1 Introduction

The pyrimidine and its derivatives have a wide variety of applications in different fields, prominently for biological treatment process,[1‐8]such as antitumor, anticancer, anti-inflammatory, anti-hypertensive, antibacterial and antifungal agents, etc. The five-membered heterocyclic ring substituted in pyrimidine derivatives have moderate inhibition against the bacterial species.[9] In order to improve the antibacterial activity, we have synthesized twenty pyrimidine derivatives.

The pyrimidine and its derivatives have been synthesized by using various methods. In continuation of this work, novel pyrimidine derivatives were synthesized by using the reported procedure for the first step.[10,11]These pyrimidine derivatives have a large number of reactive sites to produce the substituted derivatives.[12‐18]

Herein, the reaction was carried out only in the 5 th position of the pyrimidine ring, because the ethanolic group which is present in the position 5 is easily removed by boiling in presence of catalyst and hydrazine carbothioamide.[19,20] The synthesized 10 derivatives of 5-(5-amino-1, 3, 4-thiadiazole-2-yl)-3,4-dihydro-6-methyl-4-phenyl-pyrimidin-2(1H)-one and 10 derivatives of 3,4-dihydro-5-(5-mercapto-4H-1,2,4-triazol-3-yl)-6-methyl-4-phenylpyrimidin-2(1H)-one are subjected to in vitro study of antibacterial activities by using three different species namely, Pseudomonas aeruginosa (Gram –ve), Staphylococcus aureus (Gram + ve) and Escherichia coli (Gram –ve). The inhibition values are compared with standard drug, ciprofloxacin. All the synthesized compounds were characterized by using elemental analysis, mass spectra, 1H and 13C NMR spectra.

2 Experimental

Melting points were determined using open capillary method and are uncorrected. The compounds are checked for homogeneity by TLC on silica gel-G using pet ether and ethyl acetate as eluent in 3:5 ratio. The IR spectra were recorded on FT-IR THERMO NICOLET AVATAR 370 spectrometer using KBr disc. The 1H and 13C NMR were recorded on Bruker Avance-III 400 MHz - NMR spectrometer using DMSO- d 6. Elemental analyses were recorded on elemental vario EL III instrument. The mass spectra were recorded on Joel GC-mate spectrometer. All the compounds gave satisfactory micro analytical results.

2.1 General Procedure

The synthesize 5-(hydrazine carbothioamide)-3,4-dihydro-6-methyl-4-phenyl pyrimidin-2(1H)-one (Scheme 1) 2(a–j), an equimolar mixture of compound 1a (2.61 g, 0.01 mol) and thiosemicarbazide (0.91 g, 0.01 mol) in acetone was refluxed for 10–12 h and allowed to cool. The yellow crude solid was purified by recrystallization from alcohol. M.p.: 139–141 ∘C. [Yield: 2.4 g, 82%] Analysis: Calculated (%) for C13 H 15 O 2 N 5S: C, 51.17; H, 4.94; N, 22.50; S, 10.47. Found (%): C, 51.10; H, 4.85; N, 22.24; S, 10.94. GCMS: m/z 305 [M +]. FT-IR (KBr, cm −1): 3365, 3241, 3116 (NH), 3079 (Ar–H), 2978 (CH), 1724 (C =O), 1385 (C–N), 1219 (C =S), 1089 (N–N). 1H NMR (400 MHz, DMSO- d 6): δ 2.251 (s, 3H), 5.152 (d, J = 3.2 Hz, 1H), 6.501 (s, 2H), 7.213–7.336 (m, 5H), 7.702 (d, J = 2.8 Hz, 1H), 8.175 (d, J = 6.4 Hz, 2H,), 9.149 (s, 1H). 13C NMR (400 MHz, DMSO- d 6): δ 17.72, 59.17, 99.33, 126.21, 127.23, 128.34, 148.25, 151.71, 152.16, 165.33, 178.40.

Scheme 1
figure 1

Synthesis of 5-(hydrazine carbothioamide)-3,4-dihydro-6-methyl-4-phenyl pyrimidin-2 (1H)-one 2(a–j).

