Antibacterial and DNA cleavage activity of carbonyl functionalized N-heterocyclic carbene-silver(I) and selenium compounds

Accepted Manuscript Antibacterial and DNA Cleavage Activity of Carbonyl Functionalized NHeterocyclic Carbene-Silver(I) and Selenium Compounds

Rosenani A. Haque, Muhammad Adnan Iqbal, Faisal Mohamad, Mohd. R. Razali PII:

S0022-2860(17)31449-7

DOI:

10.1016/j.molstruc.2017.10.092

Reference:

MOLSTR 24463

To appear in:

Journal of Molecular Structure

Received Date:

09 August 2017

Revised Date:

25 October 2017

Accepted Date:

25 October 2017

Please cite this article as: Rosenani A. Haque, Muhammad Adnan Iqbal, Faisal Mohamad, Mohd. R. Razali, Antibacterial and DNA Cleavage Activity of Carbonyl Functionalized N-Heterocyclic Carbene-Silver(I) and Selenium Compounds, Journal of Molecular Structure (2017), doi: 10.1016/j. molstruc.2017.10.092

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ACCEPTED MANUSCRIPT Highlights   

Synthesis of carbonyl functionalized benzimidazolium salts (I-IV). Synthesis of NHC-Ag-NHC, NHC-Ag-OAc and NHC-Se type compounds (V-VII). Antimicrobial testing of salt (IV) and respective Ag and Se compounds (V-VII).

ACCEPTED MANUSCRIPT Antibacterial and DNA Cleavage Activity of Carbonyl Functionalized NHeterocyclic Carbene-Silver(I) and Selenium Compounds

Rosenani A. Haque1, Muhammad Adnan Iqbal1,2, Faisal Mohamad3 and Mohd. R. Razali1‡ ‡Corresponding

1School

of Chemical Sciences, Universiti Sains Malaysia, Penang, Malaysia

2Department 3School

author: [email protected]

of Chemistry, University of Agriculture, Faisalabad, Pakistan

of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia

Abstract The article describes syntheses and characterizations of carbonyl functionalized benzimidazolium salts, I-IV. While salts I-III are unstable at room temperature, salt IV remained stable and was further utilised to form N-heterocyclic carbene (NHC) compounds of silver(I), V and VI, and selenium compound, VII respectively. Compounds IV-VII were tested for their antibacterial potential against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Salt IV shows a very low inhibition potential (minimum inhibitory concentration, MIC 500 µg/mL) compared to the respective silver(I)-NHC, V and VI (MIC 31.25 µg/mL against both, E. coli and S. aureus) and selenium compound, VII (MIC 125 µg/mL against E. coli and 62.50 µg/mL against S. aureus). In DNA cleavage abilities, all the test compounds cleave DNA in which the VII cleaves the DNA at the faster rate. Meanwhile, the silver(I)-NHC complexes V and VI act at the same mode and pattern of DNA cleavage while VII is similar to IV.

Keywords: N-Heterocyclic carbene (NHC), silver(I), selenium, antibacterial, DNA cleavage. 1

ACCEPTED MANUSCRIPT

1. Introduction Antimicrobial drugs play an important role in the human health care by treating bacterial infections as well as by preventing their further spread in the body [1]. However, the bacteria have an ability to induce resistance against the drugs after prolong usage and hence the newer drugs with better inhibition ability are usually required [2]. It has been explored that the metal-based antimicrobial agents play a better role against the bacterial resistance and may act as future antimicrobial drugs [3]. Although, the coordination compounds show antimicrobial activities better than the organic compounds, they however lose their effect before reaching the target site due to releasing metal ions at the faster rate [4]. This problem has been resolved by introducing metal complexes of N-heterocyclic carbenes (NHCs) [5] since the NHCs release metal ions at slower rate compared to the other coordination compounds which sustain their biological effects for longer time [4]. Metal complexes of NHCs are rapidly growing in medicinal chemistry due to the fact of their diverse biological potential [6]. However, in the recent years, the increasing numbers of research publications have been mainly focused on the antibacterial [6d, 7] and anticancer [8] applications of the silver(I)- and Au(I)-NHC complexes. The use of silver and gold in medicinal chemistry is perhaps due to their relatively compatible relationship with the biological systems compared to the other transition elements [9]. On the other hand, various compounds based on selenium have also been tested and found to have an attractive antimicrobial activity [10]. In addition to these interesting findings, evidences have been found that the carbonyl functionalized compounds have also played a significant role against the bacterial growth [11]. Hence, we report here the syntheses of carbonyl functionalized benzimidazolium salt, IV and respective silver(I)-NHC, IV and V as well as selenium compounds, VII. The 2

ACCEPTED MANUSCRIPT purposes of this work are to investigate, whether the carbonyl functionality shows any role against bacterial inhibition in vitro as well as to test the antibacterial efficiency of silver(I) compared to selenium, since the silver(I) complexes (V and VI), along with selenium compounds (VII) have been derived from the same pro-ligand IV. The results are then compared with those reported before in literatures.

