World Inventia Publishers

S. Shobha Rani et al. J Sci Res Pharm, 2017;6(Suppl 1):17-22

JSRP Full Proceedings | National Conference on “Emerging Trends and Innovations in Pharmaceutical Sciences (ETIPS - 2017)” JNTU, Hyderabad, Telangana, INDIA.

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World Inventia Publishers

Journal of Scientific Research in Pharmacy http://www.jsrponline.com/

Vol. 6, Suppl 1, 2017 ISSN: 2277-9469

USA CODEN: JSRPCJ

Research Article Full Proceeding Paper

SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL 3-AMINO-BENZOFURAN DERIVATIVES AS POTENTIAL ANTIMICROBIAL, CHORISMATE MUTASE INHIBITORS

Mallikarjuna Rao, V a, b, S. Shobha Rani b*, A. Srinivasa Rao c, Srishylam. B a

a Medicinal Chemistry Division, GVKBIO Sciences, Pvt, Lt, IDA Nacharam, Hyderabad-500076, Telangana, INDIA. * b Centre for Pharmaceutical Sciences, IST, Jawaharlal Nehru Technological University, Hyderabad-500085, Telangana, INDIA.

c Shri Vishnu College of Pharmacy, Bhimavaram-534202, West Godawari Dt, Andhra Pradesh, INDIA.

Received on: 05-10-2017; Revised and Accepted on: 08-11-2017

ABSTRACT

Novel series of 2-(5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzofuran-3-amine analogues were isolated and conformed

spectroscopically for in vitro Mycobacterium Tuberculosis Chorismate Mutase (*MtbCM or CM) inhibition, antibacterial, antifungal screening. All the

isolated compounds were screened to have good antibacterial activity and compound 7c have exhibited well (MIC value of 3.9 and 7.8 µg/mL) by well

diffusion method. Most of the screened compounds were showed potential inhibition of chorismate mutase (CM) in vitro and compound 6a was exhibited

86% inhibition when tested with 30 µM. 3-amino-benzofuran as a new scaffold for the discovery of promising inhibitors of CM. CM is present in

microorganisms to produce essential amino acids to survive but not in the humans. All the synthesized compounds exhibited promising inhibition of CM

and anti-microbial activity, which was good sign to proceed further to extent the research on 3-aminobenzofuran analogues. All the isolated compounds

were isolated in pure form and characterized by 1HNMR, FT-IR and Mass Spectral analysis.

KEYWORDS: 3-Amino-benzofuran, 1,3,4-oxadiazole, Benzofuran, Hydrazones, Antibacterial activity, antifungal activity and chorismate mutase (CM).

INTRODUCTION

Over the last few decades the fluoroquinolone antibiotics

have been a mainstay in the treatment of bacterial diseases and the most notable member of this family ciprofloxacin, possesses potent antimicrobial activity despite recent evidences of bacterial strains having fluoroquinoloneresistance [1, 2]. Despite the availability of a range of effective antibiotics and drugs, the threat of infectious diseases or death due to microbial infections, still poses considerable health problems. Benzofuran [10] class of fused heterocycles has been of great interest to explore a new class of broad spectrum antibiotics against pathogens. In recent years there has been an increased interest on substituted benzofuran as antibacterial, anti-fungal, and anti-cancer agents. It has been demonstrated that aryl hydrazones are an important and promising class of anti-infective agents [3-6]. A series of phenyl hydrazones [7-9] have been reported to have good anti-bacterial. These results have encouraged us to extend our research further to evaluate as antibacterial, antifungal, CM inhibition activity.

Oxadiazole derivatives belong to an important group of heterocyclic compounds. Among wide variety of heterocyclic compounds that have been explored for developing pharmaceutically important molecules, 1,3,4-oxadizole derivatives have played vital role in medicinal chemistry. Large number of synthetic compounds with 1,3,4-oxadiazole nucleus have been screened for antibacterial, anti-tubercular, anticancer activities. In recent years 1,3,4-oxadiazoles have shown anti-viral activity [11].

