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Synthesis, Characterization, Antitumor, Antibacterial and Urease Inhibitory Activity of a Small Series of N-tosyl benzimidazoles.

Byline: Naghmana Rashid, Almas Kiran, Zaman Ashraf, Moazzam Hussain Bhatti, Bushra Mirza, Hammad Ismail, Muhammad Rafiq and Jerry P. Jasinski

Summary: Benzimidazole derivatives exhibited a broad range of biological activities, e.g., antimicrobial, antiviral, anthelmintic, anti-inflammatory, anticancer and as an anti-ulcer/proton pump inhibitor. Keeping in view the large number of reported drugs containing benzimidazole moiety on one hand and sulfonamide on the other hand, a small series of N-tosyl benzimidazoles (4a-e) have been synthesized. The present work describes the synthesis, characterization and bio-evaluation of five new N-tosyl benzimidazoles with the objective to develop new compounds with improved anticancer, antibacterial and urease inhibitory activities. The substituted 1,2-phenylenediamines in the first step were condensed with aliphatic carboxylic acids to synthesize the substituted benzimidazoles. In the second step the tosyl chloride was reacted with substituted benzimidazoles in basic conditions to afford the title N-tosyl benzimidazoles (4a-e).

The screening for their antitumor activities was performed against Agrobacterium tumefaciens by following the potato disc tumor assay. The compound (4e) exhibited excellent antitumor activity with IC50 values 474.45ugml-1 compared to other synthesized compounds. Antibacterial activity results revealed that compounds 4d and 4e having methyl and ethyl substitution respectively at the imidazole ring showed excellent zone inhibition against both gram positive and gram negative strains. The urease inhibitory activity results showed that derivative 4e exhibited highest potential to inhibit the urease enzyme compared to all other derivatives. Based upon our investigation it is proposed that compound (4e) may serve as lead structure to design more potent biological active compounds having multitargets inhibition activities.

Keywords: Benzimidazole, Sulfonamides, N-tosyl benzimidazoles, Anticancer activity, Antitumor activity, Urease inhibitory activity, Antibacterial activity, X-ray crystallography

Introduction

Benzimidazole is a significant heterocyclic moiety for the discovery of new drugs. A wide range of 1,2-disubstituted benzimidazoles have been synthesized with a variety of substitutions at different positions on the phenyl ring of the benzimidazoles [1]. Benzimidazole and its derivatives have been researched in pharmaceutical industry as an NSAID (non-steroidal anti-inflammatory drug) drug [2]. Benzimidazole derivatives have also provided a broad range of biological activities, e.g., antimicrobial [3], antiviral [4], anthelmintic, anti-inflammatory [5,6], anticancer [7,8] and as an anti-ulcer/proton pump inhibitor [9]. It has also been reported that benzimidazole derivatives bearing sulfonamide moiety possess carbonic anhydrase inhibitory activity [10-12]. Benzimidazole derivatives are also found to be active in the treatment of several diseases such as epilepsy, diabetes, and tuberculosis.

Recently benzimidazole containing compounds such as regioisomers of 5(6)-bromo-1-[(phenyl)sulfonyl]-2-[(4-nitrophenoxy)methyl]-1H-benzimidazole have been reported as an antitubercular agents [13]. The potent anticancer, antibacterial, antifungal and antiprotozoal activities were also reported for halogenated 2-trifluoromethyl- and 2-pentafluoroethyl-benzimidazoles [14-16].

The tosyl substituted benzimidazoles have also been reported to possess significant biological activity Scheme-I. Omeparazole 1 (prilosec, losec) and esomeprazole (Nexium) drugs are used to control the gastric acid secretions and are also used as an antimicrobial drug. The benzimidazole derivative 2 having piperazine ring at 2 position is reported as 5-HT3 receptor antagonists is potent against viruses [17]. Sulfonamide containing benzimidazole derivative 3, a non-peptidic LHRH (luteinizinghormone-releasing hormone) antagonist is expected to be effective for the oral treatment of dysfunctional sex steroid production [18]. A number of clinically used drugs possess benzimidazole nucleus and potential of sulfonyl moiety in drugs is also well-established. Keeping in view the importance of these two moieties a series of benzimidazoles containing sulfonyl functionality i.e. tosyl benzimidazoles were synthesized and characterized.

