Dendrimers have wide range of applications in industry, Pharmaceutical and medicine. Oxadiazole compounds also have many applications in Pharmaceutical and medicine. Modification of dendrimers which have many branches like NH2 functional groups with heterocyclic derivatives make them capable to show variety properties. In this article, PEI-dend-4N(Ts)(2-(methyl)-5-aryl-1,3,4 oxadiazole) ]] 8a-f have been synthesized from PEI (Poly ethylene imine) dendrimer which modified with 1,3,4 oxadiazole derivatives and investigated their antimicrobial activitie

Avval, M. M.

Shashikanth, S.

Srinivasa Murthy, V.

University of Mysore - Digital Repository of Research, Innovation and Scholarship (ePrints@UoM)

          ISSN: 0975-8585  May-June    2014  RJPBCS 5(3)  Page No. 441 Research Journal of Pharmaceutical, Biological and Chemical Sciences  Synthesis and Antimicrobial activity Evaluation of Poly ethylene Imine (PEI) dendrimer modified with 1,3,4 oxadiazole derivatives.                              Maziar Mansouji Avval1*, V.Srinivasa Murthy2,  S.Shashikanth1.  1 Department of Studies in Chemistry, University of Mysore, Manasagangorti 570 006, India. 2 BNM Institute of Technology, Bhanashankari, Bangalore 560 076, India.   ABSTRACT  Dendrimers have wide range of applications in industry, Pharmaceutical and medicine. Oxadiazole compounds also have many applications in Pharmaceutical and medicine. Modification of dendrimers which have many branches like –NH2 functional groups with heterocyclic derivatives make them capable to show variety properties. In this article, PEI-dend-4[N[(Ts)(2-(methyl)-5-aryl-1,3,4 oxadiazole) ]] 8a-f have been synthesized from PEI (Poly ethylene imine) dendrimer which modified with 1,3,4 oxadiazole derivatives and investigated their antimicrobial activities.  Keywords: Poly ethylene imine(PEI) Dendrimer. 1,3,4 Oxadiazole. Antimicrobial.                       *Corresponding author            ISSN: 0975-8585  May-June    2014  RJPBCS 5(3)  Page No. 442 INTRODUCTION  Dendrimers are an interesting class of molecules, because they are relatively easily may be modified at their surface to obtain specific biological properties. Therefore by appropriate variation of the surface motifs, dendrimers with specific biological properties can be constructed and their biological properties can be modulated in endless ways. The biological properties of dendrimers are to a large extent determined by the nature of the surface groups, opening up for the possibility to specifically tailor the dendrimers for the desire biological effects. Dendrimers can also be synthesized to possess intrinsic biological properties that can be useful for treatment and other medical interventions. Dendrimers offer a range of advantages with respect to mediating biological effects in the living organism.   Oxadiazoles and their analogues can be considered as simple membered heterocycles possessing one oxygen and two nitrogen atoms. 1,3,4 oxadiazoles is the only isomers not containing a nitrogen-oxygen bond and are thermally stable neutral aromatic molecules[1] and its estimated resonance energy is 167.4 KJ/mol. 1,3,4 oxadiazole are associated with potent. Pharmacological activity due to the presence of toxophoric linkage[2] considerable evidence have been accumulated to demonstrate the efficacy of 1,3,4 oxadiazoles including anti  protelytic, analgesic[3]anti malarial, anti cetylcholine esterase[4], anti inflammatory[5][6]  diuretic[7] [8]  , anti pyretic[9] and anti emetic activities.   MATERIALS AND METHODS  All the solvents and reagents were used as AR grade and used as such without further purification. The NMR spectra were recorded on Agilent 400 MHz spectrometer using DMSO-d6, CDCl3 solvents. Silica gel column chromatography was performed using Merck silica gel (100-200 mesh) and Merck made TLC were used for reaction monitoring. General procedure for the synthesis of DEN-4[N[(Ts)(1,3,4 oxadiazole)]].  