*Corresponding author: Dr. S. Shobha Rani Centre for Pharmaceutical Sciences, IST, Jawaharlal Nehru Technological University, Hyderabad-500085, Telangana, INDIA. * E-Mail: [email protected], [email protected]

Fig. 1: Shikimate pathway, Chorismate to Prephenate by Chorismate Mutase

Tuberculosis (TB) still remains a leading cause of death worldwide due to a number of factors such as long duration of treatment (6 - 9 months), increased incidence of (multi or extensive) drug resistance, co-morbidity with HIV-AIDS and declined effort in anti-infective drug discovery research. Indeed, TB kills more than two million people a year worldwide. Thus the discovery [12], development and introduction of new treatments for tuberculosis have become an


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essential goal of current pharmaceutical research. Mycobacterium tuberculosis chorismate mutase (MtbCM or CM) catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway to form phenylalanine and tyrosine after chorismate being formed by the action of chorismate synthase, an enzyme of the shikimate pathway main trunk (Fig-1). In bacteria, MtbCM plays a key role in the synthesis of aromatic amino acids necessary for the survival of organism and therefore inhibition of MtbCM may hinder the supply of nutrients to the organism. Due to the absence of this pathway in animals but not in bacteria CM is considered as a promising target for the identification of new drugs [14]. However, only a few small molecules have been reported to possess inhibitory activity against CM [13–16]. In continuation of our efforts on the identification of novel inhibitors of CM, we became interested in evaluating the library of small molecules based on 3-amino-benzofuran framework.

MATERIAL AND METHODS

Chemistry: In the present work, proposed hybrids were synthesized

utilizing the reaction sequence as shown in Scheme 1 and 2. Alkylation

of 2-hydroxy benzonitrile using equimolar quantity of ethyl bromoacetate in presence of K2CO3/Cs2CO3in dry dimethyl formamide gave an excellent yield of ethyl 3-amino benzofuran-2-carboxylate3. Ethyl 3-amino-benzofuran-2-carboxylate 3 on stirring with excess hydrazine hydrate in ethanol at reflux temperature gave its hydrazide derivative 4 in good yield. Compound 4 on condensation with 3,4-dichloro benzaldehyde in presence of catalytic amount of glacial acetic acid (or)activated 4Å molecular sieves to give hydrazone compound 5.Hydrazone intermediate was further converted into its derivatives as sulphonamides (6a-6d) with sulfonyl chlorides in presence of DiPEA, DMAP in dichloromethane at ambient temperature for 5 days. All the sulphonamide compounds were treated with [Bis(trifluoroacetoxy) iodo]benzene in dry dichloromethane at room temperature for 4 h to give Oxadiazole derivatives 7a-7d. Allthe final compounds were purified by flash chromatography on combiflash using Red Sep 12g catridge and 5 to 50% ethyl acetate in hexanes as an eluent. Compound 4 was treated with 3,4-dichloro benzoyl chloride in presence of TEA, DMAP in dichloromethane at ambient temperature for 12 h to give compound 10. This was further treated with Deoxo-Fluor®, TEA, K2CO3 in dry DCM at 0 °C to 25 °C for 2 h to give compound 11.

Experimental Section:

Commercially available chemicals and solvents were purchased from Sigma Aldrich and were used without further purification. All the reactions were conducted in oven dried glassware under an argon atmosphere, THF was dried using Na metal and

benzophenone, DMF was used as dry solvent and stored on molecular sieves (4Å). TEA was stored over KOH. reactions were monitored by thin layer chromatography (TLC) on silica gel plates (60 F254), visualizing with UV-light (254 nm) or iodine spray and then flash column chromatography was carried out with Merck silica gel 100-200





mesh as stationary phase. NMR spectra were recorded on a Bruker 400 spectrometer (or otherwise stated) at 400 MHz for 1H NMR in CDCl3 or DMSO-d6, or else stated. Signals are reported as m (multiplet), s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), dt (doublet of triplet), br s (broad singlet), br d (broad doublet) and coupling constants are reported in Hertz (Hz). Chemical shifts (d) are reported in ppm relative to the residual solvent peak. Elucidations of chemical structures were based on 1H NMR, NOESY experiments. MS spectra were recorded on a Micromass LC-TOF instrument by using electrospray ionization (ESI).Concentrations are reported in gram per 100 mL. Melting points were measured on a Büchie M 565 and FT-IR values were recorded on Shimadzue IR affinity-1 using KBr pellet.