Furthermore the synthesized tosyl benzimidazoles were also screened for their antitumor and antibacterial activities against selected strains.

Experimental

Reagents and chemicals

Instrumentation

FTIR spectra (4000-400cm-1) were recorded using KBr pellets in Nicolet iS10 (Thrmo Scientific) spectrophotomer. 1HNMR spectra were recorded using AVANCE AV-III 300 Hz NMR spectrophotometer. EIMS were carried out by using JEOL JMS 600-H. X-ray crystallography was carried out at Keene State College (Keene, NH) whereby individual crystals of compounds 4a and 4b were mounted on a CryoLoop (Hampton Research) and placed in a -100AdegC compressed air stream on an Agilent Gemini-EOS Single Crystal Autodiffractometer. Crystallographic data were collected using graphite monochromated 0.71073 A Mo-K[alpha] radiation and integrated and corrected for absorption using the CrysAlisRed [19] software package. The structures were solved using the OLEX2 software package [20] using Superflip [21] for structure solution and Shelx 2013 [22] for structure refinement.

All other pertinent crystallographic details such as I, ranges, and R-factors can be found in Table-1. Melting points were measured on a Gallen Kamp, cat. NO. MPD350.BM 3.5, Sayno, U.K instrument.

General procedure for the preparation of compounds (3a-e)

The equimolar amounts of 1,2-phenylenediamine and monobasic acids (formic acid, acetic acid and propionic acid separately) were refluxed for 4 hours in 4N HCl. After the completion of reaction the mixture was cooled at room temperature, diluted with water (250 ml) and brought to pH 6.5 with Na2CO3. The products were precipitated out, filtered them and recrystallized in (ethanol/water 1:3) to afford the desired substituted benzimidazoles. This general procedure was followed for the synthesis of title benzimidazoles (3a-e).

1H-benzimidazole (3a)

Yield: 69 %; m.p: 167 AdegC; IR (KBR); 1652 (C=N), 1003 (C-N), and 3300 (N-H) cm-1; 1HNMR (DMSO) I': 7.55 (dd, 2H, J=5.7;3.3Hz) , 7.17 (dd, 2H, J=6.9;3.9Hz); 13CNMR (DMSO) I': 143 (C-2), 112 (C-4,7), 102 (C-5, 6), 128 (C-8, 9) ppm. EIMS: m/z 118.0 (M+, 100%).

2-Methyl-1H-benzimidazole (3b)

Yield: 40 %; m.p:170 AdegC; IR (KBR); 1621 (C=N), 1027 (C-N), and 3273 (N-H) cm-1 ; 1HNMR (DMSO) I': 2.46 (s, 3H), 7.41 (dd, 2H, J=6.9;3Hz), 7.08 (dd, 2H, J=6.9;3.9 Hz); CNMR (DMSO) I': 145 (C-2), 113 (C-4, 7), 102 (C-5, 6), 130 (C-8, 9), 31 (C-1') ppm; EIMS: m/z 132 (M+, 100%).

6-Nitro-1H-benzimidazole (3c)

Yield: 78%; m.p: 195 AdegC; IR (KBR); 1338-1517 (NO2), 1629 (C=N), 1065 (C-N), and 3452 (N-H) cm-1 ; 1HNMR (DMSO) I': 8.08 (s, 1H ) , 8.63 (d, 1H, J=2.1Hz),8.19 (d, 1H, J=9 Hz), 7.96 (d, 1H, J=9 Hz ); 13CNMR (DMSO) I': 143 (C-2), 113 (C-4), 155 (C-5) , 111 (C-6), 102 (C-7) , 129 (C-8, 9) ppm; EIMS: m/z 164 (M+, 100%).

