- Email: [email protected]
New Dihydropyrimidines as Antimicrobial Agents
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 4 (2017) 5657–5662
www.materialstoday.com/proceedings
ICMDA 2016
New Dihydropyrimidines as Antimicrobial Agents Kundan Lala*, L.J. Paliwala, M.B. Bagadeb Post Graduate Department of Chemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440 033, Maharashtra, India. bPost Graduate Teaching Department of Chemistry, Seth Kesarimal Porwal College, Kamptee - 441 001, Maharashtra, India.
*a
Abstract A series of novel dihydropyrimidines having benzo chromone and substituted phenyl ring along with pyrimidine nucleus have been synthesized under microwave irradiation. Synthesized compounds were characterized by physical, chemical and spectroscopic methods. Solvent free conditions, simple procedures, with high purity of the products in a very less time make this method highly valuable. The compounds were screened for antimicrobial properties to check their efficacy against few bacterial species. Results of the invitro antimicrobial activities showed that the compounds possess very promising antibacterial properties. © 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON MULTIFUNCTIONAL MATERIALS FOR DEVICE APPLICATIONS(ICMDA-2016). Keywords: p-Fluroaniline; Microwave Irradiation (MWI), Chloropyrimidine; Chromen-4-one; Antibacterial Activity; Well-Diffusion.
*Corresponding author Tel.:+91 9960723698 Email address:[email protected]
2214-7853 © 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON MULTIFUNCTIONAL MATERIALS
FOR DEVICE APPLICATIONS(ICMDA-2016).
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Kundan Lal et al/ Materials Today: Proceedings 4 (2017) 5657–5662
1. Introduction The growing resistance of the microbial species against available drugs has created a major problem for the human society. The current scenario requires invention of new drugs with increased potency against the pathogens. Literature survey reveals that drugs containing heterocyclic moiety showed great potency against the dangerous microbes. Various heterocyclic moieties are synthesized in pharmaceutical companies to counter the growing resistance [1]. Among heterocyclic groups compounds possessing nitrogen containing compounds showed a wide variety of drugs against various pathogens [2]. In the nitrogen containing heterocyclic compounds pyrimidine and their derivatives play a vital role in the treatment of various diseases [3-4]. Different pyrimidine derivatives possess broad spectrum properties and show various biological activities such as analgesic[5-7], anti-HIV [8], anti-viral [9], anti-inflammatory [10-12], antimicrobial [13-15], anti-tubercular [16-18], antitumour[19-20], anticonvulsant and antioxidant [21], anticancer [22-23], antimalarial [24] and anti amoebic activity [25]etc, Apart from the biological activity of heterocyclic compounds the different protocols has also been taken into practice to enhance the yield. Environment friendly approach has been used extensively since the beginning of the 21st century for the synthesis of heterocyclic compounds [26]. Among various methods, microwave assisted synthesis has been found to be of great interest as it not only environmental friendly but also give high yield with less time for the reaction [27-30]. In accordance with the accessibility of the earlier drugs having pyrimidine nucleus used for the treatment in bacterial diseases, the current work is another progress [31]. Here we would like to report the microwave assisted synthesis of some new pyrimidine derivatives with benzo chromone with pyrimidine nucleus along with substituted phenyl ring. These new compounds have been evaluated for their biological activities against various species of gram positive and gram negative bacteria. Apart from adopting the environment friendly approach the potency of the synthesized compounds has been enhance by incorporating the chromone nucleus and various electron donation and electron withdrawing groups along with pyrimidine nucleus. 2. Experimental 2.1 Materials All the chemicals were obtained commercially, mostly from S.D. Fine, Merck and were used without purification. 2.2 Equipment Melting points were determined by the open capillary method and are uncorrected. The progress of the reaction as well as purity of compounds were monitored by TLC using silica coated aluminium TLC plates (TLC Silica Gel 60 F254, Merck, Germany) by different eluent systems. The spots were visualized by keeping the dry plates in iodine vapors and in UV light. IR spectra were recorded on Perkin Elmer Spectrometer (RX-IFTIR) in KBr discs. 1H and 13C NMR spectra were recorded in DMSO-d6 on Bruker Avance II 400 MHz NMR Spectrometer. Mass spectra were scanned on Expression CMS (ESI) spectrometer. Elemental analyses were carried out with Elementar Vario EL III elemental analyzer. The microwave reactions were performed in the CEM Focused MicrowaveTM Synthesis System. 2.3 General procedure for preparation of 3-(2-chloro-6-substituted phenyl-3,4-dihydropyrimidin-4-yl)-4Hbenzo[h]chromen-4-one 2a-f Conventional Procedure A: 4-(4-Oxo-4H-benzo[h]chromen-3-yl)-6-substituted phenyl-3,4-dihydropyrimidin-2(1H)one 1 (0.01mol) was taken in a round bottom flask with phosphoryl chloride (POCl3) (15 mL) and was refluxed at 100 °C for 5 h on water bath. After the completion of reaction, the reaction mixture was poured into cold water and was further neutralized by using 5% NaHCO3 solution. Obtained product was filtered, washed with distilled water, dried and was recrystallized from ethanol. Microwave Assisted Reaction A: A mixture of 4-(4-Oxo-4H-benzo[h]chromen-3-yl)-6-substituted phenyl-3,4dihydropyrimidin-2(1H)-one 1 (1mmol) and alumina (0.5 g) was mix well together and transferred to Erlenmeyer flask and to it phosphoryl chloride (POCl3) (0.5 mL) was added. The flask was then capped and was irradiated under microwave irradiation at 175 W at 110 °C for 10-12 min. After the completion of reaction (monitored by TLC), the reaction mixture was filtered to remove alumina from the product and was poured into cold water. The product was further neutralized by using 5% NaHCO3 solution. Obtained product was filtered, washed with distilled water, dried and was recrystallized from ethanol.
