Synthesis and in vivo diuretic activity of some novel pyrimidine derivatives

Article abstract of DOI:10.3109/14756366.2011.557022

A series of 1,6-dihydropyrimidine-2-amine derivatives and 1,6-dihydropyrimidine-2-thiol derivatives were synthesized by the reaction of substituted 1,3-diphenylprop-2-en-1-one (chalcones) with guanidine hydrochloride and thiourea, respectively. All the sy

Full text of DOI:10.3109/14756366.2011.557022

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(6): 819–826
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.557022
ReseaRch aRtIcle
Synthesis and in vivo diuretic activity of some novel
pyrimidine derivatives
Jaseela Majeed and M. Shaharyar
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi-110062, India
abstract
Aseriesof1,6-dihydropyrimidine-2-aminederivativesand1,6-dihydropyrimidine-2-thiolderivativesweresynthesized
by the reaction of substituted 1,3-diphenylprop-2-en-1-one (chalcones) with guanidine hydrochloride and thiourea,
respectively. All the synthesized compounds were in good agreement with elemental and spectral data. The
synthesized compounds were screened in vivo for diuretic activity. The four compounds 2d, 2e, 3d and 3e, which
showed moderate to good diuretic activity, were evaluated for their toxicity studies and none of the compounds
showed any toxicity of the liver as compared with control. However, compounds 3eand 3d showed diuretic properties
more than that of standard (acetazolamide) and were long acting. Overall, compound 3e, 6-(2,6-dichlorophenyl)-4-
(pyridin-2-yl)-1,6-dihydropyrimidine-2-thiol, was found to be the most promising candidate of the series.
Keywords: Pyrimidines, diuretic activity, chalcones, toxicity studies
Introduction
therefore, there is an urgent clinical need for the devel-
opment of selective diuretics (high ceiling [loop]/
potassium sparing/osmotic) devoid of many of the
unpleasant side effects viz hypokalemia, hyperurice-
mia, hypercholestremia, etc. associated with current
diuretic regimens. Because of the lack of oral activity
and toxicities associated with current diuretic regimen,
research efforts were focused on the development of
orally effective agents. In medicinal chemistry, pyrimi-
dine derivatives have been very well known for their
therapeutic applications. e presence of a pyrimidine
base in thymine, uracil and cytosine, which are essential
building blocks of nucleic acids, DNA and RNA, is one
possible reason for their activity. ey possess broad
range of pharmacological activities such as anti-cancer5,
anti-viral6, anti-HIV7, anti-hypertensive8, anti-con-
vulsant9, anti-tubercular10, diuretic11, anti-bacterial12,
anti-fungal13 and anti-epileptics14 properties, and many
classes of chemotherapeutic agents containing pyrimi-
dine nucleus are in clinical use15. However, pyrimidines
are still least explored compounds for diuretic profile
although a few promising diuretic drugs possess this
ring16,17.Triamterene(2,4,7-triamino-6-phenylpteridine)
Hypertension is a very common chronic condition associ-
ated with increased mortality and multiple morbidities1.
Blood pressure (BP) is directly associated with risks of
several types of cardiovascular diseases, and the asso-
ciation of BP with disease risk are continuous with large
proportion of most populations having non-optimal
BP values2. Recently, in World Health Organization−
International Society of Hypertension guidelines for the
management of hypertension have also described the
importance of diuretics as one of the most valuable class
of drugs3. Diuretics reduce both systolic and diastolic
BPs in the great majority of hypertensive patients. ey
are as effective as most other antihypertensive drugs and
also enhance the antihypertensive efficacy of multidrug
regimens and can be useful in achieving BP control. Joint
National Committee on Prevention, Detection, Evaluation
and Treatment of High Blood Pressure (JNC 6) guidelines,
which were issued in 1997, and JNC 7 guidelines, which
wereissuedin2003,bothrecommenddiureticsasfirst-line
drugs in the treatment of uncomplicated hypertension4.
From the literature, it is clear that diuretic plays a
significant role in the management of hypertension;
Address for Correspondence: M. Shaharyar, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University,
New Delhi-110062, India. E-mail: yarmsy@yahoo.co.in
(Received 25 August 2010; revised 12 January 2011; accepted 14 January 2011)
819

820 J. Majeed and M. Shaharyar
is a mild, clinically effective potassium sparing diuretic
either used alone or as an adjunct to thiazide and loop
diuretics. It is orally active but has very poor water solu-
bility and may be contraindicated in people with kidney
problems, diabetes and in elderly patients.
until neutral. e resulting chalcone was purified by
recrystallization with ethanol.
