Synthesis, characterization and biological evaluation of novel 2,4,6-trisubstituted bis-pyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.05.055

A new series of 2,4,6-trisubstituted bis-pyrimidines were synthesized and evaluated for in vitro antiamoebic activity against HM1:IMSS strain of Entamoeba histolytica. Out of 16 compounds 8 compounds have shown IC₅₀ values in the range of 0.10-

Full text of DOI:10.1016/j.ejmech.2011.05.055

European Journal of Medicinal Chemistry 46 (2011) 4669e4675
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, characterization and biological evaluation of novel
2
,4,6-trisubstituted bis-pyrimidine derivatives
a
,
d
,
, Faisal Hayat , Sayeed Mukhtar d, Attar Salahuddin a,
a
b,
*
Humaira Parveen
c
c
a
Andleeb Khan , Fakhrul Islam , Amir Azam
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Chemical Research Department, R&D I, Ranbaxy Laboratories Limited, Gurgaon, Haryana 122001, India
Department of Medical Elementology and Toxicology, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
Department of Chemistry, Faculty of Science, University of Tabuk, Tabuk, Saudi Arabia
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 January 2011
Received in revised form
A new series of 2,4,6-trisubstituted bis-pyrimidines were synthesized and evaluated for in vitro anti-
amoebic activity against HM1:IMSS strain of Entamoeba histolytica. Out of 16 compounds 8 compounds
have shown IC50 values in the range of 0.10e1.86
mM. Bis-pyrimidine having methyl-, methoxy-, thio-
20 May 2011
methyl- and dimehyl-phenyl substituents, exhibited higher antiamoebic activity than the reference drug
Accepted 23 May 2011
Available online 30 May 2011
metronidazole (IC50 ¼ 1.9
m
M). The toxicological studies of active compounds on PC12-rat pheochor-
0
omocytoma cell line showed that all compounds were non-toxic at a concentration of 100
m
M. 4-4 -
Benzene-1,3-diylbis[6-(4-methylphenyl-2-(piperidin-1-yl)pyrimidine] (4c) was found most active
IC50 ¼ 0.10 M) and least toxic among all the compounds.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Bis-chalcones
Bis-pyrimidines
(
m
Antiamoebic activity
Entamoeba histolytica MTT assay
1
. Introduction
spectrum of biological activities [6e25]. In view of these observations
and as a part of our ongoing program devoted to the synthesis of
diverse heterocycles as antiamoebic agents [26,27], we have
synthesized a new series of organic molecules containing two
pyrimidine rings and have evaluated the same against HM1:IMSS
strain of E. histolytica, a protozoan responsible for amoebiasis. On the
basis of their activity and favourable therapeutic indexes, these
compounds were identified as viable leads for further studies.
Amoebiasis is the most aggressive protozoal disease and consid-
ered to be the second or third leading cause of death amongst the
parasitic diseases [1]. Entamoeba histolytica, a protozoan parasite, is
the causative agent of amoebiasis and amoebic dysentery. Though
ubiquitous in distribution, this parasite is more prevalent in tropical
and subtropical regions [2]. Metronidazole is known to be highly
effective amoebicide and is considered to be the drug of choice for the
treatment of amoebiasis, but this drug has been shown to be muta-
genic in a microbiological system and carcinogenic to rodents [3e5].
Repeated treatment of E. histolytica infection with commonly used
antiamoebic drugs results in not only increasing the toxicity potential
but also leads to the development of clinical resistance. Therefore,
new effective agents with less toxicity against amoebiasis are
urgently required. Five membered heterocyclic compounds natural
as well as synthetic are important for their biological activities.
Compounds with pyrimidine ring are of interest due to their broad
2. Chemistry
To synthesize the 2,4,6-trisubstituted bis-pyrimidine derivatives
(3ae3h and 4ae4h), teraphthaldicarboxaldehyde was reacted with
different substituted aromatic acetophenone (aeh) in NaOH and
methanol to yield the corresponding bis-chalcones 2ae2h [28]
Scheme 1. Pyrrolidine-1-carboxamidine hydrochloride and piperi-
dine-1-carboxamidine hydrochloride were synthesized by reflux-
ing pyrrolidine and piperidine, respectively, with S-methyl
isothiourea sulphate in water according to reported procedure [29].
The bis-chalcones 2ae2h were further cyclized with imidine
hydrochlorides in the presence of sodium isopropoxide (synthe-
sized in situ by adding sodium metal in isopropanol) to afford 2,4,6-
trisubstituted bis-pyrimidines 3ae3h and 4ae4h as shown in
Scheme 1. All the synthesized compounds were well characterized
Abbreviation:
mM, Micromole.
*
Corresponding author. Department of Chemistry, Jamia Millia Islamia, Jamia
Nagar, New Delhi 110025, India. Tel.: þ91 11 2698 1717/3253; fax: þ91 11 2698
0229/1232.
E-mail address: humaira_chem@yahoo.co.in (H. Parveen).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.055

4670
H. Parveen et al. / European Journal of Medicinal Chemistry 46 (2011) 4669e4675
R
CH3
O
H
O
O
H
H
N
( )n
n = 0, 1
2
+
a-h
NH
(b)
. H SO
(a)
H CS
2
4
3
2
NH2
R
R
H N
2
HCl .
N
(
)n
O
O
2a-2h
HN
n = 0, 1
(c)
R
R
N
N
N
N
Where R = Groups of different aromatic
ketones
N
N
a = H, b = 4-Cl, c = 4-Me, d = 3,4-diMe,
e = 4-S-Me, f = 4-OMe, g = 3,4-diOMe,
h = 3,4,5-triOMe
(
)n
( )n
(
3a-3h), n = 0
4a-4h), n = 1
(
ꢁ
Scheme 1. General synthesis of 2,4,6-trisubstituted bis-pyrimidines (3a-3h) and (4a-4h). Reagents and conditions : (a) Different acetophenones (a-h), 10% aq NaOH, methanol, 0 C-
rt, 30 min. (b) (i) Piperidine or pyrrolidine, S- methylisothiourea sulphate, water, reflux, 15 min (ii) Barium chloride, reflux, 15 min. (c) Pyrrolidine-1-carboxamidine hydrochloride
(
for 3a-3h) or piperidine-1-carboxamidine hydrochloride (for 4a-4h), sodium isopropoxide, isopropanol, reflux, 8h.
by spectroscopic methods such as IR, NMR, Mass and elemental
analysis.
suggested the condensation of substituted acetophenones with ter-
1
ephthaldicarboxaldehyde. In H NMR spectra, the proton of
unsaturated carbonyl compounds showed two doublets in the
range of 7.51e7.69 ppm for H- and 7.77e7.88 ppm for H- with
a,
b
3
. Pharmacology
d
a
d
b
coupling constant in the range of 15.6e16.2 Hz, it is inferred that
they are trans isomers. The rest of signals for aromatic protons have
appeared in the expected regions. The structures of all these
compounds (2ae2h) were further confirmed by C NMR spectra. A
characteristic signal for the chalcone (C]O) appeared in the range of
All newly synthesized bis-pyrimidine derivatives (3ae3h) and
4ae4h) were screened in vitro against HM1:IMSS strain of
(
13
E. histolytica by microdilution method [30]. All the experiments
were carried out in triplicate at each concentration level and
repeated thrice. Cytotoxicity of active compounds has been studied
by MTT assay on PC12-rat pheochoromocytoma cell line [34]. The
results of biological activity are summarized in Tables 1 and 2.
d
d
189.85e190.83 ppm. The signals at
143.41e145.66 ppm revealed the presence of a, b
d
121.56e122.87 ppm and
-unsaturated keto
function in bis-chalcones (2ae2h).
