Synthesis, crystal structures, characterization and biological studies of nitrile-functionalized silver(I) N-heterocyclic carbene complexes

Accepted Manuscript Synthesis, crystal structures, characterization and biological studies of nitrilefunctionalized silver(I) N-heterocyclic carbene complexes Rosenani A. Haque, Sze Yii Choo, Srinivasa Budagumpi, Amirul Al-Ashraf Abdullah, Mohamed B. Khadeer Ahamed, Amin M.S. Abdul Majid PII: DOI: Reference:

S0020-1693(15)00211-X http://dx.doi.org/10.1016/j.ica.2015.04.023 ICA 16516

To appear in:

Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

21 July 2014 12 April 2015 20 April 2015

Please cite this article as: R.A. Haque, S.Y. Choo, S. Budagumpi, A.A-A. Abdullah, M.B. Khadeer Ahamed, A.M.S. Abdul Majid, Synthesis, crystal structures, characterization and biological studies of nitrile-functionalized silver(I) N-heterocyclic carbene complexes, Inorganica Chimica Acta (2015), doi: http://dx.doi.org/10.1016/j.ica. 2015.04.023

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Synthesis, crystal structures, characterization and biological studies of nitrilefunctionalized silver(I) N-heterocyclic carbene complexes

Rosenani A. Haquea,*, Sze Yii Chooa, Srinivasa Budagumpib, Amirul Al-Ashraf Abdullahc, Mohamed B. Khadeer Ahamedd, Amin M.S. Abdul Majidd

a

The School of Chemical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia b

Centre for Nano and Material Sciences, Jain University, Jain Global Campus, Bangalore 562 112, Karnataka, India c

The School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia d

EMAN Research and Testing Laboratory, The School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia

* Corresponding author:

Dr. Rosenani A. Haque, PhD, The School of Chemical Sciences Universiti Sains Malaysia 11800 USM, Penang, Malaysia E-mail: [email protected] H/P: +6019 4118 262, +604 653 3578

1

Abstract

The synthesis of a series of silver(I) complexes (3a-d), [1-(2’/3’-methylbenzyl)-3(2’’/3’’-benzonitrile)benzimidazol-2-ylidenesilver(I)]PF6, of nitrile-functionalized Nheterocyclic carbenes (NHCs) based on benzimidazole core and their characterization and biological evaluations have been reported. The NHC precursors (1a-d and 2a-d) have been synthesized from successive N-alkylation of benzimidazole with appropriate substituted benzyl halides. Their corresponding bis-NHC silver(I) complexes, 3a-d, have been prepared from the reaction of the nitrile-functionalized benzimidazolium salts with silver(I) oxide in methanol/acetonitrile at mild reaction conditions in good yield (65-73%). Both, salts and complexes, were characterized by 1H and 13CNMR, FT-IR spectroscopic and elemental analysis techniques. The molecular structures of the silver(I)-NHC complexes, 3a and 3c, were elucidated by X-ray diffraction technique. Both the complexes displayed weak π-π stacking interactions between benzimidazole and benzyl benzene rings. All four silver(I) complexes exhibited potent antimicrobial activity against a Gram negative (E. coli) and Gram positive (S. aereus and B. subtilis) bacteria. Also the IC50 values of both, salts (2a-d) and complexes (3a-d) have been determined by an MTT-based assay method against the human colon cancer cell line, HCT 116. Complexes 3a-d displayed three- to five-fold increase in the anticancer activity with the IC50 values 18.8±1.3, 20.6±1.5, 19.2±1.0, 14.9±0.8 µM compared to the anticancer potential of salts 2a-d (64.3±2.5, 58.3±4.5, 83.9±1.5, 72.4±1.0 µM), respectively.

Key words: π-π stacking interaction; Antibacterial study; Anticancer agent; N-heterocyclic carbene; HCT 116; X-ray diffraction.