Synthesis of 2-(2-cyanophenoxy)acetate (2): To a stirred suspension of 2-Hydroxy benzonitrile 1 (20 g,

167.89 mmol), K2CO3 (69.61 g, 503.69 mmol) in dry acetone (250 mL) under argon atmosphere was added ethyl bromoacetate (33.64 g, 201.46 mmol) dropwise. After stirring for 2h at 50 °C, the reaction mixture was evaporated to dryness. The crude compound obtained was partitioned between ethyl acetate and cold water. The combined organic extracts were washed with brine solution and dried over anhydrous sodium sulphate. After filtration and removal of the volatiles under reduced pressure, the residue obtained was purified by flash column chromatography using petroleum ether/ethyl acetate (10:1) as an eluent to afford 2(29.64 g, 86% yield). 1HNMR (400 MHz, CDCl3) δ 7.59 (d, J=1.5, 7.8 Hz, 1H), 7.54 - 7.49 (m, 1H), 7.06 (t, J=7.6 Hz, 1H), 6.84 (d, J=8.3 Hz, 1H), 4.76 (s, 2H), 4.26 (q, J=6.8 Hz, 2H), 1.28 (t, J=7.1 Hz, 3H); FT-IR 2238 (CN), 1721 (C=O) cm-1.

Synthesis of ethyl 3-aminobenzofuran-2-carboxylate (3): To a stirred suspension of ethyl 2-(2-cyanophenoxy)acetate

2(29 g, 141.31 mmol), K2CO3 (39.06 g, 282.63 mmol) in dry N,N-dimethylformamide (300 mL) under argon atmosphere was heated at 140 °C for 4h. The reaction mixture was evaporated to dryness. The crude compound obtained was partitioned between ethyl acetate and cold water. All the combined organic extracts were washed with brine solution and dried over anhydrous sodium sulphate. After filtration and removal of the volatiles under reduced pressure, the residue obtained was purified by flash column chromatography (silica-gel, 100-200 mesh) using petroleum ether/ethyl acetate (7:3) as an eluent to give 3 (21.54 g, 74% yield). 1HNMR (400 MHz, CDCl3) δ 7.56 (d, J=7.8 Hz, 1H),

7.46 (d, J=3.9 Hz, 2H), 7.26 (m, 1H), 4.98 (b s, 2H), 4.45 (q, J=6.8 Hz, 2H), 1.44 (t, J=7.1 Hz, 3H); FT-IR 1712 (C=O) cm-1. HRMS m/z 206.2315 (M+H)+.

Synthesis of3-aminobenzofuran-2-carbohydrazide (4): To a stirred suspension of ethyl 3-aminobenzofuran-2-

carboxylate 3 (21 g, 102.33 mmol), hydrazine hydrate (80% aq solution, 25.58 mL, 409.32 mmol) in ethanol (350 mL) were heated to reflux for 2h. The reaction mixture was evaporated to dryness. The crude compound obtained was partitioned between diethyl ether and cold water. All the combined organic extracts were washed with brine solution and dried over anhydrous sodium sulphate. After filtration and removal of the volatiles under reduced pressure, the residue obtained was purified by flash column chromatography (silica-gel, 100-200 mesh) using chloroform/methanol (8:2) as an eluent to give 4 (12.36 g, 63% yield). 1HNMR (400 MHz, DMSO-d6) δ 9.17 (b s, 1H), 7.84 (d, J=7.8 Hz, 1H), 7.46 - 7.37 (m, 2H), 7.29 - 7.20 (m, 1H), 5.95 (s, 2H), 4.31 (b s, 2H); FT-IR 2238 (CN), 1658 (C=O) cm-1; HRMS m/z 192.2415 (M+H)+.