Assignment of selected characteristic IR bands provides signifi-
cant indications for the formation of the bis-pyrimidine (3ae3h and
4
. Results and discussion
4
ae4h). All the compounds showed sharp bands in the region
ꢀ
1
4.1. Synthesis
1533e1598 cm due to C]N strech which confirmed the formation
ring closure. In addition, the absorption bands at 1233e1295 cm
ꢀ1
The synthesis of 2,4,6-trisubstituted bis-pyrimidine derivatives
3ae3h and 4ae4h) was performed in a manner as outlined in
were attributed to CeN stretch vibrations, which also confirm the
formation of desired pyrimidine ring in all the compounds 3ae3h
and 4ae4h. The structure of the compounds was further confirmed
(
Scheme 1. In the IR spectra of bis-chalcones (2ae2h) the appearance
ꢀ
1
ꢀ1
1
13
of characteristic bands at 1650e1661 cm and 1572e1589 cm
due to unsaturated carbonyl group and C]C, respectively,
by
H
NMR and
C NMR. The appearance of singlet at
a,
b
d
7.35e7.41 ppm showed the CeH proton of desire pyrimidine ring in

H. Parveen et al. / European Journal of Medicinal Chemistry 46 (2011) 4669e4675
4671
Table 1
,4,6-Trisubstituted Bis-Pyrimidines (3ae3h), their antiamoebic activity against
HM1:IMSS strain of Entamoeba histolytica and toxicity profile.
compounds. The signals due to the phenyl ring, pyrimidine and
piperidine ring resonate at their usual position, and the values are
given in the experimental section.
2
R
R
4.2. Antiamoebic activities
Preliminary experiments were carried out to determine the
N
N
N
N
in vitro antiamoebic activity of all the compounds 3ae3h and 4ae4h
by microdilution method using HM1:IMSS strain of E. histolytica. The
results are summarized in Tables 1 and 2. The data are present in
terms of percent growth inhibition relative to untreated controls, and
plotted as probit values as a function of drug concentration. The
antiamoebic effect was compared with the most widely used anti-
amoebic medication metronidazole which had a 50% inhibitory
N
N
3a-3h
S. No.
R
Antiamoebic activity
Toxicity Profile
concentration (IC50) of 1.9
that in bis-pyrimidine derivatives, when the R group was a phenyl
ring the pyrrolidine substituted compound (3a) showed IC50 4.76 M.
Substitution of the phenyl ring with chloro group (3b) dimethoxy
group (3g) and trimethoxy group (3h) also did not affect the activity.
Substitution with thiomethyl group (3e), monomethoxy group (3f),
mono- (3c) and dimethyl group (3d), increased the activity. Analmost
similar trend was also observed in piperidine substituted
compounds. The phenyl ring substituted compound showed IC50
mM in our experiments. The results showed
IC50
(
m
M)
S.D.a (ꢂ)
IC50
(
m
M)
Safety Index
3
3
3
3
3
3
3
3
a
b
c
d
e
f
H
4-Cl
4-Me
3,4-DiMe
4-SMe
4-OMe
2,5-DiOMe
4.76
9.57
0.16
0.89
1.54
0.88
5.81
2.12
0.012
0.028
0.013
0.012
0.009
0.011
0.031
0.006
N.D.b
N.D.
>100
>100
>100
>100
N.D.
N.D.
N.D.
m
>625
>112.36
>64.94
>113.64
N.D.
g
h
3,4,5-TriOMe
N.D.
N.D.
a
Standard Deviation. The compounds with bold font IC50 values are more active
2
.86
and trimethoxy group (4h) had no effect on the activity. Monomethyl
4c) dimethyl (4d), thiomethyl group (4e) and monomethoxy (4f)
mM. Substitution with chloro group (4b), dimethoxy group (4g)
than metronidazole.
b
Not detected.
(
showed increased activity. In general the activity profiles in both the
pyrrolidine and piperidine substituted compounds are almost
similar. Therefore out of sixteen compounds screened in vitro for
antiamoebic activity, 8 compounds (3ce3f) and (4ce4f) were found
more active than the reference drug metronidazole.
all the compounds 3ae3h and 4ae4h. Pyrrolidine and piperidine
functionality appeared in the H NMR spectra as multiplets at
1
d
1.52e3.86 ppm for the compounds 3ae3h and 4ae4h. Further
evidence for the formation of pyrimidines analogues was obtained
13
from
161.8e162.8 ppm was attributed to C]N and C]C respectively in
all the compounds 3ae3h and 4ae4h. The characteristic signal for
CeH of pyrimidine ring found in the range of 102.1e105.7 ppm
C
NMR spectra.
A
signal at
d
164.4e166.5 and
d
4.3. Toxicity profile
d
clearly favoured the formation of pyrimidine nucleus in all the
Compounds 3ce3f and 4ce4f was tested to find the toxic effects
on PC12-rat pheochoromocytoma cell line. No one inhibited cell
growth at a concentration of 100 mM. To investigate the selectivity
Table 2
of the compounds, the “safety index” (SI) was calculated and
defined as the toxicity IC50/protozoal IC50, where toxicity IC50 is
defined as the concentration of compound that kills 50% of the
PC12-rat pheochoromocytoma cell line and protozoal IC50 is the
concentration that kills 50% of amoeba protozoa. This allows to
estimate which compound might be efficacious or toxic against
human cells and potentially in vivo. The numerical results for each
compound are given in Tables 1e2.
2,4,6-Trisubstituted Bis-pyrimidines (4ae4h), their antiamoebic activity against
HM1:IMSS strain of Entamoeba histolytica and toxicity profile.