2

1. Introduction In the last years, there have been major advances in the design and syntheses of Nheterocyclic carbene (NHC)-based transition metal complexes to access a library of compounds suitable as catalysts [1] for various transformation reactions as well as drugs for dangerous diseases such as cancer [2]. Especially, late transition metal chemistry of functionalized NHC ligands has attracted tremendous interest which offers additional opportunities for the design and development of complexes having hybrid ligands containing at least one NHC ligand [3]. This type of stabilizing, spectator chelating hybrid NHC ligands result from the combination of a NHC moiety and an additional donor functional group. The formation of a chelate constructed by coordination of the NHC and extra donor moiety reduces the tendency of the rapid ligand dissociation, which brings about additional stability to the complex [4]. This enables the functionalized-NHC ligands and their complexes as some of the most popular scaffolds in both, catalysis and medicinal organometallic chemistry. During the last decade, silver(I)-NHC complexes gained considerable attention as novel potential anticancer and antibacterial agents. A great number of review articles [5] have been devoted for the design and synthesis of silver(I)-NHC complexes active as antibacterial and anticancer agents since these small molecules have been identified as suitable control ligands in bioorganometallic chemistry. These small molecules can show high specificity toward their biological targets followed by treating the infected site. However, the design concepts for such silver(I)-NHC based drugs are still in the beginning stage; mainly because of lack of understanding in their 3-dimensional structure activity relationships. Although it was demonstrated that biologically relevant silver(I) and other group XI metal-NHC complexes could easily be synthesized, isolated and characterized [6], it seems that their structure activity relationships have been largely ignored until recently. This is because of the two main reasons; firstly, the difficulty in delivery of these candidates at required sites and secondly, uncontrolled dissociation of the molecule to release silver(I) ions randomly. These questions can be answered by exploring the structures of silver(I)NHC complexes developed for antibacterial and/or anticancer therapy and their stability in 3

the biological fluids. On the other hand, Ott et al reported [7] a series of linear mono- and bis-NHC gold(I) complexes displayed potent cytotoxic activity against MCF-7 and HT29 evaluated by bioassays. It is concluded that the complexes are able to behave as strong inhibitors of the linear synthetic dodecapeptide which is a structural mimic of the C-terminal portion of enzyme thioredoxin reductase. Among the biologically relevant silver(I)-NHC based complexes, few of them, namely

1-methyl-3-(p-cyanobenzyl)-benzimidazol-2-ylidenesilver

acetate

[8],

methyl

caffeine-derived silver acetate [9] and their structural analogues have been studied for their antibacterial and anticancer studies. Latter complex displayed remarkable activity in in vitro and in vivo studies against tobramycin-resistant bacteria Burkholderia dolosa, named as SCC1 and trademarked as Silvamist®. From our group, a series of publications highlighting the antibacterial and anticancer potential of bis-NHC silver(I) and palladium(II) complexes against several pathogens have been reported [10]. Surprisingly, silver(I) complexes with functionalized NHC ligands displayed promising anticancer potentials against human derived colon cancer cells [11]. In continuation of our study on nitrile-functionalized NHC silver(I) complexes, herein we report a series of nitrile-functionalized benzimidazolium salts and their respective silver(I)-NHC complexes, their syntheses, crystal structures, characterizations and in vitro antibacterial and anticancer evaluations. 2. Experimental 2.1. General considerations All solvents used were of reagent grade and were used without further purification. o(bromomethyl)toluene, m-(bromomethyl)toluene, 2-cyanobenzyl bromide, 3-cyanobenzyl bromide, benzimidazole, potassium hydroxide and silver(I)-oxide were purchased from Sigma-Aldrich and were used without further purification. 1-(2-methylbenzyl)-1Hbenzimidazole and 1-(3-methylbenzyl)-1H-benzimidazole were synthesized according to literature method [12] with slight modifications. 1H and 13C NMR spectra were obtained at room temperature on a Bruker 300 and 500 MHz Ascend spectrometer from solutions in DMSO-d6 or CD3CN using TMS as an internal reference. The FT-IR spectra of the compounds were recorded in potassium bromide disks using a Perkin Elmer 2000 system 4

spectrometer in the range 4000 to 400 cm-1. The melting points were assessed by using a Stuart Scientific SMP-1 (UK) instrument. All reported compounds were analyzed for Carbon, Hydrogen and Nitrogen by the CHN microanalysis using a Perkin Elmer 2400 LS Series CHN/S analyser. For X-ray single crystal structure analysis, Bruker SMART APEX22009 CCD area-detector diffractometer used for the data collection. SAINT Bruker-2009 used for the cell refinement, SAINT used for the data reduction and SHELXTL (Sheldrick, 2008) used to solve the structure. Calculations, structure refinement, molecular graphics and the material for publication were achieved using the SHELTXL and PLATON (Spek, 2009) software packages. Structure were solved by direct methods and refined by full-matrix leastsquares against F2. Biological evaluations, antimicrobial and anticancer activity tests, were carried out following the reported methods [10]. Solubility studies were carried out using a Hitachi U-2000 Spectrophotometer, Hitachi Ltd, Tokyo, Japan, and turbidity stuidies were measured using a Lovibond® turbidity meter. 2.2. Synthesis of benzimidazolium salts 2.2.1.

