IN VITRO EVALUATION OF THE ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF SOME NEW N-(2-DIALKYLAMINOETHYL)BENZANILIDES

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1 38 FAMACIA, 200, Vol. 58, IN VITO EVALUATION OF THE ANTIBACTEIAL AND ANTIFUNGAL ACTIVITY OF SOME NEW N-(2-DIALKYLAMINOETHYL)BENZANILIDES DIANA CAMELIA NUŢĂ *, CAMEN BALOTESCU CHIFIIUC 2, ALEXANDU VASILE MISSI, ILEANA CONELIA CHIIŢĂ, CAMELLINA DANIELA BĂDICEANU University of Medicine and Pharmacy Carol Davila Bucharest, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 6 Traian Vuia, , Bucharest, omania 2 Microbiology-Immunology Department, Faculty of Biology, University of Bucharest, Ale. Portocalelor -3, sect. 5, Bucharest, omania *corresponding author: diananuta@yahoo.com Abstract The use of most antimicrobial agents is presently limited worldwide, not only by the rapidly developing drug resistance, but also by the unsatisfactory status of present treatment of bacterial and fungal infections and drug side effects. Therefore the development of new and different antimicrobial drugs is an important objective and many research studies are directed towards the design of new agents. ecently substituted anilides have received considerable attention because some of them are generally considered good bacterostatic and fungistatic substances. The present study is a follow-up paper to the previous articles dealing with the biologically active amides as potential antimicrobial drugs. In our laboratory we obtained new N-(2-dialkylaminoethyl)benzanilides which were tested to determinate the minimal inhibitory concentration (MIC) in order to evaluate their in vitro antimicrobial and antifungal activity. The importance of substituents in the benzoyl ring and in the anilide moiety for optimal activity have been noted. ezumat Utilizarea medicamentelor antimicrobiene este limitată, din cauza dezvoltării rapide a rezistenţei la medicamentele respective, a profilului nesatisfăcător al actualului arsenal terapeutic utilizat pentru tratarea infecţiilor microbiene şi fungice şi efectelor adverse ale acestor medicamente. De aceea dezvoltarea de noi medicamente antimicrobiene reprezintă un obiectiv important, foarte multe proiecte de cercetare având drept scop proiectarea de noi substanţe. Anilidele divers substituite au revenit recent în atenţie deoarece mai mulţi reprezentanţi sunt consideraţi substanţe cu un bun potenţial bacteriostatic şi fungistatic. Acest studiu continuă studiile în care s-au cercetat amidele cu acţiune biocidă ca potenţiale medicamente antimicrobiene. Am obţinut în laborator noi N- (dialchilaminoetil)benzanilide care au fost testate în vederea determinării concentraţiei minime inhibitorii (CMI) pentru a evalua activitatea antimicrobiană şi antifungică in vitro a acestora. Am observat importanţa substituenţilor pe structura benzanilidică pentru apariţia unui efect biologic optim. Keywords: N-(2-dialkylaminoethyl)benzanilides, antimicrobial and antifungal activity

2 FAMACIA, 200, Vol. 58, 39 Introduction Anilides are bacterostatic and fungistatic biocides, used as preservatives at relatively low concentrations against a variety of bacterial and fungal species, in paper, plastics and paints, and in a lesser degree as antiseptics in medicinal soaps and deodorants. They also have limited use as agricultural preservatives or pesticides [5]. Some of them (dichlorsalicylanilide, bromsalicylanilide, dibromsalan, tribromsalan, methabromsalan, flusalan) are active against Gram-positive bacteria, many of which are important pathogenic or odorcausing bacteria on the skin. These anilides generally have little efficacy against Gram-negative bacteria and fungi. Tribromsalan demonstrates persistence on the skin, bacterial or fungal growth inhibition, but its use has been limited as antiseptics because it causes irritation. Little or no activity has been reported against viruses or other microorganisms. Some anilides have resonable safety profiles, others are restricted in use due to toxicity concerns. Their mechanism of action seems to be by adsorbing to and destroying the semipermeability of the cytoplasmic membrane, leading to microbial cell death. Anilides have been shown to cause disruption of the proton force across the bacterial surface and interruption of membrane functions, including active antiseptic and sometimes antifungal transport and energy metabolism. These effects may be due to interference with proteins or the phospholipid bilayer of the membrane to disrupt its structure and function. A new mechanism of action of these compounds, discovered in 998, stated that they act as inhibitors of the two-component regulatory system (TCS) in bacteria [5]. The anilides used for their properties have the disadvantage of impossible oral administration because of their irritative effect on the gastric mucosa due to salycilic acid derivative structure. They also have the disadvantage of a low solubility in water, which also makes the administration difficult, therefore the number of pharmaceutical forms being thus reduced. We obtained in our laboratory new anilides and tested them for their antimicrobial features; they have the nitrogen atom from the amide group substituted with a dialkylaminoethyl chain, the salts (hydrochlorides) being easily obtained. These compounds have the advantage of a good solubility in water, so they can be used as aqueous solutions [,2,].

