SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF NEW BENZOXAZOLE AMINO ACID ESTER DERIVATIVES

Headnote

ABSTRACT

In view of the fact that large number of derivatives of benzoxazole have been found to exhibit a wide variety of pharmacological activities. In the current research work, new amino acid ester derivatives of benzoxazole heterocyclic moiety were synthesized. Benzoxazole-2-thiol was S-alkylated by ethyl bromoacetate in presence of triethylamine base to get ethyl acetate ester derivatives (Ethyl 2-(benzo[d]oxazol-2-yl thio) acetate) (Compound 1) which is then deesterified using alkaline solution to get carboxylic acid derivative (2-(benzo[d]oxazol-2-yl thio) acetic acid) (Compound 2) which coupled to different amino acid esters (glycin ethyl ester HCl, L-tyrosin ethyl ester HCl, L-phenyl alanine ethyl ester HCl and ethyl-paraaminobenzoate) (Compounds a-d respectively) by conventional solution method to get the final target compounds (Ethyl 2-(2-(benzo[d]oxazol-2-yl thio) acetamido) acetate, Ethyl 2-(2-(benzo[d]oxazol-2-yl thio) acetamido )-3-(4-hydroxyphenyl) propanoate, Ethyl 2-(2-(benzo[d]oxazol-2-yl thio) acetamido)-3-phenylpropanoate and Ethyl 4-(2-(benzo[d]oxazol-2-yl thio) acetamido) benzoate) (Compounds 3, 4, 5 and 6 respectively). The identification and characterization of the synthesized compounds were confirmed by melting point, thin layer chromatography (TLC), FT-IR and elemental microanalysis (CHN). All synthesized final target compounds were screened for antimicrobial activity (antibacterial and antifungal activities).

Keywords: Heterocyclic derivatives; benzoxazole derivatives; Amino acid derivatives; Antimicrobial activity.

INTRODUCTION

The main objective of the medicinal chemistry is to synthesize the compounds that show promising activity as therapeutic agents with lower toxicity.1 Biologically active benzoxazole derivatives have been known for long time, since they are the isosters of naturally occurring cyclic nucleotides and they may easily interact with the biopolymers of the organisms.2 Benzoxazole (I) and Benzoxazole-2-thiol (II) (figure 1) have been reported to show a broad spectrum of biological activities.3 Notable among these are antihistaminic4, antibacterial, antifungal5, cyclooxygenase inhibiting6, antitumor7, antiulcer8, anticonvulsant9, hypoglycemic10, anti-inflammatory11,12 and cytotoxic activity13-15. In the present work four different amino acid esters were incorporated to benzoxazole-2-thiol moiety by using bromoethylacetate as spacer. The final target compounds were all screened for antimicrobial activity.

CHEMISTRY

Scheme 1 shows the synthetic pathway of the intermediate and final target compounds which involved:

The synthesis of four different amino acid esters (glycin ethyl ester HCl (compound a), L-tyrosin ethyl ester HCl (compound b), L-phenyl alanine ethyl ester HCl (compound c) and ethyl-p-aminobenzoate (compound d)).

Synthesis of intermediate compounds 1 and 2 by S-alkylation of benzoxazole-2-thiol by ethyl bromoacetate in presence of triethylamine base to get ethyl acetate ester derivatives (Ethyl 2-(benzo[d]oxazol-2-yl thio) acetate) (Compound 1) which is then deesterified (saponification) using alkaline solution ( 1N NaOH), and then acidified with (1N HCl) to get carboxylic acid derivative (2-(benzo[d]oxazol-2-yl thio) acetic acid) (Compound 2).