Synthesis

of

2-((3-(2-methylbenzyl)-2,3-dihydro-1H-benzimidazol-1-yl)methyl)

benzonitrile hexafluorophosphate (2a) To a stirring solution of 2-bromomethylbenzonitrile (0.196 g, 1 mmol) in 1,4-dioxane (10 mL) was added a solution of 1-(2-methylbenzyl)-1H-benzimidazole (0.222 g, 1 mmol) in 1,4-dioxane (10 mL). The reaction mixture was refluxed for 2 days, after this time, boiling reaction mixture yielded white solid of benzimidazolium bromide salt 1a. The solid so obtained was filtered and washed with fresh 1,4-dioxane and diethyl ether. Later, the bromide salt was directly converted into its hexafluorophosphate counterpart by salt metathesis reaction. To a stirring methanolic solution of 1a was added dropwise a solution of KPF6 (0.276 g, 1.5 mmol) in methanol (20 mL) and stirred for 4 h to precipitate a white solid which is designated as 2a. So formed precipitate was filtered, washed with distilled water (2 x 3 mL) to remove unreacted KPF6, then washed with diethyl ether and dried at room temperature. Yield: 89.5%. M.P.: 187.9 °C. 1H NMR (500 MHz, d6-DMSO): δ 2.36 (3H, s, CH3-Ar), 5.82 (2H, s, CH2-methylbenzyl), 6.05 (2H, s, CH2-benzonitrile), 7.21 (2H, m, ArH), 7.33 (2H, m, Ar-H), 7.45 (1H, d, J = 8.0 Hz, Ar-H), 7.63 (1H, t, J = 8.0 Hz, Ar-H), 7.69 5

(1H, m, Ar-H), 7.76 (1H, t, J = 7.5 Hz, Ar-H,), 7.85 (2H, d, J = 8.0 Hz, Ar-H), 7.88 (1H, m, Ar-H), 7.99 (1H, m, Ar-H), 9.87 (1H, s, benzimidazolium 2-CH). 13C{1H} NMR (125 MHz, d6-DMSO): δ 18.7 (CH3-Ar), 48.6 (CH2-methylbenzyl), 49.2 (CH2-benzonitrile), 116.9 (nitrile-C≡N), 110.7, 113.8, 114.2, 126.5, 127.00, 127.2, 128.5, 128.8, 129.5, 130.8, 131.4, 133.8, 134.0, 136.7, 137.0 (Ar-C), 143.5 (benzimidazolium C2’). FTIR (KBr disk) cm-1: 2953, ~3030 ν(C-H, aliphatic and aromatic), 2229 ν(C≡N, benzonitrile), 1608 ν(C=N, benzimidazole), 1068 ν(C-N, benzimidazole). Anal. Calcd for C23H20N3F6P1: C 57.2, H 4.2, N 8.7. Found: C 57.7, H 4.6, N 8.2. 2.2.2.

Synthesis

of

2-((3-(3-methylbenzyl)-2,3-dihydro-1H-benzimidazol-1-yl)methyl)

benzonitrile hexafluorophosphate (2b) This compound was prepared in a manner analogous to that for salt 2a, only with 1(3-methylbenzyl)-1H-benzimidazole

instead

of

1-(2-methylbenzyl)-1H-benzimidazole.