2-Methyl-6-nitro-1H-benzimidazole (3d)

Yield: 47 %; m.p:220 C; IR (KBr); 1338-1517 (NO2), 1629 (C=N), 1065 (C-N), and 3452 (N-H) cm-1 ; 1HNMR (DMSO) I': 2.55 (s, 3H ) , 8.35 (d, 1H, J=2.1 Hz),8.05 (dd, 1H, J=9; 2.4 Hz), 7.62 (d, 1H, J=9); 13CNMR (DMSO) I': 143 (C-2), 117 (C-4), 152 (C-5),115 (C-6), 110 (C-7), 129 (C-8, 9), 40 (C-1') ppm; EIMS: m/z 176.9 (M+, 100%).

2-Ethyl-6-nitro-1H-benzimidazol (3e)

Yield: 46 %; m.p: 155 AdegC; IR (KBR); 1341-1516 (NO2), 1625 (C=N), 1062 (C-N), and 3435 (N-H) cm-1 ; 1HNMR (DMSO) I': 2.90 (q, 2H, J=7.8 Hz), 1.33 (t, 3H, J=7.8 Hz), 8.36 (d, 1H, J=2.1 Hz),8.05 (dd, 1H, J=9; 2.4 Hz), 7.62 (d, 1H, J=9 Hz); 13CNMR (DMSO) I': 146 (C-2), 142 (C-5), 129 (C-8, 9), 118 (C-4), 116 (C-6), 110 (C-7), 60 (C-2'), 30 (C-1') ppm; EIMS: m/z 190.9 (M+, 100%).

General procedure for the preparation of compounds (4a-e)

The substituted benzimidazoles (3a-e) (10mmol) were dissolved in 10% NaOH (25 ml). A concentrated solution of 4-methylbenezenesulfonyl chloride (19mmol) in acetone (5ml) was added slowly with continuous stirring to the benzimidazoles solution. The reaction mixture was stirred until white precipitates were appeared in the reaction flask. The crude product was filtered off, washed with water and recrystallized from ethanol to afford the N-tosyl benzimidazoles. This general procedure was adopted for the synthesis of the N-tosyl benzimidazoles (4a-e).

1-(Toluene-4-sulfonyl)-1H-benzimidazole (4a)

Yield: 54%; m.p: 118AdegC; Rf: 0.24 (n-Hexane / Ethyl acetate 8:2); IR (KBr);1594 (C=N), 1256 (C-N), and 1161-1373 (S=O) cm-1 ; 1HNMR (DMSO) I': 8.82 (s, 1H), 7.40 (m), 8.03 (d, 2H, J=8.1 Hz), 7.74 (d, 2H, J=7.5 Hz), 2.33 (s,3H); 13CNMR (DMSO) I': 146, 143, 133, 129, 128, 119, 117,110, 30 ppm; EIMS: m/z 272 (M+, 81.8%).

2-Methyl-1-(toluene-4-sulfonyl)-1H-benzimidazole

(4b) Yield:72 %; m.p: 120 AdegC; Rf: 0.29 (n-Hexane / Ethyl acetate 8:2); IR (KBr);1597 (C=N), 1274 (C-N), and 1173-1371 (S=O) cm-1 ; 1HNMR (DMSO) I': 2.78 (s, 3H), 7.37 (m, 4H), 7.96 (d, 2H, J=6 Hz), 7.44 (d, 2H, J=6 Hz), 2.33 (s,3H); 13CNMR (DMSO) I': 146 (C-2), 130 (C-4, 7),128 (C-5, 6), 143 (C-8, 9), 60 (C-1') 133 (C-a), 119 (C-b, f),110 (C-c, e), 30 (C-a') ppm; EIMS: m/z 285.9 (M+, 100%).

