Synthesis, anti-bacterial, anti-asthmatic and anti-diabetic activities of novel N-substituted 2-(4-styrylphenyl)-1 H -benzimidazole and N-substituted-3[4-(1 H -benzimidazole-2-yl)-phenyl]-acrylic acid tert -butyl ester

Synthesis of a series of novel substituted benzimidazole derivatives by the condensation of o - phenylenediamine ( OPDA, 1) with 4-bromobenzoic acid (2) and subsequent reactions of the benzimidazole with different electrophilic reagents is reported. The latter compounds were reacted with styrene and tert -butyl acrylate following Heck Coupling. All the compounds synthesized were screened for their potential anti-bacterial, anti-asthmatic and anti-diabetic properties, which exibibited some promising results towards testing organism in vivo


Introduction
Benzimidazoles are among the important heterocyclic compounds found in several natural and non-natural products such as Vitamin B12 1 , marine alkaloid kealiiquinone 2 , benzimidazole nucleosides 3 etc.Some of their derivatives are marketed as anti-fungal agents such as Carbendazim 4 , anti-helmintic agents such as Mebendazole and thiabendazole 5 and anti-psychotic drug such as Pimozide 6 and other derivatives have been found to possess some interesting bioactivities such as anti-tubercular 7 , anti-cancer 8 etc.Recently, we have also published some series of biologically active benzimidazoles 9 .Owing to the immense biological importance of benzimidazole derivatives, we synthesized some substituted alkenylbenzimidazoles and screened them for biological activity.
The condensation of o-phenylenediamine (OPDA) (1) with 4-bromobenzoic acid (2) was carried out in presence of polyphosphoric acid at 180 o C for 4 h to obtain the known 2-(4-bromophenyl)-1H-benzimidazole (3) 10 (Scheme-1). + Scheme 1 Initially, we thought of alkylating the benzimidazole -NH with suitable electrophilic reagents to generate N-alkylatedbenzimidazoles. In this regard, compound 3 was alkylated with different alkylating agents in N, N'-dimethylformamide and in presence of sodium hydride as base to obtain the corresponding alkylated derivatives 4a 10c , 4b, 4c 10a and 4d 10a (Scheme 2).It is noteworthy to mention here that, in an alternative approach, compound 4a has also been prepared by condensing N-methyl-OPDA dihydrochloride 11 (1a) with 4-bromobenzoic acid (2) in the presence of polyphosphoric acid at 180 o C (Scheme 3).The structure of compound 4a obtained via 1a was confirmed by its physical and spectral characteristics and also by comparison with the compound obtained by direct methylation of compound 3. Compounds 4a -4d were then reacted with tert-butylacrylate in presence of tri-otolylphosphine, triethylamine and palladium acetate as catalyst under Heck coupling conditions 12 to get 5a-5d (Scheme 4).

Biological activity
All the compounds prepared herein were screened for their potential biological activities such as, anti-bacterial activity 14 against Staphylococcus aureus (gram positive) and Salmonella typhimurium (gram negative) bacterial strains 15 at concentration 500, 200, 100, 10 and 0.1µg/ml.Cephalexin was used as a reference standard.The results of the anti-bacterial activity screening of the tested compound are summarized in Table 1 & Table 2. Most of the compounds tested were found to have good anti-bacterial activity against Salmonella typhimurium, however, they were found to have poor activity against Staphylococcus aureus.Also they were tested against PDE -IV for potential anti-asthmatic effect, and against DPP-IV and PTP1B for potential antidiabetic effects.No activity was found.The anti-asthmatic activity was carried out using Phosphodiesterase IV enzyme (PDE-IV) 16 (Table 3) and the primary screening of the compounds was done at 1uM concentration using human PDIV enzyme, where Rolipram & Ariflo were used as standard compounds.
The anti-diabetic activity was carried out with dipeptidyl peptidase (DPP-IV) 17 enzyme (Table 3) and the primary screening of the compounds was carried at 300 nM concentration using recombination human DPP-IV enzyme by the use of 1-(2-amino -3,3-dimethylbutanoyl pyrrolidine -2-carbonitrile as the standard compound at 100 nM.Similarly, the PTP1B 18 (Inhouse compound, also for anti-diabetic) activity (Table 3) was done using the test compounds at 30 µM with the standard compound N-

Protocol for PDE-IV-inhibition assay
Phosphodiesterase IV enzyme converts [ 3 H] cAMP to the corresponding [ 3 H] 5'-AMP in proportion to the amount of Phosphodiesterase IV present.The [ 3 H] 5'-AMP then was quantitatively converted to free [ 3 H] adenosine and phosphate by the action of snake venom 5'nucleotidase hence the amount of [ 3 H] adenosine liberated is proportional to Phosphodiesterase IV activity.
The assay was performed at 34 o C in a 200 mL total reaction mixture.The reaction mixture contained 25 mM of tris-buffer, 10 mM MgCl 2 , 1 µM cAMP (cold) and [ 3 H] cAMP(0.1µCi)stock solutions of the compounds to be investigated were prepared in dimethyl sulfoxide in concentrations such that the dimethyl sulfoxide content in the test samples did not exceed 0.05% by volume to avoid affecting the Phosphodiesterase IV activity.Compounds were then added in the reaction mixture (25µL/tube).The assay was initiated by addition of enzyme mix (75µL) and the mixture was incubated for 20 minutes at 34 o C. The reaction was stopped by boiling the tubes for 2 min at 100 o C in a water bath.After cooling on ice for 5 minutes and addition of 50 µg 5'nucleotidase snake venom from Crotalus atrox incubation was carried out again for 20 min at 34 o C. The un-reacted substrate was separated from ( 3 H) adenosine by addition of Dowex AG 1X-8 (400 µL), which was pre equilibrated in (1:1) water:ethanol.Reaction mixture was then thoroughly mixed, placed on ice for 15 minutes, vortexed and centrifuged at 14,000 rpm.for 2 min.After centrifugation, a sample of the supernatent (150 µL) was taken and added in 24 well optiplates containing scinillant (1 mL) and mixed well.The samples in the plates were then determined for radioactivity in a Top Counter and the Phosphodiesterase IV activity was calculated.Phosphodiesterase IV enzyme was present in quantities that yield < 30% total hydrolysis of substrate (linear assay conditions).Rolipram and Cilomilast were used as a standard in all assays.