Synthesis of 3-amino-N'-(3,4-dichlorobenzylidene)benzofuran-2-carbohydrazide (5):

To a stirred suspension of 3-aminobenzofuran-2-carbohydrazide 4(3g, 15.69mmol), 3,4-dichloro benzaldehyde (3.02g, 17.26 mmol), glacial acetic acid (0.941g, 15.69 mmol) in absolute ethanol (60 mL) were heated to reflux for 2h. The reaction mixture was evaporated to dryness. The crude compound obtained was partitioned between diethyl ether and cold aq NaHCO3 solution. All the combined organic extracts were washed with brine solution and dried over anhydrous sodium sulphate. After filtration and removal of the volatiles under reduced pressure, the residue obtained was purified by flash column chromatography (silica-gel, 100-200 mesh) using petroleum ether/ethyl acetate (6:4)as an eluent to give 5 (2.86 g, 52.5% yield). 1H NMR (400 MHz, DMSO-d6) δ11.80 (s, 1H), 8.44 (s, 1H), 8.00 - 7.89 (m, 2H), 7.75 - 7.65 (m, 2H), 7.49 (d s, 2H), 7.29 (dd, J=2.8, 5.2, 7.8 Hz, 1H), 6.36 (s, 2H); HRMS m/z 348.3012 (M+H)+, m/z 350.3212 (M+H+2)+.Regioselectivity of amine/hydrazide with aldehyde to form an imine was further conformed based on the NOESY spectrum and it is clearly indicated the absence of imine proton correlation with phenyl ring protons and we conclude the substitution regioselectivity is on compound 5.

Fig. 3: Regioselectivity of Amine/Hydride with Aldehyde to form an Imine

Synthesis of 4-bromo-N-(2-(2-(3,4-dichlorobenzylidene)hydrazine carbonyl)benzofuran-3-yl)benzene sulfonamide 6a:

To a stirred solution of compound 5 (100 mg, 0.288 mmol) in THF was added 4-bromo-benzene-1-sulfonyl chloride (109 mg, 0.432 mmol) at 0 °C followed by DiPEA (88 mg, 0.72 mmol) and DMAP (12 mg 0.098 mmol), the reaction mass was allowed to stir at RT for 16 h. The reaction mixture was concentrated to afford residue which was purified by reverse phase preparative chromatography (Grace C-18 column, 0.1% formic acid in water and ACN as mobile phase) to afford 6a (86 mg, 0.151 mmol, 53% yield) as a white solid. Melting Range 240 - 243 °C.1H NMR (300 MHz, DMSO-d6) δ 12.32 (br s, 1H), 10.18 (br s, 1H), 8.26 (br s, 1H), 8.03 - 7.85 (m, 1H), 7.81 - 7.41 (m, 7H), 7.32 (br s, 1H). HRMS m/z 568.2861 (M+H)+, 570.2794 (M+H+2)+.

N-(2-(2-(3,4-dichlorobenzylidene)hydrazinecarbonyl)benzofuran-3-yl)-4-methylbenzene sulfonamide 6b:

108 mg of 6b (0.215 mmol, 75% yield) as a yellow solid. Melting Range 302 - 306 °C,1H NMR (300 MHz, DMSO-d6) δ 13.08 (br s, 1H), 8.25 - 8.15 (m, 1H), 7.94 (br d, J = 8.1 Hz, 1H), 7.87 - 7.82 (m, 1H), 7.76 - 7.62 (m, 3H), 7.47 - 7.40 (m, 1H), 7.32 (br t, J = 7.7 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.11 (br t, J = 7.5 Hz, 1H), 2.26 (s, 3H). HRMS m/z 503.2586 (M+H)+, m/z 505.2641 (M+H+2)+.

N-(2-(2-(3,4-dichlorobenzylidene)hydrazine-1-carbonyl)benzofuran-3-yl)ethanesulfonamide 6c:

94 mg of 6c (0.213 mmol, 75% yield) as a yellow solid. Melting Range198 - 202 °C,1H NMR (300 MHz, DMSO-d6) δ 12.16 (br s, 1H), 10.42 (br s, 1H), 8.38 (s, 1H), 8.11 (s, 1H), 7.86 - 7.48 (m,4H), 7.32 - 7.28 (m, 2H), 3.41 (q, 2H), 1.18 (t, 3H). HRMS m/z 441.3101 (M+H)+, m/z 443.3106 (M+H+2)+.