Dendrimer dissolved in dry DMF and added at 0oC to 60% NaH under N2 atmosphere.1,3,4 oxadiazole dissolved in dry DMF and added to dendrimer mixture drop wise in 15 min. The reaction mixture heated to 80oC overnight then extracted with EtOAc and worked up.             1                             2                        3                     4  Sheme1: i) Tosyl Chloride/MDC,TEA/RT, 2h  ii)Tolene/NaOH 20%/0oC, 2h iii)Toluene/Acetonitrile/ethylenediamine/Reflux.            ISSN: 0975-8585  May-June    2014  RJPBCS 5(3)  Page No. 443           5a-f                           6a-f                                                                    7a-f  R:a:4Me,   R:b:4Cl,  R:c:4F,  R:d:4OMe,  R:e:4Br, R:f:H                        Scheme 2:  iv)Chloro acetyl chloride/THF/reflux  v) POCl3/reflux   7a-f + 4 8a-f  R:a:4Me,   R:b:4Cl,  R:c:4F,  R:d:4OMe,  R:e:4Br, R:f:H                        Scheme 3: vi) DMF/NaH 60%/0oc/reflux.  Typical procedure for the synthesis of PEI-dend-4[N[(Ts)(2-(methyl)-5-aryl-1,3,4 oxadiazole) ]] 8a-f.  Dendrimer (4),(0.2gr-0.0023 mol) dissolved in 4 ml of dry DMF was added to 60% NaH (14.4gr-0.6mol) in dry DMF (10ml) under nitrogen atmosphere. The reaction mixture heated to 80oC for 4 h and cooled to 0oC.   2-(Chloromethyl)-5-aryl-1,3,4 oxadiazole (7a-f),(0.2gr-0.0118mol) dissolved in 4 ml dry DMF and added to dendrimer solution drop wise in 15 min. The reaction mixture heated to 80oC overnight. The completion of the reaction was monitored by TLC and after completion, quenched by iced water then extracted with 2x10ml EtOAc and washed with 3x20ml water and brine solution then passed over Na2SO4, concentrated under reduced presser to give crude product. The crude product was purified by column chromatography to afford 74% yield product.           ISSN: 0975-8585  May-June    2014  RJPBCS 5(3)  Page No. 444 DEN-4[N[(Ts)(2-(methyl)-5-(4-methylphenyl)-1,3,4 oxadiazole)]] 8a . 1H NMR (CDCl3): 2.07(s, 1H), 2.17-2.21(t, 2H), 2.32-2.37(dd, 6H), 2.62(t, 2H), 5.10(s, 2H), 7.31-7.41(dd, 4H), 7.62-7.64(d, 2H), 7.86-7.88(d, 2H). DEN-4[N[(Ts)(2-(methyl)-5-(4-Chlorophenyl)-1,3,4 oxadiazole)]] 8b . 1H NMR (CDCl3): 2.01(s, 1H), 2.13-2.17(t, 2H), 2.31(s, 3H), 2.59(t, 2H), 5.08(s, 2H), 7.39-7.49(dd, 4H), 7.52-7.54(d, 2H), 7.66-7.68(d, 2H). DEN-4[N[(Ts)(2-(methyl)-5-(4-Fluorophenyl)-1,3,4 oxadiazole)]] 8c . 1H NMR (CDCl3): 2.03(s, 1H), 2.15-2.17(t, 2H), 2.35 (s, 3H), 2.60(t, 2H), 5.12(s, 2H), 7.38-7.48(dd, 4H), 7.66-7.68(d, 2H), 8.62-8.64(d, 2H). DEN-4[N[(Ts)(2-(methyl)-5-(4-methoxyphenyl)-1,3,4 oxadiazole)]] 8d . 1H NMR (CDCl3): 2.10(s, 1H), 2.2-2.26(t, 2H), 2.48(s, 3H), 2.69(t, 2H), 3.81(s, 3H), 5.30(s, 2H), 7.41-7.51(dd, 4H), 7.72-7.74(d, 2H), 8.01-8.03(d, 2H). DEN-4[N[(Ts)(2-(methyl)-5-(4-Bromophenyl)-1,3,4 oxadiazole)]] 8e . 1H NMR (CDCl3): 2.03(s, 1H), 2.11-2.13(t, 2H), 2.24(s, 3H), 2.58(t, 2H), 5.15(s, 2H), 7.29-7.39(dd, 4H), 7.32-7.34(d, 2H), 7.42-7.44(d, 2H). DEN-4[N[(Ts)(2-(methyl)-5-(phenyl)-1,3,4 oxadiazole)]] 8f . 1H NMR (CDCl3):  2.00(s, 1H), 2.18-2.22(t, 2H), 2.68(t, 1H), 5.11(s, 2H), 7.49-7.59(m, 5H), 7.59-7.61(d, 2H), 8.11-8.13(d, 2H).  Biology  Primary Screening by Agar well diffusion method  The  antibacterial  activity of the newly synthesized compounds were evaluated by agar well diffusion method [10]. All the microbial cultures were adjusted to 0.5 McFarland standards, which is visually comparable to a microbial suspension of approximately 1.5×108 CFU/ml. 20 ml of Muller Hinton agar media was poured into each Petri plate and plates were swabbed with 100 ml inoculate of the test microorganisms and kept for 15 min for adsorption. Using sterile cork borer of 8 mm diameter, wells were bored into the seeded agar plates and these were loaded with a 100 µl volume with concentration of 1.0 mg/ml of each compound reconstituted in the dimethyl formamide (DMF). All the plates were incubated at 37 ˚C for 24 h. Antibacterial activity of all the synthesized compounds was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (Hi antibiotic zone scale). The dimethyl formamide (DMF) solvent and gentamicin 10µg/well (standard antibiotic) were used negative and positive control  respectively. The experiments were performed in triplicates.  