Kundan Lal et al/ Materials Today: Proceedings 4 (2017) 5657–5662
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2.4 General procedure for the Synthesis of 3-(2-(4-fluorophenylamino)-6-phenyl-3,4-dihydropyrimidin-4-yl)-4H benzo [h]chromen-4-one 3a-f Conventional Procedure B: A mixture of 3-(2-chloro-6- substituted phenyl-3,4-dihydropyrimidin-4-yl)-4Hbenzo[h]chromen-4-one 2a-f (0.01mol) and p-fluoro aniline (0.01 mol) in ethanol (25 mL) was refluxed for 20 h. After the completion of reaction, the reaction mixture was poured into cold water and was further neutralized by using 5% NaHCO3. Obtained product was filtered, washed with distilled water, dried and was recrystallized from ethanol: acetic acid (2:1). Microwave Assisted Reaction B: A mixture of 3-(2-chloro-6- substituted phenyl-3,4-dihydropyrimidin-4yl)-4H-benzo[h]chromen-4-one 2a-f (1mmol) and alumina (0.5 g) was mix well together and transferred to Erlenmeyer flask and to it p-fluoro aniline (1 mmol) was added. The flask was then capped and was irradiated under microwave irradiation at 175 W at 110 °C for 10-12 min. After the completion of reaction, the reaction mixture was filtered to remove alumina from the product and was poured into ice cold water. The obtained product was further neutralized by using 5% NaHCO3 solution. Product was filtered, washed with distilled water, dried and was recrystallized from ethanol: acetic acid (2:1). The physical and chemical properties has been reported in Table 1 Table 1. Physical Properties of 3-(2-chloro-6-substituted phenyl-3,4-dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 2a-f and 3-(2-(4fluorophenylamino)-6-phenyl-3,4-dihydropyrimidin-4-yl)-4H benzo [h]chromen-4-one 3a-f Compound
2a 2b 2c 2d 2e 2f 3a 3b 3c 3d 3e 3f
R1
H H H H H H H H H H H H
R2
H NO2 H H H H H NO2 H H H H
R3
H H Cl OH Br NO2 H H Cl OH Br NO2
Yield (%) Conventional(Reflux)
MWI
50 60 48 50 60 65 60 62 58 70 68 65
70 75 70 70 70 80 80 88 75 85 80 90
Reaction Time Conventional(h) MWI(min) 5 5 5 5 5 5 20 20 20 20 20 20
12 10 12 12 12 10 18 16 15 15 15 18
3. Biological Activity The antimicrobial activities were determined by using well diffusion method [32] by measuring zone of inhibition in mm. The microbial strains were obtained from the microbiology laboratory, S.K.Porwal College, Kamptee. MHA (Mueller-Hinton Agar) was used as microbial growth medium. The media were prepared according to the instructions given by the manufacturer. A loopful of culture was inoculated from the stock culture in 5 mL of MHA broth and the broth was incubated at 35 °C in incubator for 6-8 h. After incubation, this culture was used for the inoculation of MH test agar plates. Medium was autoclaved and was maintained at 45-50 °C in constant temperature water bath. 0.5 ml of 6-8 h old test organism was transferred to petridish of 100 mm size (sterilized in oven at 180 °C for 1 h) using sterile micropipette. MH test agar medium maintained at 45-50 °C was poured and mixed properly. Petri plates were allowed to set at room temperature. A 10 mm borer was used to prepare wells in agar plates. By using micropipette 10 μl of the test sample was transferred to each well. Plates were immediately kept at 4 °C in refrigerator for 1 h for diffusion of the samples and then shifted to 35 °C in incubator. The zone of inhibition was measured in millimetres by the end of the incubation period of 24 h at 35 oC in incubator. Ciprofloxacin was used as a standard drug for antibacterial screening. DMSO was used as control as it does not show any inhibition. All newly synthesized compounds 2a-f and 3a-f were screened to check their antimicrobial activities against two Gram positive strains (S.aureus, and B.Subtillis) and two Gram negative strains (E.coli and K. aerogenes) at concentration of 10 μl. The results have been summarized in Table 2.