Synthesis of 1,6-dihydropyrimidine-2-amine derivatives
(2a-f) A mixture of chalcone (1 mole), guanidine hydro-
chloride (1.5 mole) and sodium hydride (3.0 mole) in Di
Methyl Formamide (DMF) (100 mL) was refluxed for 6–8
hours. After completion of reaction, mixture was poured
into ice cold water, a solid so separated was filtered
and purified by column chromatography to afford pure
compounds.
Diuretic agents such as furosemide and triam-
terene have been reported for their hepatotoxicities18,19
erefore, the need of toxicity studies is significant.
.
Among the pyrimidines, aminopyrimidines and
thiopyrimidines are broadly found in bioorganic and
medicinal chemistry with applications in drug discovery
and developments. ey are reported to possess broad
spectrum of biological activities as well20,21. In pursuit
of this goal, it was proposed to carry out the synthesis
and biological screening of aforesaid heterocycles with
appropriate substitution, with improved efficacy and
decreased toxicity.
Synthesis of 1,6-dihydropyrimidine-2-thiol derivatives
(3a-f) A mixture of chalcone (0.01 mole), thiourea (0.012
mole) and sodium methoxide (0.025 mole) in ethanol
(30 mL) was refluxed for 3–6 hours. e reaction mixture
was concentrated and cooled. e solid separated out
was filtered and recrystallized from DMF or water.
Materials and methods
3-Phenyl-1-(pyridin-2-yl)prop-2-en-1-one (1a) Yield: 76%;
mp: 160–162°C; Rf: 0.9556; FTIR (KBr) υmax cm−1: 1726
(C=O), 1648 (CH=CH), 1576 (C=N); H NMR (CDCl3) δ
(ppm): 8.84 (2H, d, Ar H-3,5), 8.52 (1H, d, Ar H-6′), 8.41
(1H, m, Ar H-4), 8.01 (2H, d, Ar H-2,6), 7.94 (1H, m, Ar
H-4′), 7.86 (1H, d, Hβ), 7.76 (2H, d, Ar H-3’,5′),7.23 (1H,
d, Hα),. Anal. Calcd. for C14H11NO: C, 71.98; H, 5.64; N,
22.38. Found: C, 71.73; H, 5.61; N, 22.30.
All the chemicals used were laboratory grade and pro-
cured from E.Merck (Germany) and S.D Fine Chemicals
(India). Diagnostic kits for the estimation of biochemi-
cal parameters such as serum glutamic oxaloacetic
transaminase (SGOT) and serum glutamic pyruvic
transaminase (SGPT) were purchased from local sup-
plier manufactured by Ranbaxy Diagnostics Ltd., New
Delhi, India. Melting points were determined by open
tube capillary method and are uncorrected. in-layer
chromatography plates (silica gel G 60 F254, 0.2 mm)
were used to confirm the purity of commercial reagents
used, compounds synthesized and to monitor the
reaction. Two different solvent systems toluene:ethyl
acetate:formic acid (5:4:1, v/v/v) and benzene:acetone
(9:1, v/v) were used for thin-layer chromatography.
e spots were located under iodine vapors and ultra-
violet light. Infrared (IR) spectra were obtained on a
Perkin–Elmer 1720 Fourier transform infrared (FTIR)
spectrometer (KBr pellets). 1H NMR spectra were
recorded on a Bruker AC 400 MHz, spectrometer using
Tetra Methyl Silane (TMS) as an internal standard in
DMSO-d6/CDCl3. e FAB mass spectra were recorded
on a JEOLSX 102/DA-6000 Mass Spectrometer. All the
biochemical estimations were carried out using stan-
dard kits in semi auto-analyzer Screen Master 3000.
1
3-(4-Methoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one
(1b) Yield: 80%; mp: 128–130°C; Rf: 0.8837; FTIR (KBr)
υmax cm−1: 1734 (C=O), 1658 (CH=CH), 1582 (C=N), 1172
(–OCH3); 1H NMR (CDCl3) δ (ppm): 9.10 (1H, d, Ar H-6′),
8.65 (1H, m, Ar H-4′), 8.44–8.52 (2H, m, Ar H-3, 5), 8.10
(2H, d, Ar H-2,6), 8.04 (2H, d, Ar H-3′,5′), 7.95 (1H, d,
Hβ), 6.94 (1H, d, Hα), 3.84 (3H, s, OCH3). Anal. Calcd. for
C15H13NO2: C, 75.30; H, 5.48; N, 5.85. Found: C, 75.28; H,
5.46; N, 5.83.