R
R
N
N
N
N
5. Conclusion
N
N
The sixteen 2,4,6-trisubstituted bis-pyrimidines (3ae3h and
ae4h) were synthesized by the cyclization of bis-chalcones (2ae2h)
4
4
a-4h
with pyrrolidine-1-carboxamidine hydrochloride and piperidine-1-
carboxamidine hydrochloride respectively. The in vitro antiamoebic
activity was examined using HM1:IMSS strain of E. histolytica and
results showed that out of the 16 compounds 8 compounds having
monomethoxy substitution, thiomethyl group and mono and
dimethyl group, exhibited higher antiamoebic activity than the
S. No.
R
Antiamoebic activity Toxicity Profile
IC50
(m
M) S.D.a (ꢂ) IC50
(
m
M) Safety Index (SI)
4
4
4
4
4
4
4
4
a
b
c
d
e
f
H
4-Cl
4-Me
3,4-DiMe
4-SMe
4-OMe
2,5-DiOMe
3,4,5-TriOMe
Metronidazole
2.86
5.33
0.10
0.53
1.86
0.52
7.39
2.41
9
0.009
0.004
0.014
0.005
0.006
0.010
0.008
0.005
0.020
N.D.b
N.D.
>100
>100
>100
>100
N.D.
N.D.
N.D.
>1000
>188.68
>53.76
>192.31
N.D.
reference drug metronidazole (IC50 ¼ 1.9
mM). The MTT assay
revealed that all the compounds are non-toxic to PC12-rat pheo-
0
choromocytoma cells. 4-4 -Benzene-1,3-diylbis[6-(4-methylphenyl-
g
h
2-(piperidin-1-yl)pyrimidine] (4c) was found most active and least
N.D.
N.D.
>52.63
>100
toxic among all the compounds. The present study suggested that the
newly synthesized 2,4,6-trisubstituted bis-pyrimidines are new leads
in antiamoebic chemotherapy. These molecules can be very useful for
further optimization work in amoebicidal chemotherapy, in order to
a
Standard Deviation. The compounds with bold font IC50 values are more active
than metronidazole.
b
Not detected.

4672
H. Parveen et al. / European Journal of Medicinal Chemistry 46 (2011) 4669e4675
3 3 3
4H, AreH), 2.47 (s, 3H, CH ), 2.34 (s, 3H, CH )
); 13C NMR (CDCl
discover and develop better and yet safer therapeutic agents for
amoebiasis.
d
(ppm): 189.66 (C]O), 144.76 (C-
b
), (138.77, 136.67, 133.42, 127.76,
þ
3
Aromatic), 121.67 (C-a), 21.50 (CH ); ESI-MS m/z: [M 1] 367.1.
6
. Experimental protocol
0
0
6
.1.4. (2E,2 E)-3,3 -benzene-1,4-diylbis[1-(3,4-dimethylphenyl)prop-
2-en-1-one] (2d)
Yield 82%; solid (Ethanol); mp: 200 C; Anal. calc. for C28
All the chemicals were purchased from Aldrich Chemical
ꢁ
Company (USA). Precoated aluminium sheets (silica gel 60 F254
,
H O
26 2
: C
ꢀ1
Merck Germany) were used for thin-layer chromatography (TLC) and
spots were visualized under UV light. Elemental analyses were per-
formed on Heraeus Vario EL III analyzer at Central Drug Research
Institute, Lucknow, India. The results were within ꢂ0.3% of the
theoretical values. Melting points were determined on MEL-TEMP
capillary melting point apparatus and are uncorrected. IR spectra
were recorded on PerkineElmer model 1600 FT-IR RX1 spectro-
85.25, H 6.64%; found: C 85.28, H 6.63%; IR nmax (cm ): 3061 (AeH),
1
2931 (CeH), 1656 (C]O), 1579 (C]C); H NMR (CDCl
8.01e7.91 (m, 4H, AreH), 7.86e7.81 (2H, m, AreH), 7.79 ( d, 2H,
J ¼ 15.6 Hz, H ), 7.77e7.71 (m, 2H, AreH),7.68 (d, 2H, J ¼ 15.6 Hz, H ),
7.62e7.55 (m, 4H, AreH), 2.47 (s, 6H, CH ); C NMR
(CDCl (ppm): 190.75 (C]O), 145.66 (C- ), (138.22, 136.76, 134.11,
128.88, Aromatic), 122.87 (C- ); ESI-MS m/z: [M 1] 395.2.
3
) d(ppm):
b
a
13
), 2.28 (s, 6H, CH
3 3
3
)
d
b
þ
a
1
13
photometer as KBr discs. H NMR and C NMR spectra were recorded
on Bruker AVANCE 400 spectrometer using CDCl or DMSO-d as
0
0
3
6
6.1.5. (2E,2 E)-3,3 -benzene-1,4-diylbis[(1-(4-methylthio)phenyl)
solvent with TMS as internal standard. Splitting patterns are desig-
nated as follows; s, singlet; d, doublet; m, multiplet. Chemical shift
values are given in ppm. ESI-MS was recorded on a MICROMASS
QUATTRO II triple quadrupole mass spectrometer.
prop-2-en-1-one] (2e)
ꢁ
Yield 74%; solid (Ethanol); mp: 185e190 C; Anal. calc. for
ꢀ
1
C
26
H
22
O
2
S
2
: C 72.52, H 5.15%; found: C 72.54, H 5.18%; IR
n
max (cm ):
3068 (AreH), 2917 (C-H), 1653 (C]O), 1589 (C]C); H NMR (CDCl
(ppm): 8.12e7.94 (m, 4H, AreH), 7.84 ( d, 2H, J ¼ 16.2 Hz, H
7.79e7.71 (m, 4H, AreH),7.68 (d, 2H, J ¼ 16.2 Hz, H ), 7.31e7.23 (m,
(ppm): 189.85 (C]
1
3
)
d
b
),
6.1. General procedure for the preparation of bis-chalcones
a
13
(2ae2h)
3 3
4H, AreH), 2.42 (s, 3H, eSCH ); C NMR (CDCl ) d
O), 144.84 (C-
b
), (138.22,136.78,134.22,127.52, Aromatic), 121.64 (C-
), ESI-M m/z: [M 1] 431.1.
þ
An aqueous solution of sodium hydroxide (60%, 150 mL) was
a), 14.98 (SCH
3
added slowly to a methanolic solution (200 mL) of appropriate
acetophenone (33 mmol). After cooling the solution to room
0
0
6.1.6. (2E,2 E)-3,3 -benzene-1,4-diylbis[1-(4-methoxyphenyl)prop-2-
temperature,
a
methanolic suspension (50 mL) of tereph-
en-1-one] (2f)
ꢁ
thaldicarboxaaldehyde (2.2 g, 16.4 mmol) was added. The reaction
mixture was stirred at room temperature for 20 h, and then it was
poured into a mixture of ice and hydrochloric acid (pH was adjust to
about 2). The resulting solid was filtered, dissolved in dichloro-
methane (150 mL), and washed with a saturated solution of sodium
hydrogen carbonate (2 ꢃ 100 mL). The solvent was evaporated to
dryness and the residue was purified by column chromatography
using dichloromethane as eluent.
