 mmol). The product obtained was
3.2.10. Synthesis of 4-benzhydryl-N-(3-methoxyphenyl)piperazine-
1-carbothioamide (10b)
white fluffy solid (0.593 g). 1H NMR (DMSO-d6, 400 MHz)
d: 9.17 (s,
This was obtained from 1-benzhydrylpiperazine (4) (0.5 g,
1.98 mmol), 3-methoxyphenylisothiocyanate (0.354 g, 1.98 mmol)
and triethylamine (0.601 g, 5.94 mmol). The product obtained was
1H, –NH), 7.45–7.40 (m, 6H, Ar–H), 7.33–7.29 (t, 4H, Ar–H), 7.22 (t,
2H, Ar–H), 7.11 (m, 2H, Ar–H), 4.35 (s, 1H, –CH–), 3.89 (br s, 4H,
–CH2–), 2.36 (br s, 4H, –CH2–). MS (ESI, þ ion): m/z ¼ 406.3. IR (KBr,
cmꢁ1): 3287, 1319. Anal. calcd. for C24H24ClFN3S (in %): C 71.08, H
5.97, N 10.36, S 7.91. Found. C 71.05, H 5.99, N 10.33, S 7.94.
white crystalline solid (0.702 g). 1H NMR (DMSO-d6, 400 MHz)
d:
9.23 (s,1H, –NH), 7.46 (d, 4H, Ar–H), 7.33 (t, 4H, Ar–H), 7.22–7.15 (m,
3H, Ar–H), 6.89 (t, 2H, Ar–H), 6.67 (m, 1H, Ar–H), 4.39 (s, 1H, –CH–),
3.73 (s, 3H, –OCH3), 3.37 (br s, 4H, –CH2–), 2.36 (br s, 4H, –CH2–).
MS (ESI, þ ion): m/z ¼ 418.2. IR (KBr, cmꢁ1): 3250, 1338. Anal. calcd.
for C25H27N3OS (in %): C 71.91, H 6.52, N 10.06, S 7.68. Found. C
71.95, H 6.50, N 10.08, S 7.72.
3.2.16. Synthesis of 4-benzhydryl-N-(2,4-
dichlorophenyl)piperazine-1-carbothioamide (10h)
This was obtained from 1-benzhydrylpiperazine (6) (0.5 g,
1.98 mmol), 2,4-dichlorophenylisothiocyanate (0.404 g, 1.98 mmol)

C.S.A. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 1223–1229
1227
and triethylamine (0.601 g, 5.94 mmol). The product obtained was
white fluffy solid (0.704 g). 1H NMR (DMSO-d6, 400 MHz)
: 9.20 (s,
140
120
100
80
d
1H, –NH), 7.63 (s, 1H, Ar–H), 7.50–7.45 (m, 5H, Ar–H), 7.36–7.29 (m,
5H, Ar–H), 7.22 (t, 2H, Ar–H), 4.35 (s, 1H, –CH–), 3.46 (br s, 4H,
–CH2–), 2.40 (br s, 4H, –CH2–). MS (ESI, þ ion): m/z ¼ 456.4. IR (KBr,
cmꢁ1): 3264, 1348. Anal. calcd. for C24H23Cl2N3S (in %): C 63.16, H
5.08, N 9.21, S 7.02. Found. C 63.13, H 5.11, N 9.25, S 7.05.
60
40
20
0
3.3. Biology
We tested the effect of novel synthesized compounds 8(a–d),
9(a–d) and 10(a–h) on cell proliferation using five different cell
lines in a cell toxicity assay and the percentage cell survived at the
No. of days (1)
Fig. 1. MTT assay for human normal skin fibroblast cell line.
dose level of 100 mM for 24 h was determined. Percentage cell
survival for tested compounds against NF-103, MCF-7, HepG-2, HT-
29 and HeLa cells are tabulated in Table 2. The extent of inhibition
of carcinoma cell lines by 8(a–d), 9(a–d) and 10(a–h) were sche-
matically presented in Figs. 1–5.
medium and incubated for 48 h before addition of test compound.
Cells were then exposed to known concentrations of the compound
to be tested (100
37 ꢀC. After drug exposure, the culture medium was removed and
200 l of MTT reagent (diluted in culture medium, 1 mg/ml) was
added. After incubating for 4 h, the MTT/medium was removed and
DMSO (200 l) was added to dissolve the formazan crystals.
mM expressed as final concentration) for 24 h at
Stock solutions (100 mM) of test compounds were prepared in
dimethylsulfoxide (DMSO) and stored at ꢁ20 ꢀC. These concen-
trated solutions were added immediately to cell culture wells on
the day of experimentation. The final DMSO concentration was 0.1%
in each well and it showed no interference with the biological
activities tested.
m
m
Absorbance of the colored solution was measured on a microtiter
plate reader using a test wavelength of 570 nm. Results were
evaluated by comparing the absorbance of the wells containing
compound treated cells with the absorbance of wells containing
0.1% DMSO alone (solvent control). Conventionally, cell viability
was estimated to be 100% in the solvent control. All assays were
performed in triplicate and mean ꢂ SD values were used to esti-
mate cell viability.