2. Experimental 2.1 Materials and methods All the experiments were carried out under aerobic conditions unless stated otherwise. All the chemicals were used as received. Melting points of the synthesized compounds were determined using Stuart Scientific SMP-1 (UK) instrument. FT-IR spectral patterns were recorded by FT-IR Perkin Elmer-2000 spectrometer using KBr disc method [12]. FT-NMR (1H and

13C)

spectra of the synthesized compounds were collected by Bruker 500 MHz

UltrashieldTM spectrometer either in deuterated dimethyl sulfoxide (DMSO-d6) or deuterated acetonitrile (CD3CN) solvents.

2.2 Syntheses of salts 2.2.1 Synthesis of 3-acetyl-1-propyl-benzimidazolium bromide (I) N-propylbenzimidazole (1.60 g, 10 mmol) was dissolved in 25 mL of 1,4-dioxane and acetyl bromide (1.48 mL, 20 mmol) was then added dropwise in the consistently stirring solution under nitrogen environment. Instantly, yellow to orange flakes appeared in the reaction medium. Reaction mixture was stirred overnight, filtered and washed with fresh 1,4-dioxane (6×3 mL) to remove the yellow coloration. The white precipitates obtained were dried at room temperature. Yield 89% (2.53g). FT-IR (KBr disc, ν cm-1): 3130, 3027 (Csp2-H aromatic stretch); 2912, 2780 (Csp3-H aliphatic stretch); 1750 (C=O stretch); 1606, 1566 (C=C;C=N 3

ACCEPTED MANUSCRIPT aromatic stretch); 1467, 1368, 1329 (CH2 bending). 1H NMR (500 MHz, DMSO-d6, δ ppm): 0.88 (t, 3H, 1 × CH3), 1.87 (s, 3H, 1 × CH3 of acetyl), 1.93 (sext, 2H, 1 × CH2), 4.50 (t, 2H, 1 × N-CH2), 7.59 (m, 2H, Ar-H), 7.87 (m, 1H, Ar-H), 8.08 (m, 1H, Ar-H), 9.88 (s, 1H, 1 × NCHN). 13C NMR (125.1 MHz, DMSO-d6, δ ppm): 10.6, 21.0, 22.1 (CH3 of acetyl), 47.8 (NCH2), 113.4, 114.8, 126.0, 126.4, 130.0, 131.0 (Ar-C), 141.4 (NCHN), 171.9 (C=O).

2.2.2 Synthesis of 3-acetyl-1-benzyl-benzimidazolium bromide (II) N-benzylbenzimidazole (1.30 g, 6.2 mmol) was dissolved in 25 mL of acetonitrile and acetyl bromide (0.74 mL, 10 mmol) was then added dropwise in the consistently stirring solution under nitrogen. Within a minute white precipitates appeared in the yellowish reaction medium. Reaction mixture was stirred overnight, filtered and washed with fresh acetonitrile (3×2 mL) to remove the yellow coloration. White precipitates obtained were dried at room temperature. Yield 74% (1.51g). FT-IR (KBr disc, ν cm-1): 3087, 3048 (Csp2-H aromatic stretch); 2971, 2858, 2746 (Csp3-H aliphatic stretch); 1758 (C=O stretch); 1607, 1551 (C=C;C=N aromatic stretch); 1483, 1446, 1444, 1360, 1323 (CH2 bending). 1H NMR (500 MHz, DMSO-d6, δ ppm): 1.88 (s, 3H, 1 × CH3 of acetyl), 5.86 (s, 2H, 1 × N-CH2), 7.33 (m, 1H, Ar-H), 7.35 (m, 2H, Ar-H), 7.53 (m, 4H, Ar-H), 7.85-7.91 (m, 2H, Ar-H), 10.12 (s, 1H, 1 × NCHN).

13C

NMR (125.1 MHz, DMSO-d6, δ ppm): 21.0 (CH3 of acetyl), 49.7 (N-CH2),

113.4, 115.0, 126.5, 128.2, 128.8, 128.9, 130.74, 131.0, 134.3 (Ar-C), 141.7 (NCHN), 172.0 (C=O).