Yield 78%; solid (Ethanol); mp: 220 C; Anal. calc. for C26
C 78.37, H 5.57%; found: C 78.31, H 5.52%; IR
(AreH), 2914 (CeH), 1657 (C]O), 1583 (C]C); H NMR (CDCl
H
22
O
4
:
ꢀ
1
n
max (cm ): 3069
1
3
)
b
),
d
(ppm) 8.20e7.92 (m, 4H, AreH), 7.86 ( d, 2H, J ¼ 16 Hz, H
7.76e7.73 (m, 4H, AreH), 7.71 (d, 2H, J ¼ 16 Hz, H ), 7.45 (d, 1H,
J ¼ 6Hz, AreH), 7.03e6.95 (m, 3H, AreH), 3.84 (s, 3H, eOCH ), 3.80
(ppm): 190.87 (C]O), 143.76 (C-
), (137.87, 136.80, 134.92, 127.23, Aromatic), 122.45 (C- ), 55.19
a
3
1
3
3 3
(s, 3H, eOCH ); C NMR (CDCl ) d
b
(
a
þ
3
OCH ), ESI-MS m/z: [M 1] 399.1.
0
0
6
.1.1. (2E,2 E)-3,3 -benzene-1,4-diylbis(1-phenylprop-2-en-1-one) (2a)
ꢁ
0
0
Yield 88%; solid (Ethanol); mp: 100e110 C; Anal. calc. for
6.1.7. (2E,2 E)-3,3 -benzene-1,4-diylbis[1-(3,4-dimethoxyphenyl)
C
24
H
18
O
2
: C 85.18, H 5.36%; found: C 85.09, H 5.29%; IR
n
max
H
prop-2-en-1-one] (2g)
Yield 78%; solid (Ethanol); mp: 189 C; Anal. calc. for C28
73.35, H 5.72%; found: C 37.31, H 5.78%; IR
ꢀ
1
1
ꢁ
(
cm ): 3064 (AreH), 2921 (CeH), 1650 (C]O), 1589 (C]C);
H
26
O
6
: C
ꢀ
1
NMR (CDCl
3
)
d
(ppm): 8.00e7.93 (m, 4H, AreH), 7.88 (d, 2H,
), 7.76e7.72 (m, 4H, AreH), 7.69 (d, 2H, J ¼ 15 Hz, H ),
(ppm): 190.83 (C]O),
n
max (cm ): 3068 (AreH),
1
J ¼ 15 Hz, H
b
a
2927(CeH), 1654 (C]O), 1572 (C]C); H NMR (CDCl
8.18e7.96 (m, 4H, AreH), 7.84 ( d, 2H, J ¼ 15.6 Hz, H ), 7.68e7.53 (m,
3H, AreH), 7.51 (d, 2H, J ¼ 15.6 Hz, H ), 7.27e7.18 (m, 3H, AreH), 3.83
3
) d(ppm)
13
7.57e7.35 (m, 6H, AreH); C NMR (CDCl
3
)
d
b
143.41 (C-
b
), (136.07,134.70,130.02,128.51, Aromatic),122.52 (C-
a
).
a
13
þ
ESI-MS m/z: [M 1] 339.1.
3 3 3
(s, 6H, eOCH ), 3.80 (s, 6H, eOCH ); C NMR (CDCl ) d(ppm): 189.76
(
C]O), 143.87 (C-
b
), (138.44, 136.60, 134.82, 127.56, Aromatic),
0
0
þ
6
.1.2. (2E,2 E)-3,3 -benzene-1,4-diylbis[1-(4-chlorophenyl)prop-2-
en-1-one] (2b)
Yield 76%; solid (Ethanol); mp: 220 C; Anal. calc. for C24
122.09 (C-
a), 56.87 (OCH
3
), ESI-MS m/z: [M 1] 459.1.
ꢁ
0
0
H
16
O
2
Cl
2
6.1.8. (2E,2 E)-3,3 -benzene-1,4-diylbis[1-(3,4,5-trimethoxyphenyl)
prop-2-en-1-one] (2h)
ꢀ
1
:
(
d
C 70.77, H 3.96%; found: C 70.88, H 3.86%; IR
AreH), 2929 (CeH), 1655 (C]O), 1575 (C]C); H NMR (CDCl
(ppm): 8.16e8.09 (m, 4H, AreH), 7.94e7.89 (m, 3H, AreH), 7.83 ( d,
H, J ¼ 15.6 Hz, H ), 7.77 (d, 2H, J ¼ 7.6 Hz, AreH),7.71 (d, 2H,
J ¼ 15.6 Hz, H ), 7.53e7.46 (m, 4H, AreH); C NMR (CDCl
90.65 (C]O), 144.56 (C- ), (138.17, 136.50,134.12,127.51, Aromatic),
n
max (cm ): 3068
1
ꢁ
3
)
Yield 78%; solid (Ethanol); mp: 180 C; Anal. calc. for C30
H
30
O
8
: C
ꢀ1
69.49, H 5.83%; found: C 69.42, H 5.88%; IR
n
max (cm ): 3067(AreH),
1
2
b
2925 (CeH), 1658 (C]O), 1576 (C]C); H NMR (CDCl
8.12e7.91 (m, 4H, AreH), 7.86 ( d, 2H, J ¼ 15.6 Hz, H ), 7.66e7.55 (m,
2H, AreH),7.52 (d, 2H, J ¼ 15.6 Hz, H ), 7.25e7.22 (m, 2H, AreH), 3.92
3
) d(ppm)
13
a
3
)
d(ppm):
b
1
b
a
13
þ
122.66 (C-
a
). ESI-MS m/z: [M 1] 408.0.
3 3 3
(s, 6H, OCH ), 3.88 (s, 6H, OCH ); C NMR (CDCl ) d(ppm): 190.46
(
(C-
C]O), 144.67 (C-
b
), (138.87, 136.44 134.82, 127.57, Aromatic), 121.56
0
0
þ
6
.1.3. (2E,2 E)-3,3 -benzene-1,4-diylbis[1-(4-methylphenyl)prop-2-
a
), 61.84 (OCH
3
), 55.67 (OCH
3
). ESI-MS m/z: [M 1] 519.2.
en-1-one] (2c)
Yield 76%; solid (Ethanol); mp: 165e170 C; Anal. calc. for
ꢁ
6.2. General procedure for the synthesis of compounds (3ae3h)
ꢀ
1
C
26
H
22
O
2
: C 85.22, H 6.05%; found: C 85.30, H 6.00%; IR
n
max (cm ):
1
3
066 (AreH), 2922 (CeH), 1661 (C]O), 1583(C]C); H NMR (CDCl
3
)
To a solution of 1.0 equiv. of pyrrolidine-1-carboxamidine
hydrochloride in 50 mL of isopropanol, 1.1 equiv. of sodium metal
was added. The reaction mixture was refluxed for 2 h and then
d
(ppm): 7.89e7.80 (m, 4H, AreH), 7.77 ( d, 2H, J ¼ 16.2 Hz, H
b
),
7
.72e7.71(m, 4H, AreH),7.69 (d, 2H, J ¼ 16.2 Hz, H ), 7.57e7.18 (m,
a

H. Parveen et al. / European Journal of Medicinal Chemistry 46 (2011) 4669e4675
4673
0
different bis-chalcones (2ae2h, 1.0 equiv.) were added to it and
6.2.5. 4-4 -Benzene-1,3-diylbis[6-(4-methylthiophenyl-2-(pyrrolidin-
refluxed for 8 h. The solvent was removed from the reaction mixture
under reduced pressure. Water was added and the aqueous phase
was extracted with chloroform and washed with brine solution. The
organic phase was dried over anhydrous sodium sulphate, filtered
and concentrated. The crude product was purified by crystallization
from methanol or ethanol or sometimes by column chromatography
on silica gel (2% methanol in chloroform to afford the pure
compounds.