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Table 1 Invitro Antimicrobial Activity of synthesized compounds 2a-f and 3a- f. Compound
Zone of inhibition (in mm) Gram Negative
2a 2b 2c 2d 2e 2f 3a 3b 3c 3d 3e 3f Ciprofloxacin
E. coli 14 18 12 10 18 20 9 18 16 10 16 20 22
Gram Positive K. aerogenes 9 4 3 11 11 9 6 8 9 5 13 10 20
S. aureus 11 17 22 10 15 18 9 14 15 22 14 21 22
B. subtilis 10 20 10 8 20 18 10 20 17 14 19 17 22
4. Results and Discussion Herein, we are reporting the synthesis and invitro antibacterial study of 3-(2-chloro-6-substituted phenyl3,4-dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 2a-f and 3-(2-(4-fluorophenylamino)-6- substituted phenyl3,4-dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 3a-f. At first, 3-(2-chloro-6-substituted phenyl-3,4dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 2a-f have been synthesized from 4-(4-oxo-4Hbenzo[h]chromen-3-yl)-6-substitued phenyl-3,4-dihydropyrimidin-2(1H)-one 1a-f by mixing with phosphoryl chloride(POCl3) and was refluxed/ irradiated under microwave radiation. Obtained products were basified by using 5% NaHCO3 solution to get the desired products (Scheme 1). Further, a mixture of 3-(2-chloro-6- substituted-3,4dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 2a-f and p-fluoro aniline was mixed and was reflux/irradiated under microwave radiation Scheme 1. After the completion of reaction, the reaction mixture was poured into ice cold water and was neutralized using 5% NaHCO3 solution. The obtained products were further characterized on the basis of spectral data (IR, MS, 1H and 13C NMR). FTIR Spectra of 3-(2-chloro-6-phenyl-3,4-dihydropyrimidin-4yl)-4H-benzo[h]chromen-4-one 2a showed characteristic band at 1069 cm-1 and peak at 768 cm-1 showing the presence –C-Cl. Peaks observed at 3198 and 1633 cm-1 confirms the presence of NH and -C=O (Chromone). Molecular ion peaks at m/z 386.2 [M+, 100 %], 388.7 [M+2, 30 %] were observed which also explained the existence of chlorine in the compound and the ratio of the two consecutive peaks was found to be 3:1 lend credence to the structure. The 1H NMR spectrum showed characteristic doublet at 9.15 ppm (J =2.5Hz) due to NH proton. Active methylene protons of CH appeared as doublet at 7.0 ppm (J =7.2Hz). The aromatic protons have been appeared as multiplet between 7.9-7.3 ppm. The doublet of =CH occurred at 8.4 ppm (J =7.5). The 13C NMR spectrum recorded in DMSO-d6 showed characteristic peak at 189.1 due to –C=O (chromone). The peaks depicted between 137.4-113.8 are assigned to aromatic carbons. The peaks at 89.0, 55.30and 44.0 are due to–CH and -C=CH- and –C-Cl of pyrimidine ring respectively. Similarly, compound 3-(2-(4-fluorophenylamino)-6-phenyl-3,4dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 3(a) showed characteristic band at 1633 cm-1 due to C=O (chromone) stretching vibration. A broad peak at 3364cm-1 was observed due to NH of pyrimidine ring. One sharp peak observed at 1212 cm-1(m) showed the presence of Ar-F in the aromatic ring. Molecular ion peak at m/z 462.3 [M+, 95 %], also lend credence to the structure for 3(a). The 1H NMR spectrum recorded in DMSO-d6 showed characteristic singlet at 9.1 ppm and a broad peak appeared at 10.0 ppm assigned to NH of pyrimidine and pfluroaniline respectively. Active methylene protons -CH appeared as doublet at 8.3 ppm ( J =7.0) and aromatic protons appeared as multiplet between 8.5-7.4 ppm. The doublet of =CH occurred at 6.5 ppm (J =7.5). The 13C NMR spectrum recorded in DMSO-d6 showed characteristic doublet at 114.50 ppm (J=7Hz) appear for –Corth-F (fluroaniline) also doublet of doublet appeared at 130.08, 127.83, 128.89 and 168.64 ppm with (J= 29.3, J= 9.8Hz, J= 9.2 and J= 246.3 respectively) appear for –C-F bonding with reference to various positions. Similarly, peak at 156.5 assigned to –C=O (pyrimidine). Compounds like 2b, 2d, 3b and 3d showed prominent peaks for the NO2
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groups at 1340(s), 1530(s) and 1500(s). Presence of Br and F groups also observed at 1040 and 1130 cm-1 respectively. Mass spectrum showed M+ (100%) and M+2(97%) for bromine and peaks of M+ (99.2%) and M++2(27%) confirms the presence of chlorine. The peaks depicted in the region 129.7-114.5 are assigned to aromatic carbon. The peaks at 89.14 and 55.20 are due to –CH and -C=CH- of pyrimidine ring respectively. Table 1 also showed that adopted method drastically increases the yield from 48-65% to 70-80% in case of 2a-f and 58-70% to 80-90% with 3a-f. Also the time for completion of the reaction has been reduced very much from 5 h to 10-12 min in case of 2a-f and 20 h to 15-18 min and the easy protocol make this method very useful for the others. Considering the invitro antimicrobial activity of compounds 2a-f and 3a-f, compounds 2b, 2c, 2e, 2f, 3b, 3e and 3f showed prominent activity against S.aureus, B.Subtillis and E.coli and moderate activity against K. aerogenes. Other compounds showed moderate activity. The presence of the electron with drawing and electro negative group enhance the antibacterial properties of the compounds.