2.2.3 Synthesis of 3-octanoyl-1-benzyl-benzimidazolium bromide (III) N-benzylbenzimidazole (1.0 g, 4.8 mmol) was dissolved in 25 mL of acetonitrile and octanoyl bromide (0.82 mL, 4.8 mmol) was then added dropwise in the consistently stirring solution under nitrogen. Reaction mixture was stirred overnight, cooled in ice bath, filtered 4

ACCEPTED MANUSCRIPT and washed with cold fresh acetonitrile (3×2 mL) to remove the yellow coloration. White precipitates obtained were dried at room temperature. Yield 75% (0.3g). FT-IR (KBr disc, ν cm-1): 3126, 3102, 3062 (Csp2-H aromatic stretch); 2979, 2929, 2856 (Csp3-H aliphatic stretch); 1712 (C=O stretch); 1613, 1550 (C=C;C=N aromatic stretch); 1496, 1448, 1378, 1322, 1307 (CH2 bending). 1H NMR (500 MHz, Acetonitrile-d3, δ ppm): 0.85 (t, 3H, 1 × CH3), 1.22-1.29 (m, 8H, 4 × CH2), 1.52 (m, 2H, 1 × CH2), 4.60 (t, 2H, 1 × CH2), 5.70 (s, 2H, 1 × N-CH2), 7.34 (m, 1H, Ar-H), 7.39 (m, 2H, Ar-H), 7.62 (m, 4H, Ar-H), 7.92-7.94 (m, 2H, Ar-H), 9.52 (s, 1H, 1 × NCHN). 13C NMR (125.1 MHz, acetonitrile-d3, δ ppm): 13.3, 22.3, 24.6, 28.6, 28.7, 31.4, 33.5, 50.4 (N-CH2), 113.1, 117.1, 126.7, 128.8, 129.0, 131.0, 133.5, 140.4 (NCHN), 174.7 (C=O).

2.2.4 Synthesis of 3-benzyl-1-(2-oxobutyl)-benzimidazolium bromide (IV) N-benzylbenzimidazole (1.37 g, 6.62 mmol) was dissolved in 70 mL of 1,4-dioxane and 1bromo-2-butanone (1.0 g, 6.62 mmol) was then added dropwise in the consistently stirring solution. The reaction mixture was left to reflux for 24 h. White precipitates appeared in the reaction medium, which then cooled to room temperature and filtered. The white precipitates were washed with fresh 1,4-dioxane (3×5 mL) following by diethyl ether (3×5 mL). Yield 89% (2.10g). FT-IR (KBr, ν cm-1); 3133, 3064, 3037 (Csp2-H aromatic stretch), 2987, 2944 (Csp3-H aliphatic stretch), 1730 (C=O stretch), 1614, 1560 (C=C;C=N aromatic stretch), 1482, 1452, 1428, 1370 (CH2 bending). 1H NMR (500 MHz, DMSO-d6, δ ppm); 1.04 (t, 7.5 Hz, 3H, 1 × CH3), 2.77 (q, 2H, 1 × CH2), 5.76 (s, 2H, 1 × N-CH2-C=O), 5.89 (s, 2H, N-CH2aromatic), 7.39-7.45 (m, 3H, aromatic-H), 7.52 (d, J = 5.0 Hz, 2H, aromatic-H), 7.66 (m, 2H, aromatic-H), 8.02 (m, 2H, aromatic-H), 9.82 (s, 1H, NCHN). 13C NMR (125.1 MHz, DMSOd6, δ ppm); 6.9 (1 × CH3), 32.4 (1 × CH2), 49.8 (1 × N-CH2-C=O), 54.7 (1 × N-CH2-

5

ACCEPTED MANUSCRIPT aromatic), 113.8, 114.0, 126.6, 126.7, 128.2 (2 × aromatic-C), 128.7, 129.0 (2 × aromatic-C), 130.3, 131.8, 133.9 (12 × aromatic-C), 143.2 (1 × NCHN), 202.7 (1 × C=O).

2.3 Synthesis of metal complexes 2.3.1 Synthesis of acetyl(1-benzyl-3-(2-oxobutyl)-benzimidazol-2-yl)silver(I) (V) Compound IV (0.50 g, 1.39 mmol) was dissolved in 50 mL of methanol along with silver acetate (0.46 g, 2.78 mmol). The reaction mixture was stirred at room temperature for 48 h, covered from light. The solvent was then removed to facilitate the formation of white powder. To the solid obtained, 100 mL of dichloromethane was added. The mixture was vigorously stirred and filtered through a pad of celites. The filtrate was evaporated to obtain a brownish sticky fluid, washed with diethyl ether (2×3 mL) and dried under vacuum which was then the light brown crystalline obtained. Yield 77% (0.48g). FT-IR (KBr, ν cm-1); 3064, 3029 (Csp2-H aromatic stretch), 2975, 2936 (Csp3-H aliphatic stretch), 1718 (C=O stretch), 1568 (C=N stretch), 1482, 1397, 1332 (CH2 bending). 1H NMR (500 MHz, CDCl3, δ ppm); 0.89 (t, 7.5 Hz, 3H, 1 × CH3), 2.01 (s, 3H, 1 × CH3 of acetate), 2.67 (q, 2H, 1 × CH2), 5.34 (s, 2H, 1 × N-CH2-C=O), 5.52 (s, 2H, N-CH2-aromatic), 7.19-7.30 (m, 9H, aromatic-H).