1-yl)pyrimidine] (3e)
Yield 72%; solid (Ethanol); mp: 105e110 C; Anal. calc. for
: C 70.10, H 5.88, N 13.62%; found: C 70.15, H 5.91, N
ꢁ
36 36 6 2
C H N S
ꢀ1
13.66%; IR
(C]C), 1285 (CeN); H NMR (DMSO-d
nmax (cm ): 3066 (AreH), 2954(CeH), 1575 (C]N),1435,
1
6
) d (ppm) 8.13e7.96 (m, 4H,
AreH), 7.78e7.69 (m, 4H, AreH), 7.37 (s, 2H, pyrimidine) 7.33e7.23
(m, 4H, AreH), 3.83e3.71 (m, 8H, pyrrolidine), 2.41 (m, 6H, SMe),
13
2.04e1.74 (m, 8H, pyrrolidine); C NMR (DMSO-d6)
C]N pyrimidine), 162.7 (C]C pyrimidine), 161.4 (N¼CeN pyrimi-
dine), 140.1, 141.5, 130.7129.3, 126.6, 122.7, 117.6 (AreC), 104.4 (CeH
d(ppm): 164.5
(
0
6
.2.1. 4-4 -Benzene-1,3-diylbis[6-phenyl-2-(pyrrolidin-1-yl)
pyrimidine] (3a)
Yield 71%; solid (Ethanol); mp: 123 C; Anal. calc. for C34
C 77.83, H 6.15, N 16.02%; found: C 77.89, H 6.13, N 16.05%; IR
pyrimidine), 47.6, 25.7 (CH
m/z: [M 1] 617.8.
2
pyrrolidine), 16.7 (SCH3 phenyl). ESI-MS
ꢁ
þ
H
32
N
6
:
n
max
ꢀ1
0
(
cm ): 3056 (AreH), 2973 (C-H), 1576 (C]N),1436, (C]C), 1286
CeN); 1H NMR (DMSO-d
(ppm) 8.12e7.76 (m, 4H, AreH),
.68e7.61 (m, 4H, AreH), 7.54e7.33 (m, 6H, AreH), 7.37 (s, 2H,
pyrimidine), 3.81e3.72 (m, 8H, pyrrolidine), 2.02e1.78 (m, 8H,
eCH (ppm): 164.7 (C]N
pyrrolidine); 13C NMR (DMSO-d
pyrimidine), 162.3 (C]C pyrimidine), 161.5 (N¼CeN pyrimidine),
40.1, 138.7, 130.5, 127.8, 126.9, 123.7, 122.5, 115.6 (AreC), 104.7
6.2.6. 4-4 -Benzene-1,3-diylbis[6-(4-methoxyphenyl-2-(pyrrolidin-
(
6
)
d
1-yl)pyrimidine] (3f)
ꢁ
7
Yield 66%; solid (Ethanol); mp: 131 C; Anal. calc. for C36
36 6 4
H N O :
C 73.95, H 6.21, N 14.37%; found: C 73.91, H 6.29, N 14.39%; IR
n
max
ꢀ1
2
6
)
d
(cm ): 3075 (AreH), 2945 (CeH), 1567 (C]N),1436, (C]C), 1286
1
(CeN); H NMR (DMSO-d
7.94e7.83 (m, 4H, AreH), 7.35 (s, 2H, pyrimidine) 7.18e7.02 (m, 4H,
AreH), 3.89e3.83 (m, 8H, pyrrolidine), 3.84 (s, 3H, OCH ), 2.06e1.72
(ppm): 164.5 (C]N
6
) d (ppm) 8.19e7.97 (m, 4H, AreH),
1
þ
(
CeH pyrimidine), 47.2, 25.3 (CH
2
pyrrolidine). ESI-MS m/z: [M 1]
3
13
5
25.2.
6
(m, 8H, pyrrolidine); C NMR (DMSO-d ) d
pyrimidine), 162.4 (C]C pyrimidine), 161.6 (N¼CeN pyrimidine),
6.2.2. 4-4’-Benzene-1,3-diylbis[6-(4-chlorophenyl-2-(pyrrolidin-1-yl)
140.7, 139.2, 130.7, 129.2, 126.1, 123.4, 122.6, 115.3 (AreC), 102.2 (CeH
pyrimidine] (3b)
Yield 73%; solid (Ethanol); mp: 180 C; Anal. calc. for
Cl : C 68.80, H 5.09, N 14.16%; found: C 68.85, H 5.12, N
4.11%; IR
C]C), 1279 (CeN); H NMR (DMSO-d
AreH), 7.97e7.83 (m, 4H, AreH), 7.49e7.43 (m, 4H, AreH), 7.35 (s,
H, pyrimidine), 3.83e3.74 (m, 8H, pyrrolidine), 2.05e1.76 (m, 8H,
eCH (ppm): 164.6 (C]N
pyrrolidine); 13C NMR (DMSO-d
pyrimidine), 162.7 (C]C pyrimidine), 161.2 (N¼CeN pyrimidine),
40.7, 138.7, 136.1, 130.6, 129.6, 126.2, 123.6, 122.6, 115.1 (AreC),
pyrimidine), 55.5 (eOCH
z: [M 1] 585.2.