3.3.1. Cell culture
NF-103, HeLa, HepG-2, MCF-7 Cell lines were cultured in DMEM
with 10% FCS, HT-29 cultured in Mc Coy’s Medium with 10% FCS,
CRL-170 cultured in RPMI with 10% FCS and 1% v/v antibiotic. The
proliferation of skin fibroblast cells (NF-103), breast carcinoma cells
(MCF-7), hepatocellular carcinoma cells (HepG-2), cervix carci-
noma cells (HeLa) and colon carcinoma cells (HT-29) can be assayed
at different time intervals besides 24 h. The positive control used in
the experiment was wells with DMEM (10% FBS and 1% v/v anti-
biotic added) added while the negative controls used were wells
with DMEM (1% v/v antibiotic and no FBS added). Cell cultures used
were procured from the Department of Biological Sciences,
National University of Singapore, Singapore. The absorbance was
measured at 570 nm with a microtiter plate reader.
4. Results and discussion
Piperizine derivatives were shown to inhibit growth inhibition
of human erythroleukemia K5-62 cells and myeloid leukemia HL-
60 cells [26] and also shown to inhibit topoisomarase II activity
[27]. Wilson et al reported the interaction of DNA with unfused
aromatic system containing terminal piperazino substituents [9].
Bisdioxopiperazines have been reported for their anti-tumor effects
against two experimental lung cancer models in vivo [28]. Recently,
a new class of sulfonamides has been used in the treatment of
diseases arising from abnormal cell growth and proliferation
[29,30].
3.3.2. In vitro cell viability assay – MTT assay
The potential effects on cell viability were investigated by using
the MTT assay [25] [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide] as an indicator of metabolically active cells.
A known number of MCF-7 or HT-29 or HeLa or HepG-2 cells were
In view of the above findings, the anticancer activity was
checked by carrying out the reactions of 1-benzhydrylpiperazine
transferred into 96 well plates in a volume of 200
ml of culture
Table 2
140
120
100
80
Evaluation of cytotoxicity towards carcinoma cells (% cell survival) for the synthe-
sized compounds
Compound
NF-103
MCF-7
HepG-2
HeLa
HT-29
8a
8b
8c
8d
9a
9b
9c
9d
10a
10b
10c
10d
10e
10f
10g
10h
60.95
60.44
57.42
54.90
58.61
56.10
53.99
55.07
59.70
57.76
55.93
59.13
56.84
54.79
52.73
53.53
64.03
61.73
68.09
41.11
44.18
68.69
71.43
61.01
54.49
58.71
63.70
69.79
73.57
66.94
43.91
45.61
70.14
72.63
66.16
47.76
46.10
61.85
66.16
62.85
61.02
64.51
48.75
71.80
69.48
66.16
45.43
59.20
61.89
65.15
61.89
47.69
46.80
53.92
64.25
70.31
56.79
72.83
66.04
63.07
69.75
73.90
48.37
45.45
76.27
79.20
69.27
43.64
47.50
63.40
70.24
71.98
62.10
64.82
55.59
70.79
69.27
73.77
47.39
68.18
60
40
20
0
Control
8d
9a
No. of days (1)
10g
10h
Fig. 2. MTT assay for MCF-7 cell line.

1228
C.S.A. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 1223–1229
140
120
100
80
140
120
100
80
60
60
40
40
20
20
0
0
Control
8d
9a
No. of days (1)
10c
10g
Control
8d
No. of days (1)
9a
10g
Fig. 3. MTT assay for HepG-2 cell line.
Fig. 5. MTT assay for HT-29 cell line.
with different sulfonyl chlorides/acid chlorides/isothiocyanates
containing substituted aromatic rings.
From benzamide derivatives 9(a–d), compound 9a exhibits
44.18%, 46.10%, 46.80%, 47.50% inhibitory activity against MCF-7,
HepG-2, HeLa, HT-29 carcinoma cell lines, respectively. The
remaining compounds 9(b–d) exhibit less inhibitory activity. The
potent inhibition of compound 9a might be due to the presence of
isoxazole moiety.
High activity of compound 9a is connected with quite different
distribution of charge in the isoxazole ring compared to other
analogues. Rigidity of the isoxazole ring is essential for a more
efficient binding to the active site of the enzyme. Replacing the
isoxazole ring by morpholine 9b and pyrrolidine 9c resulted in the
loss of the activity. Similarly, replacing cyclopropyl ring 9d still
decreases the antiproliferative activity.