13C

NMR (125.1 MHz, CDCl3, δ ppm); 7.3 (1 × CH3), 33.4 (1 × CH2), 53.4 (1 × N-CH2-C=O), 57.3 (1 × N-CH2-aromatic), 111.4, 112.2, 124.3, 124.5, 127.2 (2 × aromatic-C), 128.4, 129.1 (2 × aromatic-C), 133.5, 134.4, 133.1 (12 × aromatic-C), 178.0 (Ag-acetate carbonyl), 189.8 (Ag-Ccarbene), 203.3 (1 × C=O).

2.3.2

Synthesis

of

bis(1-benzyl-3-(2-oxobutyl)-benzimidazol-2-yl)silver(I)

hexaflourophosphate (VI) Compound IV (1.0 g, 2.80 mmol) was dissolved in 150 mL of methanol along with silver oxide (0.64 g, 2.80 mmol). The reaction mixture was stirred at room temperature for 48 h, 6

ACCEPTED MANUSCRIPT covered from light. The reaction mixture was then filtered through a pad of celites and to the colorless filtrate, 2 equivalents of potassium hexaflourophosphate (1.03 g, 5.2 mmol) was added. The reaction mixture was then left to stir for 2 h. Light yellow precipitates appeared after adding distilled water (25 mL) were filtered followed by washed with distilled water (3× 5 mL) to remove the excess of potassium hexaflourophosphate. The obtained yellow powder then left dried at room temperature. Yield 54% (0.61.0g). FT-IR (KBr, ν cm-1); 3114, 3064, 3033 (Csp2-H aromatic stretch), 2979, 2940, 2878 (Csp3-H aliphatic stretch), 1730 (C=O stretch), 1614, 1513 (C=C;C=N aromatic stretch), 1479, 1455, 1397, 1355 (CH2 bending). 1H NMR (500 MHz, DMSO-d6, δ ppm); 1.02 (t, 7.0 Hz, 6H, 2 × CH3), 2.72 (q, 4H, 2 × CH2), 5.70 (s, 4H, 2 × N-CH2-C=O), 5.85 (s, 4H, 2 ×N-CH2-aromatic), 7.29-7.43 (m, 14H, aromatic-H), 7.68 (m, 4H, aromatic-H). 13C NMR (125.1 MHz, DMSO-d6, δ ppm); 7.0 (2 × CH3), 32.5 (2 × CH2), 48.1 (2 × N-CH2-C=O), 51.7 (2 × N-CH2-aromatic), 111.4, 112.2, 119.0, 123.5, 126.2, 127.4, 128.1, 132.8, 131.4, 132.8 (aromatic-C), 190.6 (Ag-Ccarbene), 204.4 (2 × C=O).

2.3.3 Synthesis of 1-(3-benzyl-2-selenoxobenzimidazol-2-yl)butan-2-one (VII) Compound IV (0.15 g, 0.42 mmol) was dissolved in 30 mL of distilled water. Sodium carbonate anhydrous (0.12 g, 0.84 mmol) and selenium metal (0.05 g, 0.84 mmol) were then added. The reaction mixture was then left to reflux for 8 h. A red colored oily layer formed after 3 h of reaction progress. After 8 hours the reaction mixture was stopped, cooled down to the room temperature and 100 mL of chloroform was then added. The chloroform layer was separated using separating funnel as a red colored solution. The separated layer was evaporated using rotary evaporator at 70 oC (70 mbar) to obtain the compound as red coloured sticky fluid which then washed with diethyl ether (2×3 mL). The compound was further dried under vacuum for 3 d to facilitate the formation of reddish sticky fluid. Yield 7

ACCEPTED MANUSCRIPT 73% (0.11g). FT-IR (KBr, ν cm-1); 3084, 3061, 3031 (Csp2-H aromatic stretch), 2973, 2931 (Csp3-H aliphatic stretch), 1715 (C=O stretch), 1612, 1604, 1496 (C=C;C=N aromatic stretch), 1458, 1404, 1362, 1335 (CH2 bending). 1H NMR (500 MHz, CDCl3, δ ppm); 1.15 (t, 7.0 Hz, 3H, 1 × CH3), 2.62 (q, 2H, 1 × CH2), 5.31 (s, 2H, 1 × N-CH2-C=O), 5.71 (s, 2H, 1 ×N-CH2-aromatic), 7.15-7.36 (m, 7H, aromatic-H), 7.85 (m, 2H, aromatic-H).

