Research Journal of Chemical Sciences ______ ______________________________ ______ ____ ISSN 2231 - 606X Vol. 2 ( 4 ), 40 - 44 , April (201 2 ) Res.J.Chem.Sci. International Science Congress Association 40 Synthesis, Antibacterial and Antifungal Activities of some new Bipyrazolic Tripodal Derivatives RADI Smaail 1* Toubi Yahya 1 , Hamdani Imad 1 Hakkou Abdelkader 2 , Souna Faiza 2 , Himri Imane 2 and Bouakka Mohammed 2 1 Laboratoire de Chimie Organique, Macromoléculaire et Produits Naturels (URAC 25), Facultés des Sciences, Oujda, MOROCCO 2 Laboratoire de Biochimie, Département de Biologie, Facultés des Sciences, Oujda, MOROCCO Available online at: www.isca.in (Received 1 9 th February 201 2 , revised 23 r d February 201 2 , accepted 5 th March 201 2 ) Abstract A series of novel bipyrazolic tripodal derivatives were prepared in one step, in good and excellent yields. The in vitro antibacterial and antifungal activities of these products and their starting materials were screened against two fungal strai ns (Saccharomyces cerevisiae and Fusarium oxysporum f.sp albedinis) and against bacterial strains (Echerichia coli). Structur e - activity relationship (SAR) reflects the effect of substituted drugs. A considerable activity was recorded with respect to th e Fusarium oxysporum f.sp albedinis with MIC = 7,05. Keywords : Bipyrazole; structure – antibacterial activity relationships, antibacterial activity, antifungal activity . Introduction The important progression of microbial infections and the recrudescence of resistance towards the antibiotics used nowadays incite the researchers to make more efforts to discover and synthesize new m olecules with systemic activity. This activity must be efficient and less toxic for the host cells. In the agricultural environment, the oxysporum Fusarium species is one of the most important fungi organisms in the cultivated soil. It constitutes on its o wn 40 to 70% of the total Fusaran flora. It is represented by a set of much varying forms regarding morphology and physiology. These forms behave either as saprophytes or parasites of different plants, among these forms there may be various degrees of viru lence. The number of pathogenic forms of oxysporum Fusarium is estimated to 80. Some special forms are capable of producing disease in plants belonging to more than one family, such as oxysporum Fusarium f.sp apii which attacks celery and peas, oxysporum F usarium f.sp vasinfectum which is pathogenic for cotton, tobacco and alfalfa, oxysporum Fusarium f.sp lycopersici which attacks tomatoes, and oxysporum Fusarium f.sp melonis which attacks melon . The oxysporum Fusarium f.sp albedinis causal agent of vascu lar fusarium of date palm (Bayoud) constitute with oxysporum Fusarium f.sp cubense causal agent of fusarium of banana trees, the two most serious diseases. The discovery, development and synthesizing a new efficient, active and less toxic molecule for systemic activities was the aim and subject of many researchers. This is evident from the large number of scientific articles and reviews 1 - 5 . In this context, pyrazole derivatives are well established in the literature as important biologically active heterocyclic compounds. These derivatives are the subject of many research studies due to their widespread potential biological activities such as anti - inflammatory 6 , anti - anxiety 7,8 , antipyretic 9 , antimicrobial 10 , antiviral 11 , antitumor 12,13 , anticonvulsa nt 14 , antihistaminic 15 , antidepressant 16 , insecticides 17 , and fungicides 17 . On the other hand, it was found that pyrazole - 3 - carboxylate derivatives have shown potent and selective anti - viral/anti - tumour activity 18 . Also, pyrazole - 4 - carboxylates act as int ermediates for agricultural microbicides and herbicides 19 . Besides it has been observed those pyrazole - 4 - carboxylates when subjected to in vitro anti - bacterial screening showed activity against some strains of g ram - positive bacteria 20 . In our recent work, a series of acyclic and macrocyclic pyrazole compounds containing one, two, three or four