O
O
R3 R2
O HN
NH O
R1
NH2
R3
POCl3
R2
NaHCO3
O HN
(ia) or (ii a)
N
R1 F
Cl 2a-f
1a-f
C2H5OH NaHCO3
(i b) or (ii b)
O
R3 R2
O
2a. and 3a. R1=H; R2=H; R3=H; 2b. and 3b.R1=H; R2=NO2; R3=H; 2c. and 3c. R1= H; R2=H; R3=Cl; 2d. and 3d.R1=H; R2=H; R3=OH 2e. and 3e. R1=H; R2=H; R3=Br; 2f. and 3f. R1=H; R2=H; R3=NO2
HN
N
R1
NH F
3a-f
(i a) Reflux 5h , 50-70% (ii a) POCl3+Al2O3, MWI 200 W, 110oC, 10-12 min, 70-80% (i b) Reflux 20 h, 60-70% (ii b) Al2O3, MWI 120 W, 100oC, 15-17 min, 80-90%
Scheme 1
Scheme 1
5. Conclusions Microwave Synthesizer is proved to be an useful device to carryout environmental friendly synthesis of dihydropyrimidine derivatives with high yields along good purity in a very short span of time. The current protocol is highly useful for the synthesis of 3-(2-chloro-6-substituted phenyl- 3,4-dihydropyrimidin-4-yl)-4Hbenzo[h]chromen-4-one 2a-f and 3-(2-(4-fluorophenylamino)-6-substitutedphenyl-3,4-dihydropyrimidin-4-yl)-4Hbenzo [h]chromen-4-one 3a-f. Besides the green chemistry process synthesized molecules were evaluated for their antimicrobial properties using well diffusion method. Most of the compounds show their efficacy against S.aureus and B.subtilis (Gram +ve) bacteria and E.coli (Gram-ve) bacterial species. Apart from them compounds 2e, 2f, 3b, 3e and 3f shows good antibacterial properties at the very low concentration of 10 μl in comparison of the standard drug Ciprofloxacin. Antimicrobial properties attributed due to the presence of electron withdrawing and electron donating group. Acknowledgements The authors are thankful to, SAIF Chandigarh for 1H and 13C NMR analysis, CIL Chandigarh for IR analysis, Saif Cochin for Elemental Analysis, Syn Zeal Research Solutions, Gandhinagar for Mass Analysis, Department of Microbiology, S.K.Porwal College for conducting microbial activities, Head, Department of Chemistry, Rashtrasant
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Tukadoji Maharaj Nagpur for providing laboratory facilities. The author is grateful to UGC, New Delhi for financial support. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26]
G.Bacolini, Topics.Heterocycl.Syst.Synth. React. 1(1996)103-110. A.T.Balaban, D.C.Oniciu, A.R.Katritzky, Chem.Rev. 104(2004) 2777-2812. A.Domling,Curr.Opin. Chem. Biol. 4(2000) 318-323. M.Chauhan, R. Kumar, Bioorg. Med. Chem. 21(2013) 5657-5668. G. Khanage, A. Raju, P.B. Mohite, R. B. Pandhare, Adv. Pharm. Bull. 3(2013) 13-18. R. Sawant, V. Sarode, Iran. J.Pharm .Res. 10(2011) 733-739. P. Mondal, S. Jana, A. Balaji, R. Ramakrishna, L. Kanthal. J. Young. Pharm. 4(2012) 38-41. Tian Y, Du D, D. Rai, L. Wang, H. Liu, P. Zhan, E.D. Clercq, C. Pannecouque, X. Liu, Bioorg. Med. Chem. 22(2014) 2052-2059. Y.P. Lv, X.Y. Wang, B.A. Song, S. Yang, K. Yan, G.F. Xu, P.S. Bhadury, F.Liu , L.H. Jin, D.Y. Hu. Molecules.12(2007) 965-978. M.M. Alam, M. Akhter, A. Husain, A. Marella, O.P. Tanwar, R. Ali, S.M. Hasan, H. Kumar, R. Haider, M. Shaquiquzzaman, Acta.Pol.Pharm. 