13C

NMR

(125.7 MHz, CDCl3, δ ppm); 7.3 (1 × CH3), 33.7 (1 × CH2), 50.5 (1 × N-CH2-C=O), 55.0 (1 × N-CH2-aromatic), 109.5, 110.5, 120.3, 122.3, 123.1, 127.1, 127.4, 127.9, 128.5, 135.4, 168.6 (Se-Ccarbene), 203.4 (1 × C=O).

2.4 Antibacterial study The potential of IV-VII against bacterial proliferation was tested by performing minimum inhibitory concentration (MIC) test as described before [13]. Gram negative bacteria, Escherichia coli ATCC 25922 and Gram positive, Staphylococcus aureus were used in this study. Preparations of bacterial suspension were carried out by inoculating a loopful of a fresh colony from Luria Bertani (LB) agar into a fresh LB broth and were incubated at 37 oC

for overnight growth. An amount of 100 µL overnight bacteria culture was again

transferred into a fresh 10 mL LB broth and incubated at 37 oC until the turbidity was equal to 0.5 McFarland standard. The bacteria culture was then used to adjust the final tested MIC concentration of each sample. The tested MIC concentrations were in the range of 1.0 mg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.50 µg/mL, 31.25 µg/mL and 15.63 µg/mL. The growth and inhibition of bacteria were observed after overnight incubation at 37 oC.

2.5 DNA cleavage activity DNA cleavage activity was performed by mixing and incubating samples (salt/complexes) with circular pDsRED plasmid (Clontech Lab, USA). Each of the reaction 8

ACCEPTED MANUSCRIPT mixtures contained 1 μL of pDsRED plasmid (0.6 μg/mL), 1 μL of either salt or complex (100 μg), 8 μL of the sterile distilled water. The mixture was then incubated at 37°C for 8 hours. Prior to gel electrophoresis, 5 μL of each sample was mixed with 2 μL of 6x loading buffer (0.35% bromophenol blue, 0.35% xylene cyanol, 0.6 orange G and 20% sucrose in H2O) and loaded into 0.7% agarose gel. Gel electrophoresis was carried out for 30 minutes at 90 V in 0.5x TAE buffer (20 mM Tris-Cl pH 8.0, 10mM acetic acid, 0.5 mM EDTA). The gel was stained in ethidium bromide solution (1μg/mL) for 15 minutes and followed by destaining in water for 10 minutes. The image was photographed by using gel imaging documentation system under UV light mode.

3. Results and Discussion 3.1 Synthesis Attempts to prepare N-acylated/alkylated benzimidazoles (A-C, Scheme 1) were performed in the presence of a base, potassium hydroxide in DMSO [8a]. However, despite various attempts, the syntheses of A and B remained unsuccessful through this route with the reactants are recovering after the reaction procedure (Scheme 1).

9

ACCEPTED MANUSCRIPT O O N Br

N

R

R

(A)

N

NH

O

O R

Br

N

N

R

(B)

N Br

N

R

R

(C)

Scheme 1: Attempts in synthesizing of N-acylated/alkylated benzimidazoles, A-C. Syntheses of Nacylated/alkylated benzimidazoles of types A and B are remained unsuccessful. The N-alkyled benzimidazoles of type C were successfully prepared. R = methyl or hexyl (A); R = methyl or isopropyl (B) and R = propyl or benzyl (C).

This failure in preparing A and B could be due to the fact that carbonyl group reacts with amine in the presence of base or ethanol [10b,14]. Hence, the syntheses of required compounds I-III were carried out using N-alkylated benzimidazoles of type C (Scheme 2). Compounds I-III were found to be moisture sensitive. In order to assess the moisture sensitivity of these compounds, a small quantity of each was placed on a filter paper separately and was placed in open environment to be observed consistently for 7 days. Compound I hydrolysed within three days whereas compounds II and III were found stable for the specified period. The NMR analysis of hydrolysed material showed the de-attachment of alkylated material (as R-OH) from the benzimidazole ring. Besides, attempts for complexation of I-III with metal source (Ag2O) unsuccessful with neither crystal nor precipitation are formed. 10

ACCEPTED MANUSCRIPT

O

O N

Br 1,4-dioxane

N Br-

I R

O

O N

N

N

Br

N Br-

Acetonitrile

II O

O N

Br

N Br-

Acetonitrile

III Scheme 2: Syntheses of N-acylated benzimidazolium salts (I-III). All compounds were synthesized by stirring the reaction mixtures at room temperature under nitrogen environment. Each of these compounds is remained unstable towards air and moisture.