Yield: 91.8%. M.P.: 186.5 °C. 1H NMR (500 MHz, d6-DMSO): δ 2.31 (3H, s, CH3-Ar), 5.77 (2H, s, CH2-methylbenzyl), 6.06 (2H, s, CH2-benzonitrile), 7.21 (1H, m, Ar-H), 7.32 (3H, m, Ar-H), 7.53 (1H, d, J = 7.5 Hz, Ar-H), 7.66 (3H, m, Ar-H), 7.78 (1H, m, Ar-H), 7.87 (1H, m, Ar-H), 7.99 (2H, m, Ar-H), 9.97 (1H, s, benzimidazolium 2-CH). 13C{1H} NMR (125 MHz, d6-DMSO): δ 21.7 (CH3-Ar), 49.7 (CH2-methylbenzyl), 51.2 (CH2-benzonitrile), 117.7 (nitrile-C≡N), 112.0, 114.6, 115.0, 126.2, 128.0, 129.6, 130.2, 130.6, 132.0, 132.3, 134.3, 134.8, 137.5, 139.3 (Ar-C), 144.3 (benzimidazolium C2’). FTIR (KBr disk) cm-1: ~2950, 3035 ν(C-H, aliphatic and aromatic), 2228 ν(C≡N, benzonitrile), 1611 ν(C=N, benzimidazole), ~1115 ν(C-N, benzimidazole). Anal. Calcd for C23H20N3F6P1: C 57.2, H 4.2, N 8.7. Found: C 57.1, H 4.3, N 8.3. 2.2.3.

Synthesis

of

3-((3-(2-methylbenzyl)-2,3-dihydro-1H-benzimidazol-1-yl)methyl)

benzonitrile hexafluorophosphate (2c) This compound was prepared in a manner analogous to that for salt 2a, only with 3bromomethylbenzonitrile instead of 2-bromomethylbenzonitrile. Yield: 79.7%. M.P.: 188.8 °C. 1H NMR (500 MHz, d6-DMSO): δ 2.37 (3H, s, CH3-Ar), 5.79 (2H, s, CH2methylbenzyl), 5.84 (2H, s, CH2-benzonitrile), 7.25 (2H, m, Ar-H), 7.34 (2H, m, Ar-H), 7.66 (3H, m, Ar-H), 7.87 (2H, d, J = 8.0 Hz, Ar-H), 7.96 (2H, m, Ar-H), 8.03 (1H, s, Ar-H), 9.81 6

(1H, s, benzimidazolium 2-CH).

13

C{1H} NMR (125 MHz, d6-DMSO): δ 18.7 (CH3-Ar),

48.6 (CH2-methylbenzyl), 49.1 (CH2-benzonitrile), 118.4 (nitrile-C≡N), 111.9, 113.9, 126.5, 126.9, 128.5, 128.9, 130.2, 130.8, 131.0, 131.5, 131.9, 132.5, 133.1, 135.5, 136.7 (Ar-C), 143.1 (benzimidazolium C2’). FTIR (KBr disk) cm-1: 2970, 3037 ν(C-H, aliphatic and aromatic), 2230 ν(C≡N, benzonitrile), 1610 ν(C=N, benzimidazole), ~1100 ν(C-N, benzimidazole). Anal. Calcd for C23H20N3F6P1: C 57.2, H 4.2, N 8.7. Found: C 57.1, H 4.8, N 8.8. 2.2.4.

Synthesis

of

3-((3-(3-methylbenzyl)-2,3-dihydro-1H-benzimidazol-1-yl)methyl)

benzonitrile hexafluorophosphate (2d) This compound was prepared in a manner analogous to that for salt 2a, only with 3bromomethylbenzonitrile