N-(2-(2-(3,4-dichlorobenzylidene)hydrazine-1-carbonyl)benzofuran-3-yl)ethanesulfonamide 6d:

81 mg of 6d (0. 19 mmol, 66% yield) as a yellow solid. Melting Range 213 - 216 °C, 1H NMR (300 MHz, DMSO-d6) δ 11.16 (br s, 1H), 10.02 (br s, 1H), 8.36 (s, 1H), 8.24 (s, 1H), 7.91 - 7.56 (m, 4H), 7.36 - 7.22 (m, 2H), 3.01 (s, 3H). HRMS m/z 427.2642 (M+H)+, m/z 429.2589 (M+H+2)+.

Synthesis of 4-bromo-N-(2-(5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzofuran-3-yl)benzenesulfonamide 7a:

A dried round-bottomed flask was charged with compound 6a (50 mg, 0.088 mmol), [Bis(trifluoroacetoxy)iodo] benzene (BTI, 45.5 mg, 0.105 mmol)in anhydrous DCM (5 mL) at room temperature. This mixture was stirred at room temperature and monitored the reaction progress by thin-layer chromatography (TLC). When the reaction was complete, the reaction mixture was evaporated under reduced pressure to give residue, which was purified directly by loading on to silica gel and eluted with 0 - 45% of ethyl acetate in petroleum ether to afford compound 7a (18 mg, 0.031 mmol,36% yield). Melting Range 184 - 188 °C.1H NMR (400 MHz, DMSO-d6) δ 10.49 (br s, 1H), 8.12 (s, 1H), 7.92 - 7.81 (m, 4H), 7.68 - 7.21 (m, 6H); LC/MS (ESI): 566.26 [M+H]+ 568.24 (M+H+2)+, HRMS (TOF, ESI+) m/z 566.2212 (M+H)+, 568.2216 (M+H+2)+.

N-(2-(5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzofuran-3-yl)-4-methylbenzene sulfonamide 7b:

26 mg of 7b (0.052 mmol, 53% yield) as a yellow solid. Melting Range 302 - 306 °C, 1H NMR (300 MHz, DMSO-d6) δ 10.42 (br s, 1H), 7.96 - 7.84 (m, 2H), 7.76 - 7.53 (m, 6H), 7.51 - 7.18 (m, 3H), 2.39 (s, 3H); LC/MS (ESI): 501.36 [M+H]+ 503.35 (M+H+2)+, HRMS (TOF, ESI+) m/z 501.3468 (M+H)+, 503.3398 (M+H+2)+.

N-(2-(5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzofuran-3-yl)ethanesulfonamide7c:

22 mg of 7c (0.050 mmol, 45% yield) as yellow solid. Melting Range 216 - 219 °C;1H NMR (300 MHz, DMSO-d6) δ 11.06 (br s, 1H), 7.94 - 7.82 (m, 2H), 7.62 - 7.55 (m, 3H), 7.28 – 7.38 (m, 2H), 3.48 (q, 2H), 1.26 (t, 3H). LC/MS (ESI): 439.28 [M+H]+ 441.316 (M+H+2)+, HRMS (TOF, ESI+) m/z 439.2802 (M+H)+, 441.2806 (M+H+2)+.

N-(2-(5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzofuran-3-yl)methanesulfonamide 7d

19 mg of 7d (0.044 mmol, 39% yield) as yellow solid. Melting Range 248 - 251 °C, 1H NMR (300 MHz, DMSO-d6) δ 10.94 (br s, 1H), 7.91 - 7.86 (m, 2H), 7.64 - 7.51 (m, 3H), 7.30 - 7.41 (m, 2H), 2.96 (s, 3H). LC/MS (ESI): 425.26 [M+H]+ 427.30 (M+H+2)+, HRMS (TOF, ESI+) m/z 425.2580 (M+H)+, 427.2601 (M+H+2)+.