69(2012) 1077-1085. K. M. Amin, M.M. Hanna, H.E. Ab-Youssef, R.F. George, Eur. J. Med. Chem. 44(2009) 4572-4584. Adhikari, B. Kalluraya, V. Sujith, K. Gouthamchandra, R. Jairam, R. Mahmood, R. Sankolli, Eur. J. Med. Chem. 55(2012) 467-474. L.J. Paliwal, M.B. Bagade, K. Lal, Am. J. Pharm.Tech. Res. 4(2014) 646-656. K. Rana, A. Arora, S. Bansal, R. Chawla, Indian J. Pharm. Sci. 76(2014) 339-347. A.P. Keche, G.D. Hatnapure, R.H. Tale, A.H. Rodge, S.S. Birajdar, V.M. Kamble, Bioorg. & Med. Chem. Lett. 22(2012) 3445-3448. N.R. Kamdar, D.D. Haveliwala, P.T. Mistry, S.K. Patel, Eur. J. Med. Chem. 45(2010) 5056-5063. B. Mohan, R B.V. Kumar, S.C. Dinda, D. Naik, S.P. Sreenivasan, V. Kumar, D.N. Rana, P.S. Brahmkshatriya, Bioorg. & Med. Chem. Lett. 22(2012) 7539-7542. Z. Liu, Y. Wang, H. Lin, D. Zuo, L. Wang L, Y. Zhao, P. Gong, Eur.J.Med. Chem. 85(2014) 215-217. A. Gangjee, A. N. Ojas, M.A. Yu Jhnat, Thorpe J.E, L.C. Bailey-Downs, Bioorg. Med. Chem. 21(2013) 1312-1323. K.N. Mohana, B.N.P. Kumar, L. Mallesha, Drug Invention Today. 5(2013) 216-222. N. Kaur, K. Kaur, T. Raj, G. Kaur, A. Singh, T. Aree, S.J. Park, T.J. Kim, N. Singh, D.O. Jang, Tetrahedron. 71(2015) 332-337. K. Rana, A. Arora, S. Bansal, R. Chawla, Indian J.Pharm. Sci. 76(2014) 339-347. K. Singha, H. Kaura, K. Chibaleb, J. Balzarinic, Eur.J.Med.Chem. 66(2013) 314-323. H. Parveen, F. Hayat, A. Salahudin, A. Azam, Eur.J.Med.Chem. 45(2010) 3497-3503. S.G. Khanage, S.A. Raju, P.B. Mohite, R.B. Pandhare, Adv.Pharm.Bull. 2(2012)213-222. A.Dastan , A.Kulkarnia, B. Torok, Green Chem. 14 (2012)17-37.
[27] V.Santagada, F.Frecentese, E.Perissutti, F.Fiorino, B.Severino, G.Caliendo, Med. Chem.9 (2009)340-358. [28] M.Phukan, K.J.Borah, R. Borah Green Chem. Lett. Rev. 2(2009)249-253. [29] P.T.Anastasa, E.S.Beacha, GreenChem. Lett. Rev. 1(2007)9-24. [30] H.Villar, M.Frings, C.Bolm, Chem. Soc. Rev. 36(2007)55-56. [31] K.Lal, L.J.Paliwal, M.B.Bagade, Lett. Org. Chem.13 (2016)255-262. [32] A.L. Barry, Bio. Abstr. 64(1977) 25183.
ScienceDirect Materials Today: Proceedings 4 (2017) 5657–5662
www.materialstoday.com/proceedings
ICMDA 2016
New Dihydropyrimidines as Antimicrobial Agents Kundan Lala*, L.J. Paliwala, M.B. Bagadeb Post Graduate Department of Chemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440 033, Maharashtra, India. bPost Graduate Teaching Department of Chemistry, Seth Kesarimal Porwal College, Kamptee - 441 001, Maharashtra, India.
*a
Abstract A series of novel dihydropyrimidines having benzo chromone and substituted phenyl ring along with pyrimidine nucleus have been synthesized under microwave irradiation. Synthesized compounds were characterized by physical, chemical and spectroscopic methods. Solvent free conditions, simple procedures, with high purity of the products in a very less time make this method highly valuable. The compounds were screened for antimicrobial properties to check their efficacy against few bacterial species. Results of the invitro antimicrobial activities showed that the compounds possess very promising antibacterial properties. © 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON MULTIFUNCTIONAL MATERIALS FOR DEVICE APPLICATIONS(ICMDA-2016). Keywords: p-Fluroaniline; Microwave Irradiation (MWI), Chloropyrimidine; Chromen-4-one; Antibacterial Activity; Well-Diffusion.
*Corresponding author Tel.:+91 9960723698 Email address:[email protected]
2214-7853 © 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON MULTIFUNCTIONAL MATERIALS
FOR DEVICE APPLICATIONS(ICMDA-2016).