5/6-nitro-1-(toluene-4-sulfonyl)-1H-benzimidazole

(4c) Yield: 60 %; m.p: 150 AdegC; Rf: a=0.23, b=0.63 (n-Hexane / Ethyl acetate 8:2); IR (KBr); 1338-1517 (NO2), 1587 (C=N), 1300 (C-N), and 1173-1371 (S=O) cm-1 ; 1HNMR (DMSO) I': 8.32 (d, 1H, J=1.8 Hz), 8.62 (d, 1H, J= not clear), 8.02 (d, 1H, J=9 Hz), 8.10 (d, 2H, J=9.3 Hz), 7.50 (d, 2H, J=8.1Hz), 2.36 (s,3H); 13CNMR (DMSO) I': 146 (C-2), 130 (C-4, 7),128 (C-5, 6), 143 (C-8, 9), 133 (C-a), 119 (C-b, f),110 (C-c, e), 30 (C-a') ppm; EIMS: m/z 316 (M+, 39.4%).

2-Methyl-1-[(4-methylphenyl) sulfonyl]-5/6-nitro-1H-benzimidazole

(4d) Yield: 61 %; m.p: 153 AdegC; Rf: a=0.25, b=0.12 (n-Hexane / Ethyl acetate 8:2); IR (KBr); 1348-1519 (NO2), 1596 (C=N), 1270 (C-N), and 1173-1371 (S=O) cm-1 ; 1HNMR (DMSO) I': 2.83 (s, 3H), 8.25 (d, 1H, J=1.5 Hz), 8.45 (d, 1H, J= not clear), 8.15 (d, 1H, J=9 Hz), 8.02 (d, 2H, J=8.1 Hz), 7.48 (d, 2H, J=7.8 Hz), 2.36 (s,3H); 13CNMR (DMSO) I': 149 (C-2), 121 (C-4),155 (C-5), 120 (C-6), 116 (C-7), 148 (C-8, 9), 24 (C-1'), 131 (C-a), 128 (C-b, f),127 (C-d), 123 (C-c, e), 33 (C-a') ppm; EIMS: m/z 330.9 (M+, 100%).

2-Ethyl-5/6-nitro-1-(toluene-4-sulfonyl)-1H-benzimidazole (4e)

Yield: 61 %; m.p: 136AdegC; Rf: a= 0.37, b=0.22 (n-Hexane / Ethyl acetate 8:2); IR (KBr); 1347-1525 (NO2), 1595 (C=N), 1276 (C-N), and 1164-1376 (S=O) cm-1 ; 1HNMR (DMSO) I': 2.34 (q, 2H), 3.18(t, 3H), 8.25 (d, 1H, J=2.1 Hz), 8.75 (d, 1H, J= not clear), 7.90 (d, 1H, J=9 Hz), 8.00 (d, 2H, J=8.1 Hz), 7.46 (d, 2H, J=8.1 Hz), 2.36 (s,3H); 13CNMR (DMSO) I': 149 (C-2), 121 (C-4),155 (C-5), 120 (C-6), 116 (C-7), 148 (C-8, 9), 24 (C-1'), 16 (C-2'), 131 (C-a), 128 (C-b, f),127 (C-d), 123 (C-c, e), 33 (C-a') ppm; EIMS: m/z 345.0 (M+, 40.6%).

Potato Disc Antitumor Assay

The N-tosyl benzimidazoles (4a-e) were tested for their antitumor activities by using potato disc antitumor method [23]. In this assay a 48h old culture of an AT-10 strain of Agrobacterium tumefaciens was used. Inoculum with three concentrations (1000, 100, 10ugml-1) of each test sample containing bacterial culture was prepared. Red skinned potatoes were surface sterilized in 0.1% mercuric chloride (HgCl2) solution in distilled water, for 7-10 minutes and then washed with autoclaved distilled water inside LFH. Potato cylinders were made with the help of a sterilized cork borer (8 mm). These cylinders were cut into (5 mm x 8 mm) thick discs and were placed on solidified agar plates (10 discs per plate). Then 50 ul of inoculums were applied to the top of each disk and each petriplate was wrapped with parafilm strips to avoid contamination and loss of moisture during the incubation period. These petri-plates were placed in a 28AdegC incubator for 21 days.