pyrazole rings were prepared and demonstrated several applications 21,22 . The present study was carried out to investigate the antibacterial and antifungal inhibitio ns of several bipyrazole - 3 - carboxylates which were synthesized in excellent yields. We targeted to study the structure – activity relationship by altering the methyl part and the carboxylate moiety at the 3 - position of the pyrazole rings and substitutions at the 2 - position of the aniline ring. Herein we report, for the first time, the screening results of the antibacterial and antifungal activities of novel bipyrazolic tripodal derivatives and their starting materials. Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 4 ), 40 - 44 , April (201 2 ) Res.J.Chem.Sci International Science Congress Association 41 Material and Methods General: All solvents and other chemicals, obtained from usual commercial sources, were of analytical grade and used without further purification. The NMR spectra were obtained with a Bruker AC 300 spectrometer. Elemental analyses were performed by Microanalysis Ce ntral Service (CNRS). Molecular weights were determined on a JEOL JMS DX - 300 m ass s pectrometer. General procedure for the preparation of compounds 5 - 12: The products 5 - 12 were prepared by the addition of each: aniline, pyridin - 2 - amine, 2 - nitrobenzenamine and 2 - methylbenzenamine to 3 or 4 . To a solution of the substituted hydroxymethylpyrazole 3 or 4 (10 mmol) in acetonitrile (25 ml) was added the amine derivatives (10 mmol) and the mixture was continued under stirring at room temperature for 4 - 5 days. The crude material was evaporated, washed with water and CH 2 Cl 2 and purified by silica gel column flash - chromatography to give the target product. Synthesis of N, N - bis((3,5 - dimethyl - 1H - pyrazol - 1 - yl)methyl) - 2 - methylphenylamine 11 : White powder. Yield 90%. Mp 104 - 107°C. 1 H RMN (300MHz, CDC l3 ) δ ppm: 2,10 (s, 3H, phenyl - CH 3 ), 2,30 (s, 12H, pyrazol - CH 3 ), 5,40 (s, 4H, N - CH 2 - N), 6,65 (s, 2H, pyrazol), 6,60 - 7,20 (m, 4H, C 6 H 4 ). 13 C RMN (75 MHz, CDC l3 ) δ ppm: 11,50 (pyrazol - CH 3 ), 14( - O - CH 2 - CH 3 ), 59,65 (N - CH 2 - N), 109 (pyrazol, CH), 114 - 120 - 129(CH 2 , C 6 H 5 ), 140 (pyrazol, CH - CH 3 ), 143 (C=N), 145 (C 6 H 5 , C - N), 162,5 (C=O). Anal. Calcd. for C 19 H 25 N 5 : C 70.56, H 7.79, N 21.65, Found: C 70.29, H 7.83, N 21.55; m/z (M+): 323.44 Synthesis of N, N - bis ((3 - carboxyethyl - 5 - methyl - 1H - pyrazol - 1 - yl)methyl) - 2 - methyl phenylamine 12 : White powder. Yield 88%. Mp 142 - 144 °C. 1 H RMN (300MHz, CDC l3 ) δ ppm: 1,40 (t, 6H, - O - CH 2 - CH 3 ), 2,10 (s, 3H, phenyl - CH 3 ), 2,30 (s, 6H, pyrazol - CH 3 ), 4,40 (q, 4H, - O - CH 2 - CH 3 ), 5,50 (s, 4H, N - CH 2 - N), 6,65 (s, 2H, pyrazol, CH), 6,60 - 7,20 (m, 4H, C 6 H 4 ). 13 C RMN (75 MHz, CDC l3 ) δ ppm: 11,50 (pyrazol - CH 3 ), 14 ( - O - CH 2 - CH 3 ), 59,50 (N - CH 2 - N), 61( - O - CH 2 - CH 3 ), 109 (pyrazol, CH), 114 - 120 - 129(CH 2 , C 6 H 5 ), 140 (pyrazol, CH - CH 3 ), 143 (C=N), 145 (C 6 H 5 , C - N), 162,5 (C=O). Anal. Calcd. for C 23 H 29 N 5 O 4 : C 62.85, H 6.65, N 15.93, Found: C 62.71, H 6.56, N 16.02; m/z (M+): 439.5 Results and Discussion Chemistry: The synthesis of target drugs was illustrated in scheme - 1. Compounds (3, 5 - dimethy l - 1 H – pyrazol – 1 - yl ) methanol 3 and (3 - carboxyethyl - 5 - methyl - 1 H - pyrazol - 1 - yl) methanol 4 were already reported by several old and recent works. 23 - 26 The target bipyrazoles 5 - 12 were prepared, respectively, by condensation of two equivalents of 3 or 4 with on e equivalent of amino aryl derivatives (ii - v) (commercially available) under gentle conditions (room temperature, atmospheric pressure, 4 - 5 days), using anhydrous acetonitrile as solvent. The reaction is very slow but selective at room temperature. Recent ly, we have reported 27 the synthesis and antitumor activity of some compounds 5 - 10 against three human cancer cell lines including breast (MDA - MB231), prostate (PC3) and colorectal (LoVo) cancers. The structures of the all newly products 11 