Synthesis of final target compounds wich is: Ethyl 2-(2-(benzo[d]oxazol-2-yl thio) acetamido) acetate (Compound 3), Ethyl 2- (2-(benzo[d] oxazol-2-yl thio) acetamido )-3- (4- hydroxyphenyl) propanoate (Compound 4), Ethyl 2-(2-(benzo[d]oxazol-2-yl thio) acetamido)-3 -phenyl propanoate (Compound 5) and Ethyl 4-(2-(benzo[d] oxazol-2-yl thio) acetamido) benzoate (Compound 6) by coupling of free carboxyl group of compound 2 with free amine group of the synthesized amino acid esters (compounds a-d) to get the final target compounds (3-6).

Conventional solution method were used as a coupling method and using dicyclohexalecarbodiimide (DCC) and 1-hydroxybenzotriazole (HoBt) as a coupling reagent in this method in order to obtained higher yield product and prevent racemization. Scheme 2 shows mechanism of coupling reaction mediated by DCC and HoBt.16

RESULTS AND DISCUSSION

The current research work involves following steps of reactions starting from the synthesis of intermediate compounds (amino acid esters (compounds a-d), compound 1 and compound 2) followed by coupling reaction of free carboxyl group of compound 2 with free amine group of the synthesized amino acid esters (compounds a-d), using coupling as in peptide synthesis.

In the present work, the method used was the direct coupling with DCC/HOBt. The use of DCC is particularly convenient, since it can be simply added to the solution containing the amine and carboxyl components to be coupled. It reacts rapidly with the free carboxylic acid present to form an "active ester" intermediate (isourea), which may react with amine component directly or may proceed through an intermediate (symmetrical anhydride) as in scheme 2. Coupling reactions mediated by DCC can be also modified by the addition of other reagents, such as HOBt. This addition will lead to the formation of HOBt active esters which in turn may; accelerate coupling reactions, suppress byproduct formation and suppress racemization.

The products were confirmed by chromatographic and spectral data. The melting points of all compounds were observed different from ingredients melting points which were confirmed the synthesis of products. The purity of the synthesized compounds was checked by observing single spot on TLC plate. All synthesized compounds were give only single spot which is mean they were obtained in pure form. The melting points and Rf values of all synthesized compounds were given in table 1. The structures of the synthesized compounds were confirmed by spectral analysis (FT-IR & CHN).

FT-IR (table 2) shows characteristic band at 1728 cm-1 for carbonyl stretching vibration of ester group of compound 1, while shows a broad hydroxyl stretching vibration of free carboxyl group of compound 2 extending from 3412- 2500cm-1 and strong absorption peak at 1701cm-1 of the carbonyl stretching vibration of free carboxyl and absence of 1728cm-1 peak for carbonyl stretching vibration of ester.

Also the spectral data revealed that the spectra of all synthesized final target compounds (compounds 3-6) showed that the absorption intensity of the carboxylic carbonyl group diminished and shifted to the right near 1650 cm-1 which is indicative of formation of amide bond and no absorption peak was observed for carboxylic OH group and presence of distinct, strong and un-obscure NH stretching vibration single band between 3275-3325 cm-1 indicating that carboxylic site reacted with selected amine forming amide.

Elemental microanalysis (CHN) of the final target synthesized compounds (compounds 3-6) was done in France laboratories and shown in table 3.

Biological activity

Biological activity (antimicrobial activity) study were designed as a comparative study of the synthesized final target compounds (compounds 3-6) in relation to Ofloxacin as a control in antibacterial activity study (table 4) and to Ketoconazol as a control in antifungal activity study (table 5), compounds 3-6 were tested against a panel of microorganisms including gram positive bacteria (S aureus and Streptococcus pyogen), gram negative bacteria (Proteus spp.) for studying antibacterial activity and tested against (Candida albicans and Aspergillus spp.) for antifungal activity study.

Concerning the antibacterial activity study and from results in table 4 show that compound 6 has significant (p=0.05) activity against gram positive bacteria (S aureus and Streptococcus pyogen), while compounds 5, 4 and 3 showed good activity against the same strains of bacteria in comparable to Ofloxacin. All tested compounds (3-6) showed good activity against gram negative bacteria (Proteus spp.) in comparable to Ofloxacin.