Protocol for the DPP-IV assay DPPIV inhibition measurement in vitro:
DPPIV activity was determined by the cleavage rate of 7-amino-4-methyl coumarin (AMC) from synthetic substrate Glycyl-Prolyl-AMC.In brief, the assay was conducted by adding 10 ng of human recombinant Dipeptidyl peptidase IV enzyme (DPPIV, available commercially from R & D Systems) in 50 µl of the assay buffer (25 mM Tris, p H 7.4, 140 mM NaCl, 10 mM KCl, 1% BSA) to 96 well black flat bottom micro-titer plates.The reaction was initiated by adding 50 µl of 100 µM substrate Gly-Pro-AMC.The incubation was carried out in the kinetic mode at 30 o C for 30 min.Fluorescence was measured using Fluorostar at excitation filter of 380 nm and emission filter of 460 nm) Test compounds and solvent controls were added as 1 µl additions.Test compounds were dissolved in DMSO and tested at 300 nM concentration.Percent inhibition was calculated with respect to the solvent control sample (no test compound added).Dipeptidyl peptidase (i.e., anti-diabetic).

Conclusions
In conclusion, we synthesized a series of novel substituted benzimidazole derivatives by the condensation of OPDA with 4-bromobenzoic acid and subsequent reactions of the benzimidazole with different electrophilic reagents.All the compounds thus obtained were reacted with styrene and tert-butyl acrylate following Heck Coupling to obtain the substituted alkenylbenzimidazole derivatives.Some of the compounds were found to have good anti-bacterial activity against Salmonella typhimurium, however they were found to have less activity against S. aureus.These compounds were also tested against PDE-IV for potential anti-asthmatic effect, and against DPP-IV and PTP1B for potential anti-diabetic effects.Unfortunately, the results were disappointing.

Experimental Section
General Procedures.Melting points are uncorrected and were recorded on a MRVIS Series, Lab India Instrument.TLC analysis was done using pre-coated silica gel plates and visualization was done using Iodine / UV lamp.IR spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrometer.

General procedure for the synthesis of compounds 4a-4d
To a solution of 2-(4-bromo-phenyl)-1H-benzimidazole (3, 2 mmole) in dimethyl formamide (10 ml) was added sodium hydride (60 %, 2.4 mmole) lot wise at 0 o C.After completion of addition the temperature of the reaction mixture was slowly raised to room temperature and stirred at this temperature for 1 h.The reaction mixture was again cooled to 0 o C and the respective alkyl halide (2.4 mmole) was added at 0 o C. The temperature of the reaction mixture was then allowed to warm to room temperature and stirred for 2 h.After completion of the reaction, water (50 ml) was slowly added to reaction mixture and extracted with ethyl acetate (2 x 25 ml).The organic layer was washed with water (2 x 25 ml), brine and dried over anhydrous magnesium sulfate and concentrated under vacuum to yield the corresponding N-substituted derivatives 4a-4d.The crude compounds were recrystallized from hot aq.ethanol to obtain pure products.(See Table 4).

General procedure for the synthesis of compounds 5a-5g under Heck coupling conditions
To a solution of compounds 4a-4d (2.5 mmole) in DMF (50 ml) was added styrene or tert-butyl acrylate (3 mmole), triethylamine (3 mmole), tri-(o-tolyl) phosphine (0.125 mmole) and palladium acetate (0.125 mmole).The reaction mixture was then heated to 100-110 o C for 3 h.The reaction mixture was then allowed to cool to room temperature and filtered through hyflo.Evaporation of the solvent yielded crude products, which were subjected to column chromatography to isolate the pure products.(See Table 4).

Table 1 .
Antibacterial activity of compounds against Staphylococcus aureus

Table 2 .
Antibacterial activity of compounds against Salmonella typhimurium

Table 3 .
Anti-diabetic & anti-asthmatic activity of compounds Symbols: --Total Inhibition, no growth of organism; P -Poor growth compared to controls; + -Medium growth compared to controls; ++ -Confluent growth, no inhibition.

Table 4 .
1H &13C-NMR spectra were recorded on a Varian Mercury Vx SWBB 300MHz spectrometer with CDCl 3 as solvent unless otherwise mentioned.Elemental analysis was carried out on a Perkin-Elmer Series-II CHNS/O Analyzer 2400.o-phenylenediamine, alkylating and acylating agents were obtained from commercial suppliers.Styrene and tert.-Butylacrylate were obtained from Aldrich.All the solvents used were of commercial grade only.Physical and analytical data of compounds 3