The number of tumors was counted after staining with the Lugol's solution (10% KI and 5% I2) with the help of a dissecting microscope and the percentage inhibition was determined as follows:

(Equation)

Antibacterial Activities

In vitro evaluation of the antibacterial activity of the compounds was carried out by agar well diffusion assay against six different Gram positive and Gram negative bacteria [24]. Antibacterial activity was determined by using a Mueller Hinton Agar (MHA). The fresh inoculums of these bacteria were prepared and diluted by sterilized normal saline. The turbidity of these cultures was adjusted by using 0.5Mc-Farland. A homogeneous bacterial lawn was developed by sterile cotton swabs. The inoculated plates were bored by a 6 mm sized borer to make the wells. Sample dilutions were prepared by dissolving each sample (1.0mg) in 1.0 mL of DMSO used as negative control in this bioassay. The equimolar concentration of Levofloxacin (1.0mg/ml), a broad spectrum antibiotic (positive control) was prepared. These plates were incubated at 37 AdegC for 24 hours. Antibacterial activity of the compounds was determined by measuring the diameter of zone of inhibition (mm, +- standard deviation).

Urease Inhibitory Activity

The urease inhibitory activity of the synthesized N-tosyl benzimidazoles was determined by measuring the amount of ammonia produced by the indophenols method described by Weatherburn [25]. The reaction mixtures, comprising 20 uL of enzyme (Jack bean urease, 5 U/mL) and 20uL of test compounds in 50uL buffer (100mM urea, 0.01 M K2HPO4, 1mM EDTA and 0.01 M LiCl2, pH 8.2), were incubated for 10 min at 37 AoC in 96-well plate. Briefly, 40uL each of phenol reagents (1%, w/v phenol and 0.005%, w/v sodium nitroprusside) and 70uL of alkali reagent (0.5%, w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The absorbance at 625nm was measured after 30min, using a microplate reader (OPTIMax, Tunable). All reactions were performed in triplicate. The urease inhibition activities were calculated according to the following formula:

Urease inhibition activity (%) = (ODcontrol - ODsample x 100) / ODcontrol

Where ODcontrol and ODsample represent the optical densities in the absence and presence of sample respectively. Thiourea was used as the standard inhibitor for urease.

Results and discussion

Chemistry

Substituted benzimidazoles (3a-e) were synthesized by following the already reported method [26] with slight modifications. 1,2-phenylene diamine (1a-b) were reacted with monobasic acids (2a-c) in presence of 4N HCl to obtain substituted benzimidazoles (3a-e) Scheme II. N-tosyl benzimidazoles (4a-e) were synthesized by reacting 2-alkyl substituted-benzimidazoles with tosyl chloride in 10% sodium hydroxide and acetone solution according to the methods reported earlier [27-28]. The molecular structure of 4b was determined by single X-ray diffraction technique.1HNMR of 4c, 4d and 4e revealed a characteristic set of signals for aromatic protons indicating the presence of regioisomers. The 1HMNR spectra of 4a and 4b were also consistent with the proposed structures. Ar-H (4, 5, 6, and 7) displayed multiplet signals in range of 7.23-7.40 ppm.

Moreover, Ar-H (2', 3') for N-tosyl substitution displayed the doublet at 8.10 ppm (J=9.3 Hz) for 4c, 8.02 ppm (J=8.1Hz) for 4d, 8.00 ppm (J=8.1Hz) for 4e, 8.03ppm (J=8.1Hz) for 4a and at 7.96 ppm (J=6 Hz) represented ortho coupling. FTIR spectra of (3a-e) showed a strong peak for N-H in the range of 3500-3300cm-1 whereas it was not observed in (4a-e) which confirmed the formation of desired N-tosyl benzimidazoles (4a-e). X-ray crystallography exhibited a monoclinic crystal system for 4b with a moiety formula C15H14N2O2S, Mr=286.34.