In thioureas 10(a–h), compound 10g exhibits 43.91%, 45.43%,
48.37%, 47.39% inhibitory activity against MCF-7, HepG-2, HeLa,
HT-29 carcinoma cell lines, respectively. Compound 10h showed
45.61%, 45.45% inhibitory activity against MCF-7, HeLa carcinoma
cell lines, respectively. The –NH– group in thioureas is essential for
binding with the enzyme and the nature of the amide side chain is
important for the exhibition of anticancer activity. Relatively high
activity of compounds 10g and 10h can be related to the presence of
the electron withdrawing fluoro and chloro substituent on the
aromatic ring. Structure activity relationship can be drawn for the
derivatives 10(d–h) containing electronegative atoms, which
reveals that, compound 10g has more electronegative fluorine atom
compared to 10(d–f) having chlorine atom. In the same aspect,
compound 10h has disubstituted chlorine atom exhibits relatively
good inhibition compare to 10(d–f) having mono substituted
chlorine atom. Introducing electron donating methoxy groups
10(a–c) to the phenyl ring of the substituent at ortho, meta and para
position resulted in the loss of the activity. However, the presence
of electron withdrawing fluorine atom 10g in the phenyl ring
increased the antiproliferative efficacy.
The novel synthesized compounds were tested for their effect
on cellular viability against human carcinoma cell lines MCF-7,
HepG-2, HeLa and HT-29. All the synthesized compounds were
tested for their cytotoxicity against normal skin fibroblast cells.
Assays were performed in vitro on exponentially growing cells. The
activity was evaluated by measuring the levels of surviving cells
after incubation for 24 h with the test samples, using the MTT
colorimetric assay, based on the ability of metabolically active cells
to convert the pale yellow MTT to a blue formazan product, which is
quantifiable spectrophotometrically.
Among the sulfonamide analogues 8(a–d), compound 8d
exhibits 41.11%, 47.76%, 47.69%, 43.64% inhibitory activity against
MCF-7, HepG-2, HeLa, HT-29 carcinoma cell lines, respectively,
whereas compounds 8(a–c) showed less inhibitory activity. From
the data obtained, it is obvious that the nature of the N-terminal on
the 1-benzhydrylpiperazine exerts a striking effect on the anti-
proliferative activity. Within the few variations considered in this
study, the presence of a trifluoroethyl alkyl chain afforded a clear
beneficial effect with regard to antiproliferative properties. With
the exception of 8b as alkyl camphor sulfonyl counterpart on 1-
benzhydrylpiperazine was less active compare to 8d. The good
inhibition by compound 8d could be attributed to the presence of
electron withdrawing trifluoroethyl group. Introduction of an
aromatic ring resulted in compounds (8a and 8c) completely
devoid of activity.
140
120
100
80
5. Conclusion
60
From our experimental results, it could be concluded that the
introduction of alkyl trifluoroethyl containing sulfonyl group, iso-
xazole ring in the amide moiety and substitution of fluoro, chloro in
the aromatic ring bearing thiourea group on the 1-benzhy-
drylpiperazine system has great potential to get novel anti-
proliferative compounds. From this work we were able to identify
a few active molecules which are capable of inhibiting the growth
of human cancer cell lines in vitro. These compounds exhibited
a relatively weak cytotoxicity. Our results suggest that, the anti-
proliferative properties induced by the 1-benzhydrylpiperazine
40
20
0
Control
8d
9a
No. of days (1)
10g
10h
Fig. 4. MTT assay for HeLa cell line.

C.S.A. Kumar et al. / European Journal of Medicinal Chemistry 44 (2009) 1223–1229
1229
[14] S. Gubert, M.A. Braso, A. Sacristan, J.A. Ortiz, Arzneim-Forsch. 37 (1987)
1103–1107.
[15] M. Kajino, Y. Wada, Y. Nagai, A. Nagaoka, K. Meguro, Chem. Pharm. Bull. 37 (8)
(1989) 2225–2228.
framework might involve distinct mechanisms. Hence, there is
a need for further investigations to clarify the features underlying
the antiproliferative activities of these new derivatives.
[16] J.R. Shanklin Jr., C.P. Johnson, A.G. Proakis, R.J. Barrett, J. Med. Chem. 34 (10)
(1991) 3011–3022.
[17] Y. Nomura, T. Yamakawa, K. Nishioka, T. Omura, N. Miyake, M. Masaki,
H. Nohira, Chem. Pharm. Bull. 43 (2) (1995) 241–246.
Acknowledgements
[18] A. Hiristo, R. Robert, E. BartlettUlrich, S. Paul, Eur. J. Med. Chem. 24 (1989)
227–232.
[19] M. Gillard, C. Van Der Perren, N. Moguilevsky, R. Massingham, P. Chatelain,
Mol Pharmacol. 61 (2) (2002) 391–399.
