SYNTHESIS AND ANTIMICROBIAL EVALUATION OF 2-(4-FLUORO BENZYLTHIO)-N-(SUBSTITUTED PHENYL)PYRIMIDINE-4-AMINES

SYNTHESIS AND ANTIMICROBIAL EVALUATION OF 2-(4-FLUORO BENZYLTHIO)-N-(SUBSTITUTED PHENYL)PYRIMIDINE-4-AMINES

N.M. Goudgaon*, Y. Rohini Reddy and B.U. Sheshikant

Department of Post Graduate Studies and Research in Chemistry, Gulbarga University, Gulbarga - 585106, Karnataka, India

ABSTRACT: Reaction of 4-fluorobenzylchloride with 2-thiouracil (1) gave 2-(4-fluorobenzylthio)pyrimidin-4(3H)-one (2), which on chlorination with POCl₃ furnished 4-chloro-2-(4-fluorobenzylthio)-4-chloropyrimidine (3). This intermediate when treated with various substituted anilines gave desired targeted compounds 4(a-k) in 50-90% yield. Structural assignments of the synthesized compounds were based on their IR, ¹H NMR, Mass and analytical data. The antimicrobial evaluation of newly synthesized compounds was carried out by cup-plate method. The investigation of antimicrobial screening reveals that the compounds 4b, 4g, 4c and 4f showed good activity against bacterial strain B. subtilis. Compounds4a, 4e, 4b, 4c, 4f, 4g and 4h wereactive against bacterial strain P. aeruginosa. Compounds 4a and 4c were active against fungul strain A. niger. Compounds 4e, 4b and 4j showed good activity against fungal strain A. flavus. All the synthesized compounds showed excellent antifungal activity against T.viridae. Remaining compounds exhibited moderate to poor activity against bacterial and fungal strains when compared to standard drugs Gentamycin and Fluconazole respectively. So, further we have carried out the antifungal screening of all the synthesized compounds at different concentrations against T. viridae to determine their IC₅₀ values. Compounds 4e, 4b, 4g, 4i, 4d, 4f and 4j have shown better IC₅₀ values

Pyrimidines, Anilines, Antibacterial activity, Antifungal activity

INTRODUCTION:Pyrimidines have a long and distinguished history extending from the days of their discovery as important constituents of nucleic acids to their current use in medicinal chemistry. Pyrimidine derivatives are well known to exhibit various biological and pharmacological activities such as antitumour ¹, anti-HIV ^{2, 3}, antimicrobial^4-6, antidepressants ⁷, hypnotics and sedatives ⁸, analgesicand anti-inflammatory ⁹, antioxidant ¹⁰ and acid-pump antagonist ¹¹ activities

The presence of fluorine increases thermal stability, lipophilicity and bioavailability ¹². Although the utility of fluorine substituents is there, only few methods are available for carbon-fluorine bond formation ^13-14, which indicates the unavailability of suitable fluorination methods.

As incorporating fluorine into system is not easy, we have reacted different fluorine substituted reagents with 2-thiouracil derivatives so as to get pyrimidine molecule bearing fluorine on its substituent. In view of potent pharmacological activity associated with pyrimidines and enhancement of activity by introducing fluorine in the molecule, We herein report the synthesis and antimicrobial activities of 2-(4-fluorobenzylthio)-N-(substituted phenyl)pyrimidine-4-amines (Scheme 1).

MATERIALS AND METHODS: All the solvents and chemicals were obtained from S. D. Fine-Chem Ltd. Mumbai and were purified by standard procedures. Melting points were determined in open capillary and were uncorrected. IR spectra in KBr disc were recorded on Perkin-Elmer-Spectrum-one FT-IR spectrophotometer (ν_max in cm^-1). ¹H NMR spectra were recorded in DMSO-d₆ with a BRUKER NMR 500 MHz Spectrophoto-meter using TMS as internal standard (chemical shift in δ or ppm). Mass spectra were recorded on LCMS 2010A, SHIMADZU Mass Spectrophoto-meter.

Purity of the compounds was checked by TLC using silica gel ‘G’ plates obtained from Whatman Inc, and a fluorescent indicator.