5658
Kundan Lal et al/ Materials Today: Proceedings 4 (2017) 5657–5662
1. Introduction The growing resistance of the microbial species against available drugs has created a major problem for the human society. The current scenario requires invention of new drugs with increased potency against the pathogens. Literature survey reveals that drugs containing heterocyclic moiety showed great potency against the dangerous microbes. Various heterocyclic moieties are synthesized in pharmaceutical companies to counter the growing resistance [1]. Among heterocyclic groups compounds possessing nitrogen containing compounds showed a wide variety of drugs against various pathogens [2]. In the nitrogen containing heterocyclic compounds pyrimidine and their derivatives play a vital role in the treatment of various diseases [3-4]. Different pyrimidine derivatives possess broad spectrum properties and show various biological activities such as analgesic[5-7], anti-HIV [8], anti-viral [9], anti-inflammatory [10-12], antimicrobial [13-15], anti-tubercular [16-18], antitumour[19-20], anticonvulsant and antioxidant [21], anticancer [22-23], antimalarial [24] and anti amoebic activity [25]etc, Apart from the biological activity of heterocyclic compounds the different protocols has also been taken into practice to enhance the yield. Environment friendly approach has been used extensively since the beginning of the 21st century for the synthesis of heterocyclic compounds [26]. Among various methods, microwave assisted synthesis has been found to be of great interest as it not only environmental friendly but also give high yield with less time for the reaction [27-30]. In accordance with the accessibility of the earlier drugs having pyrimidine nucleus used for the treatment in bacterial diseases, the current work is another progress [31]. Here we would like to report the microwave assisted synthesis of some new pyrimidine derivatives with benzo chromone with pyrimidine nucleus along with substituted phenyl ring. These new compounds have been evaluated for their biological activities against various species of gram positive and gram negative bacteria. Apart from adopting the environment friendly approach the potency of the synthesized compounds has been enhance by incorporating the chromone nucleus and various electron donation and electron withdrawing groups along with pyrimidine nucleus. 2. Experimental 2.1 Materials All the chemicals were obtained commercially, mostly from S.D. Fine, Merck and were used without purification. 2.2 Equipment Melting points were determined by the open capillary method and are uncorrected. The progress of the reaction as well as purity of compounds were monitored by TLC using silica coated aluminium TLC plates (TLC Silica Gel 60 F254, Merck, Germany) by different eluent systems. The spots were visualized by keeping the dry plates in iodine vapors and in UV light. IR spectra were recorded on Perkin Elmer Spectrometer (RX-IFTIR) in KBr discs. 1H and 13C NMR spectra were recorded in DMSO-d6 on Bruker Avance II 400 MHz NMR Spectrometer. Mass spectra were scanned on Expression CMS (ESI) spectrometer. Elemental analyses were carried out with Elementar Vario EL III elemental analyzer. The microwave reactions were performed in the CEM Focused MicrowaveTM Synthesis System. 2.3 General procedure for preparation of 3-(2-chloro-6-substituted phenyl-3,4-dihydropyrimidin-4-yl)-4Hbenzo[h]chromen-4-one 2a-f Conventional Procedure A: 4-(4-Oxo-4H-benzo[h]chromen-3-yl)-6-substituted phenyl-3,4-dihydropyrimidin-2(1H)one 1 (0.01mol) was taken in a round bottom flask with phosphoryl chloride (POCl3) (15 mL) and was refluxed at 100 °C for 5 h on water bath. After the completion of reaction, the reaction mixture was poured into cold water and was further neutralized by using 5% NaHCO3 solution. Obtained product was filtered, washed with distilled water, dried and was recrystallized from ethanol. Microwave Assisted Reaction A: A mixture of 4-(4-Oxo-4H-benzo[h]chromen-3-yl)-6-substituted phenyl-3,4dihydropyrimidin-2(1H)-one 1 (1mmol) and alumina (0.5 g) was mix well together and transferred to Erlenmeyer flask and to it phosphoryl chloride (POCl3) (0.5 mL) was added. The flask was then capped and was irradiated under microwave irradiation at 175 W at 110 °C for 10-12 min. After the completion of reaction (monitored by TLC), the reaction mixture was filtered to remove alumina from the product and was poured into cold water. The product was further neutralized by using 5% NaHCO3 solution. Obtained product was filtered, washed with distilled water, dried and was recrystallized from ethanol.