3-(4-Fluorophenyl)-1-(pyridin-2-yl)prop-2-en-1-one
(1c) Yield: 84%; mp: 143–145°C; Rf: 0.9302; FTIR (KBr)
υmax cm−1:1727 (C=O), 1654 (CH=CH), 1533 (C=N); 1H
NMR (CDCl3) δ (ppm): 8.75 (1H, d, Ar H-6′), 8.65 (2H, d,
Ar H-3, 6), 8.24(1H, d, Hβ), 8.15 (1H, s, Ar H-2), 7.82 (1H,
m, Ar H-4′), 7.61 (1H, m, Ar H-5), 7.36 (2H, d, Ar H-3′,5′),
7.25 (1H, d, Hα),. Anal. Calcd. for C14H10FNO: C, 74.00; H,
4.44; N, 6.16. Found: C, 73.86; H, 4.41; N, 6.14.
Methods
Chemistry
3-(2-Chlorophenyl)-1-(pyridin-2-yl)prop-2-en-1-one
(1d) Yield: 81%; mp: 167-169°C; Rf: 0.8837; FTIR (KBr)
υmax cm−1: 1726 (C=O), 1642 (CH=CH), 1536 (C=N), 853
Synthesis of substituted 1,3-diphenylprop-2-en-1-ones
(1a-f) An equimolar mixture of 2-acetyl pyridine and
respective aryl aldehyde was stirred in ethanol (25mL).
To this mixture, an aqueous solution of NaOH (40%,
10 mL) was added at once and the reaction mixture was
stirred for 40 min at room temperature. e mixture
was kept overnight at room temperature, poured onto
crushed ice and then acidified with dilute HCl. e solid
separated was filtered and washed carefully with water
1
(C-Cl); H NMR (CDCl3) δ (ppm): 8.76 (1H, d, Ar H-6′),
7.92 (2H, d, Ar H-3,5), 7.68 (1H, d, Hβ), 7.66 (1H, m, Ar
H-4′),7.52 (2H, d, Ar H-3′,5′),7.42 (1H, d, Hα), 7.02 (2H, d,
Ar H-4,6). Anal. Calcd. for C14H10ClNO: C, 69.00; H, 4.14;
N, 5.75. Found: C, 68.89; H, 4.12; N, 5.73.
3-(2,6-Dichlorophenyl)-1-(pyridin-2-yl)prop-2-en-1-one
(1e) Yield: 68%; mp: 152–154°C; Rf: 0.9048; FTIR (KBr)
Journal of Enzyme Inhibition and Medicinal Chemistry

Diuretic activity of some novel pyrimidine derivatives 821
υmax cm−1: 1723 (C=O), 1648 (CH=CH), 1536 (C=N), 852
(C–Cl); H NMR (CDCl3) δ (ppm): 8.78 (1H, d, Ar H-6′),
8.34 (1H, d, Hβ), 7.98 (1H, m, Ar H-4′), 7.84 (2H, d, Ar
H-3,5), 7.56 (1H, d, Ar H-4), 7.46 (2H, d, Ar H-3′,5′),7.36
(1H, d, Hα). Anal. Calcd. for C14H9Cl2NO: C, 60.46; H,
3.26; N, 5.04. Found: C, 60.33; H, 3.24; N, 5.02.
ring-A), 5.92 (1H, d, H-6 pyrimidine proton), 5.81 (1H, d,
H-5 pyrimidine proton), 4.5 (2H, s, Ar–NH2). FABMS m/z:
285 (M+1). Anal. Calcd. for C15H13ClN4: C, 63.27; H, 4.60; N,
19.68. Found: C, 63.17; H, 4.57; N, 19.65.
1
6-(2,6-Dichlorophenyl)-4-(pyridin-2-yl)-1,6-dihydro-
pyrimidin-2-amine (2e) Yield: 76%; mp: 203–205°C; Rf:
0.7130; FTIR (KBr) υmax cm−1: 3410 (NH2), 3216 (NH),
3-(3,4-Dimethoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one
(1f) Yield: 74%; mp: 136–138°C; Rf: 0.7627; FTIR (KBr)
υmax cm−1: 1734 (C=O), 1647 (CH=CH), 1412 (C=N), 1173
(–OCH3); 1H NMR (CDCl3) δ (ppm): 9.14 (1H, d, Ar H-6′),
8.37 (1H, m, Ar H-4′), 8.11 (2H, d, Ar H-3′,5′), 7.86 (1H,
d, Hβ), 7.64 (1H, s, Ar H-2), 7.32 (2H, d, Ar H-5, 6), 7.11
(1H, d, Hα), 3.87–3.95 (6H, s, 2 × –OCH3). Anal. Calcd. for
C16H15NO3: C, 71.36; H, 5.61; N, 5.20. Found: C, 71.28; H,
5.56; N, 5.18.
