Synthesis and cytotoxicity evaluation of some novel 1-(3-Chlorophenyl) piperazin-2-one derivatives bearing imidazole bioisosteres

Article abstract of DOI:10.1071/CH13031

A series of substituted 3-chlorophenylpiperazinone derivatives were synthesised using L-778123 (an imidazole-containing FTase inhibitor) as a model by bioisosteric replacement of the imidazole ring. The final compounds were evaluated against two human cancer cell lines including A549 (lung cancer) and HT-29 (colon cancer) by MTT assay. The results showed that substitution of imidazole ring with 1-amidinourea, semicarbazide, and thiobiuret led to improvement of cytotoxic activity against both cell lines.

Full text of DOI:10.1071/CH13031

CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH13031
Synthesis and Cytotoxicity Evaluation of Some Novel
1-(3-Chlorophenyl)piperazin-2-one Derivatives
Bearing Imidazole Bioisosteres
A
C D
,
A C D
, ,
Saeed Ghasemi, Simin Sharifi,
Soodabeh Davaran,
A
A C
,
A B E
, ,
Hosein Danafar, Davoud Asgari,
and Javid Shahbazi Mojarrad
A
Department of Medicinal Chemistry, Faculty of Pharmacy, Tabriz University of Medical
Sciences, Tabriz 51664, Iran.
B
Tuberculosis and Lung Disease Research Center, Tabriz University of Medical
Sciences, Tabriz 51664, Iran.
C
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical
Sciences, Tabriz 51664, Iran.
D
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University
of Medical Sciences, Tabriz 51664, Iran.
E
Corresponding author. Email: jvshahbazi@yahoo.com
A series of substituted 3-chlorophenylpiperazinone derivatives were synthesised using L-778123 (an imidazole-containing
FTase inhibitor) as a model by bioisosteric replacement of the imidazole ring. The final compounds were evaluated against
two human cancer cell lines including A549 (lung cancer) and HT-29 (colon cancer) by MTT assay. The results showed
that substitution of imidazole ring with 1-amidinourea, semicarbazide, and thiobiuret led to improvement of cytotoxic
activity against both cell lines.
Manuscript received: 19 January 2013.
Manuscript accepted: 22 February 2013.
Published online: 25 March 2013.
Introduction
advanced stages of human clinical trials for the treatment of
hematological cancers and solid tumours (Fig. 1).[3,8–10]
Previous studies showed that although the substitution of an
imidazole ring with other groups such as pyridine in FTIs can
improve cytotoxic activity, farnesyltransferase inhibitor activity
decreased. On the other hand, a survey of cancer cell lines has
shown that cell lines without mutant Ras proteins were also
sensitive to FTIs (more than 70 % of the cell lines).[3,4] These
results show that other mechanisms exist in addition to inhibi-
tion of the farnesyltransferase (FTase) enzyme.[11–14]
Cancer is an important cause of death in developed countries,
and the introduction of new anticancer agents is immediately
required due to problems with existing drugs, such as toxicities
and drug resistance.[1]
Research shows that the mutation of ras genes is responsible
for 30 % of human cancers.[2] The activation of signal transduc-
tion pathways by Ras proteins has a crucial role for cellular
growth. They undergo post-translational modifications by
several sequential enzymatic steps. Farnesyltransferase is a zinc
metalloenzyme, which adds a fanesyl group to the carboxyl
terminal of a group of membrane-bound small G-proteins, such
as Ras proteins. It has been shown that farnesyltransferase
inhibitors (FTIs) can stop Ras protein farnesylation and suppress
the growth of Ras-dependent tumour cells.[3,4] Therefore, the
search for FTIs for the treatment of cancer has recently generated
considerable interest. FTIs consist of three main classes; pepti-
domimetics, farnesyl pyrophosphate (FPP) analogues, and a
class that has features of the other two classes. The potent
peptidomimetic FTIs had a free thiol group. Free thiols are
associated with several adverse drug effects, therefore the
development of FTIs is clearly directed towards non-thiol FTIs.