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2.2 General procedure for synthesis of 5-(5-amino-1, 3, 4-thiadiazole-2-yl)-3, 4-dihydro-6-methyl-4-phenyl- pyrimidin-2(1H)-one (Scheme 2), 3(a–j)

Hydrazine carbothioamide 2a (3.05 g, 0.01 mol) was dissolved in 5 mL conc. H2 SO 4. This solution was stirred at RT and left overnight. It was then poured in crushed ice. The resulting suspension was kept in ammoniacal water for 2 h, filtered and purified by recrystallization from alcohol as white crystals. M.p.: 174–176 ∘C. [Yield: 2.7 g; 81%]. 1H NMR (400 MHz, DMSO- d 6): δ 2.258 (s, 3H), 4.004 (s, 2H), 5.159 (d, J = 3.2 Hz, 1H), 7.227–7.347 (m, 5H), 7.701 (d, J = 2 Hz, 1H), 9.151 (s, 1H). 13C NMR (400 MHz, DMSO- d 6): δ 17.03, 59.15, 99.30,126.20, 127.20, 128.34, 144.82, 148.27, 152.10, 165.32. FT-IR (KBr, cm −1): 3354, 3227, 3110 (NH), 3027 (Ar–H), 2976 (CH), 1689 (C =O), 1460 (C =N), 1225 (C–S), 1378 (C–N), 1098 (N–N). GCMS: m/z 287 [M +]. Analysis: Calculated (%) for C13 H 13 ON 5S: C, 54.38; H, 4.56; N, 24.39; S, 11.13. Found (%): C, 54.35; H, 4.56; N, 24.64; S, 11.68.

2.3 General procedure for Synthesis of 3,4-dihydro-5-(5-mercapto-4H-1,2,4-triazol-3-yl)-6-methyl-4-phenyl pyrimidin-2(1H)-one (Scheme 3) 4(a–j)

The carbothioamide 2a (3.05 g, 0.01 mol) was added into (8 g in 100 mL) 8% NaOH; it was refluxed for 4 h. The reaction mixture was cooled to room temperature and acidified with dilute acetic acid, then filtered and washed well with water and purified by recrystallization from alcohol as shiny crystals. M.p.:119–121 ∘C, [Yield: 2.42 g; 80%]. 1H NMR (400 MHz, DMSO- d 6): δ 2.304 (s, 3H), 3.217 (s, 1H), 5.507 (d, J = 3.6 Hz, 1H), 6.975 (s, 1H), 7.268–7.338 (m, 5H), 7.766 (d, J = 2.4 Hz, 1H), 9.217 (s, 1H). 13C NMR (400 MHz, DMSO- d 6): δ 17.74, 59.14, 99.24, 126.22, 127.21, 128.33, 144.84, 148.29, 152.15, 155.11, 165.32. FT-IR (KBr, cm −1): 3423 (NH), 3027 (Ar–H), 2968 (CH), 2235 (SH), 1654 (C =O), 1590 (C =N), 1373 (C–N), 1057 (N–N). GCMS: m/z 287 [M +]. Analysis: Calculated (%) for C13 H 13 ON 5S: C, 54.38; H, 4.56; N, 24.39; S, 11.13. Found (%): C, 54.41; H, 4.22; N, 24.35; S, 11.53.

3 Results and Discussion

Compounds 3(a–j) and 4(a–j) were synthesized as per the Schemes 1, 2 and 3. The final compound 3a was prepared by the reaction of hydrazine carbothioamide 2a, conc.H2 SO 4 and NH3, whereas 4a was prepared by refluxing hydrazine carbothioamide 2a with NaOH. Hydrazine carbothioamide 2a was synthesized by reaction of pyrimidine ethyl ester 1 with thiosemicarbazide in acetone, followed by condensation reaction.

Scheme 2
figure 2

Synthesis of 5-(5-amino-1, 3, 4-thiadiazole-2-yl)-3, 4-dihydro-6-methyl-4-phenyl pyrimidin-2(1H)-one 3(a–j).

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Scheme 3
figure 3

Synthesis of 3,4-dihydro-5-(5-mercapto-4H-1,2,4-triazol-3-yl)-6-methyl-4-phenyl pyrimidin-2(1H)-one 4(a–j).

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The pyrimidine ethyl ester 1a was prepared by the reaction of benzaldehyde, ethylacetoacetate and urea or thiourea in the presence of mineral acid, followed by Biginelli reaction. The structures of the synthesized compounds were confirmed by IR, 1H and 13C NMR, GC-MS and CHN analysis. Formation of 2a was confirmed by the presence of N–H stretching peaks at 3365, 3241 cm −1 and 3116 cm −1 and C =O stretching peaks at 1724 cm −1 in IR and singlet at δ 6.50 for NH2 group in 1H NMR spectrum.