3
), 47.5, 25.6 (eCH
2
pyrrolidine). ESI-MS m/
ꢁ
þ
C
1
(
34
H
30
N
6
2
n
ꢀ
1
0
max (cm ): 3067 (AreH), 2973 (C-H), 1596 (C]N), 1476,
6.2.7. 4-4 -Benzene-1,3-diylbis[6-(2,5-dimethoxyphenyl-2-
1
6
)
d
(ppm) 8.14e7.99 (m, 4H,
(pyrrolidin-1-yl)pyrimidine] (3g)
ꢁ
Yield 70%; solid (Ethanol); mp: 148 C; Anal. calc. for C38
40 6 4
H N O :
2
C 70.79, H 6.25, N 13.03%; found: C 70.81, H 6.29, N 13.09%; IR
n
max
ꢀ1
2
6
)
d
(cm ): 3077 (AreH), 2976 (CeH), 1535 (C]N),1465, (C]C), 1244
1
(CeN); H NMR (DMSO-d
7.92e7.81 (m, 4H, AreH), 7.41(s, 2H, pyrimidine) 7.25e7.08 (m, 4H,
AreH), 3.84 (s, 6H, 2XOCH ), 3.86 (s, 6H, 2XOCH ), 3.81e3.72 (m, 8H,
pyrrolidine); 2.05e1.70 (m, 8H, pyrrolidine) C NMR (DMSO-d
(ppm): 164.8 (C]N pyrimidine), 162.1 (C]C pyrimidine), 160.5
6
) d (ppm) 8.17e7.99 (m, 4H, AreH),
1
1
04.5 (CeH pyrimidine), 47.4, 25.7 (eCH
2
pyrrolidine). ESI-MS m/z:
3
3
þ
13
[M 1] 593.1.
6
)
d
0
6
.2.3. 4-4 -Benzene-1,3-diylbis[6-(4-methylphenyl-2-(pyrrolidin-1-yl)
(N¼CeN pyrimidine), 154.0, 139.2, 130.8, 129.7, 125.9, 123.6, 122.5,
pyrimidine] (3c)
Yield 69%; solid (Ethanol); mp: 150 C; Anal. calc. for C36
C 78.23, H 6.57, N 15.21%; found: C 78.25, H 6.59, N 15.26%; IR
117.5 (AreC), 103.2 (CeH pyrimidine), 55.4 (eOCH3), 47.9, 25.3
ꢁ
þ
H
36
N
6
:
(eCH
2
pyrrolidine). ESI-MS m/z: [M 1] 645.3.
n
max
ꢀ1
0
(
cm ): 3087 (AreH), 2997(C-H), 1543 (C]N), 1413, (C]C), 1278
CeN); 1H NMR (DMSO-d
(ppm) 8.09e7.96 (m, 4H, AreH),
.72e7.68 (m, 4H, AreH), 7.37 (s, 2H, pyrimidine), 7.22e7.11 (m, 4H,
AreH), 3.82e3.76 (m, 8H, pyrrolidine), 2.03e1.77 (m, 8H, CH
pyrrolidine); 13C NMR (DMSO-d6)
(ppm): 164.5 (C]N pyrimi-
dine), 162.8 (C]C pyrimidine), 161.3 (N¼CeN pyrimidine), 140,
41.4, 130.6, 129.3, 126.9, 122.4, 117.7 (AreC), 104.8 (CeH pyrimi-
6.2.8. 4-4 -Benzene-1,3-diylbis[6-(3,4,5-trimethoxyphenyl-2-
(
6
)
d
(pyrrolidin-1-yl) pyrimidine] (3h)
ꢁ
7
Yield 71%; solid (Ethanol); mp: 114 C; Anal. calc. for
2
C
40
H
44
N
6
O
4
: C 68.16, H 6.29, N 11.92%; found: C 68.19, H 6.25, N
ꢀ1
d
11.98%; IR
nmax (cm ): 3055 (AreH), 2917 (CeH), 1587
1
(C]N),1464, (C]C), 1243 (CeN); H NMR (DMSO-d
8.11e7.94 (m, 4H, AreH), 7.66 (s, 4H, AreH), 7.38 (s, 2H, pyrimidine)
6
)
d
(ppm)
1
dine), 47.3, 25.6 (CH
2
pyrrolidine), 21.3 (CH
3
phenyl). ESI-MS m/z:
3.88e3.82 (m, 8H, pyrrolidine), 3.84 (s, 12H, OCH3), 3.82 (s, 6H,
þ
13
[M 1] 553.3.
OCH
3
), 2.03e1.74 (m, 8H, pyrrolidine);
6
C NMR (DMSO-d )
d
(ppm): 164.5 (C]N pyrimidine), 162.7(C]C pyrimidine), 161.4
0
6
1
.2.4. 4-4 -Benzene-1,3-diylbis[6-(3,4-dimethylphenyl-2-(pyrrolidin-
(N¼CeN pyrimidine), 140.3, 130.6, 129.9, 126.9, 123.6, 122.7, 115.5
-yl)pyrimidine] (3d)
Yield 71%; solid (Ethanol); mp: 180 C; Anal. calc. for C38
(AreC), 103.9 (CeH pyrimidine), 60.9 (OCH
3
), 55.5 (2XOCH
pyrrolidine). ESI-MS m/z: [M 1] 705.3.
3
), 47.5,
ꢁ
þ
40 6
H N : C
25.3 (eCH
2
7
8.59, H 6.94, N 14.47%; found: C 78.55, H 6.91, N 14.49%; IR
n
max
ꢀ
1
(
cm ): 3056 (AreH), 2986 (C-H), 1597 (C]N), 1432 (C]C), 1235
CeN); 1H NMR (DMSO-d
(ppm) 8.15e7.96 (m, 4H, AreH),
.75e7.73 (m, 4H, AreH), 7.38 (s, 2H, pyrimidine), 7.23e7.14 (m, 4H,
AreH), 3.83e3.78 (m, 8H, pyrrolidine), 2.35 (s, 6H, CH ) 2.01e1.76 (m,
(ppm): 164.4 (C]N
pyrimidine), 162.7 (C]C pyrimidine), 161.5 (N¼CeN pyrimidine),
40.3, 141.6, 130.5, 129.4, 126.7, 122.8, 117.4 (AreC), 104.6 (CeH
6.3. General procedure for the synthesis of compounds (4ae4h)
(
7
6
) d
To a solution of 1.0 equiv. of piperidine-1-carboxamidine hydro-
chloride in 50 mL of isopropanol, 1.1 equiv. of sodium metal was
added. The reaction mixture was refluxed for 2 h and then different
bis-chalcones (2ae2h, 1.0 equiv.) were added to it and refluxed for
8 h. The solvent was removed from the reaction mixture under
reduced pressure. Water was added and the aqueous phase was
extracted with chloroform and washed with brine solution. The
3
13
8
2
H, CH pyrrolidine); C NMR (DMSO-d6) d
1
pyrimidine), 47.4, 25.7 ( eCH
m/z: [M 1] 581.3.
2
pyrrolidine), 21.7 (CH
3
phenyl). ESI-MS
þ

4674
H. Parveen et al. / European Journal of Medicinal Chemistry 46 (2011) 4669e4675
ꢀ
1
organic phase was dried over anhydrous sodium sulphate, filtered
and concentrated. The crude product was purified by crystallization
from methanol or ethanol or sometimes by column chromatography
on silica gel (2% methanol in chloroform to afford the pure
compounds.
