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Journal of Enzyme Inhibition and Medicinal Chemistry Volume 26, 2011 - Issue 6
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Research Article

Synthesis and in vivo diuretic activity of some novel pyrimidine derivatives

Jaseela MajeedDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi-110062, India.
&
M. ShaharyarDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi-110062, India.Correspondence[email protected]
Pages 819-826 | Received 25 Aug 2010, Accepted 14 Jan 2011, Published online: 07 Mar 2011

Abstract

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 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 3e and 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.

Introduction

Hypertension is a very common chronic condition associated with increased mortality and multiple morbiditiesCitation1. Blood pressure (BP) is directly associated with risks of several types of cardiovascular diseases, and the association of BP with disease risk are continuous with large proportion of most populations having non-optimal BP valuesCitation2. 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 drugsCitation3. Diuretics reduce both systolic and diastolic BPs in the great majority of hypertensive patients. They 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 were issued in 2003, both recommend diuretics as first-line drugs in the treatment of uncomplicated hypertensionCitation4.

From the literature, it is clear that diuretic plays a significant role in the management of hypertension; therefore, there is an urgent clinical need for the development of selective diuretics (high ceiling [loop]/potassium sparing/osmotic) devoid of many of the unpleasant side effects viz hypokalemia, hyperuricemia, 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, pyrimidine derivatives have been very well known for their therapeutic applications. The 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. They possess broad range of pharmacological activities such as anti-cancerCitation5, anti-viralCitation6, anti-HIVCitation7, anti-hypertensiveCitation8, anti-convulsantCitation9, anti-tubercularCitation10, diureticCitation11, anti-bacterialCitation12, anti-fungalCitation13 and anti-epilepticsCitation14 properties, and many classes of chemotherapeutic agents containing pyrimidine nucleus are in clinical use15. However, pyrimidines are still least explored compounds for diuretic profile although a few promising diuretic drugs possess this ringCitation16,Citation17. Triamterene (2,4,7-triamino-6-phenyl pteridine) 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 solubility and may be contraindicated in people with kidney problems, diabetes and in elderly patients.

Diuretic agents such as furosemide and triamterene have been reported for their hepatotoxicitiesCitation18,Citation19. Therefore, 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. They are reported to possess broad spectrum of biological activities as wellCitation20,Citation21. 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.

Materials and methods

All the chemicals used were laboratory grade and procured from E.Merck (Germany) and S.D Fine Chemicals (India). Diagnostic kits for the estimation of biochemical parameters such as serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were purchased from local supplier manufactured by Ranbaxy Diagnostics Ltd., New Delhi, India. Melting points were determined by open tube capillary method and are uncorrected. Thin-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. The spots were located under iodine vapors and ultraviolet 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. The FAB mass spectra were recorded on a JEOLSX 102/DA-6000 Mass Spectrometer. All the biochemical estimations were carried out using standard kits in semi auto-analyzer Screen Master 3000.

Methods

Chemistry

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 (25 mL). 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. The mixture was kept overnight at room temperature, poured onto crushed ice and then acidified with dilute HCl. The solid separated was filtered and washed carefully with water until neutral. The 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 hydrochloride (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.

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. The reaction mixture was concentrated and cooled. The solid separated out was filtered and recrystallized from DMF or water.

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); 1H 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.

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.

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 (C-Cl); 1H 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) υmax cm−1: 1723 (C=O), 1648 (CH=CH), 1536 (C=N), 852 (C–Cl); 1H 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.

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.

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 (C=N); 1H 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-(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.

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 δ ppm): 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 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.

6-(2,6-Dichlorophenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidin-2-amine (2e)

Yield: 76%; mp: 203–205°C; Rf: 0.7130; FTIR (KBr) υmax cm−1: 3410 (NH2), 3216 (NH), 3085 (ArH), 1612 (C=N); 1H NMR (DMSO-d6, in δ ppm): 8.4 (1H, d, Ar H-6′ ring-B), 7.87 (1H, d, NH pyrimidine 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-dihydropyrimidin-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-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 str); 1H NMR (CDCl3, in δ ppm): 9.72 (1H, s, S–H), 7.74 (1H, d, NH pyrimidine proton), 6.7–8.1 (11H, m, aromatic 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-dihydropyrimidine-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 (C–N str); 1H 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), 1635 (C=N), 1580 (aromatic C=C str), 1524 (C–N str); 1H 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 protons). 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.