RESULT AND DISCUSSION: Reaction of 2-thiouracil (1) with 4-flourobenzylchloride in aq. NaOH gave compound 2 in 85% yield having m.p. 158-160˚C (Scheme 1). ¹H NMR spectra displayed a singlet at δ 4.39 due to two protons of CH₂ and two doublet peaks, one at δ 7.24 due to C₅H of pyrimidine and another at δ 7.39 due to C₆H of pyrimidine ring and multiplet from δ 7.25-7.90 due to four aromatic protons confirms the formation of compound 2.

SCHEME 1

Further formation of compound 2 is confirmed by the presence of absorption bands at 1708, 1574cm^-1 assigned to C=O, C=N with 687 cm^-1 characterized by C-F stretching were shown in its IR spectrum.  Chlorination of compound 2 with POCl₃ yielded 4-chloro-2-(4-fluorobenzylthio)pyrimidine (3) in 90% yield, m.p. 44-46˚C. Formation of this compound 3 was confirmed by the presence of absorption peaks at 1575, 736 and 707 cm^-1 due to C=N, C-Cl and C-F in its IR spectrum.

Further confirmation of compound 3 is by the presence of singlet at δ 4.4 due to two protons of SCH₂PhF, aromatic protons signal as a multiplet at δ 7.14 -7.16, characteristic absorption of C₅Hand C₆H of pyrimidine ring as a doublet at δ 7.40  and δ 8.60 respectively in its ¹H NMR spectrum. This intermediate on reaction with various aromatic amines in presence of piperidine in methanol furnished the desired targeted compounds (4a-k) in 50 - 80% yield. Compound 4a was obtained in 50% yield having m.p. 104˚-106˚C. The IR (cm^-1) spectrum of compound 4a showed absorptions at 3081, 1575 and 707 cm^-1  due to the presence of aromatic NH, C=N and C-F groups.

¹H NMR displayed singlet at δ 4.34 due to two protons of SCH₂PhF, multiplet in the region δ 6.77-7.40 due to eight aromatic protons and a singlet at δ 6.42 due to NH proton and two doublet peaks at δ 6.20 and δ 8.18 due to C₅H and C₆H of pyrimidine ring.

Final confirmation of compound 4a is by the appearance of molecular ion peak at m/z = 330 (M⁺¹, 100%) in its mass spectrum. Physical data of all the synthesized compounds are tabulated in Table 1.

TABLE 1: PHYSICAL DATA OF SYNTHESIZED COMPOUNDS (2, 3 & 4a-k)

 EXPERIMENTAL:

Synthesis of 2-(4-fluorobenzylthio)pyrimidine-4(3H)-one (2): 2-Thiouracil (1) (0.075 mole) and NaOH (0.1 mole) were dissolved in hot water (450 ml) and then mixed with 4-fluorobenzylchloride (0.1 mole). The temperature was increased until all the 4-fluorobenzylchloride went into the solution for about one hour. On cooling the separated solid product was collected by filtration, washed with water, dried and crystallized from ethanol to afford compound 2.

Yield: 85%. M.P: 158-160 ^oC. IR(cm^-1): 1708 (C=O), 1574 (C=N), 687 (C-F). ¹H NMR (DMSO-d₆): δ4.39 (s, 2H, SCH₂ PhF), 7.24 (d, 1H, C₅H of pyrimidine), 7.25-7.90 (m, 4H, ArH), 7.39 (d, 1H, C₆H of pyrimidine). Anal. Calcd. for C₁₁H₉FN₂OS: C, 51.76; H, 3.55; N, 10.97. Found: C, 51.72; H, 3.51; N, 10.92%.

Synthesis of 2-(4-fluorobenzylthio)-4-chloro pyrimidine (3): A mixture of 2-(4-fluorobenzylthio)pyrimidine-4(3H)-one (2) (0.01 mole) and POCl₃ (0.1 mole) was refluxed for 3 hours. Excess of POCl₃ was removed under reduced pressure and the reaction mixture was treated with ice/water. The separated solid was extracted with ether (3 x 100 ml) and the ether extract was washed with 5% aq.NaHCO₃ solution (1 x 25 ml), water (1 x 25 ml) and then dried over anhydrous sodium sulphate and after solvent evaporation yielded the crude compound 3 was recrystallized from EtOH.

