Synthesis and in vitro antiproliferative activity of 2-methyl-3-(2- piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives against human cancer cell lines

Article abstract of DOI:10.1007/s12272-012-0105-z

A series of new 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin- 4-one derivatives 6a-j were synthesized by a nucleophilic substitution reaction of 2-methyl-3-(2-piperazin-1-ylethyl)-pyrido[1,2-a]pyrimidin-4-one with various sulfonyl chlorides. The compounds were characterized by different spectral studies. All the compounds were evaluated for their antiproliferative effect using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method against four human cancer cell lines (K562, Colo-205, MDA-MB 231, IMR-32) for the time period of 24 h. Among the series, compounds 6d, 6e and 6i showed good activity on all cell lines except K562, whereas the other compounds in the series exhibited moderate activity. Compound 6d could be a potential anticancer agent and therefore deserves further research.

Full text of DOI:10.1007/s12272-012-0105-z

Arch Pharm Res Vol 35, No 1, 51-57, 2012
DOI 10.1007/s12272-012-0105-z
Synthesis and In Vitro Antiproliferative Activity of 2-Methyl-3-(2-
piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one Derivatives
against Human Cancer Cell Lines
Lingappa Mallesha1, Kikkeri N. Mohana1, Bantal Veeresh2, Ravi Alvala2, and Alvala Mallika3
2
1Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India, Department of
3
Pharmacology, G Pullareddy College of Pharmacy, Mehdipatnam, Hyderabad, India, and Institute of life sciences, Uni-
versity Hyderabad Campus, Gachibowli, Hyderabad, India
(Received February 15, 2011/Revised July 9, 2011/Accepted July 31, 2011)
A series of new 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives
6a-j were synthesized by a nucleophilic substitution reaction of 2-methyl-3-(2-piperazin-1-yl-
ethyl)-pyrido[1,2-a]pyrimidin-4-one with various sulfonyl chlorides. The compounds were
characterized by different spectral studies. All the compounds were evaluated for their anti-
proliferative effect using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay method against four human cancer cell lines (K562, Colo-205, MDA-MB 231,
IMR-32) for the time period of 24 h. Among the series, compounds 6d, 6e and 6i showed good
activity on all cell lines except K562, whereas the other compounds in the series exhibited
moderate activity. Compound 6d could be a potential anticancer agent and therefore deserves
further research.
Key words: Piperazine, Sulfonyl chlorides, MTT assay, Antiproliferative activity, Human
cancer cell lines
city drawbacks. These drawbacks often prevent suc-
cessful treatment and are responsible for reduced
INTRODUCTION
Cancer is becoming the most significant health survival times (Mencher and Wang, 2005; Jimeno and
hazard around the world. Development of resistance Hidalgo, 2006).
against the existing anticancer drugs keeps the re-
Piperazines are currently the most important build-
search window open for the search of newer anti- ing blocks in drug discovery with a high number of
cancer molecules. Cancer is a serious pathology and a positive hits encountered in biological screens of this
substantial number of new antineoplastic agents have heterocycle and its congeners. Piperazine and its ana-
been discovered. Considerable insight has been gained logues are important pharmacophore that can be
into the mechanisms by which many of these com- found in biologically active compounds across a num-
pounds affect cellular growth and this knowledge has ber of different therapeutic areas such as antifungal
been used to design new chemotherapic drugs (Verweij (Upadhayaya et al., 2004), antibacterial, antimalarial,
and De Jonge, 2000; Verdecchia et al., 2001). Currently, antipsychotic agents (Chaudhary et al., 2006), and anti-
combined anticancer therapies or multi-acting drugs tumour activity against colon, prostate, breast, lung,
are clinically preferred to traditional cytotoxic treat- and leukemia tumors (Hulme and Cherrier, 1999).
ment with the aim of overcoming resistance and toxi- The new class of sulfonamides (Reddy et al., 2004) has
been used in the treatment of diseases arising from
abnormal cell growth and proliferation. Most cancers
Correspondence to: Lingappa Mallesha, Department of Studies
are characterized by uncontrolled cell proliferation,
in Chemistry, University of Mysore, Manasagangotri, Mysore
lack of cell differentiation and loss of contact inhibi-
570 006, India
tion, which all confer upon the tumor cell a capability
of invading local tissues and metastasizing. Recently,
Tel: 91-997-2033777
E-mail: mallesha83@gmail.com
51