and

1-(3-methylbenzyl)-1H-benzimidazole

instead

of

2-

bromomethylbenzonitrile and 1-(2-methylbenzyl)-1H-benzimidazole, respectively. Yield: 88.3%. M.P.: 189.1 °C. 1H NMR (500 MHz, d6-DMSO): δ 2.32 (3H, s, CH3-Ar), 5.74 (2H, s, CH2-methylbenzyl), 5.86 (2H, s, CH2-benzonitrile), 7.22 (1H, t, J = 7.5 Hz, Ar-H), 7.33 (2H, d, J = 7.5 Hz, Ar-H), 7.37 (1H, s, Ar-H), 7.67 (3H, m, Ar-H), 7.89 (2H, t, J = 6.5 Hz, Ar-H), 7.97 (2H, m, Ar-H), 8.04 (1H, s, Ar-H), 9.99 (1H, s, benzimidazolium 2-CH). 13C{1H} NMR (125 MHz, d6-DMSO): δ 20.9 (CH3-Ar), 49.1 (CH2-methylbenzyl), 50.1 (CH2-benzonitrile), 118.4 (nitrile-C≡N), 111.9, 113.8, 125.4, 126.9, 128.8, 129.4, 130.2, 131.0, 131.9, 132.5, 133.2, 133.7, 135.5, 138.4 (Ar-C), 143.0 (benzimidazolium C2’). FTIR (KBr disk) cm-1: 2922, 3056 ν(C-H, aliphatic and aromatic), 2236 ν(C≡N, benzonitrile), 1610 ν(C=N, benzimidazole), 1110 ν(C-N, benzimidazole). Anal. Calcd for C23H20N3F6P1: C 57.2, H 4.2, N 8.7. Found: C 57.9, H 4.5, N 8.5. 2.3. Synthesis of silver(I)-NHC complexes 2.3.1.

Synthesis

of

bis-{2-((3-(2-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-

yl)methyl)benzonitrile}silver(I) hexafluorophosphate (3a) In the first approach, a suspension of silver oxide (0.116 g, 0.5 mmol) and benzimidazolium bromide salt 1a (0.418 g, 1 mmol) in methanol (20 mL) were stirred in a round-bottom flask in the absence of light at room temperature for 2 days. After this time, the

7

mixture was filtered through a pad of celite to remove the unreacted silver oxide. To the resulting colourless solution was added dropwise a solution of KPF6 (0.276 g, 1.5 mmol) in methanol (10 mL), with further stirring for 4 h. The obtained white solid, 3a, was filtered and washed with distilled water to remove unreacted KPF6 and was left to dry at room temperature. So formed complex was recrystallized from acetonitrile-diethyl ether mixture to afford complex 3a in pure form. In the second approach, a suspension of silver oxide (0.116 g, 0.5 mmol) and benzimidazolium hexafluorophosphate salt 2a (0.483 g, 1 mmol) in methanol (20 mL) were stirred in a round-bottom flask in the absence of light at room temperature for 2 days. Later the same work up was done as mentioned in first approach. Yield of the complexes obtained from first approach found a bit better than the second approach. Yield: 68.6%. M.P.: 191.3 °C. 1H NMR (500 MHz, d6-DMSO): δ 2.18 (6H, s, 2 x CH3-Ar), 5.57 (4H, s, 2 x CH2methylbenzyl), 5.77 (4H, s, 2 x CH2-benzonitrile), 6.78 (2H, d, J = 7.5 Hz, Ar-H), 7.00 (2H, t, J = 7.5 Hz, Ar-H), 7.12 (6H, m, Ar-H), 7.44 (6H, m, Ar-H), 7.56 (2H, t, J = 7.25 Hz, ArH), 7.67 (4H, m, Ar-H), 7.83 (2H, d, J = 7.5 Hz, Ar-H).

13

C{1H} NMR (125 MHz, d6-

DMSO): δ 18.9 (CH3-Ar), 50.0 (CH2-methylbenzyl), 50.4 (CH2-benzonitrile), 117.1 (nitrileC≡N), 110.4, 112.3, 112.5, 124.5, 126.1, 127.0, 127.9, 128.0, 128.9, 130.5, 133.4, 133.6, 133.9, 135.9, 139.2 (Ar-C), 190.2 and 191.8 [d, 1J (C-109Ag)= 213 Hz] and [d, 1J (C-107Ag)= 183 Hz]

(benzimidazol-2-ylidene C2-Ag). FTIR (KBr disk) cm-1: 2941, 3021 ν(C-H,

aliphatic and aromatic), 2224 ν(C≡N, benzonitrile), 1587 ν(C=N, benzimidazole), 1105 ν(CN, benzimidazole). Anal. Calcd for C46H38Ag1N6F6P1: C 59.6, H 4.1, N 9.1. Found: C 59.4, H 4.2, N 9.4. 2.3.2.