Synthesis of 3,4-dichloro-N-(2-(5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzofuran-3-yl)benzamide 11:

To a stirred solution of compound 9 (114 mg, 0.598 mmol), triethylamine (241 mg, 2.392 mmol) in dry DCM (4 mL) was added compound 8 (250 mg, 1.196 mmol) at 0 °C and continued to stir at room temperature for 6 h. After completion of the reaction by TLC, the reaction mixture was added Deoxo-Fluor (264 mg, 1.196 mmol), K2CO3 (162 mg, 1.196 mmol), and triethylamine (121 mg, 1.196 mmol) at room temperature and continued to stir for another 2 h. After completion of the reaction by TLC, the reaction mixture was evaporated under reduced pressure to give residue, which was purified directly loading on to silica gel and eluted with 0 - 10% of methanol in dichloromethane to afford compound 11 (44 mg, 0.084 mmol, 14% yield). Melting Range 284 - 287 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (br s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.85 - 7.88 (m, 2H), 7.71 - 7.62 (m, 3H), 7.55 - 7.28 (m, 3H); LC/MS (ESI): 520.19 [M+H]+ 522.24 (M+H+2)+.

Biology: Anti-microbial activity:

The antimicrobial activity of the 3-amino-benzofuranhybrids were determined using well diffusion method [17] against different pathogenic reference strains procured from the Microbial Type Culture Collection (MTCC), CSIR-Institute of Microbial Technology, Chandigarh, India. The pathogenic reference strains were seeded on the surface of the Muller-Hinton agar Petri plates, with0.1 mL of previously prepared

microbial suspensions individually containing 1.5 x 108 cfu mL-1 (equal to 0.5 McFarland). Wells of6.0 mm diameter were prepared in the medium plates using a corkborer and the synthesized Benzofuran 1,3,4-oxadiazole hybrids ata dose range of 125 - 0.97 µg well-1 was added in each well under sterile conditions in a laminar air flow chamber. Standard antibiotic solution of Ciprofloxacin at a dose range of 125 - 0.97 µg well -1served as positive control, while the well containing DMSO served as negative control. The plates were incubated for 24 h at 37 °C and the well containing the least concentration showing the inhibition zone was considered as MIC. All experiments were carried out in duplicates and mean values are represented.

Similarly antifungal activity of the synthesized 3-amino-benzofuran hybrids were determined using well diffusion method [17] against different Candida strains such as C. albicans MTCC 183,C. albicans MTCC 227, C. albicans MTCC 854, C. albicans MTCC 1637,C. albicans MTCC 3017, C. albicans MTCC 3018, C. albicans MTCC3958, C. albicans MTCC 4748, C. albicans MTCC 7315, C.parapsilosis MTCC 1744, C. aaseri MTCC 1962, C. glabrata MTCC3019, C. krusei MTCC 3020 and Issatchenkia hanoiensis MTCC 4755procured from the Wells of6.0 mm diameter were prepared in the media plates using a corkborer and the synthesized compounds dissolved in 10% DMSO at adose range of 125 - 0.97 µg/mL were added in each well under sterile conditions in a laminar air flow chamber. Standard antibiotic solutions of Miconazole at a dose range of 125 - 0.97 µg well-1,served as positive control, while the well containing DMSO served as negative control. The plates were incubated for 24 h at 30 oC for different Candida strains. The well containing the least concentration showing the inhibition zone is considered as the minimum inhibitory concentration. All the experiments were carried out in duplicates and mean values are represented.