[20] C. Anil Kumar, S. Nanjunda Swamy, S.L. Gaonkar, Basappa, B.P. Salimath,
K.S. Rangappa, Med. Chem. 3 (2007) 269–276.
[21] C. Anil Kumar, S. Jayarama, Basappa, B.P. Salimath, K.S. Rangappa, Invest. New.
Drugs 25 (2007) 343–350.
[22] B.S. Priya, C. Anil Kumar, S. Nanjunda Swamy, Basappa, S. Naveen,
K.S. Rangappa, Bioorg. Med. Chem. Lett. 17 (2007) 2775–2780.
[23] C.S. Ananda Kumar, S. Nanjunda Swamy, N.R. Thimmegowda, S.B. Benaka
Prasad, G.W. Yip, K.S. Rangappa, Med. Chem. Res. 16 (2007) 179–187.
[24] C.S. Ananda Kumar, S. Naveen, S.B. Benaka Prasad, N.R. Thimmegowda,
N.S. Lingegowda, M.A. Sridhar, J. Shashidhara Prasad, K.S. Rangappa, J. Chem.
Crystallogr. 37 (2007) 727–731.
[25] D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofziger,
M.J. Currens, D. Seniff, M.R. Boyd, Cancer Res. 48 (1998) 4827–4833.
[26] R. Gillet, P. Jeannesson, P.H. Sefreoui, M.L. Amould-Guerin, S. Kirkiacharian,
J.C. Jardillier, F. Pieri, Cancer Chemother. Pharmacol. 41 (1998) 252–255.
[27] J.P. Braybrooke, K.J. O’Byrne, D.J. Propper, A. Blann, M. Saunders, N. Dobbs,
C. Han, J. Woodhull, K. Mitchell, J. Crew, K. Smith, R. Stephens, T.S. Ganesan,
D.C. Talbot, A.L. Harris, Clin. Cancer Res. 6 (2000) 4697–4704.
[28] D.Y. Lu, B. Xu, J. Ding, BMC Pharmacol. 4 (2004) 32.
The authors are grateful to UGC, Govt. of India for financial
support to K.S.R. under the project UGC-SAP (Phase I) vide No. F.
540/10/2004-05 (SAP I) UGC vide No. DV6/69/UGC/2006-07. The
CHNS data from the instrument granted by DST-FIST programme
Council of Scientific and Industrial Research (CSIR), for financial
support under CSIR-SRF to K. Vinaya is greatly acknowledged.
References
[1] C. Sawyers, Nature 432 (2004) 294–297.
[2] Q. Li, W. Xu, Curr. Med. Chem. Anticancer Agents 5 (2005) 53–63.
[3] S.K. Mencher, L.G. Wang, BMC Clin. Pharmacol. 5 (2005) 3–9.
[4] A. Jimeno, M. Hidalgo, Crit. Rev. Oncol. Hematol. 59 (2006) 150–158.
[5] W.O. Foye, T.L. Lemke, D.A. William, Principles of Medicinal Chemistry, fourth
ed (1995).
[6] N. Haga, T. Ishibashi, A. Hara, Y. Abiko, Pharmacology 31 (1985) 208–217.
[7] H. Sashida, Y. Abiko, Biochem. Pharmacol. 34 (1985) 3875–3880.
[8] T. Toyo-oka, T. Kamishiro, M. Masaki, T. Masaki, Jpn. Heart J. 23 (1982)
829–834.
[29] J.C. Medina, B. Shan, H. Beckmann, R.P. Farrell, D.L. Clark, R.M. Learned,
D. Roche, A. Li, V. Baichwal, C. Case, P.A. Baeuerle, T. Rosen, J.C. Jaen, Bioorg.
Med. Chem. Lett. 8 (1998) 2653–2656.
[30] B. Shan, J.C. Medina, E. Santha, W.P. Frankmoelle, T.C. Chou, R.M. Learned,
M.R. Narbut, D. Stott, P. Wu, J.C. Jaen, T. Rosen, P.B.M.W.M. Timmermans,
H. Beckmann, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 5686–5691.
[9] W.D. Wilson, H.J. Barton, F.A. Tanious, S.B. Kong, L. Strekowski, Biophys. Chem.
35 (1990) 227–243.
[10] C. Hulme, M.P. Cherrier, Tetrahedron Lett. 40 (1999) 5295–5299.
[11] M. Yoshida, Y. Maehara, K. Sugimachi, Clin. Cancer Res. 5 (1999) 4295–4300.
[12] C.C. Guo, H.P. Li, X.B. Zhang, Bioorg. Med. Chem. 11 (2003) 1745–1751.
[13] C.C. Guo, R.B. Tong, K.L. Li, Bioorg. Med. Chem. 12 (2004) 2469–2475.