Yield: 90%. M.P: 44-46 ^oC. IR(cm^-1): 1575 (C=N), 707 (C-F), 736 (C-Cl). ¹H NMR (DMSO-d₆): δ 4.4 (s, 2H, CH₂),7.14-7.16 (m, 4H, ArH ), 7.40 (d, 1H, C₅H of pyrimidine), 8.6 (d, 1H, C₆H of pyrimidine). Anal. Calcd. for C₁₁H₈N₂ClFS: C, 53.33; H, 3.25; N,11.31. Found: C, 53.28; H, 3.21; N, 11.27%.

Synthesis of 2-(4-fluorobenzylthio)-N-(substituted phenyl)pyrimidine-4-amines (4a-k): To a solution of 2-(4-fluorobenzylthio)-4-chloropyrimidine(3) (0.001 mole) in methanol (20 ml) and catalytic amount of piperidine, appropriate aromatic primary amine (0.001 mole) was added and refluxed for 12-15 hours on water bath. Concentrated the reaction mixture under reduced pressure and the residue triturated with little crushed ice and neutralized the aqueous layer with 0.1N HCl. Solid separated was filtered, washed with cold water and recrystallized from ethanol to yield the target compounds (4a-k).

Spectral data of compounds (4a-k):

2-(4-Fluorobenzylthio)-N-(2-fluorophenyl) pyrimidine-4-amine (4a): Yield: 50%. M.P: 103-106ºC. IR (cm^-1): 3081 (NH), 1575 (C=N), 707 (C-F). ¹H NMR (DMSO-d₆): δ 4.34 (s, 2H, SCH₂PhF), 6.77-7.40 (m, 8H, ArH ), 6.42 (s, 1H, NH), 6.20 (d, 1H, C₅H of pyrimidine), 8.18 (d, 1H, C₆H of pyrimidine). Mass: m/z = 330 (M⁺¹, 100%). Anal. Calcd. for C₁₇H₁₃N₃F₂S: C, 61.99; H, 3.97; N, 12.75. Found: C, 61.95; H, 3.93; N, 12.71%.

2-(4-Fluorobenzylthio)-N-(4-bromo-3-trifluoro methylphenyl)pyrimidin-4-amine (4b): Yield: 62%. M.P: 195-198ºC. IR (cm^-1): 3078 (NH), 1624 (C=N), 697 (C-F). ¹H NMR (DMSO-d₆): δ 4.418 (s, 2H, SCH₂PhF), 7.23-7.83 (m,7H, ArH), 8.23 (d, 1H, C₆H of pyrimidine), 8.377 (s, 1H ,NH), 6.70 (d, 1H, C₅H of pyrimidine). Mass: m/z= 455 (M⁺², 100%). Anal. Calcd. for C₁₈H₁₂N₃BrF₄S: C, 47.18; H, 2.63; N, 9.17. Found: C, 47.14; H, 2.59; N, 9.13%.

2-(4-Fluorobenzylthio)-N-(4-fluorophenyl) pyrimidine-4-amine (4c): Yield: 65%. M.P: 102-104ºC. IR (cm^-1): 3076 (NH),1601 (C=N), 697 (C-F). ¹H NMR (DMSO-d₆): δ 4.30 (s, 2H, SCH₂PhF), 6.51(d, 1H, C₅H of pyrimidine), 7.20-7.40(m, 9H, 8ArH & NH), 7.96 (d, 1H, C₆H of pyrimidine). Mass: m/z = 330 (M⁺¹, 100%). Anal. Calcd. for C₁₇H₁₃N₃F₂S: C, 61.99; H, 3.97; N, 12.75. Found: C, 61.95; H, 3.93; N, 12.71%.

2-(4-Fluorobenzylthio)-N-(4-chlorophenyl) pyrimidine-4-amine (4d): Yield: 80%. M.P: 99-100ºC. IR(cm^-1): 3076 (NH), 1601(C=N), 697 (C-F), 733 (C-Cl). ¹H NMR (DMSO-d₆): δ 4.32 (s, 2H, SCH₂PhF), 6.94-7.40 (m,8H, ArH), 7.26 (s, 1H, NH), 6.17 (d, 1H, C₅H of pyrimidine), 7.90 (d, 1H, C₆H of pyrimidine). Mass: m/z = 347 (M⁺², 100%). Anal. Calcd. for C₁₇H₁₃N₃ClFS: C, 59.10; H, 3.78; N, 12.15. Found: C, 59.06; H, 3.73; N, 12.11%.