(cm ): 3056 (AreH), 2978 (CeH), 1598 (C]N),1446, (C]C), 1233
1
6
(CeN); H NMR (DMSO-d ) d (ppm) 8.13e7.96 (m, 4H, AreH),
7.78e7.69 (m, 4H, AreH), 7.37 (s, 2H, pyrimidine) 7.33e7.23 (m, 4H,
AreH), 3.89e3.84 (m, 8H, piperidine), 2.41 (m, 6H, -SMe), 1.69e1.53
13
6
(m, 12H, piperidine); C NMR (DMSO-d ) d (ppm): 166.5 (C]N
pyrimidine), 162.7 (C]C pyrimidine), 162.2 (N¼CeN pyrimidine),
0
6
.3.1. 4-4 -Benzene-1,3-diylbis[6-phenyl-2-(piperidin-1-yl)
137.7, 138.4, 136.8, 130.9, 129.5, 126.8, 123.3, 122.5, 119.4 (AreC),102.1
pyrimidine](4a)
Yield 68%; solid (Ethanol); mp: 133 C; Anal. calc. for C36
C 78.23, H 6.57, N 15.21%; found: C 78.20, H 6.53, N 15.28%; IR
(CeH pyrimidine), 46.5, 27.6, 24.7 (eCH
ESI-MS m/z: [M 1] 645.2.
2
piperidine)), 16.9 (SCH
3
).
ꢁ
þ
H
36
N
6
:
n
max
ꢀ
1
0
(
cm ): 3077 (AreH), 2953 (CeH), 1576 (C]N), 1486, (C]C), 1234
6.3.6. 4-4 -Benzene-1,3-diylbis[6-(4-methoxyphenyl-2-(piperidin-
1
(
7
CeN); H NMR (DMSO-d
.67e7.63 (m, 4H, AreH), 7.57e7.36 (m, 6H, AreH), 7.35 (s, 2H,
pyrimidine), 3.88e3.82 (m, 8H, piperidine), 1.71e1.56 (m, 12H,
piperidine); 13C NMR (DMSO-d
(ppm): 164.5 (C]N pyrimidine),
62.2 (C]C pyrimidine), 161.5 (N¼CeN pyrimidine), 141.4, 138.5,
30.6, 127.1, 126.7, 124.3, 123.5, 116.3 (AreC), 104.6 (CeH pyrimi-
6
)
d
(ppm) 8.14e7.74 (m, 4H, AreH),
1-yl)pyrimidine] (4f)
ꢁ
Yield 61%; solid (Ethanol); mp: 155 C; Anal. calc. for C38
40 6
H N O
2
:
C 74.48, H 6. 58, N 13.71%; found: C 74.45, H 6.53, N 13.75%; IR
n
max
ꢀ1
6
)
d
(cm ): 3022(AreH), 2965 (CeH), 1567 (C]N),1489, (C]C), 1234
1
1
1
(CeN); H NMR (DMSO-d
7.94e7.83 (m, 4H, AreH), 7.39 (s, 2H, pyrimidine) 7.18e7.02 (m, 4H,
AreH), 3.89e3.83 (m, 8H, piperidine), 3.81 (s, 3H, OCH ), 1.66e1.52
(ppm): 164.5 (C]N
6
) d (ppm) 8.19e7.97 (m, 4H, AreH),
þ
dine), 46.4, 27.6, 25.4 (eCH
2
piperidine). ESI-MS m/z: [M 1] 553.
3
13
(
m, 12H, piperidine C NMR (DMSO-d6) d
0
6
.3.2. 4-4 -Benzene-1,3-diylbis[6-(4-chlorophenyl-2-(piperidin-1-yl)
pyrimidine), 161.8 (C]C pyrimidine), 161.5 (N¼CeN pyrimidine),
pyrimidine](4b)
Yield 72%; solid (Ethanol); mp: 133 C; Anal. calc. for C36
C 69.56, H 5.51, N 13.52%; found: C 69.51, H 5.55, N 13.58%; IR
140.7, 139.5, 130.7, 129.4, 126.8, 123.3, 122.9, 115.2 (AreC),102.4 (CeH
ꢁ
34
H N
6
Cl
2
:
pyrimidine), 56.5 (eOCH
m/z: [M 1] 613.3.
3
), 47.8, 26.6, 24.9 (eCH
2
piperidine). ESI-MS
þ
n
max
1
(
cm- ): 3065 (AreH), 2923 (CeH), 1545 (C]N),1465, (C]C), 1254
CeN); 1H NMR (DMSO-d
(ppm) 8.10e8.04 (m, 4H, AreH),
.95e7.84 (m, 4H, AreH), 7.46e7.41 (m, 4H, AreH), 7.38 (s, 2H,
pyrimidine), 3.87e3.83 (m, 8H, piperidine), 1.69e1.53 (m, 12H,
piperidine); 13C NMR (DMSO-d
(ppm): 164.3 (C]N pyrimidine),
62.5 (C]C pyrimidine), 162.1 (N¼CeN pyrimidine), 140.6, 138.3,
36.5, 130.5, 129.7, 126.8, 123.9, 122.4, 117.4 (AreC), 105.7 (CeH
0
(
6
)
d
6.3.7. 4-4 -Benzene-1,3-diylbis[6-(2,5-dimethoxyphenyl-2-(piperidin-
7
1-yl)pyrimidine] (4g)
ꢁ
Yield 68%; solid (Ethanol); mp: 141 C; Anal. calc. for C40
44 6
H N O
4
:
6
)
d
C 71.41, H 6. 59, N 12.49%; found: C 71.45, H 6.53, N 12.50%; IR
n
max
ꢀ1
1
1
(cm ): 3055 (AreH), 2965 (CeH), 1533 (C]N),1485, (C]C), 1236
1
(CeN); H NMR (DMSO-d
7.92e7.81 (m, 4H, AreH), 7.41(s, 2H, pyrimidine) 7.25e7.08 (m, 4H,
AreH), 3.87e3.86 (m, 8H, piperidine), 3.84 (s, 6H, 2XOCH ), 3.83 (s,
), 1.68e1.55 (m, 12H, piperidine); C NMR (DMSO-d
(ppm): 164.6 (C]N pyrimidine), 162.5 (C]C pyrimidine), 161.7
6
) d (ppm) 8.17e7.99 (m, 4H, AreH),
þ
pyrimidine), 46.4, 27.5, 25.7 (eCH piperidine). ESI-MS m/z: [M 1]
2
6
22.2.
3
13
6
d
H, 2XOCH
3
6
)
0
6
.3.3. 4-4 -Benzene-1,3-diylbis[6-(4-methylphenyl-2-(piperidin-1-yl)
pyrimidine] (4c)
Yield 70%; solid (Ethanol); mp: 140 C; Anal. calc. for C38
C 78.59, H 6.94, N 14.47%; found: C 78.56, H 6.93, N 14.48%; IR
(N¼CeN pyrimidine), 154.3, 139.7, 130.8, 129.5, 125.3 1237, 122.5,
ꢁ
H
40
N
6
:
117.1 (AreC), 103.3 (CeH pyrimidine), 56.6, 55.7 (eOCH
3
), 47.7, 27.5,
þ
n
max
25.6 (eCH
2
piperidine), ESI-MS m/z: [M 1] 673.3.