Hence, to overcome the stability issue, salt IV was designed and synthesized (Scheme 3). This compound was found to be non-reactive towards air and moisture, soluble in methanol, ethanol and on heating in water. Salt IV was further reacted with two different sources of silver(I), Ag2O and Ag(OAc) and also selenium metal to obtain compounds, VVII, respectively. Syntheses of compounds V and VI were carried out according to the reported procedures with minor modifications [3f,7f,15]. For the formation of selenium based compound VII, the selenium metal reacts with azolium salts through carbene carbon in the presence of a weak base like K2CO3 or Na2CO3 [8b,16]. 11

ACCEPTED MANUSCRIPT N

N

1,4-dioxane, reflux

Br O H N

Br N

O

IV

a

c

b O O

O

N

Ag N

O

N

N

Ag

Se

N N

N PF6-

O

VI

V

N

O

VII

a = Ag(OAc), methanol (50 mL), stirring at RT (48 h) b = Ag2O, methanol (150 mL), stirring at RT (48 h) c = Se metal + Na2CO3, H2O (30 mL), reflux (8 h) Scheme 3: Synthesis of carbonyl functionalized ligand IV and respective complexes V-VII.

3.2 Characterization 3.2.1 FT-IR The synthesized compounds were preliminarily characterized by FT-IR. Comparing the spectral features of compounds before and after the possible bonding of silver(I) and selenium, shows some distinct changes which could be used as preliminary information for the successful incorporation of metal to the organic framework. The carbonyl vibrational 12

ACCEPTED MANUSCRIPT band in the salt IV (pro-ligand) at 1730 cm-1 shifted to the lower frequency, in V (1718 cm-1) and VII (1715 cm-1) whereas as in VI the frequency is retained (1730 cm-1). Such a shifting of specific vibrational bands has been observed by others [17].

3.2.2 FT-NMR The 1H-NMR spectra of the synthesized compounds show that the acidic proton peak (NCHN) in the 1,3-disubstituted benzimidazolium salt at 9.82 δ ppm disappeared in V-VII due to its replacement by either silver(I) or selenium [6d,18]. This provided a preliminary confirmation of the successful syntheses of desired compounds, V-VII. Furthermore, significant changes were observed in the aromatic hydrogen region, 7.19-8.02 δ ppm (aromatic-H), in complexes (V-VII) than compared to the parent salt IV. For example, in the salt IV, four chemical shifts appeared (7.39-7.45 m 3H, 7.52 d 2H, 7.66 d 2H, 8.02 m 2H δ ppm), turned to a broad multiplet (7.19-7.30 m 9H δ ppm) in V. Whereas, in VI and VII it appeared as two multiplets (7.29-7.43 m 14H, 7.68 m 4H in VI and 7.15-7.36 m 7H, 7.85 m 2H in VII). This also provided an indication for the successful syntheses of V-VII. The chemical shifts for alkyl groups appeared in the range 0.89-5.89 δ ppm both in salt IV and complexes V-VII. In 13C-NMR spectra of V-VII, the NCN carbon shifted to the downfield region (189.8 in V, 190.6 in VI, 168.6 δ ppm in VII) than compared to the salt (143.2 in IV) which indicates the successful formation of required compounds (Scheme 3). The chemical shifts for the aromatic carbons appeared at comparable positions, ranged 111.4-133.4 δ ppm. The 13C-NMR

chemical shifts for the carbonyl carbons in IV-VII appeared at around 203±1 δ

ppm.

13

ACCEPTED MANUSCRIPT 3.3 Antibacterial study The compounds IV-VII were tested for their antibacterial activity by agar and broth dilution method [13] with Gram negative, Escherichia coli ATCC 25922 and Gram positive, Staphylococcus aureus ATTC 33591 bacteria (Table 1). The test compounds show MIC values in the range of 31-500 μg/mL (Table 1). Salt IV shows mild activity with MIC 500 μg/mL than compared to respective complexes V-VII (MIC 31.25-125 μg/mL). Furthermore, the silver(I) complexes V-VI show comparable MIC values (31.25 μg/mL) against both the bacterial stains whereas the selenium adduct product, VII shows different MIC values against both the bacterial stains (125 against E. coli and 31.25 μg/mL against S. aureus). Comparing the MIC values of synthesized compounds (IV-VII) with the previously reported compounds (1-48, Figure 1), it could be noticed that the salt IV showed MIC value (500 μg/mL) better than some of the previously reported salts against E. coli. Furthermore, only the salts 41-44 show better MIC values (100-400 μg/mL) than compared to IV against S. aureus. This slightly better inhibition activity could be due to the presence of amine groups at the terminal positions of 41-44, since the molecule containing amine groups may show significant antibacterial activity [19]. Nevertheless, the MIC value of IV is not sufficient to consider it an active compound. Besides, both the silver(I) complexes (V and VI) derived from IV, show attractive inhibition values (31.25 μg/mL) than compared to the salt (IV) as well as literature complexes namely 16-17, 22-23, 28, 30, 33-34, 39 and 45-48 against E. coli (Table 1). Against S. aureus, silver(I) complexes (V and VI) remained active compared to literature complexes 16-17, 28, 34, 38-39 and 45-48. Importantly, none of the silver(I)-NHC complexes has shown MIC value better than the standard marketing drugs (Ciprofloxacin, Streptomycin, Amicillin). However, the inhibition mechanism of silver(I)-NHC might be different than these standard marketing drugs which may distinguished them and interesting for detailed biological study. 14