1
3085 (ArH), 1612 (C=N); H NMR (DMSO-d6, in δ ppm):
8.4 (1H, d, Ar H-6′ ring-B), 7.87 (1H, d, NH pyrimi-
dine proton), 7.4–7.7 (3H, m, Ar H-3′,4′,5′ ring-B),7.4
(3H, m, Ar–H ring-A), 5.91 (1H, d, H-6 pyrimidine proton),
5.82 (1H, d, H-5 pyrimidine proton), 4.6 (2H, s, Ar–NH2).
FABMS m/z: 320 (M+1). Anal. Calcd. for C15H12Cl2N4:
C, 56.44; H, 3.79; N, 17.55. Found: C, 56.33; H, 3.77;
N, 17.54.
6-(3,4-Dimethoxyphenyl)-4-(pyridin-2-yl)-1,6-dihydro-
pyrimidin-2-amine(2f) Yield: 65%; mp: 236–238°C; Rf:
0.8336; FTIR (KBr) υmax cm−1: 3412 (NH2), 3201 (NH),
3096 (Ar–H), 1412 (C=N), 1170 (OCH3); 1H NMR (DMSO-
d6, in δ ppm): 8.7 (1H, d, Ar H-6′ ring-B), 7.84 (1H, d, NH
pyrimidine proton), 7.1–7.9 (3H, m, Ar H-3′,4′,5′ ring-B),
6.4 (3H, m, Ar–H ring-A), 5.92 (1H, d, H-6 pyrimidine
proton), 5.81(1H, d, H-5 pyrimidine proton), 4.5 (2H,
s, Ar–NH2), 3.8–3.93 (6H, s, 2 × OCH3). FABMS m/z: 308
(M−2). Anal. Calcd. for C17H18N4O2: C, 65.79; H, 5.85; N,
18.05. Found: C, 65.67; H, 5.81; N, 18.03.
6-Phenyl-4-(pyridin-2-yl)-1,6-dihydropyrimidin-2-amine
(2a) Yield: 63%; mp: 210–212°C; Rf: 0.8750; FTIR (KBr)
υmax cm−1: 3410 (NH2), 3201 (NH), 3096 (Ar–H), 1536
1
(C=N); H NMR (DMSO-d6, in δ ppm): 8.8 (1H, d, Ar
H-6′ ring-B), 8.1 (5H, m, Ar-H ring-A), 7.8 (1H, d, NH
pyrimidine proton), 7.1–7.9 (3H, m, Ar H-3′,4′,5′ ring-B),
5.93 (1H, d,H-6 pyrimidine proton), 5.81 (1H, d, H-5
pyrimidine proton), 4.6 (2H, s, Ar-NH2). FABMS m/z: 250
(M+). Anal. Calcd. for C15H14N4: C, 71.98; H, 5.64; N, 22.38.
Found: C, 71.84; H, 5.61; N, 22.37.
6-(4-Methoxyphenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidin-
2-amine (2b) Yield: 72%; mp: 216–218°C; Rf: 0.5908; FTIR
(KBr) υmax cm−1: 3412 (NH2), 3201 (NH), 3096 (Ar–H), 1584
(C=N), 1174 (OCH3); 1H NMR (DMSO-d6, in δ ppm): 9.12
(1H, d, Ar H-6′ ring-B), 8.09–8.68 (3H, m, Ar H-3′,4′,5′
ring-B), 7.81 (1H, d, NH pyrimidine proton), 6.6 (4H, m,
Ar–H ring-A), 5.92 (1H, d, H-6 pyrimidine proton), 5.81
(1H, d, H-5 pyrimidine proton), 4.6 (2H, s, Ar–NH2), 3.8
(3H, s, OCH3). FABMS m/z: 278 (M−2). Anal. Calcd. for
C16H16N4O: C, 68.55; H, 5.75; N, 19.99. Found: C, 68.33; H,
5.72; N, 19.97.
6-Phenyl-4-(pyridin-2-yl)-1,6-dihydropyrimidine-2-thiol
(3a) Yield: 81%; mp: 165–167°C; Rf: 0.9455; FTIR (KBr)
υmax cm−1: 3201 (NH), 3120 (C–H str, Aromatic), 2591
(SH), 1640 (C=N), 1593 (Aromatic C=C str), 1520 (C–N
1
str); H NMR (CDCl3, in δ ppm): 9.72 (1H, s, S–H), 7.74
(1H, d, NH pyrimidine proton), 6.7–8.1 (11H, m, aro-
matic protons). FABMS m/z: 267 (M+). Anal. Calcd. for
C15H13N3S: C, 67.39; H, 4.90; N, 15.72. Found: C, 67.29;
H, 4.86; N, 15.70.