2-(4-Fluorobenzylthio)-N-(4-methoxyphenyl) pyrimidin-4-amine (4e): Yield: 70%. M.P: 118-120ºC. IR(cm^-1): 3076 (NH), 1601 (C=N), 697 (C-F). ¹H NMR (DMSO-d₆): δ 3.61 (s, 3H, OCH₃), 4.34  (s, 2H, SCH₂PhF), 6.96-7.41(m, 8H, ArH), 6.19 (d, 1H, C₅H of pyrimidine), 7.28 (s, 1H, NH ), 7.98 (d, 1H, C₆H of pyrimidine). Mass: m/z = 342 (100%). Anal. Calcd. for C₁₈H₁₆N₃OFS: C, 63.32; H, 4.72; N, 12.31. Found: C, 63.25; H, 4.67; N, 12.28%.

2-(4-Fluorobenzylthio)-N-(3-fluorophenyl) pyrimidine-4-amine (4f):Yield: 62%. M.P: 135-138ºC.IR(cm^-1): 3286 (NH), 1628 (C=N), 696 (C-F). ¹H NMR (DMSO-d₆): δ 4.34 (s, 2H, SCH₂PhF), 6.19 (d, 1H, C₅H of pyrimidine), 6.96-7.41 (m, 8H, ArH), 7.28 (s, 1H, NH), 7.98 (d, 1H, C₆H of pyrimidine). Mass: m/z = 328(M⁺¹, 100%). Anal. Calcd. for C₁₇H₁₃N₃F₂S: C, 61.99; H, 3.98; N, 12.76. Found: C, 61.95; H, 3.94; N, 12.70%.

2-(4-Fluorobenzylthio)-N-(phenyl)pyrimidine-4-amine (4g): Yield: 72%. M.P: 114-116ºC. ¹H NMR (DMSO-d₆): δ 4.31 (s, 2H, SCH₂PhF), 6.17 (d, 1H, C₅H of pyrimidine), 6.94-7.39 (m, 9H, ArH), 7.26 (s, 1H, NH), 7.96 (d, 1H, C₆H of pyrimidine). Mass: m/z = 312 (100%). Anal. Calcd. for C₁₇H₁₄N₃FS: C, 65.57; H, 4.53; N, 13.49. Found: C, 67.53; H, 4.49; N, 13.45%.

2-(4-Fluorobenzylthio)-N-(2-nitrophenyl) pyrimidine-4-amine (4h): Yield: 66%. M.P: 95-98ºC. ¹H NMR (DMSO-d₆): δ 4.36 (s, 2H, SCH₂PhF), 6.17 (d, 1H, C₅H of pyrimidine), 7.22-7.43 (m, 9H, 8ArH & NH), 7.97 (d, 1H, C₆H of pyrimidine). Mass: m/z = 356 (100%). Anal. Calcd. for C₁₇H₁₃N₄O₂FS: C, 57.29; H, 3.68; N, 15.72. Found: C, 57.25; H, 3.64; N, 15.68%.

2-(4-Fluorobenzylthio)-N-(3-nitrophenyl) pyrimidine-4-amine (4i): Yield: 62%. M.P: 118-120ºC.¹H NMR (DMSO-d₆): δ 4.33 (s, 2H, SCH₂PhF), 6.19 (d, 1H, C₅H of pyrimidine), 7.23-7.45 (m, 9H, 8ArH & NH), 11.33 (s, 1H, NH), 7.99 (d, 1H, C₆H of pyrimidine). Mass: m/z = 356 (100%). Anal. Calcd. for C₁₇H₁₃N₄O₂FS: C, 57.29; H, 3.68; N, 15.72. Found: C, 57.25; H, 3.64; N, 15.68%.

2-(4-Fluorobenzylthio)-N-(4-nitrophenyl) pyrimidine-4-amine (4j):Yield: 60%. M.P: 91-92ºC.¹H NMR (DMSO-d₆): δ 4.38 (s, 2H, SCH₂PhF), 6.93 (d, 1H, C₅H of pyrimidine), 7.26-7.85 (m, 9H, 8ArH & NH), 8.15 (d, 1H, C₆H of pyrimidine). Mass: m/z = 356 (100%). Anal. Calcd. for C₁₇H₁₃N₄O₂FS: C, 57.29; H, 3.68; N, 15.72. Found: C, 57.25; H, 3.64; N, 15.68%.