Description of the X-ray single crystal structures

The solid state structure of the compounds (4a) and (4b) were investigated by single crystal x-ray diffraction technique. The structure of 4a shows two independent molecules (A and B) in asymmetric unit as given in Fig. 1. The selected crystallographic data are summarized in Table-1 while selected geometrical parameters are listed in Table-2.

The Table-3 identifies the weak intermolecular interactions in the crystal packing. The crystal structure refines as a twin (BASF = 0.46861). As a whole, molecule is essentially non-planar although the benzimidazole ring and toluene-4-sulphonyl groups are individually planar. Dihedral angle between the mean planes of these two groups is 89.4(2)Adeg (A), 89.0(6)o (B) while the N1A/S1A/C8A/C9A and N1B/S1B/C8B/C9B torsion angles are 79.4(2)o and 82.1(2)o, respectively. The S1A--N1A and S1B--N1B bond lengths are in good agreement with data observed in the literature [27]. Crystal structure of the molecule is further stabilized by some non-classical weak intermolecular C---H...O, C-H....N interactions which link the molecules into chains along [1 0 0] and [0 0 1] forming an extended 2-dimensional supramolecular network. The compound crystallizes in the triclinic crystal system with space group P-1, a = 8.6305(11) A, b = 8.9318 (11) A, c = 17.3824 (13) A and [alpha] = 81.070 (8)Adeg, [beta] = 81.690 (9)Adeg, I3 = 80.757 (11)Adeg.

Table-1: Crystal data for C14H12N2O2S (4a) and C15H14N2O2S (4b).

###Compounds###4a###4b

###Empirical formula###C14H12N2O2S###C15H14N2O2S

###Formula weight###272.32###286.34

###Temperature (K)###173 (2)###173 (2)

###Crystal system###triclinic###monoclinic

###Space group###P -1###Cc

###Unit cell dimensions (A,Adeg)

###a###8.6305(11)###10.9624(4)

###b###8.9318 (11)###16.2917 (5)

###c###17.3824 (13)###8.0762 (3)

###[alpha]###81.070 (8)Adeg###90

###[beta]###81.690 (9)Adeg###106.089 (4)

###I3###80.757 (11)Adeg###90

###Volume (A3)###1296.7 (2)###1385.88 (9)

###Z###4###4

###Absorption coefficient u (mm-1)###0.248###0.236

###F(000)###568###600

###range for data collection (Adeg)###3.106-32.83###3.0567-32.7824

###Calculated density (mg m -3)###1.395###1.372

###Final R indexes [I>=2 (I)]###R1=-.0504, wR2=0.1181###R1=-.0391, wR2=0.0973

###Final R indexes [all data]###R1=0.1016,wR2=0.1302###R1=0.0459,wR2=0.1026

###Goodness-of-fit on F2###0.869###1.059

###Number of data collected###11589###7882

###Largest diff. peak/hole / e A-3###0.45/-0.39###0.32/-0.21

Table 2 Selected bond lengths (A) and angles (Adeg) for C14H12N2O2S (4a) and C15H14N2O2S (4b)

###C14H12N2O2S (4a)

###S1A-O1A###1.4281(17))###N1A-C1A###1.396(3)

###S1A-O2A###1.4140(19)###N1A-C7A###1.406(3)

###S1A-N1A###1.6719(19)###N2A-C1A###1.291(3)

###S1A-C8A###1.745(2)###N2A-C2A###1.387(3)

###S1B-O1B###1.4241(16)###N1B-C1B###1.388(3)

###S1B-O2B###1.4188(18)###N1B-C7B###1.403(3)

###S1B-N1B###1.674(2)###N2B-C1B###1.289(3)

###S1B-C8B###1.751(2)###N2B-C2B###1.403(3)