ꢀ
1
(
(
7
cm ): 3068 (AreH), 2943 (CeH), 1576 (C]N), 1456, (C]C), 1244
1
0
CeN); H NMR (DMSO-d
.74e7.71 (m, 4H, AreH), 7.36 (s, 2H, pyrimidine), 7.24e7.16 (m, 4H,
AreH), 3.87e3.84 (m, 8H, piperidine), 2.36 (s, 6H, CH ) 1.68e1.56
m, 12H, piperidine); 13C NMR (DMSO-d6)
(ppm): 164.7 (C]N
pyrimidine), 162.6 (C]C pyrimidine), 161.4 (N¼CeN pyrimidine),
40.1, 139.4, 130.4, 1296, 126.2, 122.5, 117.5 (AreC), 104.6 (CeH
6
)
d
(ppm) 8.12e7.99 (m, 4H, AreH),
6.3.8. 4-4 -Benzene-1,3-diylbis[6-(3,4,5-trimethoxyphenyl-2-
(piperidin-1-yl) pyrimidine] (4h)
ꢁ
3
Yield 77%; solid (Ethanol); mp: 142 C; Anal. calc. for C42
48 6 6
H N O :
(
d
C 68.83, H 6.60, N 11.47%; found: C 68.85, H 6.63, N 11.45%; IR
n
max
ꢀ1
(cm ): 3044 (AreH), 2963 (CeH), 1534 (C]N),1487, (C]C), 1241
1
1
(CeN); H NMR (DMSO-d
4H, AreH), 7.34 (s, 2H, pyrimidine) 3.88e3.82 (m, 8H, piperidine),
3.83 (s, 12H, OCH ), 3.81 (s, 6H, OCH ), 1.67e1.55 (m, 12H, piperi-
dine); C NMR (DMSO-d (ppm): 164.4 (C]N pyrimidine), 162.7
6
) d (ppm) 8.11e7.94 (m, 4H, AreH), 7.66 (s,
pyrimidine), 47.2, 27.6, 24.7 (eCH2 piperidine), 21.5 (eCH3 phenyl).
þ
ESI-MS m/z: [M 1] 581.3.
3
3
13
6
) d
0
6
.3.4. 4-4 -Benzene-1,3-diylbis[6-(3,4-dimethylphenyl-2-(piperidin-
(C]C pyrimidine), 162.7 (N¼CeN pyrimidine), 141.5, 130.8, 129.3,
1
-yl)pyrimidine] (4d)
Yield 22%; solid (Ethanol); mp: 146 C; Anal. calc. for C40
126.6, 123.7, 122.1, 115.3 (AreC), 103.5 (CeH pyrimidine), 60.3
ꢁ
H
44
N
6
:
(OCH
3
), 55.7 (2 ꢃ OCH
3 2
), 47.4, 27.7, 25.7 (eCH piperidine), ESI-
þ
C 78.91, H 7.28, N 13.80%; found: C 78.95, H 7.23, N 13.85%; IR
n
max
MS m/z: [M 1] 733.3.
ꢀ
1
(
(
7
cm ): 3066 (AreH), 2913 (CeH), 1566 (C]N), 1426, (C]C), 1239
1
CeN); H NMR (DMSO-d
.74e7.71 (m, 4H, AreH), 7.37(s, 2H, pyrimidine), 7.24e7.16 (m, 4H,
AreH), 3.85e3.83 (m, 8H, piperidine), 2.36 (s, 6H, CH ) 1.70e1.58
m, 12H, piperidine); 13C NMR (DMSO-d6)
(ppm): 165.7 (C]N
pyrimidine), 162.4 (C]C pyrimidine), 162.6 (N¼CeN pyrimidine),
39.7, 138.7, 136.9, 130.4, 129.5, 126.6, 123.2, 122.6, 118.7 (AreC),
6
)
d
(ppm) 8.12e7.99 (m, 4H, AreH),
6.4. In vitro antiamoebic assay
3
All the compounds 3ae3h and 4ae4h were screened in vitro for
antiamoebic activity against HM1:IMSS strain of E. histolytica by
microdilution method [30]. E. histolytica trophozoites were cultured
in wells of 96-well microtiter plate by using Diamond TYIS-33
growth medium [31]. The test compounds (1 mg) were dissolved
(
d
1
1
03.6 (CeH pyrimidine), 46.2, 27.1, 25.5 (eCH
2
piperidine), 21.4
þ
(
2 ꢃ CH
3
phenyl). ESI-MS m/z: [M 1] 609.3.
in DMSO (40
mL, level at which no inhibition of amoeba occurs)
[32,33]. The stock solutions of the compounds were prepared freshly
0
6
1
.3.5. 4-4 -Benzene-1,3-diylbis[6-(4-methylthiophenyl-2-(piperidin-
before use at a concentration of 1 mg/mL. Two-fold serial dilutions
were made in the wells of 96-well microtiter plate (costar). Each test
includes metronidazole as a standard amoebicidal drug, control
wells (culture medium plus amoebae) and a blank (culture medium
-yl)pyrimidine] (4e)
Yield 68%; solid (Ethanol); mp: 147 C; Anal. calc. for C38
ꢁ
40
H N
6
S
2
:
C 70.77, H 6.25, N 13.03%; found: C 70.75, H 6.23, N 13.05%; IR n
max

H. Parveen et al. / European Journal of Medicinal Chemistry 46 (2011) 4669e4675
4675
only). All the experiments were carried out in triplicate at each
concentration level and repeated thrice. The amoeba suspension was
appropriated controls taken in account. All assays were performed
in triplicate and repeated thrice.
prepared from a confluent culture by pouring off the medium at
ꢁ
3
7 C and adding 5 mL of fresh medium, chilling the culture tube on
Acknowledgements
ice to detach the organisms from the side of the flask. The number of
amoeba/mL was estimated with a haemocytometer, using trypan
This work was supported by Department of Science and Tech-
nology, SERC (Grant no. SR/FT/CS-027/2008, New Delhi, India.
blue exclusion to confirm the viability. The suspension was diluted to
5
10
organism/ml by adding fresh medium and 170 mL of this
suspension was added to the test and control wells in the plate so
that the wells were completely filled (total volume, 340 L). An
m
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down several times unless the content gets homogenized. After
0 min, the colour was read at 550 nm on an ELISA plate reader
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wavelength of 655 nm. Percentage cellular viability calculated with
mL of DMSO was added to each well by pipetting up and
(
3
1
(
[
2