52
L. Mallesha et al.
a series of arylsulfonanilides has been reported as tion of the reaction, the excess phosphorus oxychloride
potent inhibitors of various cancer cells at very low was removed under reduced pressure. The residue
concentrations (Rubenstein et al., 2001). 3-(2-Chloro- obtained was stirred in ice-cold water (100 mL) for 30
ethyl)-2-methyl-pyrido[1,2-a]pyrimidin-4-one (4) is an min, then neutralized with solid sodium carbonate
intermediate in the preparation of risperidone. Risper- and was further stirred overnight. The solid precipi-
idone contains the functional groups of benzisoxazole tated was filtered and washed with water and recry-
and piperidine as part of its molecular structure. It is stallized from ethanol. Yield 1.60 g (72%); m.p. 140-
a typical antipsychotic agent chemically classified as a 142oC; FT-IR (KBr)
benzisoxazole derivative with serotonin-5-HT2 and Cl); H-NMR (DMSO-d6, 400 MHz):
dopamine-D2 antagonist activity. In connection with CH), 7.78 (t, 1H, CH), 7.69 (d, 1H, CH), 7.15 (t, 1H,
ν
: 1654 (C=O), 1150 (C-N), 722 (C-
1
δ
9.07 (d, 1H,
such studies, the synthesis and antiproliferative acti- CH), 3.88 (t, 2H, CH2Cl), 3.23 (t, 2H, CH2), 2
vity of novel 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido CH3).
[1,2-a]pyrimidin-4-one derivatives 6a
against human
.55 (s, 3H,
-j
cancer cell lines have been reported in this paper.
Synthesis of 2-methyl-3-(2-piperazin-1-yl-ethyl)-
pyrido[1,2-a]pyrimidin-4-one (5)
A solution of 3-(2-chloroethyl)-2-methyl-4H-pyrido[1,
2-a]pyrimidin-4-one (4) (5.0 g, 22.45 mmol) and piper-
MATERIALS AND METHODS
All solvents and reagents were purchased from Sigma azine (2.13 g, 24.72 mmol) in DMF was prepared and
Aldrich Chemicals. Melting points were determined K2CO3 (9.31 g, 67.36 mmol) was added to the reaction
on an electrically heated VMP-III melting point appar- mixture. The reaction mixture was stirred for 6 h at
atus. The elemental analyses of the compounds were room temperature. The progress of the reaction was
performed on a Perkin Elmer 2400 elemental analy- monitored by TLC. Upon completion of the reaction,
ser. The FT-IR spectra were recorded using KBr discs water was added and the reaction mixture was filter-
on a Jasco FT-IR 4100 infrared spectrophotometer. ed and was finally washed with ether and dried under
1
The H-NMR spectra were recorded using a Bruker vacuum. The obtained compound was white amorphous
DRX 400 spectrometer at 400 MHz with tetramethyl- solid with 80% yield. FT-IR (KBr, cm−1)
ν
: 3365 (N
8.81
= 9.2 Hz),
= 11.2 Hz), 7.13 (t, 1H, CH, = 9.0
-H),
1
silane as the internal standard. Mass spectral data 1651 (C=O), 1160 (C
were obtained by LC MSD Trap XCT. Silica gel column (d, 2H, CH,
chromatography was performed using Merck 7734 7.51 (d, 1H, CH,
-
N); H-NMR (DMSO-d6):
δ
/
J = 14.4 Hz), 7.67 (t, 1H, CH, J
J
J
silica gel (60–120 mesh) and Merck-made TLC plates. Hz), 3.38-3.04 (m, 8H), 2.54 (s, 3H, CH3), 2.53 (t, 2H,
CH2), 2.44 (t, 2H, CH2); Anal. Calcd. for C15H20N4O
(%): C, 66.15; H, 7.40; N, 20.57. Found: C, 66.11; H,
7.32; N, 20.47.
Synthesis of 3-(1-(pyridin-2-ylamino)ethylidene)
dihydrofuran-2(3H)-one (3)
The mixture of 2-aminopyridines
1 (10 mmol) and 3-
acetyldihydrofuran-2(3H)-one (10 mmol) was reflux-
2
General procedure for the synthesis of 2-
methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]
pyrimidin-4-one derivatives 6a-j
ed in toluene (30 mL) for 12 h in the presence of a
catalytic amount of PTSA (0.02 g). The water separator
was attached between the reaction flask and the water
A solution of
2-methyl-3-(2-piperazin-1-yl-ethyl)-
condenser. The separation of an equivalent amount of pyrido[1,2-a]pyrimidin-4-one (
5
) (1.0 eq) in dry di-
water indicates the completion of reaction. The solid chloromethane was prepared and cooled to 0-5oC in an
obtained after cooling the reaction mixture was filter- ice bath. Triethylamine (3.0 eq) was added to the cold
ed and washed with toluene and then recrystallized in reaction mixture and stirred for 10 min, then various
ethanol. Yield 1.69 g (83%); m.p. 87-88oC; FT-IR (KBr, sulfonyl chlorides (1.0 eq) were added and the reaction
cm−1)
(DMSO-d6, 400 Hz):
CH), 8.25 (dd, 1H, CH), 6.83 (d, 1H, CH), 6.75 (dd, 1H, completion, the solvent was removed under reduced
ν
: 3300 (N-H), 1650 (C=O), 1150 (C-N); 1H-NMR mixture was stirred at room temperature for 8 h. The
δ
10.5 (bs, 1H, NH), 8.75 (d, 1H, progress of the reaction was monitored by TLC. Upon
CH), 4.31 (t, 2H, OCH2), 2.92 (t, 2H, CH2), 2
CH3).