###O1A-S1A-O2A###121.11(11)###O1A-S1A-N1A###105.11(10)

###O1A-S1A-C8A###109.58(10)###O2A-S1A-N1A###105.74(10)

###O2A-S1A-C8A###110.36(11)###N1A-S1A-C8A###103.25(9)

###O1B-S1B-O2B###120.84(10)###O1B-S1B-N1B###105.15(10)

###O1B-S1B-C8B###109.39(10)###O2B-S1B-N1B###106.03(10)

###O2B-S1B-C8B###110.68(11)###N1B-S1B-C8B###103.06(9)

###C15H14N2O2S (4b)

###S1-O1###1.423 (2)###N1-C1###1.416 (3)

###S1-O2###1.4273 (19)###N1-C7###1.403 (3)

###S1-N1###1.687 (2)###N2-C1###1.296 (3)

###S1-C8###1.743 (2)###N2-C2###1.397 (3)

###01-S1-O2###120.96 (12)###O1-S1-N1###106.12 (11)

###O1-S1-C8###109.75 (12)###O2-S1-N1###105.14 (11)

###O2-S1-C8###109.38 (11)###N1-S1-C8###104.02 (10)

Table-3: Weak intermolecular interactions for C14H12N2O2S (4a) and C15H14N2O2S (4b) [A and Adeg].

###D-H...A###d(D-H)/A###d(H-A)/A###d(D-A)/A###D-H-A/Adeg

###C14H12N2O2S (4a)

###C1A---H1A...O1A1###0.95###2.42###3.351(3)###166.2

###0.95###2.61###3.465(3)###150.3

###0.95###2.42###3.353(3)###166.0

###0.95###2.62###3.391(3)###138.0

###C15H14N2O2S (4b)

###C6---H6...N25###0.95###2.56###3.356(3)###142.0

###1-x, 1-y, -z; 2+x, -1+y, +z; 31-x, 2-y, 1-z; 41+x, +y, +z; 5-1/2+x, A1/2-y, -1/2+z

The crystal structure of the compound (4b) is shown in Fig. 2. The asymmetric unit reveals a single molecule of (4b). The molecule (4b) crystallizes in monoclinic crystal system with space group C c, a = 10.9624(4) A, b = 16.2917 (5) A, c = 8.0762 (3) A and [beta] = 106.089 (4)Adeg. As a whole, molecule is essentially non-planar although the benzimidazole ring and toluene-4-sulphonyl groups are individually planar. Dihedral angle between the mean planes of these two groups is 88.1(5)Adeg and the N1/S1/C8/C9 torsion angle is 95.1(2)o.

The S1-N1 bond length is in good agreement with data observed in the literature. Crystal structure of the molecule is further stabilized by some non-classical weak inter-molecular C-H....N forces which link molecules into chains along [1 0 0]. Both molecules in asymmetric unit have similar bond lengths and angles. Both molecules are essentially non-planar but individual benzimidazole and toluene-4-suphlonyl groups are planar. The dihedral angles between these groups is 79.4(2)Adeg in molecule A (N1A/S1A/C8A/C9A) and 81.2Adeg in molecule B(N1B/S1B/C8B/C9B).

Similar to 4b, the crystal structure of compound 4a is stabilized by non-classic intermolecular hydrogen bonding C- H....O and C-H....N.

Biological activities

All of the synthesized N-tosyl benzimidazoles (4a-e) were examined for antitumor activity by the potato disc method. Table-4 presented the antitumor activity results of the synthesized N-tosyl benzimidazoles (4a-e). The compound (4e) which possesses nitro functionality at benzimidazole ring exhibited excellent antitumor activity compared to other derivatives. The presence of electron withdrawing nitro group at benzimidazole ring play significant role in antitumor activity. The compound (4c) showed good antitumor activity with IC50 value 1000ugml-1 while IC50 value of most potent derivative (4e) is 474.45ugml-1.