Determination of Minimum inhibitory concentration:   The broth micro dilution method was used to determine the minimum inhibitory concentration (MIC) according to the National Committee for Clinical Laboratory Standards (NCCLS, 2001) [11].All tests were performed in a Mueller-Hinton broth for the bacterial strains. Overnight broth cultures of each strain were prepared and the final concentration in each well was adjusted to 2×106 CFU/ ml. Compounds were dissolved in dimethyl formamide (DMF) and then diluted to the highest concentration. Two-fold serial concentrations of the compounds           ISSN: 0975-8585  May-June    2014  RJPBCS 5(3)  Page No. 445 were prepared (over the range 1000–0.19 µg/ml) in a 96-well micro titer plate. In the tests, triphenyl tetrazolium chloride (TTC) (Aldrich Chemical Company Inc., USA) was also added to the culture medium as a growth indicator. The final concentration of TTC after inoculation was 0.05%. The microbial growth was determined by the absorbance at 600 nm using a universal micro plate reader after incubation at 37° C for 24 h. The MIC is defined as the lowest concentration of the compound at which the microorganism does not demonstrate visible growth.  Table1: Antibacterial activity data of PEI-dend-4[N[(Ts)(2-(methyl)-5-aryl-1,3,4 oxadiazole) ]] 8a-f.  Compound No Zone of inhibition in mm (MIC in µg/ml) Test Bacteria Gram positive bacteria Gram negative bacteria Bacillus subtilis Staphylococcus aureus Staphylococcus epidermidis E.coli Xanthomonas campestris Salmonella typhi Pseudomonas aeruginosa 1 Best 23(12.5) 27(6.25) 20(12.5) 18(25) 16(25) 20(12.5) 17(25.5) 2 Mod 12(25) 15(25) - 16(25.0)  - - 3 Low 14(25) 18(25) - - - - - 4 Best 25(12.5) 23(12.5) 21(25) 20(25) 20(12.5) 18(25) 15(25) 5 Low 12(50) 14(25) - 12(50) - - - 6 Mod 18(25) 20(25) 20(12.5) 16(12.5) - - - Gentamicin 34(2) 32(2) 29(4) 30(4) 26(8) 28(4) 20(4) Nystatin ND ND ND ND ND ND ND “-“ : No activity  , “ND” : No determined  Table 2: Antifungal activity data of PEI-dend-4[N[(Ts)(2-(methyl)-5-aryl-1,3,4 oxadiazole) ]] 8a-f.    Compound No Zone of inhibition in mm (MIC in µg/ml) Test fungi Filamentous Yeast Aspergillus niger Candida albicans 1 Best 16(6.25) 18(6.25) 2 Mod - 14(12.5) 3 Low - 12(25) 4 Best 17(6.25) 18(6.25) 5 Low 14(25) - 6 Mod - 14(25) Gentamicin ND ND Nystatin 18(6.25) 20(6.25) “-“ : No activity  , “ND” : No determined  RESULTS AND DISCUSSION  Chemistry  The desired compounds 8a-f  were  synthesized as outlined in the scheme3. Compounds 8a-f were synthesized by introducing heterocyclic compounds 7a-f (scheme2) to dendrimer 4 (scheme1).           ISSN: 0975-8585  May-June    2014  RJPBCS 5(3)  Page No. 446 The dendrimer 4 was synthesized has shown in the scheme 1.  i) A solution of 2-Chloroethylamine hydrochloride was dissolved in dichloro methane (MDC) and added drop wise to a solution of p-methyl tosylchloride (Ts-Cl) dissolved in MDC and TEA at 0o C then stirred for 2 hour in room temperature. The solid Chloroethylamine tosylate (ClEtNHTs) was collected by suction filtration and washed with distilled water and dried in vacuum. ii) ClEtNHTs was dissolved in toluene and a solution of NaOH added drop wise in a ice/salt bath. The reaction mixture stirred for 2 h then white solid tosyl aziridine collected and washed with distilled water and dried with vacuum. iii) Tosyl aziridine dissolved in toluene and acetonitrile and a solution of ethylenediamine which dissolved in acetonitrile drop wise added to tosyl aziridine solution, then refluxed at 60o C overnight. The reaction mixture cooled to room temperature and the white solid collected and washed with acetonitrile and dried by vacuum.  The heterocyclic compounds 7a-f were synthesized has shown in the scheme 2.  By refluxing 85% hydrazine hydrate and aryl esters in methanol aryl acid hydrazides 5a-f prepared. iv) Aryl acid hydrazides 5a-f treated with Chloroacetylchloride in dry THF at 0oC followed by refluxing for 1 h, for preparing N(Chloroacetyl)-aryl acid hydrazides 6a-f . v) By refluxing with phosphorous oxychloride afforded 2-(chloromethyl)-5-aryl-1,3,4 oxadiazoles 7a-f .   Modification of dendrimer has shown in the scheme3.  