1. INTRODUCTION:

Pyrimidine, being an integral part of DNA and RNA, imparts diverse pharmacological properties such as effective bactericide and fungicide.^1,2Certain pyrimidine derivatives are also known to possess antimalarial,³ antifilarial,⁴antibacterial, antifungal,^5-6anticonvulsant⁷and antihistamine⁸activity. Some of the 3,4-dihydropyrimidines (DHPM) have emerged as integral backbones of several calcium channel blockers, antihypertensive agents, adrenergic and neuropeptide antagonist.⁹Several natural marine products containing the 3,4–dihydropyrimidine core have been reported in the literature with interesting biological activities such as the anti-HIV alkaloid batzelladine B.^10,11

Fused pyrimidine derivatives have attracted attention of numerous researchers over many years, due to their important biological activities. Preclinical data from literature survey indicated that the heterocycles in association with the pyrimidine has shown good antimicrobial,^12-14antioxidant,¹⁴antitumour,¹⁵ analgesic,anti-inflammatory^16,17 and antipyretic¹⁷activities.In particular, pyrimidines^18-23 derivatives were found as potent antimicrobial agent.

Motivated by the above mentioned findings and on continuation of our investigation,²⁴ to discover new potentially active agents, we have synthesized some new pyrimidines. All the newly synthesized compounds were characterized by spectroscopic techniques and evaluated for their in vitro antifungal activity.

2. EXPERIMENTAL:

Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification. Melting points were determined by Micro control based melting point instrument and are uncorrected. All reactions were monitored by thin-layer chromatography on 0.25 mm silica gel (60GF-254) plates, using ethyl acetate: butanol: chloroform in the ratio of [1:2:1] as mobile phase and visualized with UV light. Column chromatography was performed on silica gel (200-300 mesh). Infra red (IR) spectra was recorded by using KBr pellet on a Thermo Nicolate IR-400FTIR spectrophotometer, ¹H NMR spectra was recorded on Bruker Avance-400F spectrometer (400 MHz) using tetramethylsilane as internal standard (chemical shift in δ ppm) and LC-MS with Waters Micromass Q-Tof Micro.

2.1 Chemistry

2.1.

2.1.1. Synthesis of 4-(3-Nitrophenyl)-6-oxo-2-sulfanyl-1,6-dihydropyrimidine-5-carbonitrile (1)

Compound 1 was synthesized according to reported method.²⁵

2.1.2.

2.1.3. Synthesis of 2-(3-Nitrophenyl)-4,6-dioxo-6,11-dihydro-4H-pyrimido[2,1-b]quinazoline-3-carbonitrile (2)

A mixture of 1 (0.01 mol), anthranilic acid (0.01 mol) and sodium ethoxide (0.01 mol) in ethanol was heated under reflux for 8 h. The reaction mixture was cooled and then poured on ice cold water and acidified with hydrochloric acid. The produced solid was filtered off, dried and recrystalized from DMF/Water to give compound 2.

Reaction time: 8 h; Rf value: 0.72; Yield: 58%; M.p. 206^oC; IR, cm^–1: 3374 (NH), 3075 (Ar-CH), 2888 (Aliphatic CH), 2241 (C≡N), 1701 (C=O), 1552 (C=N), 1529 (C=C), 1352 (C-NO₂), 1228 (C-N-c); ¹H NMR (DMSO-d₆) δ: 7.85-8.56 (m, 8H, Ar-H), 13.23 (s, 1H, NH); MS, m/z: 360.2 (M+1).

2.1.4. 7-(3-Nitrophenyl)-3,5-dioxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carbonitrile (3)

A mixture of 1 (0.01 mol), chloroacetic acid (0.01 mol), and fused sodiuim acetate in acetic acid (15 ml) was heated under reflux for 9 h and left to cool. The reaction mixture was then diluted with water, shaken well, and allowed to stand for 6 h. The residue was triturate with ethanol; solid product was filtered off and recrystallized from ethanol/water (1:1) to give 3.

Reaction time: 9 h; Rf value: 0.66; Yield: 49%; M.p. >250 ^oC; IR, cm^–1: 3087 (Ar-CH), 2907 (Aliphatic CH), 2221 (C≡N), 1689 (C=O), 1528 (C=N), 1481 (C=C), 1348 (C-NO₂), 1207 (C-N-c), 1098 (C-S-c); ¹H NMR (DMSO-d₆) δ: 3.94 (s, 2H, CH₂), 7.26-8.35 (m, 4H, Ar-H); MS, m/z: 315.2 (M+1).