The most frequently used replacements for cysteine are nitrogen-
containing heterocycles such as imidazole.[2,5–7] Several potent
FTIs such as tipifarnib (ZarnestraÒ), L-778123, BMS-214662,
and lonafarnib have been developed by researchers and were in
In addition, guanidine, semicarbazide, urea, and thiourea
derivatives showed potent anticancer activity with various
mechanisms.[15–19]
In the present study, we chose L-778123 as a model and
report the synthesis and evaluation of cytotoxic activity of 1-(3-
chlorophenyl) piperazin-2-one derivatives. Several classes of
bioisosteric replacement of imidazole such as 1-amidinourea,
thiobiuret, and semicarbazide were selected, synthesised and
evaluated against two human cancer cell lines including A549
(adenocarcinoma human alveolar basal epithelial) and HT-29
(human colonic adenocarcinoma) cell lines.
Results and Discussion
The intermediate 1-(3-chlorophenyl)piperazin-2-one hydro-
chloride (compound 2) was prepared according to known
methods described previously, as shown in Scheme 1.[20,21]
Journal compilation Ó CSIRO 2013
www.publish.csiro.au/journals/ajc

B
S. Ghasemi et al.
Cl
Cl
Cl
NH2
N
N
N
O
N
N
N
N
O
N
1
2
N
NH
Br
Cl
N
N
Br
O
N
O
NH2
N
N
N
S
S
O
O
3
4
Fig. 1. Chemical structures of farnesyl transferase inhibitors: Tipifarnib (1), L-778123 (2), BMS-214662 (3) and
Lonafarnib (4).
OH
8d and 8f) were also potent, with IC50 values in the range 7–
30mM. Cytotoxic activity of compound 7 didn’t change signifi-
cantly compared with L-778123. The compounds 8e and 8a
indicated good cytotoxic activity but lower than L-778123 as
the standard compound. The compound 8b showed cytotoxic
activity near 500mM. The intermediate 6 and compound 8c didn’t
reveal significant cytotoxic activity on either cell line (.1 mM).
The anticancer mechanism of the 1-(3-chlorophenyl)
piperazin-2-one derivatives are not clearly defined. Although
it is proposed that they act through inhibition of FTase and
consequently inhibit the Ras protein activation, most synthe-
sised compounds which showed poor cytotoxic activity were
potent FTIs and vice versa.[11–14,27]
There are various electron withdrawing groups such as
chloro- or nitrile- groups at the para position of the benzyl
moiety in the 1-(3-chlorophenyl)piperazin-2-one derivatives.
There are many farnesyltransferase inhibitors such as tipifarnib
which has a 4-Cl instead of a 4-CN group. These inhibitors
showed higher potency than L-778123. We guessed that substi-
tution of 4-Cl with 4-CN may improve potency. So we decided
to use 4-chlorobenzyl instead of 4-cyanobenzyl in our
derivatives.[3,4,28–31]
For cytotoxicity evaluation, lung and colon cancers were
chosen because they are the main types of cancer which cause
death worldwide, based on reports by the World Health Organi-
zation (WHO).[32,33] On the other hand, A549 (lung cancer) and
HT-29 (colon cancer) are very common cell lines in cytotoxic
activity evaluations. In addition, wild-type K-ras was found in
these cell lines.[34,35]
In this study, several novel 1-(3-chloropheny)piperazin-2-
one derivatives bearing 1-amidinourea, thiobiuret, semicarba-
zide, and biuret were synthesised, characterised and evaluated
against HT-29 and A549 cell lines to investigate structure-
cytotoxic activity relationships.
H.HCl
N
NH
O
NH2
c
N
O
a, b
HN
Cl
Cl
Cl
1
2
Scheme 1. (a) Chloroacetyl chloride, iPrAc, 08C to rt, 1 h. (b) Ethanol-
amine, iPrAc, 558C, 2 h. (c) Diisopropylazodicarboxylate (DIAD), tributyl-
phosphine, EtOAc, ꢀ108C to rt, 1 h.