2-(4-Fluorobenzylthio)-N-(2-fluoro-4-iodo phenyl)pyrimidine-4-amine (4k):Yield: 67%. M.P: 102-104ºC. IR (cm^-1): 3073 (NH), 1578 (C=N), 695 (C-F). ¹H NMR (DMSO-d₆): δ 4.34 (s, 2H, SCH₂PhF), 6.19 (d, 1H, C₅H of pyrimidine), 6.96-7.41 (m, 8H, 7ArH & NH), 7.98 (d, 1H, C₆H of pyrimidine). Mass: m/z = 456 (M⁺¹, 100%). Anal. Calcd. for C₁₇H₁₂N₃IF₂S: C, 44.85; H, 2.65; N, 9.23. Found: C, 44.80; H, 2.61; N, 9.19%.

Antimicrobial Activity: The antimicrobial activities were performed by cup plate method ¹⁵. The sample was dissolved in DMF at the concentration of 1000 µg/ml. Antibacterial activity was carried out against two gram +ve S. aureus, B. subtilis and two gram -ve P. aeruginosa and E. coli bacterial strains.

Antifungal activity was carried out against A. niger and A. flavus and T. viridae under aseptic conditions. Gentamycin and Fluconazole were used as standard drug for antibacterial and antifungal activities respectively.

The zone of inhibition was compared with standard drug after 24 hours of incubation at 25°C for antibacterial activity and 48 hours at 30°C for antifungal activity. The investigation of anti-microbial screening reveals that the compounds 4b, 4g, 4c and 4f showed good activity against bacterial strain B. subtilis. Compounds4a, 4e, 4b, 4c, 4f, 4g and 4h were active against bacterial strain P. aeruginosa. Compounds4a and 4c were active against fungul strain A. niger.

Compounds 4e, 4b and 4j showed good activity against fungal strain A. flavus. All the synthesized compounds showed excellent antifungal activity against T. viridae. Remaining compounds exhibited moderate to poor activity against bacterial and fungal strains when compared to standard drug.  Results are tabulated in Table 2.

Further the antifungal screening of all the synthesized compounds at different concentrations 1000, 750, 500, and 250 µg/ml to determine the IC₅₀ (50% inhibition concentration) values against T. viridae.

Compounds 4e, 4b, 4g, 4i, 4d, 4f and 4j have exhibited better IC₅₀ values. IC₅₀ is defined as the drug concentration that produces 50% of the maximal effect. The values are tabulated in Table 3 and percentages of inhibition are shown graphically in Figure 1.

TABLE 2: ANTIMICROBIAL ACTIVITY OF SYNTHESIZED COMPOUNDS (4a-k)

   TABLE 3:  IC₅₀ VALUES OF SYNTHESIZED COMPOUNDS (4a-k)

FIGURE 1: GRAPH SHOWING % INHIBITION AT DIFFERENT CONCENTRATIONS

CONCLUSION: The present work reports the synthesis and antimicrobial activities of                                2-(4-Fluorobenzylthio)-N-(substituted phenyl) pyrimidine-4-amines. Among the synthesized compounds 4b, 4g, 4c and 4f showed good activity against bacterial strain B. subtilis. Compounds4a, 4e, 4b, 4c, 4f, 4g and 4h wereactive against bacterial strain P. aeruginosa. Compounds4a and 4c were active against fungul strain A. niger. Compounds 4e, 4b and 4j showed good activity against fungal strain A. flavus. All the synthesized compounds showed excellent antifungal activity against T. viridae. Remaining compounds exhibited moderate to poor activity against bacterial and fungal strains when compared to standard drug.

So, further we have carried out the antifungal screening of all the synthesized compounds at different concentrations against T. viridae to determine their IC₅₀ values. Compounds 4e, 4b, 4g, 4i, 4d, 4f and 4j have shown better IC₅₀ value. Further preclinical studies of the most potent compound are warranted.