.
50 (s, 3H, pressure and the residue was mixed in water and
extracted with ethyl acetate. The organic layer was
washed with 10% ammonium chloride solution and
the organic layer was finally washed with water and
dried with anhydrous sodium sulphate. The solvent
Synthesis of 3-(2-chloroethyl)-2-methyl-4H-pyri-
do[1,2-a]pyrimidin-4-one (4)
Compound
3 (10 mmol) was refluxed in phosphorus was evaporated to obtain the crude product which was
oxychloride (20 mL) for 1 h (TLC check). After comple- purified by column chromatography over silica gel

Synthesis and Anticancer Activity of Pyrimidines
53
(60–120 mesh) using hexane:ethyl acetate (8:2) as an 2-Methyl-3-{2-[4-(4-nitro-benzenesulfonyl)-pipera-
eluent.
zin-1-yl]-ethyl}-pyrido[1,2-a] pyrimidin-4-one (6e)
FT-IR (KBr, cm−1)
ν
: 3081 (Ar-H), 1636 (C=O), 1547
1
3-[2-(4-Benzenesulfonyl-piperazin-1-yl)-ethyl]-2-
methyl-pyrido[1,2-a]pyrimidin-4-one (6a)
(NO2), 1168 (C-N); H-NMR (DMSO-d6, 400 MHz):
8.81 (d, 1H, Ar-H, = 6.54 Hz), 8.44 (d, 2H, Ar-H,
δ
J
J
=
FT-IR (KBr, cm−1)
ν
: 3022 (Ar-H), 1634 (C=O), 1157 8.88 Hz), 7.99 (d, 2H, Ar-H,
J
= 8.88 Hz), 7.85 (t, 1H,
= 10.47 Hz), 7.55 (d, 1H, Ar-H, = 8.85 Hz),
= 25.12 Hz), 7.28 (t, 1H, Ar-H, = 8.10 Hz), 4.13 (t, 2H, = 13.41
= 8.88 Hz), 3.41 (t, 2H, = 9.72 Hz), 2.92-2.84 (m, 8H), 2.36
= 15.08 Hz), 4.13 (t, 2H, (s, 3H, CH3); MS: 457.1; Anal. Calcd. for C21H23N5O5S
13.32 Hz), 3.40-3.37 (m, 4H), 2.88 (t, 2H, = 6.64 Hz), (%): C, 51.13; H, 5.07; N, 15.31. Found: C, 51.24; H,
1
(C-N); H-NMR (DMSO-d6, 400 MHz):
δ
J
8.76 (d, 1H, Ar-H,
J
J
Ar-H,
J
= 6.81 Hz), 7.86 (t, 1H, Ar-H,
J
J
7.75-7.63 (m, 5H, Ar-H), 7.55 (d, 1H, Ar-H,
Hz), 7.27 (t, 1H, Ar-H,
J
J
J
J =
J
2.85-2.82 (m, 4H), 2.36 (s, 3H, CH3); MS: 412.2; Anal. 5.20; N, 15.45.
Calcd. for C21H24N4O3S (%): C, 61.14; H, 5.86; N, 13.58.
Found: C, 61.21; H, 5.70; N, 13.75.