The synthetic route to achieve the target compounds 8a–g is
shown in Scheme 2. The ethyl N-(4-chlorobenzyl)glycine ethyl
ester 3 was synthesised in 50 % yield by the reaction of
4-chlorobenzylamine with ethyl chloroacetate. The amine
group of intermediate 3 was protected using di-tert-butyl dicar-
bonate ((Boc)2O) to yield the ester 4.[22,23] The acid 5 was
obtained by hydrolysis of ester 4 under basic conditions.[22] The
intermediate 6 was prepared from reaction of intermediate 5 and
1-(3-chlorophenyl)piperazin-2-one in the presence of N,N0-
dicyclohexylcarbodiimide (DCC) followed by acidic deprotec-
tiontoaffordintermediate6. Carbamoylimidazole 7 was prepared
from intermediate 6 in the presence of carbonyldiimidazole
(CDI). The carbamoylimidazole salt was then prepared using
methyl iodide,[24–26] before addition of the appropriate amine in
dichloromethane or acetonitrile to furnish compounds 8a–g.[24]
The results of biological activity of the compounds are
summarised as IC50 values (in mM) in Table 1. The IC50 values
of the compounds against both cell lines showed that most
compounds had significant cytotoxic activity at concentrations
lower than 500 mM. The 1-amidinourea showed the highest
cytotoxic activity against both cell lines at concentrations less
than10 mM, whichisbetter thandoxorubicinagainst the A549cell
line. The 2-thiobiuret and semicarbazide derivatives (compounds
It seems that terminal nitrogens of substituents have a key role
in the cytotoxic activity. The cytotoxic activity of compound 7
showed that all changes (substitution of 4-chlorobenzyl instead of
4-cyanobenzyl) except bioisosteric replacement didn’t change the

Bioisosteric Replacement Effects on L-778123 Cytotoxicity
C
O
Boc
N
O
O
Boc
N
H
NH2
N
OEt
OEt
c
OH
a
b
Cl
Cl
Cl
Cl
N
3
4
5
N
O
O
O
H.HCl
N
N
O
O
N
N
N
Cl
N
Cl
f
d, e
Cl
Cl
7
6
X
O
O
O
N
N
X ϭ NH2, NHCH3, N(CH3)2, NH2NH, NHCONH2, NHCSNH2, NHCNHNH2
N
Cl
g
Cl
8a–g
Scheme 2. (a) Ethyl chloroacetate, sodium carbonate, benzene, reflux overnight. (b) (Boc)2O, CH2Cl2, rt. (c) NaOH, MeOH/H2O, rt, overnight.
(d) 1-(3-chlorophenyl)piperazin-2-one, DCC, CH2Cl2, 08C to rt. (e) TFA, MeOH, rt, 1 h. (f) CDI, Et3N, CH2Cl2, rt, 48 h. (g) MeI, MeCN, rt, 24 h;
then appropriate amine, CH2Cl2 or MeCN, Et3N, rt, 24 h.
Table 1. Cytotoxic activity (IC50, lM) of intermediates 6 and 7 and
The increase in cytotoxic activity may be due to the higher
electron density on terminal nitrogens.[36–38] Anticancer activity
of potent compounds (8f and 8g) may be attributed only to
cytotoxic activity of bioisosteres of imidazole which have cyto-
toxic activity by various mechanisms such as inosine monopho-
sphate dehydrogenase inhibition by guanidine-based
compounds[15–19,27] Other substitutions didn’t significantly
improve the cytotoxic activity in comparison with the mentioned
substituents, perhaps due to lower electron density of the amide
groups. Compound 8a indicated better cytotoxic activity than
compounds 8b and 8c, which may be a result of steric effects.
In future research, more studies should be done to determine
the mechanisms of the action of most potent compounds.
compounds 8a g against HT-29 (colon cancer) and A549 (lung cancer)
]
cell lines
O
O
N
Cl
X
N
N
Cl
O
Compounds
X
–
A549
HT-29
6
.1000
105 ꢁ 2
201 ꢁ 2
307 ꢁ 4
.1000
.1000
126 ꢁ 3
198 ꢁ 2
501 ꢁ 3
.1000
26 ꢁ 3
7
Imidazole
-NH2
8a
Conclusion
8b
-NHCH3
-N(CH3)2
-NHNH2
-NHCONH2
-NHCSNH2
8c
Inconclusion,a seriesofsubstituted 1-(3-chlorophenyl)piperazin-
2-one derivatives bearing bioisosteres of imidazole were syn-
8d
22 ꢁ 5.22
124 ꢁ 1
7.3 ꢁ 0.4
3 ꢁ 0.3
1
thesised, characterised by IR, HNMR, 13CNMR, and liquid
8e
171 ꢁ 2
11 ꢁ 0.6
7.1 ꢁ 0.2
125 ꢁ 2
3 ꢁ 0.1
8f
chromatography mass spectrometry (LC-MS), and evaluated as
cytotoxic agents. The 3-chlorophenylpiperazinone derivatives
with 1-amidinourea (8g) showed higher potency with IC50 equal
to 3 mM and 7 mM against A549 and HT-29 cell lines respec-
tively, which is better than doxorubicin against A549. Thus,
compound 8g is a good lead compound for designing new
anticancer agents. The thiobiuret and semicarbazide groups can
be potent bioisosteres instead of the imidazole ring. Methyl
substitution on amine groups decreased cytotoxic activity and
the intermediate 6 (without an imidazole group) didn’t show
8g
-NHC(NH)NH2
L-778123
Doxorubicin
–
–
101 ꢁ 2
4.1 ꢁ 0.1
cytotoxic activity significantly. Replacement of imidazole with
1-amidinourea, 2-thiobiuret, and semicarbazide (compounds 8d,
8f, and 8g) resulted in better cytotoxic activity against HT-29 and
A549 cell lines in comparison with the standard compound.