6-(4-Methoxyphenyl)-4-(pyridin-2-yl)-1,6- dihydro-
pyrimidine-2-thiol (3b) Yield: 67%; mp: 152–154°C; Rf:
0.7223; FTIR (KBr) υmax cm−1: 3119 (C–H str, aromatic),
2585 (SH), 1651 (C=N), 1582 (aromatic C=C str), 1516
6-(4-Fluorophenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidin-2-
amine (2c) Yield: 78%; mp: 214–216°C; Rf: 0.8551; FTIR
(KBr) υmax cm−1: 3412 (NH2), 3201 (NH), 3096 (ArH), 1584
(C=N), 1174 (OCH3); 1H NMR (DMSO-d6, in δ ppm): 9.10
(1H, d, Ar H-6′ ring-B), 8.01–8.62 (3H, m, Ar H-3′,4′,5′
ring-B), 7.83(1H, d, NH pyrimidine proton), 6.7 (4H,
m, Ar- H ring-A), 5.94 (1H, d, H-6 pyrimidine proton),
5.82 (1H, d, H-5 pyrimidine proton), 4.6 (2H, s, Ar–NH2).
FABMS m/z: 269 (M+1). Anal. Calcd. for C15H13FN4: C,
67.59; H, 6.03; N, 19.70. Found: C, 67.48; H, 6.01; N,
19.68.
1
(C–N str); H NMR (CDCl3, in δ ppm): 9.68 (1H, s, S–H),
7.79 (1H, d, NH pyrimidine proton), 6.81–8.32 (10H, m,
aromatic protons), 3.72 (3H, s,−OCH3). FABMS m/z: 295
(M−2). Anal. Calcd. for C16H15N3OS: C, 64.62; H, 5.08; N,
14.13. Found: C, 64.57; H, 5.06; N, 14.11.
6-(4-Fluorophenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidine-2
-thiol (3c) Yield: 73%; mp: 186–188°C; Rf: 0.8778; FTIR
(KBr) υmax cm−1: 3082 (C–H str, aromatic), 2598 (S–H),
1
1635 (C=N), 1580 (aromatic C=C str), 1524 (C–N str); H
6-(2-Chlorophenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidin-2-
amine (2d) Yield: 68%; mp: 208–210°C; Rf: 0.8792; FTIR
(KBr) υmax cm−1: 3411 (NH2), 3215 (NH), 3089 (Ar–H), 1536
(C=N), 851 (C–Cl); 1H NMR (DMSO-d6, in δppm): 8.7 (1H,
d, Ar H-6′ ring-B), 7.8 (1H, d, NH pyrimidine proton),
7.5–7.9 (3H, m, Ar H-3′,4′,5′ ring-B), 6.8 (4H, m, Ar- H
NMR (CDCl3, in δ ppm): 9.13 (1H, s, S–H), 7.72 (1H, d,
NH pyrimidine proton), 6.52–8.18 (10H, m, aromatic pro-
tons). FABMS m/z: 286 (M+1). Anal. Calcd. for C15H12FN3S:
C, 63.14; H, 4.24; N, 14.73. Found: C, 63.04; H, 4.21;
N, 14.70.
© 2011 Informa UK, Ltd.

822 J. Majeed and M. Shaharyar
weight in 5 mL of 0.5% carboxy methyl cellulose + 0.9%
NaCl solution) and acetazolamide23–26 (45 mg/kg body
weight in 5 mL of 0.5% carboxy methyl cellulose + 0.9%
NaCl solution)27. e excreted urine was collected, mea-
sured and studied for cumulative urine output, diuretic
action, diuretic activity, Lipschitz value and electrolyte
excretion (Na+, K+ and Cl−). Sodium and potassium are
estimated by using lab model Mediflame photometer.
Chloride was estimated by titrating the urine by Volhards
method.
6-(2-Chlorophenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidine-2
-thiol (3d) Yield: 79%; mp: 173–175°C; Rf: 0.9303; FTIR
(KBr) υmax cm−1: 3122 (C–H str, aromatic), 2540 (S–H),
1618 (C=N), 1598 (aromatic C=C str), 1526 (C–N str), 751
(C–Cl); 1H NMR (CDCl3, in δ ppm): 9.99 (1H, s, S–H), 7.76
(1H, d, NH pyrimidine proton), 6.6–8.8 (10H, m, aro-
matic protons). FABMS m/z: 302 (M+1). Anal. Calcd. for
C15H12ClN3S: C, 59.70; H, 4.01; N,13.92. Found: C, 59.61;
H, 4.00; N, 13.89.