3-[2-(4-Methanesulfonyl-piperazin-1-yl)-ethyl]-2-
methyl-pyrido[1,2-a]pyrimidin-4-one (6f)
: 1635 (C=O), 1141 (C-N); 1H-NMR
3-{2-[4-(4-Chloro-benzenesulfonyl)-piperazin-1-yl]- FT-IR (KBr, cm−1)
ν
ethyl}-2-methyl-pyrido[1,2-a] pyrimidin-4-one (6b) (DMSO-d6, 400 MHz):
δ
8.90 (d, 1H, Ar-H,
= 17.12 Hz), 7.60 (d, 1H, Ar-
= 15.08 Hz), 4.22
= 13.52 Hz), 3.44-3.42 (m, 4H), 3.07-3.04 (m,
4H), 2.97 (t, 2H, = 13.52 Hz), 2.87 (s, 3H, CH3), 2.46
J = 7.08
FT-IR (KBr, cm−1)
(C-N), 720 (C-Cl); H-NMR (DMSO-d6, 400 MHz):
ν
: 3034 (Ar-H), 1635 (C=O), 1157 Hz), 7.90 (t, 1H, Ar-H,
J
1
δ
=
=
H,
J = 8.88 Hz), 7.31 (t, 1H, Ar-H, J
8.82 (d, 1H, Ar-H,
J
= 6.84 Hz), 7.89 (t, 1H, Ar-H,
J
J
(t, 2H, J
16.92 Hz), 7.78 (m, 4H, Ar-H), 7.58 (d, 1H, Ar-H,
J
8.88 Hz), 7.30 (t, 1H, Ar-H,
J
= 14.80 Hz), 4.15 (t, 2H, (s, 3H, CH3); MS: 350.1; Anal. Calcd. for C16H22N4O3S
J
= 13.28 Hz), 3.42 (t, 2H,
J = 9.48 Hz), 2.91-2.86 (m, (%): C, 54.84; H, 6.33; N, 15.99. Found: C, 54.71; H,
8H), 2.39 (s, 3H, CH3); MS: 446.1; Anal. Calcd. for 6.53; N, 15.81.
C21H23 ClN4O3S (%): C, 56.43; H, 5.19; N, 12.54.
Found: C, 56.49; H, 5.31; N, 12.31.
2-Methyl-3-{2-[4-(toluene-4-sulfonyl)-piperazin-1-
yl]-ethyl}-pyrido[1,2-a]pyrimidin-4-one (6g)
2-Methyl-3-{2-[4-(2,4,6-trimethyl-benzenesulfonyl)- FT-IR (KBr, cm−1)
piperazin-1-yl]-ethyl}-pyrido[1,2-a]pyrimidin-4-one (C-N); H-NMR (DMSO-d6, 400 MHz):
ν
: 3090 (Ar-H), 1638 (C=O), 1161
1
δ
J
8.80 (d, 1H,
= 15.08 Hz),
(6c)
Ar-H,
J
= 7.0 Hz), 7.89 (t, 1H, Ar-H,
FT-IR (KBr, cm−1)
ν
: 3046 (Ar-H), 1638 (C=O), 1161 7.59 (d, 1H, Ar-H,
J
= 8.08 Hz), 7.56 (d, 2H, Ar-H,
8.86 (d, 1H, 8.12 Hz), 7.47 (d, 2H, Ar-H, = 5.68 Hz), 7.30 (t, 1H,
= 18.85 Hz), Ar-H, = 13.64 Hz), 4.13 (t, 2H, = 13.28 Hz), 2.89 (t,
2H, = 13.24 Hz), 2.81-2.78 (m, 8H), 2.49 (s, 3H, CH3),
= 13.36 Hz), 2.27 (s, 3H, CH3); MS: 426.2; Anal. Calcd. for
= 10.41 Hz), 2.97-2.87 (m, 8H), 2.51 (s, C22H26N4O3S (%): C, 61.95; H, 6.14; N, 13.14. Found:
J =
1
(C-N); H-NMR (DMSO-d6, 400 MHz):
δ
J
Ar-H,
7.57 (d, 1H, Ar-H,
12.0 Hz), 7.07 (s, 2H, Ar-H), 4.19 (t, 2H,
3.35 (t, 2H,
J
= 6.51 Hz), 7.91 (t, 1H, Ar-H,
J
J
J
J
= 8.85 Hz), 7.26 (t, 1H, Ar-H,
J
=
J
J
J
9H, 3CH3), 2.26 (s, 3H, CH3); MS: 454.2; Anal. Calcd. C, 61.83; H, 6.32; N, 13.25.
for C24H30N4O3S (%): C, 63.41; H, 6.65; N, 12.32.
Found: C, 63.12; H, 6.71; N, 12.41.
