Design, synthesis and antimicrobial evaluation of pyrimidin-2-ol/thiol/amine analogues
BackgroundPyrimidine is an aromatic heterocyclic moiety containing nitrogen atom at 1st and 3rd positions and play an important role to forms the central core for different necessity of biological active compounds, from this facts, we have designed and synthesized a new class of pyrimidin-2-ol/thiol/amine derivatives and screened for its in vitro antimicrobial activity.Results and discussionThe synthesized pyrimidine derivatives were confirmed by IR, 1H/13C-NMR, Mass spectral studies and evaluated for their in vitro antimicrobial potential against Gram positive (S. aureus and B. subtilis), Gram negative (E. coli, P. aeruginosa and S. enterica) bacterial strains and fungal strain (C. albicans and A. niger) by tube dilution method and recorded minimum inhibitory concentration in µM/ml. The MBC and MFC values represent the lowest concentration of compound that produces in the range of 96–98% end point reduction of the used test bacterial and fungal species.ConclusionIn general all synthesized derivatives exhibited good antimicrobial activity. Among them, compounds 2, 5, 10, 11 and 12 have significant antimicrobial activity against used bacterial and fungal strains and also found to be more active than the standard drugs.Graphical abstractPyrimidine is an aromatic heterocyclic moiety containing nitrogen atom at 1st and 3rd positions and play an important role to forms the central core for different necessity of biological active compounds, from this facts, we have designed and synthesized a new class of pyrimidin-2-ol/thiol/amine derivatives and screened for its in vitro antimicrobial activity. The synthesized pyrimidine derivatives were confirmed by IR, 1H/13C-NMR, Mass spectral studies and evaluated for their in vitro antimicrobial potential against Gram positive (S. aureus and B. subtilis), Gram negative (E. coli, P. aeruginosa and S. enterica) bacterial strains and fungal strain (C. albicans and A. niger) by tube dilution method and recorded minimum inhibitory concentration in µM/ml. The MBC and MFC values represent the lowest concentration of compound that produces in the range of 96–98% end point reduction of the used test bacterial and fungal species.
<!>Background<!><!>Determination of MBC/MFC<!><!>Experimental section<!>Step i: synthesis of substituted chalcone (intermediate-I)<!>Step ii: synthesis of 4-(substituted phenyl)-6-(4-nitrophenyl)pyrimidin-2-ol/thiol/amine derivatives<!>4-(2-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (1)<!>4-(2-Chlorophenyl)-6-(4-nitrophenyl)pyrimidine-2-thiol (2)<!>4-(3-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (3)<!>4-(3-Chlorophenyl)-6-(4-nitrophenyl)pyrimidine-2-thiol (4)<!>4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (5)<!>4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidine-2-thiol (6)<!>4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-ol (7)<!>4-(3-Methoxyphenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (8)<!>4-(4-Methoxyphenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (9)<!>4-Bromo-2-(2-mercapto-6-(4-nitrophenyl)pyrimidin-4-yl)phenol (10)<!>4-(3-Bromophenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (11)<!>4-(2,4-Dichlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (12)<!>4-(4-(Dimethylamino)phenyl)-6-(4-nitrophenyl)pyrimidin-2-ol (13)<!>Conclusion