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, aromatic 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-dihydropyrimidine-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 (C–N str), 753 (C–Cl); 1H 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-dihydropyrimidine-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 (C–N str); 1H NMR (CDCl3, in δ ppm): 9.56 (1H, s, S–H), 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

Diuretic activity was measured on healthy adult albino rats weighing 180–200 g according to an adaptation of the method of Lipschitz et al.Citation22 Each group was comprised of six animals (n = 6). They were housed in standard environmental conditions (temperature: 25–30°C). The 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 collecting total excreted urine of rat kept in metabolic cages designed to separate the urine and faeces. The 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. The 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. The 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 solution). Control group received 5 mL of 0.9% NaCl solution per kilogram body weight. The test compounds are compared with two standard diuretics, urea (1 g/kg body weight in 5 mL of 0.5% carboxy methyl cellulose + 0.9% NaCl solution) and acetazolamideCitation23–26 (45 mg/kg body weight in 5 mL of 0.5% carboxy methyl cellulose + 0.9% NaCl solution)Citation27. The excreted urine was collected, measured 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.

Assessment of liver function

The compounds that have exhibited excellent diuretic profile have been selected for the study. The 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 federation of Clinical ChemistryCitation28. Alkaline phosphatase was estimated by using King methodCitation29. Total protein, albumin and globulin were also measured according to the reported methodsCitation30,Citation31.

Statistical analysis

Results of biochemical estimation were reported as mean ± SEM. The determination of significant inter-group difference was analyzed separately and one-way analysis of variance was carried out32. Dunnett’s test was used for individual comparisonsCitation33.

Result and discussion

Chemistry

The synthesis of chalcones from substituted benzaldehyde and 2-acetyl pyridine were carried out according to the Claisen–Schmidt condensationCitation34. The 1,6-dihydropyrimidine-2-amine (2a-f) and 1,6-dihydropyrimidine-2-thiol (3a-f) derivatives were synthesized from chalcones (1a-f) using guanidine hydrochloride and thiourea, respectively. The synthetic sequences leading to the formation of targeted compounds are depicted in .

Scheme 1.  Protocol of synthesis.

Scheme 1.  Protocol of synthesis.

Diuretic activity

The in vivo diuretic activity of the synthesized compounds is summarized in () and (). 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 good cumulative urine output. The urinary output of compound 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 acetazolamide (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 saline control. The 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. This 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, respectively ().

Table 1.  Diuretic activity of amino and thio pyrimidine derivatives.

The 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 output is concerned, and compounds 3d, 2e, 2d and 3c were nearly equal to urea (>1.0, means positive effects) ().

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 almost similar or more than standards, i.e. urea (2.74 ± 0.154) and acetazolamide (3.36 ± 0.294). The 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.  Effects of amino and thio pyrimidine derivatives on electrolyte excretion.

Compounds 3e and 2e were also found to have significant 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. showed that compounds 3a, 2b and 3f were potassium sparing (3.6, 3.4 and 3.3, respectively), while in rest of the compounds 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.

The extent of hepatic damage was assessed by the level of various biochemical parameters in circulation. shows the liver function tests with reference to selected compounds. The 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 (). It was clearly indicated that none of the compound showed any toxicity of the liver as compared with control.

Table 3.  Enzyme estimation of selected compounds.

To summarize, an attempt to obtain an efficacious and non-toxic diuretic, we have designed and synthesized a series of pyrimidine derivatives. The structure–activity relationship (SAR) of pyrimidine diuretics may be rationalized by assuming that the newly synthesized compounds may possess an important site that involves a basic centre of the drug, which may be N-1 or N-3 of pyrimidine nucleus or both. Groups that decrease the basic strength of pyrimidine nucleus reduce the diuretic activity. The other site involves the substituted phenyl ring at position 6, which may be hydrophobic in nature. This assumption is further supported in the structure of triamterene and its derivativesCitation35. To conclude, the SAR based on the observed results indicated that the type of aryl group substitution attached to the position 6 of pyrimidine nucleus plays a significant role for diuretic activity. It has been noticed that the substitution of the phenyl group at the position 6 of pyrimidine heterocycle with a chlorine atom seems more favorable for an active diuretic agent than the case of using a methoxy residue. Additional research on the mechanisms of these compounds and modification is underway.

The preliminary in vivo diuretic studies suggested the following SAR,

  1. Compound 3e prototype of series-1 possess strong aquaretic and saluretic activity when given orally in a single dose. However, compound 2e with NH2 group showed comparable diuretic action with acetazolamide, whereas compound 3e with–SH group showed improved activity, as compared with 2e and acetazolamide.

  2. Substitution at position 6 of pyrimidine ring system with electron-withdrawing group (Cl, F or Br) increases significant urinary excretion.

  3. Compounds with electron-releasing group such as −CH3 or −OCH3 substitution at any position of pyrimidine ring reduces diuresis.

In short, the diuretic activity may be increased by incorporating strong electron-withdrawing and large size substituent on the phenyl ring. The toxicity studies also indicated that the selected compounds are safe for administration. The results obtained from the in vivo diuretic studies demonstrate the potential of searching for diuretic agents among the amino and thiopyrimidine derivatives.

Declaration of interest

The authors report no conflicts of interest. The 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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