6-(2,6-Dichlorophenyl)-4-(pyridin-2-yl)-1,6-dihydro-
pyrimidine-2-thiol (3e) Yield: 64%; mp: 158–160°C; Rf:
0.8675; FTIR (KBr) υmax cm−1: 3125 (C–H str, aromatic),
2540 (S–H), 1614 (C=N), 1592 (aromatic C=C str), 1522
Assessment of liver function e compounds that have
exhibited excellent diuretic profile have been selected for
the study. e serum collected from the groups of albino
rats was used for estimation of biochemical parameters
to determine the functional state of the liver. SGOT and
SGPT were estimated by a ultraviolet kinetic method
based on the reference method of International federa-
tion of Clinical Chemistry28. Alkaline phosphatase was
estimated by using King method29. Total protein, albu-
min and globulin were also measured according to the
1
(C–N str), 753 (C–Cl); H NMR (CDCl3, in δ ppm): 9.71
(1H, s, S–H), 7.70 (1H, d, NH pyrimidine proton), 6.24–
8.64 (9H, m, aromatic protons). FABMS m/z: 337 (M+1).
Anal. Calcd. for C15H11Cl2N3S: C, 53.58; H, 3.30; N, 12.50.
Found: C, 53.46; H, 3.28; N, 12.47.
6-(3,4-Dimethoxyphenyl)-4-(pyridin-2-yl)-1,6-dihydro-
pyrimidine-2-thiol (3f) Yield: 61%; mp: 176–178°C; Rf:
0.9048; FTIR (KBr) υmax cm−1: 3086 (C–H str, aromatic),
2543 (S–H), 1618 (C=N), 1585 (aromatic C=C str), 1520
reported methods30,31
.
Statistical analysis
1
(C–N str); H NMR (CDCl3, in δ ppm): 9.56 (1H, s, S–H),
Results of biochemical estimation were reported as mean
SEM. e determination of significant inter-group dif-
ference was analyzed separately and one-way analysis of
variance was carried out32. Dunnett’s test was used for
individual comparisons33.
7.76 (1H, d, NH pyrimidine proton), 6.15–8.23 (9H, m,
aromatic protons), 3.8–3.93 (6H, s, 2 × OCH3). FABMS
m/z: 325 (M−2). Anal. Calcd. for C17H17N3O2S: C, 62.36; H,
5.23; N, 12.83. Found: C, 62.28; H, 5.21; N, 12.79.
Biological activity
Result and discussion
Diuretic activity was measured on healthy adult albino
rats weighing 180–200 g according to an adaptation of the
method of Lipschitz et al.22 Each group was comprised of
six animals (n= 6). ey were housed in standard envi-
ronmental conditions (temperature: 25–30°C). e rats
are fed with standard diet (Altromin® pellets) and water
ad libitum. Food and water are withdrawn 15 hours prior
to the experiment. Diuretic activity was measured by col-
lecting total excreted urine of rat kept in metabolic cages
designed to separate the urine and faeces. e cages
together with the funnel and measuring cylinder used
in the studies were coated with liquid paraffin before
each experiment to facilitate the collection of urine with
minimum loss. Each animal is placed in a metabolic
cage provided with a wire mesh bottom and a funnel to
collect the urine. Stainless-steel sieves are placed in the
funnel to retain feaces and to allow the urine to pass. Rats
were placed in metabolic cages individually as soon as
the treatments started. e urine sample was collected
for a total period of 5 h (urine collected initially of 20 min
was discarded). All the doses were administered with the
aid of an oral dosing needle. e test compounds were
administered orally at a dose of 45 mg/kg body weight in
5 mL of (0.5% carboxy methyl cellulose + 0.9% NaCl solu-
tion). Control group received 5mL of 0.9% NaCl solu-
tion per kilogram body weight. e test compounds are
compared with two standard diuretics, urea (1 g/kg body
Chemistry
e synthesis of chalcones from substituted benzalde-
hyde and 2-acetyl pyridine were carried out according
to the Claisen–Schmidt condensation34. e 1,6-dihy-
dropyrimidine-2-amine (2a-f) and 1,6-dihydropyrim-
idine-2-thiol (3a-f) derivatives were synthesized from
chalcones (1a-f) using guanidine hydrochloride and
thiourea, respectively. e synthetic sequences leading
to the formation of targeted compounds are depicted in
Scheme 1.