2.1.5. Synthesis of 4-(substituted)-1-methyl-2-(methylsulfanyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (4a-d)

Compounds 4a-d were synthesized according to reported method.²⁵

2.1.6. Synthesis of 2-hydrazinyl-4-(substituted)-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (5a-d)

A mixture of compound 4a-d (10 mmols) and hydrazine hydrate (0.96 g, 30 mmols) in absolute alcohol was refluxed till completion of the reaction. The reaction mixture was poured into crushed ice, product was isolated and crystallized using suitable solvents.

2.1.6.1. 2-Hydrazinyl-4-(3-hydroxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile(5a)

Reaction time: 6 h; Rf value: 0.57; Yield: 74%; M.p. >250 ^oC; IR, cm^–1: 3339 (OH), 3299 (NH-NH₂), 2932 (Ar-CH), 2932 (Aliphatic CH), 2207 (C≡N), 1653 (C=O), 1526 (C=N), 1526 (C=C); ¹H NMR (DMSO-d₆) δ: 3.19 (s, 3H, N-CH₃), 4.68 (s, 1H, NH), 6.89-7.49 (m, 4H, Ar-H), 8.58 (s, 1H, OH), 9.17 (s, 2H, NH₂); MS, m/z: 258.1 (M+1).

2.1.6.2. 2-Hydrazinyl-4-(4-hydroxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (5b)

Reaction time: 6 h; Rf value: 0.53; Yield: 66%; M.p. >250 ^oC; IR, cm^–1: 3471 (OH), 3326 (NH-NH₂), 2915 (Ar-CH), 2835 (Aliphatic CH), 2209 (C≡N), 1577 (C=O), 1518 (C=N), 1518 (C=C); ¹H NMR (DMSO-d₆) δ: 3.16 (s, 3H, N-CH₃), 4.77 (s, 1H, NH), 6.80 (d, 2H, Ar-H), 7.85 (d, 2H, Ar-H), 9.14 (s, 2H, NH₂), 9.97 (s, 1H, OH); MS, m/z: 258.1 (M+1), 259.1 (M+2).

2.1.6.3. 2-Hydrazinyl-1-methyl-4-(3-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile(5c)

Reaction time: 5 h; Rf value: 0.60; Yield: 69%; M.p. 247^oC; IR, cm^–1: 3312 (NH-NH₂), 2907(Ar-CH), 2907 (Aliphatic CH), 2223 (C≡N), 1664 (C=O), 1609 (C=N), 1539 (C=C), 1280 (C-NO₂); ¹H NMR (DMSO-d₆) δ: 43.19 (s, 3H, N-CH₃), 7.78-8.36 (m, 4H, Ar-H), 8.72 (s, 1H, NH), 9.25 (s, 1H, NH); MS, m/z: 287.0 (M+1),288.1 (M+2).

2.1.6.4. 2-Hydrazinyl-1-methyl-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile(5d)

Reaction time: 6 h; Rf value: 0.58; Yield: 71%; M.p. 197^oC; IR, cm^–1: 3301 (NH-NH₂), 3015 (Ar-CH), 2935 (Aliphatic CH), 2200 (C≡N), 1599 (C=O), 1524 (C=N), 1524 (C=C), 1347 (C-NO₂); ¹H NMR (DMSO-d₆) δ: 3.17 (s, 3H, N-CH₃), 8.11 (d, 2H, Ar-H), 8.32 (d, 2H, Ar-H), 9.18 (s, 2H, NH₂), 10.02 (s, 1H, NH); MS, m/z: 287.1 (M+1),288.1 (M+2).

2.2. Antimicrobial activity

2.2.1. In vitro antifungal activity

All the synthesized compounds were screened for their antifungal activity against Aspergillusniger and Candida albicans in DMF by the disc diffusion method.²⁶The sterile media (Potato Dextrose Agar Media, 15 ml) in each petri plates was uniformly smeared with cultures of fungi. Sterile discs of 6 mm diameter were placed in the petri plates, to which 100 µl solution of synthesized compounds in DMF at different concentrations such as 250, 500 and 1000 µg/ml (25, 50, 100 µg/disc) was added. The treatment also included 100 µl of DMF as negative control and amphotericin B as positive control ^27,28 for comparison. The plates were incubated at 26 ± 2 ^oC for 48-72 h and the zone of inhibition was determined.