Mycobacterium Tuberculosis Chorismate Mutase (*MtbCM or CM) inhibition activity: All the synthesized 3-amino-benzofuran derivatives were tested for their inhibitory potential against Mycobacterium tuberculosis H37Rv chorismate mutase (CM). The assay [18, 19] involved determination of activity of enzyme CM which catalyzes the conversion of chorismate to prephenate. Thus determination of activity of CM is based on the direct observation of conversion of chorismic acid to prephenate spectrophotometrically at OD274. This reaction was performed in the presence of test compounds to determine their CM inhibiting activities. A known inhibitor of CM i.e. 4-(3,5-dimeth-oxyphenethylamino)-3-nitro-5-sulfamoylbenzoic acid [20] was prepared and used as a reference compound the IC50 value of which was found to be less than 10 µM. Mycobacterium tuberculosis chorismate mutase (MtCM) gene was PCR amplified and cloned into expression vector pET22b. MtbCM was purified from over expressed culture of BL21 (DE3) harboring pET22b/ MtbCM by Ni-NTA affinity chromatography. Activity of chorismate mutase enzyme is based on the direct observation of conversion of chorismate to prephenate Spectrophotometrically at OD 274. The reaction volume of 100 µL contained 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.1 mg/mL bovine serum albumin, and 10 mM β -mercaptoethanol, and chorismic acid 4 mM. The reaction was started by adding 180 pmol of purified protein to the pre -warmed chorismic acid solution. Inhibitory screening of the test compounds against chorismate mutase activity was measured at 30 µM concentration of the effectors. The reaction was allowed to proceed at 37 °C and was terminated after 5 min with 100 µL of 1 N HCl. A blank with no enzyme for every reaction was kept as a control to account for the non enzymatic conversion of chorismate to prephenate. The percentage of enzyme inhibition caused by the test compound is calculated by the following formula (% inhibition = 100 – residual activity of CM).

RESULTS AND DISCUSSION

Chemistry: Molecular hybridization to identify a new lead compound was

achieved by isolation of 3-amino-benzofuran derivatives and all the final compounds synthesized were isolated using SiO2 chromatography and characterized by using spectroscopic analysis. Compound 5 regioselectivity was confirmed by NOESY experiments where the imine





proton interactions were clearly absent with benzofuran ring protons. So the primary amine at 3rd position on benzofuran was substituted with selected sulphonamides based on the best glide score obtained through docking studies with Chorismate Mutase. The hydrazone compounds were further converted to 1,3,4-oxadiazoles conveniently using [Bis(trifluoroacetoxy)iodo] benzene. But this reaction was tried with KMnO4 was found to be un-successful. Keeping the 2,3-substitutions on benzofuran the research can be extended to find the more suitable lead compounds for CM inhibition.

Biological activities: Anti-Bacterialactivity:

All the isolated compounds were screened for in vitro antibacterial activity [17, 21, 22] against different Gram-positive and Gram-negative bacterial strains. Among all the benzofuran derivatives screened, compounds7chave showed promising growth inhibition of gram +ve and –ve bacteria (MIC values for gram +ve bacteria is 3.9 to 7.8 µg/mL whereas for gram –ve bacteria is 62.5 µg/mL). Most of the compounds exhibited better selectivity towards gram +ve bacteria. Among the many pathogenic Candida species, most notably Candida albicans is a dimorphic fungus behaving as a commensal or an opportunistic pathogen causing both superficial and systemic infections. Among the tested compounds 6c and 7c are found have moderate

inhibition activity against Candida albicans (MTCC 3017) (MIC value of 62.5µg/mL) comparable to the standard miconazole drug. Compounds 6c and 7c has less bulky group ethyl sulphonamide is probably contributing to the antimicrobial activity. The anti-bacterial and antifungal activity results to this regard are tabulated in Table 1.

Mycobacterium Tuberculosis Chorismate Mutase inhibition activity: Compounds 6a, 6b, 6c, and 10 have showed >50% inhibition

of CM compared to other molecules when tested at 30 µM (Table 2) whereas rest of the compounds were either less active or inactive. Notably, compounds 6a and 6c containing 4-bromophenyl and ethyl sulphonamide groupshave showed consistent activities than others. Compound 6c (4-bromophenyl sulphonamide on 3-amino-benzofuran have showed extensively potent against Chorismate Mutase (86%). Based on the preliminary data generated compound 6c was chosen for further evaluation in vitro. In a dose response study, compound 6chave showed inhibition of CM in a dose dependent manner with an IC50value of 11.64 ± 0.54 µM. Thus N-(2-(2-(3,4-dichlorobenzylidene)hydrazine-1-carbonyl)benzofuran-3-yl)ethanesulfonamide framework appeared as a new scaffold for the development of novel inhibitors of Chorismate Mutase. Since tuberculosis is a leading cause of death worldwide, the present classes of compounds are of further interest as potential anti tubercular agents.