D
S. Ghasemi et al.
significant cytotoxic activity. It seems that electron density and
steric effects of terminal nitrogens of substituents are critical
factors in cytotoxic activity.
overnight. The MeOH was removed and the residual aqueous
suspension was diluted with H2O (200 mL) and washed with
Et2O (2 ꢂ 100 mL). To the residual solution (aqueous layer) was
added 1 N HCl (pH ,3) and extracted with EtOAc (3 ꢂ 50 mL).
The combined organic layers were washed with brine, dried over
anhydrous Na2SO4, and concentrated to give a colourless oil.
The title compound (4.57 g, 76%) was obtained as a colour-
less oil. nmax (KBr)/cmꢀ1: 3200, 1760, 1650; dH (500 MHz,
DMSO-d6) 7.38 (d, J 6.4, 2H, phenyl), 7.29 (d, J 6.4, 2H, phenyl),
4.39 (d, J 6.15, 2H, NCH2phenyl), 3.83 (s, 2H, COCH2N), 1.36
(s, 9H, CH3); dC (125MHz, DMSO-d6) 172.89, 154.45, 134.35,
130.51, 128.83, 128.34, 78.12, 50.58, 45.34, 27.76; m/z (ESI)
300.66 [MþH]; Anal. Calcd. for C14H18ClNO4: C 56.10, H 6.05,
N 4.67; found: C 56.36, H 6.22, N 4.33%.
Experimental
Chemistry
All reagents and solvents were purchased from Merck and
Sigma Aldrich. L-778123 was prepared according to known
methods.[20,21] Melting points were measured with an Electro-
thermal-9100 melting point apparatus and are uncorrected. The
IR spectra were recorded on a Shimadzu 4300 spectrophoto-
meter (potassium bromide disks). 1HNMR and 13CNMR spectra
were recorded on Varian unity 500, 400, and 300 spectrometers
and chemical shifts (d) are reported in parts per million (ppm)
using tetramethylsilane (TMS) as an internal standard. The mass
spectra were run on an Agilent 6410 LC-MS at 70 eV. Merck
silica gel 60 F254 plates were used for analytical TLC. Column
chromatography was performed on silica gel 60 (Merck, particle
size 0.06–0.20 mm).
4-[N-(4-Chlorobenzyl)glycyl]-1-(3-chlorophenyl)piperazin-
2-one (6)
A solution of 5 (5 g, 16.72 mmol) in 60 mL of CH2Cl2 was
added to a solution of DCC (4.6 g, 22.29 mmol) in 60 mL of
CH2Cl2. The mixture was stirred at room temperature for 10 min
N-(4-Chlorobenzyl) Glycine Ethyl Ester Hydrochloride (3)
and then
a
solution of 1-(3-chlorophenyl)piperazin-2-
one (2.35 g, 11.14 mmol) in 30 mL of CH2Cl2 was added. The
mixture was stirred at room temperature overnight. The N,N0-
dicyclohexylurea (DCU) was filtered off and the filter cake was
washed with CH2Cl2 (2 ꢂ 20 mL). The organic filtrate was
washed with 15 % aqueous citric acid solution (3 ꢂ 20 mL). The
aqueous layer was basified with 20 mL concentrated sodium
bicarbonate, and then extracted with CH2Cl2 three times. The
combined organic layers were washed with water (20 mL) and
brine (20 mL) and dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure to obtain the desired
amides. To a stirred solution of the resulting product (4.92 g,
10 mmol) in 15 mL MeOH was added trifluoroacetic acid (TFA,
15 mL, 0.19 mol) and the resulting mixture was stirred at room
temperature for 1 h. Excess reagent and solvent were evaporated
under reduced pressure. To the resulting oil was added 50 mL
EtOAc, followed by stirring for 30 min. The white precipitate
was filtered, washed with EtOAc, and dried to give 6.