3 40 FAMACIA, 200, Vol. 58, Materials and methods In our previous papers [9, 0, 2] we presented the synthesis of new compounds, N-(2-dialkylaminoethyl)benzanilides (hydrochlorides) (noted.-.26, table I), which were obtained as a result of the alkylation reaction of certain aromatic amines (2) with N-(2-chloroethyl)-N,Ndialkylamine hydrochloride (3), followed by the reaction of intermediary compounds (4) with different aromatic acid chlorides (5). The resulted amides (6) were turned into hydrochlorides (.-.26) by treating them with an etheric HCl solution (figure ). NH CO-Cl + Cl - + Cl -NH( Na 2 CO 3 NH -N( 2 (C 2 H 5 ) 3 N 4 2 NH -N( 2 4 CO- N -N( HCl + CO- N -N( 2 CO- N -NH( 2 3 Cl eagents and conditions: dry toluene, anhydrous Na 2 CO 3, 38-6h, reflux, :,5 mixture of N-(2-chlorooethyl)-N,N-dialkylamine hydrochloride and amine dry toluene, dry triethylamine, 8h, reflux, :, mixture of intermediary amine and substituted benzoyl chloride HCl/ ether, 5 o C Figure The synthesis scheme employed to obtain the target compounds Previously, the preliminary testing of the antimicrobial activity of the newly synthetised compounds was performed by the disc diffussion method [8]. Because all the tested compounds showed an antimicrobial activity in the above mentioned screening test, they were next tested for their antibacterial and antifungal activity by an in vitro quantitative method in order to determine the minimal inhibitory concentration (MIC) by using the two-fold dilution method in liquid Mueller-Hinton medium recommended for the bacterial strains and Yeast Peptone Glucose (YPG) medium for Candida albicans following the standard conditions recommended by CLSI (Clinical and Laboratory Standards Institute) [3, 4, 6, 7].

4 FAMACIA, 200, Vol. 58, 4 Antimicrobial activity evaluation The synthesized compounds were evaluated for their in vitro antimicrobial activity against Gram-positive Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis, Gram-negative Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa and also against C. albicans yeast. The microbial strains were identified by aid of VITEK I automatic system. The MIC (µg/ml) was determined by binary micro dilution method, in 96 multi-well plates [5,3,4]. The compounds were dissolved in sterile water to a final concentration of 2048 µg/ml, which was serially two -fold diluted in order to obtain the following concentrations: 024, 52, 256, 28, 64, 32, 6 µg/ml in a final volume of 200 μl of nutrient medium. Thereafter 50 μl of microbial inoculum (0.5 McFarland density) was added to all tubes, which were incubated at 37 ± C for 24 h. The MIC was recorded in each case as the minimum concentration of compound, which inhibited the visible growth of the tested microorganism. esults and discussion The new benzanilides (table I) are white or light yellow crystals and they were characterized by their physical properties (melting points, solubility). Their structures were confirmed by spectral methods, I, H-NM, 3 C-NM, and the high level of purity of these compounds was proved by HPLC analyses. The analysis results were presented in our previous papers [8-0, 2]. The quantitative assay results for the antimicrobial activity of the new anilides are presented in Table II. These results show that the tested compounds exhibited a specific antimicrobial activity, depending on the nature of the substituents and their position on the benzene ring, both concerning the microbial spectrum and the MIC values, which ranged widely between 024 and 32μg/mL. Table I The new benzanilides tested for the in vitro antimicrobial and antifungal activity Compd 2 Formula Mol. wt. m.p. ( 0 C) ecryst. solvent.. 4-Cl -H -CH 3 C 7 H 20 Cl 2 N 2 O Br -H -CH 3 C 7 H 20 BrClN 2 O CH 3 -H -CH 3 C 8 H 23 ClN 2 O H -CH 3 C 20 H 27 ClN 2 O : isopropanol 2: (continued)