Table No. 1: Antimicrobial activity of target compounds

Test Compounds

Minimum inhibitory concentration (µg/mL)

Staphylococcus aureus (MTCC

96)

Bacillus subtilis

(MTCC 121)

Staphylococcus aureus (MLS16

MTCC 2940)

Micrococcus luteus (MTCC

2470)

Klebsiella planticola

(MTCC 530)

Escherichia coli (MTCC

739)

Pseudomonas aeruginosa (MTCC 2453)

Candida albicans

(MTCC 3017)

4 >125 62.5 >125 >125 >125 >125 >125 >125

5 62.5 15.6 >125 >125 62.5 >125 >125 >125

6a 15.6 31.2 62.5 62.5 >125 >125 >125 >125

6b 31.2 15.6 >125 >125 >125 >125 >125 >125

6c >125 7.8 31.2 62.5 >125 >125 >125 62.5

6d 15.6 62.5 7.8 >125 62.5 >125 >125 >125

7a >125 62.5 3.9 >125 >125 >125 >125 >125

7b >125 62.5 31.2 >125 >125 >125 >125 >125

7c 62.5 3.9 7.8 62.5 62.5 62.5 62.5 62.5

7d 7.8 15.6 15.6 7.8 15.6 15.6 62.5 >125

10 62.5 >125 62.5 >125 15.6 >125 >125 >125

11 >125 >125 15.6 >125 62.5 >125 >125 >125

Ciprofloxaciline (standard)

0.9 0.9 0.9 0.9 0.9 0.9 0.9 N

Miconazole (standard)

N N N N N N N 7.8

Where control is DMSO, N= not applicable

Table No. 2: Chorismate mutase inhibition of target compounds

Test Compounds (Chorismate Mutase)% inhibition @ 30 µM

4 28

5 42

6a 86

6b 54

6c 61

6d 48

7a 06

7b 42

7c 26

7d 38

10 52

11 41

4-(3,5-dimeth-oxyphenethylamino)-3-nitro-5-sulfamoylbenzoic acid

99





CONCLUSION

A series of novel 3-sulphonamide 2-hydrazone substituted

Benzofuran hybrids (6a-6d&7a-7d) were synthesized starting from 3-amino-N'-(3,4-dichlorobenzylidene)benzofuran-2-carbohydrazide5 via acylation with sulfonyl chlorides and oxidative cyclisation with BTI reactions. These compounds were evaluated for their antibacterial, antifungal and Chorismate mutase inhibitions activities. Among the screened compounds 7c (MIC values 3.9 – 7.8 µg/mL) was found to be have promising antimicrobial activity against all the tested bacterial pathogens. None of these compounds have showed antifungal activity against a range of Candida strains. Compound 6a have showed promising inhibitory activities when tested at 30 µM and showed dose dependent inhibition of CM with IC50 value of11.64 ± 0.54 µM. Overall, this research has identified 3-amino-benzofuran-2-hydrazone as a new scaffold for the development of new inhibitors of Chorismate Mutase. In conclusion, we have described the design, synthesis and in vitro evaluation of novel 2-(5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)benzofuran-3-amine 7 derivatives as potential inhibitors of gram +ve and gram -ve microorganism and 4-bromo-N-(2-(2-(3,4-dichlorobenzylidene)hydrazine-1-carbonyl)benzofuran-3-yl)benzenesulfonamide 6afor Chorismate Mutase inhibition.

ACKNOWLEDGEMENTS

We are grateful to GVK Biosciences Pvt. Ltd. for the financial

support and for providing laboratory facilities. We specially thank Dr. Ravi kumar. P, Dr. Krishna. Ethiraj and Dr. Sudhir Kumar Singh for their scientific inputs, moral support, and motivation throughout my Ph.D. research work. We also thank to our analytical department for assistance in getting the spectral data.

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How to cite this article:

S. Shobha Rani et al. SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL 3-AMINO-BENZOFURAN DERIVATIVES AS POTENTIAL ANTIMICROBIAL, CHORISMATE MUTASE INHIBITORS. J Sci Res Pharm 2017;6(Suppl 1):17-22.

Conflict of interest: The authors have declared that no conflict of interest exists.

Source of support: Nil


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