3-{2-[4-(4-Methoxy-benzenesulfonyl)-piperazin-1-
yl]-ethyl}-2-methyl-pyrido[1,2-a]pyrimidin-4-one
2-Methyl-3-{2-[4-(4-trifluoromethyl-benzenesulfon- (6h)
yl)-piperazin-1-yl]-ethyl}-pyrido [1,2-a]pyrimidin- FT-IR (KBr, cm−1)
ν
: 3064 (Ar-H), 1635 (C=O), 1151
1
4-one (6d)
(C-N), 1084 (C-O); H-NMR (DMSO-d6, 400 MHz):
: 3039 (Ar-H), 1637 (C=O), 1238 8.82 (d, 1H, Ar-H,
δ
=
FT-IR (KBr, cm−1)
ν
J
= 6.96 Hz), 7.89 (t, 1H, Ar-H,
J
1
(C-F), 1160 (C-N); H-NMR (DMSO-d6, 400 MHz):
8.80 (d, 1H, Ar-H, = 7.16 Hz), 8.04 (d, 2H, Ar-H,
7.96 Hz), 7.94 (d, 2H, Ar-H,
Ar-H, = 15.48 Hz), 7.55 (d, 1H, Ar-H,
7.28 (t, 1H, Ar-H,
12.76 Hz), 3.40 (m, 4H), 3.32 (t, 2H,
δ
6.68 Hz), 7.67 (d, 2H, Ar-H,
J
= 14.64 Hz), 7.58 (d, 1H,
= 7.0 Hz), 7.17
= 13.32 Hz),
= 13.32 Hz), 2.51 (s,
J
J
=
Ar-H, J = 8.88 Hz), 7.30 (t, 1H, Ar-H, J
J
= 7.84 Hz), 7.87 (t, 1H, (d, 2H, Ar-H,
J
= 11.80 Hz), 4.15 (t, 2H, J
J
J
= 9.0 Hz), 3.86-3.33 (m, 8H), 2.90 (t, 2H, J
J
= 13.56 Hz), 4.13 (t, 2H, Ar-H,
J
=
3H, CH3), 2.38 (s, 3H, OCH3); MS: 442.2; Anal. Calcd.
J
= 21.20 Hz), for C22H26N4O4S (%): C, 59.71; H, 5.92; N, 12.66.
2.90-2.86 (m, 4H), 2.36 (s, 3H, CH3); MS: 480.1; Anal. Found: C, 59.67; H, 5.85; N, 12.43.
Calcd. for C22H23F3N4O3S (%): C, 54.99; H, 4.82; N,
11.86. Found: C, 54.79; H, 4.67; N, 11.68.

54
L. Mallesha et al.
2-Methyl-3-{2-[4-(2-nitro-benzenesulfonyl)-pipera-
A known number of MDA-MB 231, K562, Colo-205,
zin-1-yl]-ethyl}-pyrido[1,2-a] pyrimidin-4-one (6i) and IMR-32 cells were transferred into 96 well plates
FT-IR (KBr, cm−1)
ν
: 3059 (Ar-H), 1637 (C=O), 1528 in a volume of 200 L of culture medium and incu-
µ
1
(NO2), 1156 (C-N); H-NMR (DMSO-d6, 400 MHz):
8.81 (d, 1H, Ar-H, = 7.04 Hz), 8.01 (d, 2H, Ar-H,
20.32 Hz), 7.87 (d, 1H, Ar-H,
Ar-H, = 14.20 Hz), 7.55 (d, 1H, Ar-H,
7.25 (t, 1H, Ar-H, = 4.04 Hz), 4.18 (t, 2H,
Hz), 3.42-3.14 (m, 8H), 2.92 (t, 2H, = 13.28 Hz), 2.40 of MTT reagent (diluted in culture medium, 5 mg/mL)
(s, 3H, CH3); MS: 457.1; Anal. Calcd. for C21H23N5O5S was added. After incubating for 4 h, the MTT/medium
(%): C, 55.13; H, 5.07; N, 15.31. Found: C, 55.31; H, was removed and DMSO (100 L) was added to each
δ
=
bated for 48 h before the addition of the test com-
pound. Cells were then exposed to known concentra-
J
J
J
= 1.08 Hz), 7.86 (t, 2H, tions of the compound to be tested (10
= 8.92 Hz), as final concentration) for 24 h at 37oC. After drug
= 13.28 exposure, the culture medium was removed and 20 ìL
µM expressed
J
J
J
J
J
µ
5.13; N, 15.17.