Concerning the antifungal activity study and from results in table 5 show that compound 6, 5 and 3 have good activity against Aspergillus spp., while compound 4 have low activity against the same fungus spp. in comparable to Ketoconazol. Compounds 4 and 3 showed good activity against Candida albicans while compounds 6 and 5 showed moderate activity against the same fungus spp. in comparable to Ketoconazol.

CONCLUSION

The synthesis of the final target compounds (compounds 3-6) was successfully achieved by following the stated procedures as previously described. The results obtained from this investigation indicated that the strategy adapted for the synthesis of the designed derivatives was successful, since the conformity of synthesized compounds was achieved according to the data shown by the physical and chemical analysis including (TLC, melting point, FT-IR and Elemental analysis (CHN)). Most of these compounds show good antimicrobial activity comparable with marketable compounds.

EXPERIMENTAL

Chemistry

All reagents including amino acids, benzothiazole-2-thiol and anhydrous solvents were used as received from commercial suppliers (Merck-Germany, BDH-England, Fluka-Switzerland and Himedia-India). The melting points were determined by electrothermal CIA 9300 melting point apparatus in open capillaries and were uncorrected. IR spectra (KBr discs) were recorded on a Buck 500 FTIR scientific spectrophotometer (Iraq). Elemental microanalysis was done on a Carlo Erba elemental analyzer. This analysis was done in the micro analytical center (France). The purity of all compounds was established by single spot on the Thin-layer chromatography (TLC) plastic sheets silica gel 60 F5 precoated, 20 ×20 cm, layer thickness 0.2 mm. The spots on the chromatograms were localized using U.V. light (366 nm) (Whatmann, Merck, Germany). Iodine vapour was used as developing agent. The solvent systems used employed for separation composed from A: Chloroform: Methanol (3:7) and B: Methanol: Chloroform: Ether (4: 3: 3).

Synthesis of Amino acid esters (compounds a-c)17: A suspension of (50 mmol) amino acid (glycin, L-tyrosin and L-phenylalanin) in 150 ml of absolute ethanol was cooled down to -15°C then (3.7 ml, 50 mmol) thionyl chloride was added drop wise, (the temperature should be kept below -10 °C), the reaction mixture was leftat 40 °C for 3 h, then reflux started for 3 h, and leftat room temperature overnight, the solvent was evaporated to dryness under vacuum, redissolved in ethanol and evaporated, this process was repeated several times and recrystallized from ethanol / ethyl acetate (3:1) to give the product. The physical appearance, percentage yield, melting point and Rf value are listed in table 1. IR characteristics absorption bands were listed in table 2.

Synthesis of Ethyl-paraaminobenzoate (EPAB) (compound d)18: To Para-amino benzoic acid (12g, 0.088 mol) and 80ml of absolute ethanol in around bottom flask and 4ml of concentrated H2SO4 added slowly and refluxed for 2h at 60 °C on water bath, then cooled the flask for several minutes. Then added 150ml of 10% NaCO3 solution, which result in the evolution of considerable gas until the solution is neutralized. Filtered the solution and collect the precipitate. Recrystalized from rectified spirit and drying in desiccator under vacuum to get the product. The physical appearance, percentage yield, melting point and Rf values are listed in Table 1. IR characteristics absorption bands were listed in Table 2.

Synthesis of Ethyl 2-(benzo[d]oxazol-2-yl thio) acetate (compound 1)19: To a suspension of (1.81gm, 0.012mol) of 2-mercaptobenzoxazole in 75ml distilled water in 150ml round bottom flask add (1.8ml, 0.015mol) of triethyl amine liquid dropwise in 5 minutes with continues stirring then add (1.4ml, 0.012mol) of bromoethyl acetate liquid dropwise in 30min at room temperature then continues stirring at room temperature for additional 3h. White precipitate will be formed which will be filtered and washed several times with distilled water to get the product. The physical appearance, percentage yield, melting point and Rf values are listed in table 1. IR characteristics absorption bands were listed in table 2.