<p>Pyrimidine is an aromatic heterocyclic moiety containing nitrogen atom at 1st and 3rd positions and play an important role to forms the central core for different necessity of biological active compounds, from this facts, we have designed and synthesized a new class of pyrimidin-2-ol/thiol/amine derivatives and screened for its in vitro antimicrobial activity. The synthesized pyrimidine derivatives were confirmed by IR, 1H/13C-NMR, Mass spectral studies and evaluated for their in vitro antimicrobial potential against Gram positive (S. aureus and B. subtilis), Gram negative (E. coli, P. aeruginosa and S. enterica) bacterial strains and fungal strain (C. albicans and A. niger) by tube dilution method and recorded minimum inhibitory concentration in µM/ml. The MBC and MFC values represent the lowest concentration of compound that produces in the range of 96–98% end point reduction of the used test bacterial and fungal species.</p><!><p>Antimicrobial agents are one of the most important weapons in the resistance of infection caused by bacterial strains. In the past few years, increase the resistance of microorganisms toward antimicrobial agents become a serious health problem so there is a need of safe, potent and novel antimicrobial agents [1]. Pyrimidine aromatic heterocyclic moiety containing nitrogen atom at 1st and 3rd positions and play an important role to forms the central core for different necessity of biological active compounds [2]. Pyrimidine is the structural unit of DNA and RNA which play an imperative role in various existence progressions. Pyrimidines are present among the three isomeric diazines. Most abundant pyrimidine is uracil, cytosine and thymine [3]. These derivatives are also known as m-diazine or 1,3-diazone can be regarded as cyclic amine and shows the various biological activities i.e. antiviral [4, 5]; anticancer [6]; antimicrobial [7]; anti-inflammatory [8]; analgesic [9]; antioxidant [10]; antimalarial [11].</p><p>Pyrimidine is used as parent substance for the synthesis of a wide variety of heterocyclic compounds and raw material for the synthesis of new molecule [12]. Pyrimidine ring complexes with different heterocyclic moiety found to be an essential part of natural products agrochemicals and veterinary products. A large measure of antimicrobial drugs such as ciprofloxacin, chloramphenicol, griseofulvin and nystatin are available for bacterial and fungal infections [13].</p><!><p>Design of proposed pyrimidine derivatives based on literature survey</p><p>a, b Synthesis of 4-(substituted phenyl)-6-(4-nitrophenyl)pyrimidin-2-ol/thiol/amine derivatives</p><p>The physicochemical properties of synthesized 4-(substituted phenyl)-6-(4-nitrophenyl) pyrimidin-2-ol/thiol/amine derivatives</p><p>aTLC mobile phase-benzene</p><p>Antimicrobial activity (MIC = µM/ml) of synthesized 4-(substituted phenyl)-6-(4-nitrophenyl) pyrimidin-2-ol/thiol/amine derivatives</p><!><p>After recorded the MIC results of the synthesized compounds in concentration of (50, 25, 12.5, 6.25, 3.125, 1.56) µM/ml against microbial species i.e. Gram positive bacteria (S. aureus and B. subtilis), Gram negative bacteria (E. coli, P. aeruginosa and S. enterica) and fungal strain (C. albicans and A. niger) then their minimum bactericidal concentration (MBC) and fungicidal concentration (MFC) were determined by petri dish method using nutrient agar media (antibacterial) and sabouraud dextrose agar media (antifungal) by subculturing 100 μl of culture from each test tube that remained clear in the MIC determination into fresh medium. The MBC and MFC values represent the lowest concentration of compound that produces in the range of 96–98% end point reduction of the used test bacterial and fungal species [19].