well and the plates were agitated for 1 min. Absorbance
of the colored solution was measured on a multi-well
plate reader (Victor3, Perkin Emler) using a test
3-{2-[4-(3-Methoxy-benzenesulfonyl)-piperazin-1-
yl]-ethyl}-2-methyl-pyrido[1,2-a] pyrimidin-4-one wavelength of 570 nm. Results were evaluated by com-
(6j)
paring the absorbance of the wells containing com-
: 3054 (Ar-H), 1635 (C=O), 1151 pound treated cells with the absorbance of wells con-
FT-IR (KBr, cm−1)
ν
1
(C-N), 1084 (C-O); H-NMR (DMSO-d6, 400 MHz):
δ
=
=
taining 0.1% DMSO alone (solvent control). Conven-
tionally, cell viability was estimated to be 100% in the
solvent control and the assay was performed in tri-
8.81 (d, 1H, Ar-H,
7.10 Hz), 7.87 (s, 1H, Ar-H), 7.58 (d, 1H, Ar-H,
9.11 Hz), 7.56 (d, 1H, Ar-H,
Ar-H, = 7.10 Hz), 7.24 (t, 1H, Ar-H,
(d, 1H, Ar-H, = 7.10 Hz), 4.16 (t, 2H,
3.78-3.31 (m, 8H), 2.90 (t, 2H, = 13.32 Hz), 2.50 (s,
3H, CH3), 2.38 (s, 3H, OCH3); MS: 442.2; Anal. Calcd.
J
= 7.04 Hz), 7.92 (t, 1H, Ar-H,
J
J
J
= 8.91 Hz), 7.31 (t, 1H, plicate.
= 7.0 Hz), 7.11
= 13.34 Hz),
J
J
J
J
RESULTS AND DISCUSSION
J
In the present work, a series of ten new compounds
for C22H26N4O4S (%): C, 59.71; H, 5.92; N, 12.66. were synthesized. The structure of the synthesized
Found: C, 59.62; H, 5.84; N, 12.61.
compounds was established on the basis of FT-IR, 1H-
NMR, and mass spectral data. The chemical structure
and physical data of the novel compounds are given in
Table I. The elemental analyses data showed good
Biology
Drugs and solutions
The 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetra- agreement between the experimentally determined
zolium bromide (MTT) was dissolved (5 mg/mL) in a values and the theoretically calculated values within ±
phosphate buffer saline (pH 7.2) and filtered before 0.4%. 2-Methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]
use. The RPMI 1640 cell culture medium, MTT and pyrimidin-4-one derivatives 6a-j were prepared by the
fetal bovine serum (FBS) were purchased from Merck method summarized in Scheme 1.
chemicals.
The FT-IR spectra of 6a-j were recorded using KBr
pellets in the range of 4000-400 cm−1. The absorption
bands at 3022-3090 cm−1 are assigned to the aromatic
Cell lines and culture conditions
Human metastatic breast cancer (MDA-MB 231), C-H stretch. The absorption bands at 1634-1638 cm−1
human chronic myeloid leukemia (K562), human colon are due to the presence of C=O stretch. The strong
carcinoma (Colo-205) and human neuroblastoma (IMR- bands at 1084 cm−1 are assigned to the C-O stretch in
32) cell lines were procured from the National Center 6h and 6j. The absorption band at 3365 cm−1 is due to
for Cell Sciences in Pune, India. All cells were grown the N-H stretch in compound
5. The absence of N-H
in RPMI-1640 supplemented with 10% heat inacti- absorption bands in 6a-j confirmed the synthesized
vated FBS, 100 IU/mL penicillin, 100 mg/mL strepto- compounds. The strong bands at 1323-1327 cm−1 and
mycin and 2 mM-glutamine. The cultures were main- 1156-1170 cm−1, were attributed to SO2 (asym. stretch)
tained in a humidified atmosphere with 5% CO2 at and SO2 (sym. stretch), respectively (Hadi et al., 2009).
37oC.
A new band appeared at 1238 cm−1 (6d) corresponding
to the C-F stretching frequency. The strong band at
720 cm−1 is assigned to the C-Cl stretch in 6b. The
In vitro cell viability assay (MTT assay)
The potential effects on cell viability were investi- absorptions at 1547 cm−1 and 1528 cm−1 corresponded
gated by using the MTT assay (Mosmann, 1983). The to NO2 (asym. stretch) in 6e and 6i, respectively.
1
MTT assay is an indicator of metabolically active cells.
The characteristic resonance peaks in H-NMR for

Synthesis and Anticancer Activity of Pyrimidines
55
Table I. Chemical structure and physical data of 6a-j
Compound
R
Structure
Yield (%)
75
m.p. (oC)
120-122
6a
phenyl
6b
6c
6d
6e
6f
4-chlorophenyl
2,4,6-trimethyl phenyl
trifluoromethyl phenyl
4-nitrophenyl
74
78
71
79
77
73
76
80
76
179-180
156-158
158-160
162-163
180-182
153-154
174-176
158-160
170-172
methyl
6g
6h
6i
4-methylphenyl
4-methoxyphenyl
2-nitrophenyl
6j
3-methoxyphenyl
the novel compounds were reported using DMSO. The multipletes at
δ 2.79-3.86 ppm (Shafiee et al., 2009).