The title compound (5.43 g, 50 %), was obtained as a white
solid. Mp 220–2228C; nmax (KBr)/cmꢀ1: 3325, 1675, 1580; dH
(400 MHz, DMSO-d6) 7.60–7.32 (m, 8H, Phenyl), 4.22 (s, 2H,
C-3 of piperazinone), 4.14 (s, 2H, COCH2NH), 4.12 (s, 2H,
NHCH2phenyl), 3.73 (t, J 3.8, 2H, C-5 of piperazinone), 3.47
(dt, J 3.8, 2H, C-6 of piperazinone); dC (100 MHz, DMSO-d6)
165.35, 165.18, 142.07, 135.63, 135.13, 130.41, 129.19, 129.01,
127.44, 125.67, 118.17, 117.47, 49.17, 45.94, 45.12, 44.70,
42.30; m/z (ESI) 393.15 [MþH]; Anal. Calcd. for
C19H20Cl3N3O2: C 53.23, H 4.70, N 9.80; found: C 53.38, H
4.92, N 9.61 %.
A mixture of 4-chlorobenzylamine (10 mL, 82 mmol), ethyl
chloroacetate (9.21 mL, 90 mmol), and anhydrous sodium car-
bonate (13 g, 123mmol) in 50 mL of benzene was refluxed
overnight. After cooling, the salts wereremoved byfiltration. The
solvent was evaporated under reduced pressure and diethyl ether
was added to the residue and stirred for 1 h at room temperature.
The mixture was filtered and hydrochloric acid (4.0 N in iso-
propanol) was added to the filtrate. The white precipitate was
filtered, washed three times with diethyl ether, and then dried.
The title compound (10.35 g, 55 %) was obtained as a white
solid. Mp 140–1428C; nmax (KBr)/cmꢀ1: 3325, 1700; dH
(500 MHz, DMSO-d6)7.49 (t, J 1.5, 2H, phenyl), 7.47 (t, J 1.5,
2H, phenyl), 4.18 (s, 2H, NHþ2 CH2phenyl), 4.11 (s, 2H,
COCH2NHþ2 ), 4.07 (q, J 7.1, 2H, OCH2), 1.16 (t, J 7.1, 3H,
CH3); dC (125 MHz, DMSO-d6)169.06, 136.18, 130.25, 128.76,
126.21, 61.34, 52.73, 50.37, 13.41; m/z (ESI) 228.59 [MþH];
Anal. Calcd. for C11H15Cl2NO2: C 50.02, H 5.72, N 5.30; found:
C 50.18, H 5.52, N 5.41 %.
N-(tert-Butoxycarbonyl)-N-(4-chlorobenzyl) Glycine
Ethyl Ester (4)
A solution of 3 as free base (5 g, 20 mmol) and (Boc)2O (4.36 g,
20 mmol) in 40 mL of CH2Cl2 was stirred for 4 h at room tem-
perature. Then 100 mL of ethyl acetate was added to the reaction
mixture and the solution was transferred to a separating funnel.
The resulting solution was washed sequentially with 50 mL of
1NHCl,50mLsaturatedaqueoussodiumbicarbonate, and30mL
brine, andthendriedwithanhydroussodiumsulfate, filtered, and
the solvent was removed under reduced pressure to give 4.
The title compound (5.75 g, 80 %) was obtained as a colour-
less oil. nmax (KBr)/cmꢀ1: 1700, 1660; dH (500 MHz, DMSO-d6)
7.49 (d, J 8.5, 2H, phenyl), 7.34 (d, J 8.5, 2H, phenyl), 4.12 (s,
2H, NCH2phenyl), 3.80 (s, 2H, COCH2N), 3.73 (br d, J 4.5 Hz,
2H, OCH2), 1.61 (t, J 5.1, 3H, -CH2CH3), 1.47 (s, 9H, CH3); dC
(125 MHz, DMSO-d6) 167.15, 157.21, 134.20, 130.55, 128.70,
127.91, 77.87, 63.13, 51.94, 45.51, 28.15, 14.71; m/z (ESI)
329.61 [MþH]; Anal. Calcd. for C16H22ClNO4: C 58.62, H
6.76, N 4.27; found: C 58.80, H 6.62, N 4.12 %.