Synthesis of 2-(benzo[d]oxazol-2-yl thio) acetic acid (compound 2)20: To a stirred solution of (compound 1) (0.94gm, 4mmol) in a minimum volume of absolute Methanol / Tetrahydrofuran (8:2) mixture at a temperature between (18-22 °C), (6ml, 1N) NaOH solution (freshly prepared) was added dropwise over a period of 30min. Stirring was continued for additional 8h, the reaction mixture was acidified with equimolar HCl solution (1N) and ice water was added to get the product as a fine precipitate suspended in solution. The solution was kept in the freezer till the solution completely freezes and then allowed to melt; the product was filtered then washed with cooled distilled water and collected to get the product. The physical appearance, percentage yield, melting point and Rf values are listed in table 1. IR characteristics absorption bands were listed in table 2.

Synthesis of the target final compounds (compounds 3-6)21: To a stirred solution of amino acid esters that previously prepared (compounds a-d) (3mmol) in 9ml dimethylformamide (DMF), (0.33ml, 3mmol) of N-methyl morpholine (NMM) was added with stirring for 10min, then (0.627gm, 3mmol) of compound 2 in 9ml DMF was added to the reaction mixture which was cooled down to -10 °C then (1.03gm, 6.9mmol) of HoBt and (1.38gm, 6mmol) of DCC were added with stirring which was continued for 2 days at 0°C and then at room temperature for 5 days.

The crude product was evaporated to exclude DMF and redisolved in ethyl acetate from which N,N-dicyclohexyl urea (DCU) was filtered off. The filtrate was evaporated to dryness under vacuum and the residue was redisolved in ethyl acetate. The excess DCU was precipitated out and filtered. The clear filtrate was washed 3 times with 10ml of 0.1N HCl solution, 3 times with 10ml of 5% NaHCO3 solution, once with 10ml distilled water and once with 10ml of saturated NaCl solution using seperatory funnel. The ethyl acetate layer was dried using anhydrous magnesium sulfate powder and evaporated under vacuum; the resulted product was collected, recrystallized from (ethylacetate: petroleum-ether 60-80) mixture to get the final products (compounds 3-6).

The physical appearance, percentage yield, melting point and Rf values are listed in Table 1. IR characteristics absorption bands were listed in Table 2. The elemental microanalysis (CHN) values were listed in table 3.

Antimicrobial activity22

Preliminary antibacterial and antifungal activity has been carried out according to well diffusion method. According to Bauer et al 1967, the antimicrobial activity of the synthesized final compounds (3-6) were tested in-vitro against three pathogenic bacteria, these includes (Staphylococcus aureus, Streptococcus pyogenes) as gram positive bacteria and Proteus Spp. as gram negative bacteria and against two fungi (Aspergillus Spp. and Candida Spp.). They were clinically activated and maintained on nutrient agar medium for testing antibacterial activity and Sabaroud agar medium for antifungal activity. Ofloxacin was used as a standard drug for antibacterial activity and Ketoconazol was used as a standard drug for antifungal activity.

Sensitivity Assay23

All the synthesized final target compounds were dissolved in 1% Dimethylsulphoxide at concentration of (120 µg/ml). Well diffusion assay was carried out by using bacterial suspension of about (1.5 × 106 CFU/ml) obtained from McFarland turbidity standard (number 0.5).

This was used to inoculate by swabbing the surface of Mueller Hinton agar (MHA) plates. Excess liquid was air dried under a sterile hood and the impregnated discs were applied at equidistant points on top of the agar medium.

The inoculated culture medium prepared as above was immediately incubated at (30 °C) for 72 hours (fungal Spp.) or (37 °C) for 24 hours (bacteria) and the antimicrobial activity were evaluated by measuring the diameter of the inhibition zone (IZ) around the disc in mm.

ACKNOWLEDGMENTS

We are grateful to the staffand colleagues of the department of pharmaceutical chemistry in college of pharmacy, university of Baghdad.

References

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