expected resonances were assigned by their peak mul- The proton spectral data agree with respect to the
tiplicity and integration. The integration of spectra number of protons and their chemical shifts with the
shows good agreement with the synthesized com- proposed structures. The synthesized compounds were
pounds. The proton NMR spectral data of NH in
single resonance at 5.34 ppm, which is absent in the ion peak in the mass spectra.
spectra of 6a , indicating the replacement of the sul-
fonamide series. In addition, the resonance appearing
in the range of 7.07-8.90 ppm as singlets, doublets,
5 show further confirmed by the appearance of a molecular
δ
-j
Antiproliferative activity
Piperizine derivatives were shown to inhibit growth
δ
triplets and multiplates is attributed to the aromatic inhibition of human erythroleukemia K-562 cells and
protons. The piperazine protons were resonated as the presence of myeloid leukemia HL-60 cells has been

56
L. Mallesha et al.
Table II. Antiproliferative activity of 6a-j against human
cancer cells determined by MTT test
% Cell survival at10
µM
Compound
K562 Colo-205 MDA-MB 231 IMR-32
6a
6b
6c
6d
6e
6f
6g
6h
6i
−
−
−
*
*
*
51.27
46.50
59.56
60.61
63.20
30.19
41.32
53.99
62.75
36.23
100
53.24
51.53
55.14
65.58
62.27
41.49
49.52
54.59
61.25
37.61
100
35.71
47.43
50.25
85.45
64.38
33.45
46.48
49.44
61.83
49.56
100
35.58
46.51
−*
−*
−*
45.89
−*
100
6j
Control
(0.1% DMSO)
*Represents < 30% cell survival
Scheme 1. Reagent and reaction conditions: (i) Toluene,
PTSA, 12 h; (ii) POCl3, overnight; (iii) K2CO3, Piperazine,
DMF, 80oC, 6 h; (iv) sulphonyl chlorides, DCM, 8 h.
reported (Gillet et al., 1998). A new class of sulfonamides
has been used in the treatment of diseases arising
from abnormal cell growth and proliferation (Medina
et al., 1998). The antiproliferative action of compounds
6a-j was tested against four different cell lines. 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
Fig. 1. MTT assay for 6d, 6e, and 6i at 10 µM
pale yellow MTT to a blue formazan product which is compound 6d could be attributed to the presence of an
quantifiable spectrophotometrically. Percentage cell electron withdrawing trifluoromethyl group. However,
survival for tested compounds against MDA-MB 231, the presence of electron withdrawing groups in the
K562, Colo-205, and IMR-32 cells is tabulated in Table phenyl ring increased the antiproliferative efficacy.
II. The extent of inhibition of cell lines by 6d
,
6e, and From this work we were able to identify a few active
6i is schematically presented in Fig. 1.
molecules which are capable of inhibiting the growth
The results of the biological screening experiment of human cancer cell lines in vitro. The results were
revealed that within the library of compounds 6a j, expressed as percentage of cell proliferation compared
three compounds 6d 6e, and 6i showed good activity with cells in control (cells treated with vehicle, 0.1%
and the remaining compounds showed moderate ac- DMSO). The additional modification and diversifica-
tivity. Among the compounds 6a , compound 6d ex- tion of functional groups in order to improve the anti-
hibited 60.61%, 65.58%, and 85.45% (at 10 M) inhib- cancer activity is currently in progress.
itory activity against Colo-205, MDA-MB 231, and In conclusion, a series of novel 2-methyl-3-(2-pipera-
IMR-32 cell lines, respectively, whereas compounds zin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives
6d 6e, and 6i showed less inhibitory activity against 6a were synthesized and their antiproliferative
-
,
-j
µ
,
-j
K562. Compound 6e exhibited 63.20%, 62.27%, and activity has been evaluated. Antiproliferative assay
64.38% antiproliferation against Colo-205, MDA-MB results indicated that these derivatives have high
231, and IMR-32 cell lines, respectively. Similarly, antiproliferative activity against Colo-205, IMR-32,
compound 6i showed 62.75%, 61.25%, and 61.83% and MDA-MB 231. Compound 6d containing trifluoro-
antiproliferation against Colo-205, MDA-MB 231, and methyl group appeared to be the most active against
IMR-32 cell lines, respectively. The good inhibition by the IMR-32 and MDA-MB 231 cell lines. From the

Synthesis and Anticancer Activity of Pyrimidines
57
Crit. Rev. Oncol. Hematol., 59, 150-158 (2006).
experimental results, it could be concluded that the
introduction of the aryl sulfonyl moiety on the pipera-
zine system has significant potential to obtain novel
antiproliferative compounds. From this work, a few
Medina, J. C., Shan, B., Beckmann, H., Farrell, R. P., Clark,
D. L., Learned, R. M., Roche, D., Li, A., Baichwal, V.,
Case, C., Baeuerle, P. A., Rosen, T., and Jaen, J. C., Novel
antineoplastic agents with efficacy against multidrug
resistant tumor cells. Bioorg. Med. Chem. Lett., 8, 2653-
2656 (1998).