ACCEPTED MANUSCRIPT

R N

N

N

2PF6 1. R = propargyl, 2. R = pentyl, 3. R = hexyl, 4. R = methoxyethyl

R N

N

N Ag

R N

N

PF6 13. R = propyl, 14. R = butyl, 15. R = pentyl

N

5. R = propargyl, 6. R = pentyl, 7. R = hexyl, 8. R = methoxyethyl

N

N

N

n

O

N

R N

2PF6 Ag

Ag N R

N

nN

N

N

N R

O

N

R N

N

9. n = 2, 10. n = 3

Ag

2PF6

nN 2Br-

N

N

N R

Ag PF6 N

N

O

N R

16. R = propyl, 17. R = butyl, 18. R = pentyl O

11. n = 2, 12. n = 3

N R N

N

R N

PF6

N

N Br

25. R = ethyl, 26. R = pentyl, 27. R = butyronitrile

19. R = pentyl, 20. R = hexyl, 21. R = heptyl

N R

N

N R Br-

N

-

Cl-

31. R = butyl

35. R = 37. R =

36. R =

Cl R N

N

R N

N R

N

N

N

Ag PF6

Ag PF6 N

Ag

N

N

Ag

N R

N X

N

N

N

N R

N

O 38. R =

28. R = ethyl, 29. R = pentyl, 30. R = butyronitrile

22. R = pentyl, 23. R = hexyl, 24. R = heptyl

R4 R4

N

N

N Cl

N

R2 -

R1

N

N

40. R =

R3

R5

R3

R5

39. R =

32. X = Br, 33. X = PF6, 34. X = BF4

R2 R1

Ag Cl 45. R1 = H, R2 = H, R3 = H, R4 = H, R5 = H 46. R1 = CH3, R2 = H, R3 = CH3, R4 = H, R5 = CH3 47. R = R1 = CH3, R2 = CH3, R3 = H, R4 = CH3, R5 = CH3 48. R1 = CH3, R2 = CH3, R3 = CH3, R4 = CH3, R5 = CH3

41. R1 = H, R2 = H, R3 = H, R4 = H, R5 = H 42. R1 = CH3, R2 = H, R3 = CH3, R4 = H, R5 = CH3 43. R = R1 = CH3, R2 = CH3, R3 = H, R4 = CH3, R5 = CH3 44. R1 = CH3, R2 = CH3, R3 = CH3, R4 = CH3, R5 = CH3

Figure 1: Chemical structures of the compounds tested against bacteria E. coli and S. aureus.

15

Table 1: Dose dependent effect of synthesized compounds IV-VII on the growth of E.coli and S. aureus.

Escherichia coli ATCC 25922

Staphylococcus aureus ATCC 33591

MIC

MIC

(μg/mL)

(μg/mL)

1 mg/mL

500 µg/mL

250 µg/mL

125 µg/mL

62.50 µg/mL

31.25 µg/mL

15.63 µg/mL

1 mg/mL

IV

×

×

√

√

√

√

√

500

×

×

√

√

√

√

√

500

V

×

×

×

×

×

×

√

31.25

×

×

×

×

×

×

√

31.25

VI

×

×

×

×

×

×

√

31.25

×

×

×

×

×

×

√

31.25

VII

×

×

×

×

√

√

√

125

×

×

×

×

×

√

√

125

× = No Growth, √ = Growth

16

500 250 125 62.50 31.25 µg/mL µg/mL µg/mL µg/mL µg/mL

15.63 µg/mL

ACCEPTED MANUSCRIPT

Furthermore, compound VII shows lower inhibition (MIC 125 μg/mL and 62.5 μg/mL) against both types of bacteria than compared to the silver(I)-NHC complexes. Recently, two selenium compounds (49 and 50, Figure 2) have been synthesized and tested against E. coli and S. aureus [20]. The tested compounds (49 and 50) have shown very low inhibition potential (MIC 800 μg/mL) against both the bacterial stains. Compound VII, synthesized in the current work, shows a comparatively prominent inhibition (MIC 125 μg/mL against E.coli and 62.5 μg/mL against S. aureus). This might be due to the presence of carbonyl group in the molecule which is also evident from the MIC value of salt IV compared to the aforementioned literature salts. For example, a carbonyl functionalized selenium compound 51 (Figure 2) has shown a very prominent antibacterial activity against E. coli NIH (12.5 μg/mL) and S. aureus 209P (0.20 μg/mL) [21] whereas some imidazolium salts containing selenium but no carbonyl functionalities (52-57) did not show a significant antibacterial activity against E. coli and S. aureus except compound 54 which is perhaps due the presence of Cl at terminal position, since the halides play an important role against bacteria [22].