ACKNOWLEDGEMENT: The authors are thankful to SAIF IIT-Madras for providing spectral data. The authors also thank Professor and Chairman, Department of Microbiology, Gulbarga University, Gulbarga for providing bacterial and fungal strains.

 REFERENCES:

1. Grigoryan LA, Kaldrikyan MA, Melik-Ogandzhanyan RG and Arsenyan FG: Synthesis and antitumor activity of new 2-thio and 2-amino substituted Pyrimidines. Pharmaceutical Chemistry Journal 2011; 45(3):9-12.
2. Goudgaon NM and Schinazi RF: Activity of acyclic 6-(phenylselenenyl)pyrimidine nucleosides against human immune deficiency viruses in primary lymphocytes. Journal of Medicinal Chemistry 1991; 34:3305-3309.
3. Goudgaon NM, Mc Millan A and Schinazi RF: 1-(Ethoxymethyl)-6-(phenylselenenyl) pyrimidines with activity against human immune deficiency virus types 1 and 2. Antiviral Chemistry and Chemotherapy 1992; 3:263-266.
4. Sreenivas B, Akhilaa M and Mohammeda B: Synthesis and biological evaluation of pyrimidine analogs as potential antimicrobial agents. International Journal of Pharmacy and Pharmaceutical Sciences 2012; 4(2):306-310.
5. Goudgaon NM and Sheshikant BU: Synthesis of novel 2, 4-bis (substituted phenoxy)-6-(phenylthio)pyrimidine analogs and their antimicrobial activities. Journal of Pharmacy Research 2013; 7(1):75-79.
6. Goudgaon NM, Basha NJ and Patil SB: Synthesis and antimicrobial evaluation of 5-iodopyrimidine analogs. Indian journal of Pharmaceutical Sciences 2009; 71(6):672-677.
7. Siddique NA, Bawa S, Ali R, Afzal O, Akhtar MJ and Kumar R: Antidepressant potential of nitrogen-containing heterocyclic moieties: An updated review. Journal of Pharmacy and Bioallied Sciences 2011; 3(2):194-212.
8. Kimura T, teraoka S, Kuze J, Watanabe K, Ho IK and Yamamoto I: Sedative and hypnotic activities of pyrimidine nucleosides derivatives. Nucleic acids Symposium Series 1993; 29:51-52.
9. Vishal DJ, Mahendra DK and Sarita S: Synthesis and pharmacological study of some novel pyrimidines. Pelagia Research Library 2012; 3(3):343-348.
10. Kotaiah Y, Harikrishna N, Nagaraju K and Venkata Rao: Synthesis and antioxidant activity of 1,3,4-oxadiazole tagged thieno[2,3-d]pyrimidine derivatives. European Journal of Medicinal Chemistry 2012; 58:340-345.
11. Young AY, Chan SP, Myung HC, Hyunho C, Jae YS and Jae GK: Novel pyrimidines as acid pump antagonists (APAs). Bioorganic & Medicinal Chemistry Letters 2010; 20:5735-5738.
12. Kevin PB, Neil RK and Paul MN: Metabolism of fluorine containing drugs. Annual Review of Pharmacology and Toxicology 2001; 41:443-470.
13. Kirk KL: Fluorination in medicinal chemistry, methods, strategies and recent    developments. Organic Process Research Development 2008; 12(2):305-321.
14. Helio GB, Susiane C, Patrick TC, Liliane MFP, Jussara N et.al: New trifluoromethyl- containing (E)-N’-arylidene-[3-alkyl(aryl/heteroaryl)-4,5-dihydro-1H-pyrazol-1-yl]carbo hydrazides: Synthesis, crystal structure andantimicrobial/antioxidant activity. Journal of Fluorine Chemistry 2012; 135:303–314.
15. Indian Pharmacopoeia, Govt. of India, New Delhi. 3rd Ed., Appendix IV 1985; 90.
    

    ---

    How to cite this article:

    Goudgaon NM, Reddy YR and Sheshikant BU: Synthesis and antimicrobial evaluation of 2-(4-fluorobenzylthio)-n-(substituted phenyl)pyrimidine-4-amines. Int J Pharm Sci Res 2013; 4(11): 4336-42. doi: 10.13040/IJPSR. 0975-8232.4(11).4336-42

    ---

     

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---