</p><!><p>Structural requirements for the antimicrobial activity of the synthesized derivatives</p><p>Presence of electron withdrawing group (–Cl, Compounds 2, 5 and 12) on benzylidene portion improved the antimicrobial activity of the synthesized compounds against S. aureus, E. coli, B. subtilis and C. albicans.</p><p>Presence of electron withdrawing group (–Br, Compound 11) improved the antifungal activity of the synthesized compounds against A. niger.</p><p>Using 5-bromo-2-hydroxybenzaldehyde (Compound 10) improved the antibacterial activity of the synthesized compounds against Gram negative S. enterica and P. aeruginosa.</p><p>NO2 group presence on benzylidene portion of acetophenone play an important role to enhanced the antimicrobial activity against bacterial and fungal microorganism.</p><!><p>Starting materials were obtained from commercial sources and were used without any type of further purification. The completion of the chemical reaction was observed by thin layer chromatography (TLC) making use of silica gel G plates of 0.5 mm thickness as stationary phase and benzene as mobile phase for final compounds. Melting points of final compounds were determined by open capillary tubes method. The molecular structures of the compounds were characterized by 1H/13C-NMR (CDCl3, δ ppm), FT-IR and Mass spectral studies. The Mass spectral data were confirmed by Waters Micromass Q-ToF Micro instrument. 1H nuclear magnetic resonance (1H-NMR) spectra was recorded on Bruker Avance 400 MHz spectrometer in appropriate CDCl3 solvents and are expressed in parts per million (δ, ppm) downfield from tetramethyl silane (internal standard). 1H-NMR data are given as multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet) and number of protons. Infrared (IR) spectra were recorded on Bruker 12060280, Software: OPUS 7.2.139.1294 spectrometer in the range of 400–4000 using KBr pellets and the value of λ max were reported in cm−1.</p><!><p>The reaction mixture of 1-(4-nitrophenyl)ethanone (0.01 mol) and corresponding aldehyde (0.01 mol) were stirred for 2–3 h in methanol (5–10 ml) followed by drop wise addition of sodium hydroxide solution (10 ml 40%) with constant stirring at room temperature. Then reaction mixture was taken overnight at room temperature and then was poured into ice cold water and acidified with hydrochloric acid and the precipitated substituted chalcone was filtered, dried and recrystallized from methanol [20].</p><!><p>The solution of substituted chalcone (0.01 mol) [synthesized in "Step i: synthesis of substituted chalcone (intermediate-I)"] in methanol (50 ml) was added with 0.01 mol of potassium hydroxide and 40 ml of 0.25 M solution of thiourea/urea/guanidine hydrochloride and refluxed for 3–4 h. The reaction mixture was then cooled and acidified with few drops of hydrochloric acid (20 ml of 0.5 M solution) and the resultant precipitate 4-(substituted phenyl)-6-(4-nitrophenyl)pyrimidin-2-ol/thiol/amine was separated dried and recrystallized from methanol.</p><p>Spectral analysis determined by FT-IR (KBr pellets, cm−1) and 1H/13C-NMR (CDCl3, δ ppm).</p><!><p>M. Formula: C16H11ClN4O2; Yield: 75.00%; MS ES + (ToF): m/z 326 [M+ + 1]; IR (KBr pellets, cm−1): 2931 (C–H str.), 1596 (C=C str.), 700 (C–C str.), 1688 (C=N str. or N=CH str., pyrimidine ring), 1344 (C–N str., pyrimidine), 754 (C–Cl str.), 1521 (NO2 asym str.), 854 (C–N str., Ar–NO2), 3379 (NH2 asym str.); 13C-NMR (CDCl3-d