Se N O

O 50

49

O Se

N

N

Se N

N

R2

R1

Se

Cl N

N

R3 51

52. R1 = -CH3, R2 = -H, R3 = -H 53. R1 = -O-CH3, R2 = -H, R3 = -H 54. R1 = -Cl, R2 = -H, R3 = -H 55. R1 = -H, R2 = -CH3, R3 = -H 56. R1 = -H, R2 = -O-CH3, R3 = -H 57. R1 = -CH3, R2 = -CH3, R3 = -CH3

17

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Figure 2: Reported structures of the selenium compounds (49-57) tested against E. coli and S. aureus.

3.4 DNA cleavage activity The DNA cleavage ability of each sample was carried out by gel electrophoresis method (Figure 3). For this experiment, control (lane1) was merely a mixture of DNA and distilled water which represented the native form of circular DNA. The salt (IV, lane 2) has shown DNA cleavage ability since there was a shifted DNA band from supercoil (form I) to linear DNA (form III). The band of nicked DNA was also found noticeably thicker compared to the control DNA. Both the silver(I) complexes (V and VI, lanes 3 & 4, respectively) also show the DNA cleavage activities with the similar cleavage patterns to the IV. However, silver(I) complexes (V and VI) did not show the linear DNA band (form III), whereas thicker band of form II (nicked) was obviously observed. The activity of selenium compound, VI (lane 5) showed similar patterns to the IV, whereas the reaction seemed to be faster perhaps due to the presence of selenium. The DNA degradation was also observed by the presence of smearing appearance on the lane 5. In general, all the test compounds show DNA cleavage activity.

18

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Figure 3. DNA cleavage activities of salts and complexes in 0.7% agarose gel. Circular DNA of pDSRED of each treatment; (1) Control DNA, (2) Plasmid+Salt IV, (3) Plasmid+V, (4) Plasmid+VI, (5) Plasmid+VII was incubated at 37°C for 8 hours. The label of I, II and III are respectively referred as forms I (supercoil), II (nicked) and III (linear DNA).

Overall, the DNA cleavage activity experiments show that all compounds, including salts, have a significant nuclease activity. The current results provided possible antibacterial mode of actions or mechanism of the test compounds [23]. Interestingly, two types of DNA cleavage patterns were observed in the experiment as described above. The salt and selenium compound, VII show the same patterns of cleaving circular DNA (control) while the two silver(I) complexes, V and VI show the same cleaving patterns but different than IV and VII. The DNA cleavage pattern induced by IV and VII shows thicker DNA in form II and III. On the other hand, antibacterial results of IV and VII show that both have entirely different MIC values, salt IV is almost inactive compared to compound VII (see Table 1). This indicates that the ability of VII to cleave DNA remains faster than the salt IV. The treatments of silver(I) complexes (V and VI), changed the DNA patterns from circular (form I) to nicked (form II) and thicker DNA band in form I (supercoil). Induction of such unique cleaved DNA patterns by V and VI might be due to the presence of silver(I) ions. Mechanistic studies on silver(I) ions against Escherichia coli and Staphylococcus aureus have shown that silver(I) ions thickened and condensed the bacterial DNA at the center of the bacterial cell which was not observed in normal bacterial cells [23]. The condensed and thicken DNA in circular plasmid DNA is always referred to the supercoiled shape and easy to migrate in agarose gel due to this specific shape. Related to the increase of supercoil shape (form I) in plasmid DNA, it may answer the presence of thick DNA band in supercoil form when it was treated with V and VI. There was no difference in antibacterial activity shown by both V and VI. Both the silver(I) complexes were found better in showing 19

ACCEPTED MANUSCRIPT

antibacterial activity than VII. Although VII is able to degrades the DNA faster than both the silver(I) complexes, the antibacterial activity was found to be comparatively lower. In other view, the condensed DNA structure and inactivation of protein are few other mechanisms exhibited by silver(I) ions to inhibit the bacterial cell survival and replication [24]. Thus, silver(I) complexes may have more versatile and effective to inhibit bacterial growth than selenium based compounds.

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2-

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