4-[N-(4-Chlorobenzyl)-N-(1H-imidazol-1-ylcarbonyl)
glycyl]-1-(3-chlorophenyl)piperazin-2-one (7)
Intermediate 6 (4.28 g, 10 mmol) and triethylamine (1.4 mL,
10 mmol) was added to a solution of CDI (1.78 g, 11 mmol) in
30 mL CH2Cl2. The mixture was stirred for 48 h at room tem-
perature and then washed with water (2 ꢂ 30 mL). The organic
layer was dried over anhydrous Na2SO4, filtered and the solvent
was evaporated under vacuum to yield white solid.
The title compound (2.42 g, 50 %) was obtained as a white
solid. Mp 220–2228C; nmax (KBr)/cmꢀ1: 1690, 1580; dH
(300 MHz, CDCl3) 7.96 (s, 1H, imidazole), 7.77 (s, 1H, imidaz-
ole), 7.38–7.13 (m, 8H, phenyl), 7.09 (s, 1H, imidazole), 4.46 (s,
2H, NCH2phenyl), 4.42 (s, 2H, COCH2N), 4.22 (s, 2H, C-3 of
N-(4-Chlorobenzyl)-N-(tert-butoxycarbonyl) Glycine (5)
A solution of 4 (5 g, 15.23 mmol) and NaOH (1.8 g, 45 mmol) in
MeOH/H2O (50 mL, 50 : 50) was stirred at room temperature

Bioisosteric Replacement Effects on L-778123 Cytotoxicity
E
piperazinone), 3.98 (t, J 5.1, 2H, C-6 of piperazinone), 3.86 (t, J
5.4, 2H, C-5 of piperazinone); dC (75 MHz, CDCl3) 165.05,
164.55, 157.12, 150.42, 141.98, 137, 134.89, 134.38, 130.41,
130.34, 127.8, 125.9, 123.63, 120.89, 117.72, 50.44, 49.03,
48.67, 43.44, 35.86; m/z (ESI) 487.35 [MþH]; Anal. Calcd.
for C23H21Cl2N5O3: C 56.80, H 4.35, N 14.40; found: C 56.48,
H 4.62, N 14.13 %.
4-(4-Chlorobenzyl)-4-[2-[1-(3-chlorophenyl)-2-
oxopiperazin-4-yl]-2-oxoethyl] Semicarbazide (8d)
The title compound (0.2 g) was obtained in 45 % yield. Mp
201–2038C; nmax (KBr)/cmꢀ1: 3310, 3250, 1650, 1625, 1560; dH
(400 MHz, CDCl3) 7.37–7.18 (m, 8H, phenyl), 4.17 (s, 2H,
NCH2phenyl), 4.08 (s, 2H, COCH2N), 4.06 (s, 2H, C-3 of
piperazinone), 3.83 (t, J 5.6, 2H, C-6 of piperazinone), 3.77 (t,
J 5.4, 2H, C-5 of piperazinone); dC (100 MHz, CDCl3) 164.16,
163.97, 153.29, 145.49, 133.82, 131.71, 130.29, 128.55, 128.02,
126.07, 123.2, 120.76, 118.83, 53.17, 48.01, 45.17, 43.90, 42.01;
m/z (ESI): 451.29 [MþH]; Anal. Calcd. for C20H21Cl2N5O3:
C53.34, H 4.70, N 15.55; found: C 53.52, H 4.91, N 15.28 %.
General Procedure for the Synthesis of Compounds 8a–g
Methyl iodide (1.54 mL, 24.7 mmol) was added to a solution of
intermediate 7 (3 g, 6.17 mmol) in 30 mL acetonitrile and stirred
at 258C overnight. The solvent was removed under reduced
pressure to give the carbamoylimidazolium salt.
To a solution of the above carbamoylimidazolium salt (0.5 g,
0.8 mmol) in 10 mL CH2Cl2 or 10 mL acetonitrile was added to
the appropriate amine (8 mmol) and triethylamine (0.11 mL,
0.8 mmol). The mixture was stirred at room temperature for 24 h
and then washed with water (2 ꢂ 5 mL) and brine (5 mL). The
organic layer was dried over anhydrous Na2SO4, filtered,
concentrated under reduced pressure, and purified by column
chromatography on silica gel eluting with n-hexane/ethyl ace-
tate to yield compounds 8a–g.