Table-4: Antitumor activity of synthesized N-tosyl benzimidazoles (4a-e).

###% Inhibition at different

Sr.###Compound###IC50 Value

No.###Code

###concentrations

###(ug/ml)

###1000 ug/ml###100 ug/ml###10 ug/ml

1###4a###-###-###-###-

2###4b###32###21###3###>1000

3###4c###50###28###6###1000

4###4d###32###14###4###>1000

5###4e###67###21###8###474.45

Antibacterial activity of synthesized N-tosyl benzimidazoles (4a-e) was determined and results were presented in Table-5. Antibacterial activity results revealed that compound (4d) and (4e) which possesses methyl and ethyl substitution, respectively, at the imidazole ring exhibited superb zone inhibition against gram positive and gram negative strains. The compound (4e) exhibited 80% growth inhibition against Bacillus subtilis and 78% inhibition against Escherichia coli compared to the standard drug. Staphylococcus aureus, Pseudomona putida and Pseudomona aeruginosa are the most resistant bacteria among the tested microorganisms. Among all the N-tosyl benzimidazole derivatives, the most active was (4e). Antibacterial activity results revealed that the presence of a benzimidazole nucleus as well as the presence of polar or non-polar substituents greatly affected the result.

Table-5: Antibacterial activity of synthesized N-tosyl benzimidazoles (4a-e)

###Compounds

###Bacterial Strains

###4a###4b###4c###4d###4e###standard

###Proteus mirabilis###7###10###16###20###23###30

###Bacillus subtilis###6###9###13###17###20###25

###Escherichia coli###10###15###15###19###25###32

Staphylococcus aureus###-###-###-###20###26###34

###Pseudomonas putida###-###-###12###16###22###36

Pseudomonas aeruginosa###11###-###-###14###20###28

The urease inhibitory potential of the synthesized N-tosyl benzimidazoles derivatives (4a-e) was also determined and results were presented in Table-6. The derivative 4e exhibited highest potential to inhibit the urease enzyme compared to all other derivatives. The compound 4e displayed IC50 value 31.1 +- 0.85uM while IC50 value for the standard thiourea is 20.9 +- 0.92 uM. The presence of electron withdrawing substituent at benzimidazole phenyl ring and alkyl substitution at heterocyclic ring play vital role in the urease inhibitory activity.

Table-6: Urease Inhibitory activity of synthesized N-tosyl benzimidazoles (4a-e).

Compounds###IC50 uM

###4a###83.3 +- 1.32

###4b###83.5 +- 1.07

###4c###35.5 +- 0.93

###4d###158.6 +- 1.81

###4e###31.1 +- 0.85

Thiourea###20.9 +- 0.92

Conclusion

The N-tosyl benzimidazoles (4a-e) were efficiently synthesized starting from substituted o-phenylene diamine. The spectral data of compounds (4a-e) and X-ray crystallographic data in case of compounds (4a, 4b) assured the structures of the desired products. The benzimidazole derivative (4e) exhibited excellent antitumor activity compared to other derivatives withIC50 value 474.45ugml-1. The presence of electron withdrawing nitro group at benzimidazole ring play significant role in antitumor activity. In case of antibacterial activity compounds (4d) and (4e) which possesses methyl and ethyl substitution respectively at the imidazole ring exhibited significant zone inhibition against gram positive and gram negative bacteria. The compound (4e) exhibited 80% growth inhibition against Bacillus subtilis and 78% inhibition against Escherichia coli compared to the standard drug.

Antibacterial activity results revealed that the presence of polar and non-polar substituents in the synthesized derivatives had significant influenced on their activities. The urease inhibitory activity results showed that derivative 4e exhibited highest potential to inhibit the urease enzyme compared to all other derivatives. Based upon our investigation it is proposed that compound (4e) may serve as lead structure to design more potent biological active compounds having multi-targets inhibition activities.

Conflict of Interest

The authors declared no conflict of interests.

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