5 42 FAMACIA, 200, Vol. 58, Table I (continued).5. 4-Br -H -C 2 H 5 C 9 H 24 BrClN 2 O OCH 3 -H -C 2 H 5 C 20 H 27 ClN 2 O CH 3 -H -C 2 H 5 C 20 H 27 ClN 2 O Br -Cl -CH 3 C 7 H 9 BrCl 2 N 2 O CH 3 -Cl -CH 3 C 8 H 22 Cl 2 N 2 O OCH 3 -Cl -CH 3 C 8 H 22 Cl 2 N 2 O Cl -CH 3 C 20 H 26 Cl 2 N 2 O Br -Cl -C 2 H 5 C 9 H 23 BrCl 2 N 2 O Br -Cl -C 2 H 5 C 9 H 23 BrCl 2 N 2 O Br -Cl -C 2 H 5 C 9 H 23 BrCl 2 N 2 O CH 3 -Cl -C 2 H 5 C 20 H 25 Cl 2 N 2 O CH 3 -Cl -C 2 H 5 C 20 H 25 Cl 2 N 2 O OCH 3 -Cl -C 2 H 5 C 20 H 25 Cl 2 N 2 O OCH 3 -Cl -C 2 H 5 C 20 H 25 Cl 2 N 2 O (OCH 3 -Cl -C 2 H 5 C 2 H 28 Cl 2 N 2 O (OCH 3 -Cl -C 2 H 5 C 2 H 28 Cl 2 N 2 O Cl -C 2 H 5 C 22 H 30 Cl 2 N 2 O Br -CF 3 -CH 3 C 8 H 9 BrClF 3 N 2 O CH 3 -CF 3 -CH 3 C 9 H 22 ClF 3 N 2 O CF 3 -CH 3 C 2 H 26 ClF 3 N 2 O Br -CF 3 -C 2 H 5 C 20 H 23 BrClF 3 N 2 O CF 3 -C 2 H 5 C 23 H 30 ClF 3 N 2 O

6 FAMACIA, 200, Vol. 58, 43 Table II In vitro antimicrobial and antifungal activity expressed as MIC (μg/ ml) Compd. E. coli Salmone Pseudo Listeria S. Bacillus Candida lla monas aureus.. > >024 >024 > > > >024 > > > >024 > > >024 >024 >024 >024 >024 >024 > >024 >024 >024 >024 >024 >024 > > >024 > > >024 >024 > > > >024 >024 > > > > > > > >024 > >024 >024 > > > > > >024 > > > > > >024 >024 >024 > >024 >024 > > >024 >024 > > > >024 >024 > > > >024 > > > > > > > > > > > > >024 The results showed that 3 compounds (.,.2,.7) had a narrow spectrum of activity, being active only against Salmonella strains. The majority of the tested anilides exhibited a broad spectrum of activity and were generally active against Salmonella and Bacillus strains. A particularly strong antimicrobial activity was observed for N-(2-diethylaminoethyl)-N-phenyl-4- bromobenzamide (hydrochloride), N-(2-dimethylaminoethyl)-N-(3-chlorophenyl)-4-bromobenzamide (hydrochloride) and N-(2-dimethylaminoethyl)- N-(3-trifluoromethyl-phenyl)-3,4,5-trimethoxybenzamide (hydrochloride), showing moderate activity against all tested bacterial and yeast strains with MIC values ranging from 64 to 256 μg/ml. In contrast, compounds.4,.6,.2,.3 were practically inactive. Concerning the sensitivity of the tested microbial strains to our original amides, the results showed the high sensitivity of Salmonella, about 70 percent of tested compounds exhibiting an antimicrobial activity. The

7 44 FAMACIA, 200, Vol. 58, most active compounds (MIC= 32 μg/ml) are substituted by a chlorine atom in the meta position of the anilide moiety and also the substitution with methyl or methoxy groups in the benzoyl moiety seems to be very important for a high activity. An important antimicrobial activity was noticed against Bacillus strains, 5 compounds exhibiting a moderate antimicrobial activity. Chlorine and especially a trifluoromethyl substituent on the anilide moiety seems to be the most beneficial for the high activity against Grampositive microbial strains. This substitution also seems to be optimal for the high activity against Staphylococcus aureus, suggesting a possible use of these compounds in the treatment of Methicillin-resistant Staphylococcus aureus (MSA) infectious diseases, knowing that S. aureus has become resistant to many antimicrobials and MSA represents a major challenge worldwide. The antifungal activity, against Candida albicans was observed only for four compounds (.5,.0,.5,.24). Conclusions In summary, the biological evaluation of 26 different substituted N-(2-dialkylaminoethyl)benzanilides is described. The new compounds were tested for their in vitro antibacterial and antifungal activity expressed as MIC (μg/ml). From the results and discussion made above we conclude that the different substituted benzanilide derivatives are specifically effective against Gram-positive and Gram-negative bacteria, and respectively fungal strains. The in vitro antimicrobial activity depends on the substituents in the benzoyl ring and chlorine, the trifluoromethyl substituent in the anilide moiety being correlated in our studies with an optimal antimicrobial activity. eferences. Chiriţă I., Missir Al., Stecoza C., Limban C., Niţulescu M., Nuţă D., Moruşciag L., Bădiceanu C., Ilie C., Căproiu M.T., New anilides as potential antimicrobial agents. Note., Farmacia, 2008, LVI, 5, Chiriţă I, Missir Al., Moruşciag L., Limban C., Niţulescu M., Nuţă D., Stecoza C., Bădiceanu C., Ilie C., Căproiu M.T. New anilides as potential antimicrobial agents. Note 2, Farmacia, 2008, LVI, 6, Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement, M00-S7, Vol. 27 No.,eplaces M00-S6, Vol. 26 No. 3, Informational Supplement. 2007, CLSI: Wayne, PA, USA, 4. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A2. 999, CLSI: Wayne, PA, USA.

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