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2.4 General procedure for the Synthesis of 3-(2-(4-fluorophenylamino)-6-phenyl-3,4-dihydropyrimidin-4-yl)-4H benzo [h]chromen-4-one 3a-f Conventional Procedure B: A mixture of 3-(2-chloro-6- substituted phenyl-3,4-dihydropyrimidin-4-yl)-4Hbenzo[h]chromen-4-one 2a-f (0.01mol) and p-fluoro aniline (0.01 mol) in ethanol (25 mL) was refluxed for 20 h. After the completion of reaction, the reaction mixture was poured into cold water and was further neutralized by using 5% NaHCO3. Obtained product was filtered, washed with distilled water, dried and was recrystallized from ethanol: acetic acid (2:1). Microwave Assisted Reaction B: A mixture of 3-(2-chloro-6- substituted phenyl-3,4-dihydropyrimidin-4yl)-4H-benzo[h]chromen-4-one 2a-f (1mmol) and alumina (0.5 g) was mix well together and transferred to Erlenmeyer flask and to it p-fluoro aniline (1 mmol) was added. The flask was then capped and was irradiated under microwave irradiation at 175 W at 110 °C for 10-12 min. After the completion of reaction, the reaction mixture was filtered to remove alumina from the product and was poured into ice cold water. The obtained product was further neutralized by using 5% NaHCO3 solution. Product was filtered, washed with distilled water, dried and was recrystallized from ethanol: acetic acid (2:1). The physical and chemical properties has been reported in Table 1 Table 1. Physical Properties of 3-(2-chloro-6-substituted phenyl-3,4-dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 2a-f and 3-(2-(4fluorophenylamino)-6-phenyl-3,4-dihydropyrimidin-4-yl)-4H benzo [h]chromen-4-one 3a-f Compound
2a 2b 2c 2d 2e 2f 3a 3b 3c 3d 3e 3f
R1
H H H H H H H H H H H H
R2
H NO2 H H H H H NO2 H H H H
R3
H H Cl OH Br NO2 H H Cl OH Br NO2
Yield (%) Conventional(Reflux)
MWI
50 60 48 50 60 65 60 62 58 70 68 65
70 75 70 70 70 80 80 88 75 85 80 90
Reaction Time Conventional(h) MWI(min) 5 5 5 5 5 5 20 20 20 20 20 20
12 10 12 12 12 10 18 16 15 15 15 18
3. Biological Activity The antimicrobial activities were determined by using well diffusion method [32] by measuring zone of inhibition in mm. The microbial strains were obtained from the microbiology laboratory, S.K.Porwal College, Kamptee. MHA (Mueller-Hinton Agar) was used as microbial growth medium. The media were prepared according to the instructions given by the manufacturer. A loopful of culture was inoculated from the stock culture in 5 mL of MHA broth and the broth was incubated at 35 °C in incubator for 6-8 h. After incubation, this culture was used for the inoculation of MH test agar plates. Medium was autoclaved and was maintained at 45-50 °C in constant temperature water bath. 0.5 ml of 6-8 h old test organism was transferred to petridish of 100 mm size (sterilized in oven at 180 °C for 1 h) using sterile micropipette. MH test agar medium maintained at 45-50 °C was poured and mixed properly. Petri plates were allowed to set at room temperature. A 10 mm borer was used to prepare wells in agar plates. By using micropipette 10 μl of the test sample was transferred to each well. Plates were immediately kept at 4 °C in refrigerator for 1 h for diffusion of the samples and then shifted to 35 °C in incubator. The zone of inhibition was measured in millimetres by the end of the incubation period of 24 h at 35 oC in incubator. Ciprofloxacin was used as a standard drug for antibacterial screening. DMSO was used as control as it does not show any inhibition. All newly synthesized compounds 2a-f and 3a-f were screened to check their antimicrobial activities against two Gram positive strains (S.aureus, and B.Subtillis) and two Gram negative strains (E.coli and K. aerogenes) at concentration of 10 μl. The results have been summarized in Table 2.
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Table 1 Invitro Antimicrobial Activity of synthesized compounds 2a-f and 3a- f. Compound
Zone of inhibition (in mm) Gram Negative
2a 2b 2c 2d 2e 2f 3a 3b 3c 3d 3e 3f Ciprofloxacin
E. coli 14 18 12 10 18 20 9 18 16 10 16 20 22
Gram Positive K. aerogenes 9 4 3 11 11 9 6 8 9 5 13 10 20
S. aureus 11 17 22 10 15 18 9 14 15 22 14 21 22
B. subtilis 10 20 10 8 20 18 10 20 17 14 19 17 22
4. Results and Discussion Herein, we are reporting the synthesis and invitro antibacterial study of 3-(2-chloro-6-substituted phenyl3,4-dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 2a-f and 3-(2-(4-fluorophenylamino)-6- substituted phenyl3,4-dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 3a-f. At first, 3-(2-chloro-6-substituted phenyl-3,4dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 2a-f have been synthesized from 4-(4-oxo-4Hbenzo[h]chromen-3-yl)-6-substitued phenyl-3,4-dihydropyrimidin-2(1H)-one 1a-f by mixing with phosphoryl chloride(POCl3) and was refluxed/ irradiated under microwave radiation. Obtained products were basified by using 5% NaHCO3 solution to get the desired products (Scheme 1). Further, a mixture of 3-(2-chloro-6- substituted-3,4dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 2a-f and p-fluoro aniline was mixed and was reflux/irradiated under microwave radiation Scheme 1. After the completion of reaction, the reaction mixture was poured into ice cold water and was neutralized using 5% NaHCO3 solution. The obtained products were further characterized on the basis of spectral data (IR, MS, 1H and 13C NMR). FTIR Spectra of 3-(2-chloro-6-phenyl-3,4-dihydropyrimidin-4yl)-4H-benzo[h]chromen-4-one 2a showed characteristic band at 1069 cm-1 and peak at 768 cm-1 showing the presence –C-Cl. Peaks observed at 3198 and 1633 cm-1 confirms the presence of NH and -C=O (Chromone). Molecular ion