1-(4-Chlorobenzyl)-1-[2-[1-(3-chlorophenyl)-
2-oxopiperazin-4-yl]-2-oxoethyl]-biuret (8e)
The title compound (0.31 g) was obtained in 64 % yield. Mp
246–2488C; nmax (KBr)/cmꢀ1: 3300, 3200, 1740, 1680; dH
(300 MHz, CDCl3) 7.32–7.19 (m, 8H, phenyl), 4.25 (s, 2H,
NCH2phenyl), 4.16 (s, 2H, COCH2N), 4.06 (s, 2H, C-3 of
piperazinone), 3.79 (br d, J 19.8, 2H, C-6 of piperazinone),
3.71 (br d, J 19, 2H, C-5 of piperazinone); dC (75 MHz, CDCl3)
164.86, 161.47, 152.72, 150, 142.49, 134.82, 130.29, 127.55,
126.02, 123.76, 123.63, 120.89, 118.53, 117.72, 53.17, 51.96,
47.07, 42.93, 37.07; m/z (ESI) 479.29 [MþH]; Anal. Calcd. for
C21H21Cl2N5O4: C 52.73, H 4.43, N 14.64; found: C 52.41,
H 4.71, N 14.38 %.
1-(4-Chlorobenzyl)-1-[2-[1-(3-chlorophenyl)-2-
oxopiperazin-4-yl]-2-oxoethyl] Urea (8a)
The title compound (0.26 g, 58 %) was obtained as a white
solid. Mp 215–2178C; nmax (KBr)/cmꢀ1: 3300, 3250, 1740, 1680;
dH (500 MHz, DMSO-d6) 7.40–7.22 (m, 8H, phenyl), 4.41 (s, 2H,
NCH2phenyl), 4.27 (s, 2H, COCH2N), 4.22 (s, 2H, C-3 of
piperazinone), 4.09 (t, J 6.7, 2H, C-6 of piperazinone), 4.05 (t, J
6.8, 2H, C-5 of piperazinone); dC (125 MHz, DMSO-d6) 170.55,
169.5, 154.83, 146.20, 137.67, 137.09, 131.67, 130.73, 130.41,
129.52, 128.17, 118.55, 117.50, 53.85, 50.86, 50.26, 48.97, 48.78;
m/z (ESI) 436.49 [MþH]; Anal. Calcd. for C20H20Cl2N4O3:
C 55.18, H 4.63, N 12.87; found: C 55.42, H 4.82, N 12.58 %.
5-(4-Chlorobenzyl)-5-[2-[1-(3-chlorophenyl)-2-
oxopiperazin-4-yl]-2-oxoethyl]-2-thiobiuret (8f)
The title compound (0.32 g) was obtained in 63 % yield. Mp.
265–2678C; nmax (KBr)/cmꢀ1: 3425, 3325, 1740, 1640; dH
(300 MHz, CDCl3) 7.42–7.08 (m, 8H, phenyl), 4.49 (s, 2H,
NCH2phenyl), 4.26 (s, 2H, COCH2N), 4.17 (s, 2H, C-3 of
piperazinone), 3.91 (t, J 19.8, 2H, C-6 of piperazinone), 3.71
(t, J 19, 2H, C-5 of piperazinone); dC (75 MHz, CDCl3) 187.91,
165.8, 164.47, 150, 142.49, 138.48, 135.84, 134.88, 133.84,
131.26, 130.02, 128.76, 118.8, 117.72, 50.47, 49.05, 48.08,
47.30, 36.09; m/z (ESI) 495.39 [MþH]; Anal. Calcd. for
C21H21Cl2N5O3S: C 51.02, H 4.28, N 14.17; found C 51.42,
H 4.52, N 14.51 %.
1-(4-Chlorobenzyl)-1-[2-[1-(3-chlorophenyl)-2-
oxopiperazin-4-yl]-2-oxoethyl]-3-methyl Urea (8b)
The title compound (0.3 g) was obtained in 65 % yield. Mp
185–1878C; nmax (KBr)/cmꢀ1: 3350, 1700, 1660; dH (400 MHz,
CDCl3) 7.39–7.09 (m, 8H, phenyl), 4.44 (s, 2H, NCH2phenyl),
4.28 (s, 2H, COCH2N), 4.16 (s, 2H, C-3 of piperazinone), 4.01
(t, J 4.9, 2H, C-6 of piperazinone), 3.88 (t, J 5.6, 2H, C-5 of
piperazinone), 2.75 (s, 3H, CH3); dC (100 MHz, CDCl3) 165.41,
165, 154.52, 143.29, 138.41, 137.52, 132.83, 130.37, 126.37,
125.85, 124.28, 118.4, 117.50, 52.65, 48.55, 47.60, 43.89,
41.10, 40.73; m/z (ESI) 450.29 [MþH]; Anal. Calcd. for
C21H22Cl2N4O3: C 56.13, H 4.94, N 12.47; found: C 56.42,
H 4.71, N 12.81 %.