vi) Dendrimer was dissolved in dry DMF and added at 0o C to 60% NaH under N2 atmosphere.1,3,4 oxadiazole dissolved in dry DMF and added to dendrimer mixture drop wise in 15 min. The reaction mixture heated to 80o C overnight then extracted with EtOAc and worked up.  The formation of compounds 8a-f was confirmed by 1H NMR. The proton of –NH-Ts groups in dendrimer 4, (δ : 7.32), was disappeared in 8a-f and aromatic protons for compounds 7a-f with different substitute was appeared in ( δ : 7.86-7.88 2H) for Meta protons and (δ : 7.35-7.37 4H) for Ortho protons merged with Tosyl group protons.  Biology  The antimicrobial activity of compounds 8a-f was evaluated in vitro against some human pathogenic Gram positive bacteria such Bacillus subtilis (MTCC 121), Staphylococcus epidermis (MTCC 435), Staphylococcus aureus (MTCC 7433) and Gram negative bacteria E.Coli (MTCC 7440), Xanthomonas campestris (MTCC 7408), salmonella typhi (MTCC 733), Pseudomonas aeruginosa. Also compounds 8a-f were evaluated against fiamatory fungi such as aspergillus niger (MTCC 378) and yeast Candida albicans (MTCC 183).The corresponding zone of inhibition and minimum inhibitory concentrations were summarized in tables 1 and 2.           ISSN: 0975-8585  May-June    2014  RJPBCS 5(3)  Page No. 447 Results indicate that the compounds No: 1, 8d and No: 4, 8a have exhibited broad spectrum antimicrobial activity against both bacteria and fungi. Whereas that the rest of the compounds in the series have exhibited moderate antimicrobial activity when compared to positive controls. Antimicrobial spectrum indicates that the gram positive bacteria and fungi were more susceptible to the synthesized compounds than gram negative bacteria. The thorough investigation of our synthesized compounds 8a-f  pertaining to quantitative structure activity relationship highlighted that presence of more electron donating groups at Para position in phenyl ring bearing oxadiazole has influenced the antimicrobial activity, while it counterpart electron withdrawing groups has shown ins opposite effect. The compound No: 2, 8b have shown moderate activity when compared to positive controls. The  un substituted  compound No:5, 8f has shown better activity when comparing with compound No:3, 8c and compound No:6, 8e which has Fluorine and Bromo substituent.   CONCLUSION  In conclusion we have reported a facile route for the synthesis of PEI-dend-4[N[(Ts)(2-(methyl)-5-aryl-1,3,4 oxadiazole) ]] 8a-f, from PEI (poly ethylene imine) dendrimer 4 and 2-(chloromethyl)-5-aryl 1,3,4 oxadiazole) 7a-f.  The new molecular framework has shown broad spectrum antimicrobial activity which is substantiated by the presence of electron donating groups. Among the synthesized compounds 8a-f, compounds 8a and 8d has exhibited potent antimicrobial activity whereas the rest of the analogues have shown moderate activity when compared to the standard positive controls.   REFERENCES  [1] Frump, J. A Chem Rev 1971; 71, 483. [2] Rigo B and Couturier D. J Heterocycl Chem 1985; 22, 287. [3] Angilini I, Angilini L and Sparace F. British Pat 1,161,801. 1969. Through Chem. [4] Misra HK. Arch Pharma 1983; 316, 487. [5] Palaska E, Sahin G, Kelicen P, Durlu NT and Altinok G. IL Farmaco 2002; 57: 101 [6] Mullican MD, Wilson MW, Connor DT, Kostlan CR, Schrier DJ and Dyer RD. J Med Chem 1993; 36: 1090. [7] Raman K, Singh KH, salzman SK and Parmar SS. J Pharm Sci 1993; 82: 167.  [8] Thomas J. Ger Offen, 2,403, 357, 1974. Chem Abstr 1974, 81, 146153g. [9] Mishra L, Said MK, Itokawa H and Takeya K. Bioorg Med Chem 1995; 3: 1241. [10] Singh DP, Krishan Kumar and Chetan Sharma. European J Med Chem 2009; 44: 3299–3304. [11] Dragana Mitic, et al. European J Med Chem 2009; 44:1537–1544.  

Synthesis and antimicrobial activity evaluation of poly ethylene imine (pei) dendrimer modified with 1,3,4 oxadiazole derivatives

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Synthesis and antimicrobial activity evaluation of poly ethylene imine (pei) dendrimer modified with 1,3,4 oxadiazole derivatives

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