peaks at m/z 386.2 [M+, 100 %], 388.7 [M+2, 30 %] were observed which also explained the existence of chlorine in the compound and the ratio of the two consecutive peaks was found to be 3:1 lend credence to the structure. The 1H NMR spectrum showed characteristic doublet at 9.15 ppm (J =2.5Hz) due to NH proton. Active methylene protons of CH appeared as doublet at 7.0 ppm (J =7.2Hz). The aromatic protons have been appeared as multiplet between 7.9-7.3 ppm. The doublet of =CH occurred at 8.4 ppm (J =7.5). The 13C NMR spectrum recorded in DMSO-d6 showed characteristic peak at 189.1 due to –C=O (chromone). The peaks depicted between 137.4-113.8 are assigned to aromatic carbons. The peaks at 89.0, 55.30and 44.0 are due to–CH and -C=CH- and –C-Cl of pyrimidine ring respectively. Similarly, compound 3-(2-(4-fluorophenylamino)-6-phenyl-3,4dihydropyrimidin-4-yl)-4H-benzo[h]chromen-4-one 3(a) showed characteristic band at 1633 cm-1 due to C=O (chromone) stretching vibration. A broad peak at 3364cm-1 was observed due to NH of pyrimidine ring. One sharp peak observed at 1212 cm-1(m) showed the presence of Ar-F in the aromatic ring. Molecular ion peak at m/z 462.3 [M+, 95 %], also lend credence to the structure for 3(a). The 1H NMR spectrum recorded in DMSO-d6 showed characteristic singlet at 9.1 ppm and a broad peak appeared at 10.0 ppm assigned to NH of pyrimidine and pfluroaniline respectively. Active methylene protons -CH appeared as doublet at 8.3 ppm ( J =7.0) and aromatic protons appeared as multiplet between 8.5-7.4 ppm. The doublet of =CH occurred at 6.5 ppm (J =7.5). The 13C NMR spectrum recorded in DMSO-d6 showed characteristic doublet at 114.50 ppm (J=7Hz) appear for –Corth-F (fluroaniline) also doublet of doublet appeared at 130.08, 127.83, 128.89 and 168.64 ppm with (J= 29.3, J= 9.8Hz, J= 9.2 and J= 246.3 respectively) appear for –C-F bonding with reference to various positions. Similarly, peak at 156.5 assigned to –C=O (pyrimidine). Compounds like 2b, 2d, 3b and 3d showed prominent peaks for the NO2
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groups at 1340(s), 1530(s) and 1500(s). Presence of Br and F groups also observed at 1040 and 1130 cm-1 respectively. Mass spectrum showed M+ (100%) and M+2(97%) for bromine and peaks of M+ (99.2%) and M++2(27%) confirms the presence of chlorine. The peaks depicted in the region 129.7-114.5 are assigned to aromatic carbon. The peaks at 89.14 and 55.20 are due to –CH and -C=CH- of pyrimidine ring respectively. Table 1 also showed that adopted method drastically increases the yield from 48-65% to 70-80% in case of 2a-f and 58-70% to 80-90% with 3a-f. Also the time for completion of the reaction has been reduced very much from 5 h to 10-12 min in case of 2a-f and 20 h to 15-18 min and the easy protocol make this method very useful for the others. Considering the invitro antimicrobial activity of compounds 2a-f and 3a-f, compounds 2b, 2c, 2e, 2f, 3b, 3e and 3f showed prominent activity against S.aureus, B.Subtillis and E.coli and moderate activity against K. aerogenes. Other compounds showed moderate activity. The presence of the electron with drawing and electro negative group enhance the antibacterial properties of the compounds.
O
O
R3 R2
O HN
NH O
R1
NH2
R3
POCl3
R2
NaHCO3
O HN
(ia) or (ii a)
N
R1 F
Cl 2a-f
1a-f
C2H5OH NaHCO3
(i b) or (ii b)
O
R3 R2
O
2a. and 3a. R1=H; R2=H; R3=H; 2b. and 3b.R1=H; R2=NO2; R3=H; 2c. and 3c. R1= H; R2=H; R3=Cl; 2d. and 3d.R1=H; R2=H; R3=OH 2e. and 3e. R1=H; R2=H; R3=Br; 2f. and 3f. R1=H; R2=H; R3=NO2
HN
N
R1
NH F
3a-f
(i a) Reflux 5h , 50-70% (ii a) POCl3+Al2O3, MWI 200 W, 110oC, 10-12 min, 70-80% (i b) Reflux 20 h, 60-70% (ii b) Al2O3, MWI 120 W, 100oC, 15-17 min, 80-90%
Scheme 1
Scheme 1
5. Conclusions Microwave Synthesizer is proved to be an useful device to carryout environmental friendly synthesis of dihydropyrimidine derivatives with high yields along good purity in a very short span of time. The current protocol is highly useful for the synthesis of 3-(2-chloro-6-substituted phenyl- 3,4-dihydropyrimidin-4-yl)-4Hbenzo[h]chromen-4-one 2a-f and 3-(2-(4-fluorophenylamino)-6-substitutedphenyl-3,4-dihydropyrimidin-4-yl)-4Hbenzo [h]chromen-4-one 3a-f. Besides the green chemistry process synthesized molecules were evaluated for their antimicrobial properties using well diffusion method. Most of the compounds show their efficacy against S.aureus and B.subtilis (Gram +ve) bacteria and E.coli (Gram-ve) bacterial species. Apart from them compounds 2e, 2f, 3b, 3e and 3f shows good antibacterial properties at the very low concentration of 10 μl in comparison of the standard drug Ciprofloxacin. Antimicrobial properties attributed due to the presence of electron withdrawing and electron donating group. Acknowledgements The authors are thankful to, SAIF Chandigarh for 1H and 13C NMR analysis, CIL Chandigarh for IR analysis, Saif Cochin for Elemental Analysis, Syn Zeal Research Solutions, Gandhinagar for Mass Analysis, Department of Microbiology, S.K.Porwal College for conducting microbial activities, Head, Department of Chemistry, Rashtrasant
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