1-Amidino-3-(4-chlorobenzyl)-3-[2-[1-
(3-chlorophenyl)-2-oxopiperazin-4-yl]-2-oxoethyl]
Urea (8g)
The title compound (0.15 g) was obtained in 30 % yield. Mp
288–2908C; nmax (KBr)/cmꢀ1: 3400, 3320, 1700, 1650; dH
(400 MHz, CDCl3) 7.31–7.13 (m, 8H, phenyl), 4.10 (s, 2H,
NCH2phenyl), 4.04 (s, 2H, COCH2N), 4.02 (s, 2H, C-3 of
piperazinone), 3.74 (t, J 5.6, 2H, C-6 of piperazinone), 3.65 (t,
J 5.4, 2H, C-5 of piperazinone); dC (100 MHz, CDCl3) 166.67,
164.31, 159.14, 154.88, 139.66, 137.64, 136.51, 132.79, 130.23,
128.87, 128.70, 120.52, 118.50, 117.98, 52.80, 48.35, 47.54,
43.44, 41.28; m/z (ESI): 478.29 [MþH]; Anal. Calcd. for
C21H22Cl2N6O3: C52.84, H 4.65, N 14.85; found: C 53.12,
H 4.93, N 14.52 %.
1-(4-Chlorobenzyl)-1-[2-[1-(3-chlorophenyl)-2-
oxopiperazin-4-yl]-2-oxoethyl]-3,3-dimethyl Urea (8c)
The title compound (0.28 g) was obtained in 60 % yield.
Mp 218–2208C; nmax (KBr)/cmꢀ1: 1660, 1640; dH (300 MHz,
CDCl3) 7.37–7.19 (m, 8H, phenyl), 4.50 (s, 2H, NCH2phenyl),
4.38 (s, 2H, COCH2N), 4.09 (s, 2H, C-3 of piperazinone), 3.82
(t, J 12.6, 2H, C-6 of piperazinone), 3.62 (t, J 11.7, 2H, C-5
of piperazinone), 2.89 (s, 6H, CH3, dimethyl); dC (75 MHz,
CDCl3) 165.71, 163.38, 157.22, 143.77, 136.72, 130.20, 128.35,
128.05, 122.81, 122.11, 121.91, 118.91, 117.01, 51.93, 46.9,
43.28, 42.98, 39.99, 34.29; m/z (ESI) 464.39 [MþH]; Anal.
Calcd. for C22H24Cl2N4O3: C57.03, H 5.22, N 12.09; Found:
C 57.41, H 5.46, N 12.30 %.
Growth Inhibition Assay
Cytotoxicity of compounds 6, 7, 8a–g, L-778123, and doxoru-
bicin was examined by MTT assay at 1–1000 mM concentrations
against two human cancer cell lines A549 (adenocarcinoma
human alveolar basal epithelial cells) and HT-29 (human
colonic adenocarcinoma cells). Cell suspensions were seeded in

F
S. Ghasemi et al.
96-well plates with concentrations of 8000–10000 A549 cells
and 15000–20000 HT-29 cells per well and incubated for 24 h to
allow cell attachment. The cells were treated for 72 h with
various concentrations of 6, 7, 8a–g, L-778123, and doxorubi-
cin. Culture medium was replaced with 150 mL fresh media plus
50 mL MTT (3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyl tetra-
zolium bromide) reagent (2 mg mLꢀ1 in PBS). After an addi-
tional 4 h of incubation at 378C the medium was discarded.
200 mL Dimethyl sulfoxide plus 25 mL Sorenson buffer (0.1 M
NaCl, 0.1 M glycine, pH: 10.5) was added to each well and the
solution was shaken for 15 min at 378C to dissolve the purple
tetrazolium crystals. The absorbance of each well was measured
by the plate reader (SUNRISE TECAN, Austria) at a wave-
length of 570 nm. The amount of produced purple formazan is
proportional to the number of viable cells. Experiments were
performed two times in triplicate for determination of sensitivity
to each compound. The IC50 was calculated by linear regression
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