3. RESULTS AND DISCUSSION:

3.1. Chemistry

In the present work, the title compounds, 1 and 4 were synthesized according to reported method. Reflux of 1and anthranilic acid in ethanol in the presence of sodium ethoxideyielded, 2-(3-Nitrophenyl)-4,6-dioxo-6,11-dihydro-4H-pyrimido[2,1-b]quinazoline-3-carbonitrile(2).

Additionally, reflux of 1 with chloro acetic acid in acetic acid in the presence of sodium acetate yielded, 7-(3-Nitrophenyl)-3,5-dioxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carbonitrile (3) (Scheme 1). Nucleophilic substitution by hydrazine of the compounds 4a-d were carried out by refluxing with hydrazinehydrate to yield 2-hydrazinyl-4-(substituted)-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (5a-d) (Scheme 2).

Scheme 1

Scheme 2

Table 1. Antifungal activities of compounds^a

Compounds	*C. albicans*			*A. niger*
Compounds	25 µg	50 µg	100 µg	25 µg	50 µg	100 µg
Amphotericin-B	13	15	23	15	17	26
2	13	16	19	11	14	18
3	12	15	19	11	13	16
5a	10	13	16	09	12	14
5b	11	14	17	10	13	15
5c	13	14	18	12	14	16
5d	11	13	16	10	13	15

a = zone of inhibition in mm

3.2. Spectral Elucidation

All the pyrimidine derivatives were synthesized and confirmed by physical data, IR, ¹H NMR and Mass spectral data. All the compounds have shown C≡N peak in the range of 2241–2200 cm^-1, C=O peak in the range of 1701–1577cm^-1, C=N peak in the range of 1609–1518cm^-1 and C=C peak in the range of 1539–1481 cm^-1 in IR. The phenolic derivatives, 5a and 5b have shown the OH peak at 3339 and 3471 cm^-1, respectively. The nitro derivatives have shown C-NO₂peak in the range of 1352–1280 cm^-1. The compound 3does not shown for the NH stretch in IR, which is absent in 2a-d compounds. The compounds2has shown C-S-C peak. Compounds, 5a-dhave shown peak in the range of 3471–3301 cm^-1for NHNH₂.

All the compounds have shown peaks for Ar-H in the range of δ 6.80–8.56 in ¹H NMR. The phenolic derivatives, 5a and 5b,have shown singlet at δ 8.58 and δ 9.97 for Ar-OH in ¹H NMR, respectively. Additionally, ¹H NMR of 2 and 3 is lacking for SHpeak and having NH peak. The 5a-d derivatives have shown peak for NH and NH₂in ¹H NMR.All the synthesized derivatives have shown M+1 peak in mass spectra. The IR, ¹H NMR and Mass spectral data supported the structure of various synthesized compounds.

3.3. Antimicrobial activity

The newly synthesized compounds were screened for their antifungal activity against fungi (Candida albicansand Aspergillusniger). The results of preliminary antifungal testing of the compounds were reported as zone of inhibition (Table 1). The results of preliminary antifungal testing revealed that compounds 2 and 3 are showing good activity against both species of fungi. Compounds 2 exhibited potent activity against both the species. Polar substitution like hydrazine may reduce the antifungal activity.

4. CONCLUSION:

In conclusion, we have described simple and efficient protocol for the synthesis of pyrimidine derivatives (2, 3 and 5) with good yields. All the synthesized compounds (1-5) have been investigated for their in vitro antifungal activities. In the newly synthesized compounds, it is clear that the highest antifungal activity was observed in compounds 2 and 3. Polar substitution like hydrazine may reduce the antifungal activity. To summarize with, we found that the novel class of pyrimidines have emerged as a valuable lead. Few of synthesized compounds might be useful as antifungal agents in future. These new pyrimidine derivatives have proved to be promising candidates for further efficacy evaluation.

5. CONFLICT OF INTEREST:

None.

6. ACKNOWLEDGMENT:

The authors are thankful to administration of Sri Adichunchanagiri College of Pharmacy, B. G. Nagara for providing laboratory facilities. Authors are also grateful to IISC, Bangalore, India and Panjab University, Chandigarh for providing spectral analysis data. Additionally, CM Bhalgat is thankful to ICMR for providing Fellowship.

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Received on 29.10.2014 Modified on 01.11.2014

Asian J. Research Chem. 7(11): November, 2014; Page 905-908