and 12 were determined on the basis of the corresponding analytical and spectroscopic data. Biological Assays: The compounds described in this manuscript 1 - 12 were tested in vitro for their activity against: Fungal strains ( Saccharomyces cerevisiae ) isolated fr om a yeast strain. Fungal strains ( Fusarium oxysporum f.sp albedinis ) isolated from a date palm having a vascular fusariose.Bacterial strains ( Echerichia coli ). Streptomycin was used as reference compound and better standard in antibacterial assay. The activities were determined by the agar diffusion technique as previously described 28 . The agar media were inoculated with test organisms and a solution of the tested compound in DMSO/EtOH (50/50) was added to different concentration in the culture media. The growth is followed by a count of bacteria and yeast colonies and measurement of mycelium diameter. The inhibition percentage of a molecule is equal to the ratio of the colonies number or the mycelium diameter of the culture in presence of a dose of th e tested compound over the colonies number or the mycelium diameter of the reference culture multiplied by 100. The minimum inhibition concentration (MIC) is the least dose of the compound which caused inhibition of the micro organism growth. The most tested compounds show growth inhibitory action for fungus oxysporum Fusarium f.sp. albedinis . However, they have no effects on bacteria E. coli and yeast Saccharomyces cerevisiae. The level of inhibition depends on the compound nature and its conc entration. We can thus conclude that introduction of different substituent in 3 - position of the pyrazole rings and in 2 - position of the aniline ring has an impact on the antibacterial activity. Result in table - 1 shows that monopyrazolic derivatives ( 1 - 4 ) did not exhibit any interestingly effect against two fungal and bacterial strains examined. However, products containing two pyrazole rings showed differential activity against oxysporum Fusarium f.sp. albedinis , as evident by the MIC of 7,05 values. Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 4 ), 40 - 44 , April (201 2 ) Res.J.Chem.Sci International Science Congress Association 42 Scheme - 1 Structure of synthesized compounds Table - 1 Rate of inhibition of the growth of Fusarium Oxysporum Fsp. Albedinis According to the concen tration of the compounds tested Compounds Concentration (µg/ml) MIC (µg/ml) MIC (µM) 1,25 2,5 5 10 20 40 80 160 1 0 0 8,67 29,30 48,70 50,34 66,53 73,88 5 52,04 2 0 0 4,33 12,11 20,13 40 69,81 95,66 5 32,45 3 0 0 0 0 0 0 0 0 - - 4 0 0 0 0 0 0 0 0 - - 5 0 7,35 14,44 47,89 68,22 76,66 89,12 100 2,5 8,05 6 0 0 8,88 23,56 43,78 61,53 65,02 74,51 5 11,73 7 0 10,66 23,76 43,33 47,55 51,56 86,60 100 2,5 8,08 8 0 0 0 0 0 13,33 16,66 23,5 40 94,07 9 0 4,75 10,25 31,21 52,76 69,41 77,64 93,65 2,5 7,05 10 0 0 9,87 22,90 45,55 52,22 73,44 100 5 10,63 11 0 0 0 0 0 5,57 11,1 12,90 40 123,84 12 0 0 0 0 0 0 5,55 20,51 80 182,14 Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 4 ), 40 - 44 , April (201 2 ) Res.J.Chem.Sci International Science Congress Association 43 The bipyrazolic derivatives present two regions for SAR evaluation, the increased potency of compounds 9 and 10 could be attributed most probably to the electron - attracting nitro group in the pheny l region. Replacement of nitro group with an electron - donating methyl group resulted in complete loss of activity (compounds 11 - 12 ). The increased potency of compounds 5 and 6 could be attributed equally to a +Ï€ effect of nitrogen in the phenyl region. 29 In another variation, we alternated the methyl part and the carboxylate moiety at the 3 - position of the pyrazole region. As shown in Table 1, compounds: 5 (MIC = 8,05), 7 (MIC = 8,08) and 9 (MIC = 7,05) had considerably better activity than the other compo unds with carboxylate moiety; indicating methyl might be a good substitution for modification. Conclusion In conclusion, new bipyrazole derivatives drugs were prepared from easily accessible starting materials in few steps. 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