Diuretic activity
e in vivo diuretic activity of the synthesized compounds
is summarized in (Table 1) and (Table 3). Cumulative
urine excreted during 0–5 hours for each group (6 albino
rats) is a measure of urinary excretion. After 5 hours of
screening, the compounds 3e, 3d, 2e, 2d and 3c showed
goodcumulativeurineoutput. eurinaryoutputofcom-
pound 3e was highly significant 15.09 0.540 (P< 0.01),
i.e., increased by 300% with respect to control. Urinary
excretion of 3e was 301.80% and that of 3d, 2e, 2d and
3c lies in between urea (138.09%) and standard acetazo-
lamide (184.77%). Diuretic action was measured as the
ratio (%) of the total volume of urine excreted during the
5 hours following the administration of the test drug at a
dose of 45 mg/kg to the volume of urine excreted by the
Journal of Enzyme Inhibition and Medicinal Chemistry

Diuretic activity of some novel pyrimidine derivatives 823
R
CH
3
CHO
+
N
O
Claisen - Schmidt condensation
aq. NaOH
5'
4'
R
6'
1'
Hα
3'
N
2'
Ηβ
(1a-f )
O
NH2
NH2
H2N
H2N
sod methoxide
DMF,NaH
S
NH
R
A
R
B
N
B
N
A
N
NH
N
NH
NH2
( 2a-f )
SH
( 3a-f )
R= Phenyl; 4-Methoxyphenyl; 4-Flourophenyl; 2-Chlrophenyl; 2,6-Dichlorophenyl; 3,4-Dimethoxyphenyl
Scheme 1. Protocol of synthesis.
Table 1. Diuretic activity of amino and thio pyrimidine derivatives.
Diuresis
Dose (mg/Kg
Treatment body weight)
Total urinary
output (mL)
Normal saline
intake (mL)
Urinary
excretion (%) Diuretic action
Diuretic activity Lipschitz value
2a
2b
2c
2d
45
45
4.31 0.545
4.58 0.419
5.83 0.926
8.66 0.310*
9.08 0.864**
4.35 0.545
4.43 0.568
5.16 0.459
8.03 0.359*
11.56 0.844**
15.09 0.540**
6.28 0.612
5.65 0.353
7.54 0.436
8.98 0.523**
5.03 0.401
4.66 0.166
4.93 0.633
5.22 0.247
5.03 0.256
4.96 0.588
4.85 0.387
5.01 0.329
5.04 0.468
4.94 0.276
5.00 0.687
4.91 0.416
4.92 0.387
5.46 0.298
4.86 0.533
85.68
98.28
0.7
0.8
1.02
1.4
1.5
0.7
0.7
0.8
1.3
2.03
2.62
1.1
1.0
1.2
1.6
0.43
0.50
0.63
0.87
0.93
0.43
0.43
0.50
0.81
1.25
1.63
0.68
0.62
0.75
1.0
0.57
0.60
0.77
1.14
1.20
0.57
0.58
0.68
1.06
1.53
2.001
0.83
—
45
118.25
165.90
180.51
87.70
45
2e
45
2f
3a
3b
3c
45
45
91.34
45
102.99
159.32
234.00
301.80
127.90
114.83
138.09
184.77
45
3d
45
3e
45
3f
45
CONT
UREA
ACET
__
1000
45
1.0
1.19
Each value represents the mean SEM (n=6).
*P<0.05, **P<0.01 (Dunnett’s multiple comparison test).
CONT=control, ACET=acetazolamide.
© 2011 Informa UK, Ltd.

824 J. Majeed and M. Shaharyar
saline control. e diuretic action of 3e and 3d was 2.62
and 2.03, respectively, in comparison with 1.2 and 1.6 for
urea and acetazolamide, respectively. is activity was
evaluated in terms of daily diuresis and was measured as
the ratio of the diuretic action of the treated groups to the
diuretic action of the standard (acetazolamide) group. As
far as diuretic activity is concerned, compounds 3e and
3d was found to be 1.63 and 1.25, respectively, while 2e,
2d and 3c were calculated as 0.93, 0.87 and 0.81, respec-
tively (Table 1).
e Lipschitz value (the ratio T/U, in which T is the
response of the test compound, and U, that of urea
treatment, indices of 1.0 and higher are regarded as a
positive effect in terms of diuretic activity) shows that
3e is two times potent than urea as far as urinary out-
put is concerned, and compounds 3d, 2e, 2d and 3c
were nearly equal to urea (>1.0, means positive effects)
(Table 1).
Compounds (2a-f) and (3a-f) were also tested for
saluretic and kaliuretic effects in albino rat model. After
0–5 hours, compounds 3e, 2e and 3d showed a significant
increase in sodium excretion (P<0.01), i.e. 3.68 0.284,
2.99 0.150 and 2.83 0.429, respectively, which was either
almostsimilarormorethanstandards,i.e.urea(2.74 0.154)
and acetazolamide (3.36 0.294). e Na+ excretion of 2d
was also significant as compared with urea (P<0.05), i.e.