Treatment of compound 2a with conc. H2 SO 4 and NH3, furnished 5-(5-amino-1,3,4-thiadiazol-2-yl)-3,4-dihydro-6-methyl-4-phenyl pyrimidin-2(1H)-one (3a). The structure of 3a was elucidated on the basis of C-S linkage in the thiadiazole ring, which causes a sharp absorption band at 1225 cm −1 in its IR spectrum. 1H NMR spectrum shows a singlet at δ 4.00 due to NH2 functional group of the compound 3a. The mechanism for compounds 3(a–j) is shown in Scheme 4.

Scheme 4
figure 4

Mechanism of 5-(5-amino-1, 3, 4-thiadiazole-2-yl)-3, 4-dihydro-6-methyl-4-phenyl pyrimidin-2(1 H)-one 3(a–j).

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The IR and 1H NMR spectral data reveal that the carbonyl absorption band at 1689 cm −1 of NH–CO–NH group, N–N stretching band at 1098 cm −1, aliphatic C–H and aromatic C–H stretching at 2976 cm −1 and 3027 cm −1, in the pyrimidine compound 3a. Mass spectrum also supports the proposed structure by the presence of molecular ion peak at m/z 287 M +.

The structure of (4a) was elucidated on the basis of C–N linkage in the triazole ring, which causes an absorption band at 1373 cm −1 in its IR spectrum. 1H NMR spectrum shows a singlet at δ 3.21 due to SH functional group of compound 4a. The IR and 1H NMR spectral data reveal that the carbonyl absorption band at 1654 cm −1 of NH–CO–NH group, N–N stretching band at 1053 cm −1, aliphatic C–H and aromatic C–H stretching at 2968 cm −1 and 3027 cm −1, in the group of pyrimidine compound (4a). Molecular ion peak at m/z 287 M + in the mass spectrum also supports the proposed structure. The mechanism of compounds 4(a–j) is shown in Scheme 5.

Scheme 5
figure 5

Mechanism of 3,4-dihydro-5-(5-mercapto-4H-1,2,4-triazol-3-yl)-6-methyl-4-phenyl pyrimidin-2(1H)-one 4(a–j).

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All these compounds were screened for antibacterial activity by pseudomonas aeruginosa (Gram –ve), staphylococcus aureus (Gram + ve) and escherichia coli (Gram –ve). Ciprofloxacin was used as standard drug. Most of the synthesized compounds showed moderate to good inhibition at a concentration of 10 μg/mL.

3.1 Antibacterial studies

The newly synthesized pyrimidine derivatives were screened for their antibacterial activity in vitro against pseudomonas aeruginosa, staphylococcus aureus and escherichia coli, using agar well disk diffusion method. The test compounds were dissolved in DMSO to get a solution of 10 μg/mL concentration. The inhibition zones were measured in millimeters at the end of an incubation period of 18 h at 37 ∘C. Ciprofloxacin was used as a standard drug and the results are shown in Tables 1 and 2. The investigation of antibacterial screening data reveals that, all the tested compounds show moderate to good inhibition at 10 μg/mL concentration.

Table 1 Antibacterial activity of compounds 3(a–j).
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Table 2 Antibacterial activity of compounds 4(a–j).
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3.2 Comparison of anti bacterial activity

Comparison of antibacterial activity for all the synthesised compounds are shown in Figures S1–S3 (in Supplementray Information). Based on the comparative studies, 5-(5-amino-1,3,4-thiadiazol-2-yl)-3,4-dihydro-6- methyl-4-phenylpyrimidin-2(1H)-one, 3(a–j) compounds have less inhibition than the 3,4-dihydro-5-(5-mercapto- 4H-1,2,4-triazol-3-yl)-6-methyl-4-phenylpyrimidin-2(1H)- one 4(a–j) compounds at 10 μg/mL concentration. A few compounds showed very good inhibition, which are closer to the standard drug.

4 Conclusions

The investigation of antibacterial screening data for synthesized compounds reveal that, the 4(a–j) triazole substituted compounds have higher inhibition than the 3(a–j) thiadiazole substituted compounds, because the triazole ring which is substituted in the pyrimidine, enhances the inhibition of the compound, against the three species of pseudomonas aeruginosa, staphylococcus aureus and escherichia coli.