Mencher, S. K. and Wang, L. G., Promiscuous drugs com-
pared to selective drugs (promiscuity can be a virtue).
BMC Clin. Pharmacol., 5, 3-9 (2005).
active molecules (6d, 6e, and 6i) were identified which
are capable of inhibiting the growth of human cancer
cell lines in vitro. Hence, further investigations are
required to clarify the features underlying the anti-
proliferative activities of these new piperazine deriva-
tives.
Mosmann, T., Rapid colorimetric assay for cellular growth
and survival: Application to proliferation and cytotoxicity
assays. J. Immunol. Methods, 65, 55-63 (1983).
ACKNOWLEDGEMENTS
Reddy, N. S., Mallireddigari, M. R., Cosenza, S., Gumireddy,
K., Bell, S. C., Reddy, E. P., and Reddy, M. V., Synthesis
of new coumarin 3-(N-aryl) sulfonamides and their anti-
cancer activity. Bioorg. Med. Chem. Lett., 14, 4093-4097
(2004).
Rubenstein, S. M., Baichwal, V., Beckmann, H., Clark, D. L.,
Frankmoelle, W., Roche, D., Santha, E., Schwender, S.,
Thoolen, M., Ye, Q., and Jaen, J. C., Hydrophilic, pro-drug
analogues of T138067 are efficacious in controlling tumor
growth in vivo and show a decreased ability to cross the
blood brain barrier. J. Med. Chem., 44, 3599-3605 (2001).
Shafiee, A., Emami, S., Ghodsi, S., Najjari, S., Sorkhi, M.,
Samadi, N., Faramarzi, M. A., and Foroumadi, A., Synthe-
Of the authors, LM is grateful to the University
Grants Commission, New Delhi, for the financial
support under the UGC-RFSMS scheme, and would
like to thank the University of Mysore for the award
of Junior Research Fellowship.
REFERENCES
Chaudhary, P., Kumar, R., Verma, A. K., Singh, D., Yadav,
V., Chhillar, A. K., Sharma, G. L., and Chandra, R., Syn-
thesis and antimicrobial activity of
N-alkyl and N-aryl
piperazine derivatives. Bioorg. Med. Chem., 14, 1819-
1826 (2006).
sis and antibacterial activity of
N-[2-(2-naphthyl)ethyl]
piperazinyl quinolones. J. Iran. Chem. Soc., 6, 325-333
(2009).
Upadhayaya, R. S., Sinha, N., Jain, S., Kishore, N., Chandra,
R., and Arora, S. K., Optically active antifungal azoles:
Gillet, R., Jeannesson, P., Sefraoui, H., Amould-Guerin, M.
L., Kirkiacharian, S., Jardillier, J. C., and Pieri, F.,
Piperazine derivatives of butyric acid as differentiating
agents in human leukemic cells. Cancer Chemother.
Pharmacol., 41, 252-255 (1998).
Synthesis and antifungal activity of (2R,3S)-2-(2,4-difluo-
rophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-ethyl}-tetrazol-2-
yl/1-yl)-[1,2,4]-triazol-1-yl-butan-2-ol. Bioorg. Med. Chem.,
12, 2225-2238 (2004).
Verdecchia, A., Mariotto, A., Capocaccia, R., Gatta, G., Micheli,
A., Sant, M., and Berrino, F., Incidence and prevalence of
all cancerous diseases in Italy: Trends and implications.
Eur. J. Cancer, 37, 1149-1157 (2001).
Hadi, J. S., Alsalami, B. K., and Essa, A. H., Synthesis, spec-
troscopic characterization and theoretical study of Schiff
bases derived from phenylsulfonylamide. J. Sci. Res., 1,
563-568 (2009).
Hulme, C. and Cherrier, M. P., Novel applications of ethyl
glyoxalate with the ugi MCR. Tetrahedron Lett., 40, 5295-
5299 (1999).
Verweij, J. and De Jonge, M. J., Achievements and future of
chemotherapy. Eur. J. Cancer, 36, 1479-1487 (2000).
Jimeno, A. and Hidalgo, M., Multitargeted therapy: Can
promiscuity be praised in an era of political correctness?.