2.56 0.278 and 2.74 0.154, respectively (Table 2).
Compounds 3e and 2e were also found to have sig-
nificant kaliuretic property (P< 0.01), i.e. 1.72 0.156
and 1.480 0.405, respectively, similar to acetazolamide
(1.523 0.118) and with regards to Na+/K+ ratio, it was
observed that 3e is a stronger kaliuretic. Table 2 showed
that compounds 3a, 2b and 3f were potassium sparing
(3.6, 3.4 and 3.3, respectively), while in rest of the com-
pounds Na+/K+ ratio lies between 1.8 and 2.1, i.e. more
kaliuretic than potassium sparing.
Chloride was estimated by titrating the urine by
Volhards method. Chloride excretion was also increased
to similar extent of sodium.
e extent of hepatic damage was assessed by the
level of various biochemical parameters in circulation.
Table 3 shows the liver function tests with reference to
selected compounds. e estimation revealed that there
was no significant increase in SGOT and SGPT alkaline
phosphatase, and there is a decrease in protein level in
serum as compared with the control level (Table 3). It was
clearly indicated that none of the compound showed any
toxicity of the liver as compared with control.
To summarize, an attempt to obtain an efficacious
and non-toxic diuretic, we have designed and synthe-
sized a series of pyrimidine derivatives. e structure–
activity relationship (SAR) of pyrimidine diuretics may
be rationalized by assuming that the newly synthesized
compounds may possess an important site that involves
Table 2. Effects of amino and thio pyrimidine derivatives on electrolyte excretion.
Electrolyte concentration (m Eq/L)
Dose (mg/kg
body weight)
Treatment
2a
2b
2c
2d
Na+
K+
Cl−
Na+/K+
2.05
3.4
Na+/Cl−
1.85
1.83
1.93
1.93
2.10
1.64
1.97
1.77
1.94
2.0
45
45
45
45
45
45
45
45
45
45
45
45
—
0.37 0.278
1.82 0.150
1.12 0.089
2.56 0.278
2.99 0.150*
1.23 0.149
1.96 0.404
0.87 0.253
1.01 0.117
2.83 0.429*
3.68 0.284**
1.22 0.255
0.90 0.194
2.74 0.154
3.36 0.294**
0.18 0.468
0.53 0.574
0.62 0.104
1.34 0.465
1.48 0.405*
0.63 0.117
0.53 0.131
0.48 0.255
0.58 0.121
1.42 0.428*
1.72 0.156**
0.36 0.307
0.38 0.428
0.91 0.426
1.523 0.118**
0.20 0.128
0.99 0.133
0.58 0.187
1.32 0.614
1.42 0.895
0.75 0.140
0.99 0.686
0.49 0.151
0.52 0.946
1.41 0.382
1.78 0.454
0.43 0.224
0.44 0.174
0.93 0.564
1.62 0.407
1.80
1.91
2.02
1.95
3.6
2e
2f
3a
3b
3c
1.81
1.74
1.99
2.13
3.38
2.36
3.01
2.206
3d
3e
2.15
2.83
2.04
2.94
2.07
3f
CONT
UREA
ACET
1000
45
*P<0.05, ** P<0.01 (Dunnett’s multiple comparison test).
CONT=control, ACET=acetazolamide.
Table 3. Enzyme estimation of selected compounds.
Alkaline phosphatase
Total protein
SEM
Compound
SEM
SGOT SEM
31.33 0.26
31.87 0.21
33.77 0.68
32.66 0.24
31.98 0.73
SGPT SEM
27.37 0.48
27.57 0.34
27.19 0.90
26.18 0.58
26.89 0.97
Albumin SEM
3.88 0.16
3.93 0.68
3.46 0.39
4.36 0.24
3.76 0.48
Globulin SEM
2.34 0.88
2.86 0.33
2.67 0.49
2.13 0.94
2.43 0.68
Control
2d
2e
3d
3e
20.06 0.48
20.66 0.56
20.78 0.38
19.46 0.45
20.48 0.06
7.10 0.25
6.95 0.53
6.53 0.42
6.02 0.28
6.43 0.36
Values are given in mean SEM (n=6).
Journal of Enzyme Inhibition and Medicinal Chemistry

Diuretic activity of some novel pyrimidine derivatives 825
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Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.However, authors are thankful to Vice Chancellor,
Jamia Hamdard for providing the necessary facilities.
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