6, δ, ppm): 163.4, 163.6, 160.1, 148.3, 139.8, 132.4, 130.1, 129.2, 128.3, 127.4, 121.7, 95.2; 1H-NMR (CDCl3, δ, ppm): 7.13–8.25 (m, 8H, Ar–H), 6.71 (s, 1H, CH of pyrimidine ring), 4.2 (s, 2H, NH2).</p><!><p>M. Formula: C16H10ClN3O2S; Yield: 84.72%; MS ES + (ToF): m/z 343 [M+ + 1]; IR (KBr pellets, cm−1): 2858 (C–H str.), 1596 (C=C str.), 703 (C–C str.), 1665 (C=N str.), 1342 (C–N str., pyrimidine), 753 (C–Cl str.), 1521 (NO2 asym str.), 698 (C–N str., Ar–NO2), 2627 (S–H str.), 621 (C–S str.); 13C-NMR (CDCl3-d
6, δ, ppm): 182.4, 163.5, 163.2, 160.1, 147.3, 139.6, 132.2, 130.1, 129.6, 128.3, 127.4, 121.7, 106.1; 1H-NMR (CDCl3, δ, ppm): 7.35–8.34 (m, 8H, Ar–H), 8.40 (s, 1H, CH of pyrimidine ring), 3.01(s, 1H, SH).</p><!><p>M. Formula: C16H11ClN4O2; Yield: 78.78%; MS ES + (ToF): m/z 326 [M+ + 1]; IR (KBr pellets, cm−1): 2923 (C–H str.), 1607 (C=C str.), 703 (C–C str.), 1670 (C=N str.), 1351 (C–N str., pyrimidine), 732 (C–Cl str.), 1525 (NO2 asym str., phenyl ring), 674 (C–N str., Ar–NO2), 3387 (NH2 asym str.); 13C-NMR (CDCl3-d
6, δ, ppm): 163.2, 160.1, 147.2, 138.6, 132.0, 134.3, 130.1, 129.2, 128.1, 127.4, 125.3, 121.7, 95.3; 1H-NMR (CDCl3, δ, ppm): 7.26–9.02 (m, 8H, Ar–H), 6.0 (s, 1H, CH of pyrimidine ring), 4.3 (s, 2H, NH2).</p><!><p>M. Formula: C16H10ClN3O2S; Yield: 72.54%; MS ES + (ToF): m/z 343 [M+ + 1]; IR (KBr pellets, cm−1): 2991 (C–H str.), 1570 (C=C str.), 709 (C–C str.), 1701 (C=N str. pyrimidine ring), 1303 (C–N str.), 748 (C–Cl str.), 1521 (NO2 asym str.), 659 (C–N str., Ar–NO2), 2597 (S–H str.), 709 (C–S str.); 13C-NMR (CDCl3-d
6, δ, ppm): 181.4,163.5,163.2,160.1,146.3, 139.6, 132.2,130.1,129.6, 128.3,127.4, 125.3, 121.7, 103.1; 1H-NMR (CDCl3, δ, ppm): 7.83–8.25 (m, 8H, Ar–H), 7.41 (s, 1H, CH of pyrimidine ring), 3.06 (s, 1H, SH).</p><!><p>M. Formula: C16H11ClN4O2; Yield: 82.22%; MS ES + (ToF): m/z 326 [M+ + 1]; IR (KBr pellets, cm−1): 2942 (C–H str.), 1598 (C=C str.), 703 (C–C str.), 1673 (C=N str.), 1346 (C–N str., pyrimidine), 755 (C–Cl str.), 1523 (NO2 asym str.), 822 (C–N str., Ar–NO2), 3349 (NH2 asym str.); 13C-NMR (CDCl3-d
6, δ, ppm): 162.2, 160.1, 146.2, 138.6, 131.0, 134.3 130.1, 129.2, 128.1, 127.4, 124.3, 121.7, 96.3; 1H-NMR (CDCl3, δ, ppm): 7.33–8.34 (m, 8H, Ar–H), 7.85 (s, 1H, CH of pyrimidine ring), 4.14 (s, 2H, NH2).</p><!><p>M. Formula: C16H10ClN3O2S; Yield: 75.00%; MS ES + (ToF): m/z 343 [M+ + 1]; IR (KBr pellets, cm−1): 2927 (C–H str.), 1596 (C=C str.), 709 (C–C str.), 1345 (C–N str., pyrimidine), 824 (C–Cl str.), 1480 (NO2 asym str.), 698 (C–N str., Ar–NO2), 645 (C–S str.), 2602 (S–H str.); 13C-NMR (CDCl3-d
6, δ, ppm): 182.4, 163.2, 163.2, 161.1, 148.3, 139.6, 131.2, 130.1, 129.6, 128.3, 126.4, 121.7, 103.4; 1H-NMR (CDCl3, δ, ppm): 7.83–8.25 (m, 8H, Ar–H), 7.45 (s, 1H, CH of pyrimidine ring), 3.34 (s, 1H, SH).</p><!><p>M. Formula: C16H10ClN3O3; Yield: 84.72%; MS ES + (ToF): m/z 327 [M+ + 1]; IR (KBr pellets, cm−1): 2941 (C–H str.), 1595 (C=C str.), 705 (C–C str.), 1672 (C=N str.), 1342 (C–N str., pyrimidine), 756 (C–Cl str.), 1523 (NO2 asym str.), 3374 (O–H str.), 822 (C–N str., Ar–NO2), 1404 (C–O str., and O–H in plane bending vib.); 13C-NMR (CDCl3-d
6, δ, ppm): 160.4, 160.4, 153.2, 148.2, 139.1, 134.1, 131.1, 129.2, 128.2, 121.2, 88.1; 1H-NMR (CDCl3, δ, ppm): 7.43–8.56 (m, 8H, Ar–H), 6.61 (s, 1H, CH of pyrimidine ring), 5.04 (s, 1H, OH).</p><!><p>M. Formula: C17H14N4O3; Yield: 73.43%; MS ES + (ToF): m/z 322 [M+ + 1]; IR (KBr pellets, cm−1): 2947 (C–H str.), 692 (C–C str.), 1709 (C=N str. pyrimidine ring), 1344 (C–N str., pyrimidine), 784 (C–N str., Ar–NO2), 1041 (C–O–C str., –OCH3), 2839 (C–H str., R–CH3); 13C-NMR (CDCl3-d
6, δ, ppm): 163.2, 160.1, 146.2, 139.6, 131.0, 134.3 130.1, 128.1, 121.7, 119.3, 114.3, 111.3, 96.3, 55.2; 1H-NMR (CDCl3, δ, ppm): 6.33–8.44 (m, 8H, Ar–H), 6.85 (s, 1H, CH of pyrimidine ring), 4.2 (s, 2H, NH2), 3.34 (s, 1H, OCH3).</p><!><p>M. Formula: C17H14N4O3; Yield: 76.47%; MS ES + (ToF): m/z 322 [M+ + 1]; IR (KBr pellets, cm−1): 2937 (C–H str.), 1604 (C=C str.), 694 (C–C str.), 1661 (C=N str.), 1349 (C–N str., pyrimidine), 1502 (NO2 asym str., phenyl ring), 752 (C–N str., Ar–NO2), 1108 (C–O–C str., –OCH3), 2842 (C–H str., R–CH3); 13C-NMR (CDCl3-d
6, δ, ppm): 163.1, 160.1, 148.2, 139.6, 128.1, 125.3, 121.7, 114.3, 95.3; 1H-NMR (CDCl3, δ, ppm): 6.33–8.71 (m, 8H, Ar–H), 6.35 (s, 1H, CH of pyrimidine ring), 4.23 (s, 2H, NH2), 3.01 (s, 1H, OCH3).</p><!><p>M. Formula: C16H10BrN3O3S; Yield: 81.81%; MS ES + (ToF): m/z 404 [M+ + 1]; IR (KBr pellets, cm−1): 2869 (C–H str.), 1592 (C=C str.), 691 (C–C str.), 1680 (C=N str.), 1349 (C–N str., pyrimidine), 623 (C–Br str.), 1521 (NO2 asym str., phenyl ring), 844 (C–N str., Ar-NO2), 2597 (S–H str.), 623 (C–S str.); 13C-NMR (CDCl3-d
6, δ, ppm): 182.4, 163.2, 161.1, 154.3, 148.3, 139.6, 134.2, 133.1, 128.3, 121.2, 122.4, 115.2, 118.2, 103.4; 1H-NMR (CDCl3, δ, ppm): 7.93–8.35 (m, 7H, Ar–H), 8.41 (s, 1H, CH of pyrimidine ring), 3.05 (s, 1H, SH), 5.97 (s, 1H, OH).</p><!><p>M. Formula: C16H11BrN4O2; Yield: 64.00%; MS ES + (ToF): m/z 371 [M+ + 1]; IR (KBr pellets, cm−1): 3064 (C–H str.), 1596 (C=C str.), 692 (C–C str.), 1671 (C=N str.), 1342 (C–N str., pyrimidine), 1500 (NO2 asym str., phenyl ring), 783 (C–N str., Ar–NO2), 645 (C–Br str.); 13C-NMR (CDCl3-d
6, δ, ppm): 163.2, 160.1, 148.2, 139.6, 131.0, 134.3, 130.1, 129.2, 128.1, 126.3, 121.7, 95.3; 1H-NMR (CDCl3, δ, ppm): 6.11–8.41 (m, 8H, Ar–H), 7.35 (s, 1H, CH of pyrimidine ring), 4.00 (s, 2H, NH2).</p><!><p>M. Formula: C16H10ClN4O2; Yield: 87.61%; MS ES + (ToF): m/z 361 [M+ + 1]; IR (KBr pellets, cm−1):1600 (C=C str.), 695 (C–C str.), 1669 (C=N str.), 1346 (C–N str., pyrimidine), 848 (C–Cl str.), 1415 (NO2 asym str., phenyl ring), 735 (C–N str., Ar–NO2); 1H-NMR (CDCl3, δ, ppm): 6.34–8.67 (m, 7H, Ar–H), 6.15 (s, 1H, CH of pyrimidine ring), 4.30 (s, 2H, NH2); 13C-NMR (CDCl3-d
6, δ, ppm): 163.6, 160.1, 147.2, 139.4, 133.3, 135.1, 129.2,128.1, 127.3, 121.7, 95.6.</p><!><p>M. Formula: C18H16N4O3; Yield: 77.38%; MS ES + (ToF): m/z 336 [M+ + 1]; IR (KBr pellets, cm−1): 2923 (C–H str.), 1524 (C=C str.), 704 (C–C str.), 1670 (C=N str. or N=CH str., pyrimidine ring), 1348 (C–N str., phenyl ring), 733 (NO2 asym str., phenyl ring), 806 (C–N str., Ar. nitro group), 2858 (C–H str., R–CH3), 3393 (O–H str.); 13C-NMR (CDCl3-d
6, δ, ppm): 160.5, 154.3, 149.2, 139.6, 128.1, 122.7,121.3, 114.3, 87.2, 41.1; 1H-NMR (CDCl3, δ, ppm): 6.11–8.26 (m, 8H, Ar–H), 6.75 (s, 1H, CH of pyrimidine ring), 5.30 (s, 1H, OH), 2.19 (s, 6H, (CH3)2).</p><!><p>Summarizing, we may conclude that the synthesized compounds (2, 5, 10, 11 and 12) displayed appreciable antibacterial and antifungal activities against Gram positive bacteria (S. aureus and B. subtilis), Gram negative bacteria (E. coli, S. enterica and P. aeruginosa) and fungal strains (C. albicans and A. niger). The electron withdrawing group's play an important role to enhanced the antimicrobial potential of compounds 2, 5, 11 and 12 and these compound more active than standard drugs cefadroxil and fluconazole. The MBC and MFC values represent the lowest concentration of compound that produces in the range of 96–98% end point reduction of the used test bacterial and fungal species.</p>