Synthesis

of

bis-{2-((3-(3-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-

yl)methyl)benzonitrile}silver(I) hexafluorophosphate (3b) This compound was prepared in a manner analogous to that for complex 3a, only with either 2b or 1b instead of 2a or 1a, respectively. Yield: 73.9%. M.P.: 190.7 °C. 1H NMR (500 MHz, CD3CN): δ 2.13 (6H, s, 2 x CH3-Ar), 5.66 (4H, s, 2 x CH2-methylbenzyl), 5.92 (4H, s, 2 x CH2-benzonitrile), 7.04 (2H, d, J = 7.0 Hz, Ar-H), 7.21 (2H, d, J = 8.0 Hz, Ar-H), 7.42 (6H, m, Ar-H), 7.61 (6H, m, Ar-H), 7.73 (4H, d, J = 7.0 Hz, Ar-H), 7.83 (4H, d, J = 7.5 8

Hz, Ar-H). 13C{1H} NMR (125 MHz, CD3CN): δ 20.8 (CH3-Ar), 50.4 (CH2-methylbenzyl), 51.9 (CH2-benzonitrile), 117.2 (nitrile-C≡N), 110.5, 112.3, 112.6, 124.3, 124.5, 127.7, 127.9, 128.2, 128.6, 128.9, 133.3, 133.6, 133.8, 135.9, 138.0, 139.2 (Ar-C), 190.0 and 191.6 [d, 1J (C-109Ag)= 213 Hz] and [d, 1J (C-107Ag)= 184 Hz] (benzimidazol-2-ylidene C2-Ag). FTIR (KBr disk) cm-1: 2940, ~3025 ν(C-H, aliphatic and aromatic), 2225 ν(C≡N, benzonitrile), 1590

ν(C=N,

benzimidazole),

1014

ν(C-N,

benzimidazole).

Anal.

Calcd

for

C46H38Ag1N6F6P1: C 59.6, H 4.1, N 9.1. Found: C 59.1, H 4.3, N 9.6. 2.3.3.

Synthesis

of

bis-{3-((3-(2-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-

yl)methyl)benzonitrile}silver(I) hexafluorophosphate (3c) This compound was prepared in a manner analogous to that for complex 3a, only with either 2c or 1c instead of 2a or 1a, respectively. Yield: 65.4%. M.P.: 205.6 °C. 1H NMR (500 MHz, d6-DMSO): δ 2.16 (6H, s, 2 x CH3-Ar), 5.64 (4H, s, 2 x CH2-methylbenzyl), 5.97 (4H, s, 2 x CH2-benzonitrile), 6.74 (2H, d, J = 7.0 Hz, Ar-H), 6.88 (2H, d, J = 7.0 Hz, Ar-H), 7.11 (4H, m, Ar-H), 7.41 (4H, m, Ar-H), 7.47 (4H, m, Ar-H), 7.66 (4H, m, Ar-H), 7.70 (4H, m, Ar-H).

13

C{1H} NMR (125 MHz, d6-DMSO): δ 17.8 (CH3-Ar), 48.8 (CH2-methylbenzyl),

49.9 (CH2-benzonitrile), 117.2 (nitrile-C≡N), 110.5, 111.3, 123.2, 125.0, 125.4, 125.6, 126.7, 127.5, 128.8, 129.4, 130.7, 131.9, 132.1, 132.7, 133.1, 133.6, 134.7, 135.4, 136.6 (Ar-C), 166.2 (benzimidazol-2-ylidene C2-Ag). FTIR (KBr disk) cm-1: 2945, 3047 ν(C-H, aliphatic and aromatic), 2230 ν(C≡N, benzonitrile), ~1600 ν(C=N, benzimidazole), 1016 ν(C-N, benzimidazole). Anal. Calcd for C46H38Ag1N6F6P1: C 59.6, H 4.1, N 9.1. Found: C 59.9, H 4.3, N 9.2. 2.3.4.

Synthesis

of

bis-{3-((3-(3-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-

yl)methyl)benzonitrile}silver(I) hexafluorophosphate (3d) This compound was prepared in a manner analogous to that for complex 3a, only with either 2d or 1d instead of 2a or 1a, respectively. Yield: 68.1%. M.P.: 194.3 °C. 1H NMR (300 MHz, d6-DMSO): δ 2.15 (6H, s, 2 x CH3-Ar), 5.71 (4H, s, 2 x CH2-methylbenzyl), 5.80 (4H, s, 2 x CH2-benzonitrile), 7.07 (4H, m, Ar-H), 7.12 (4H, m, Ar-H), 7.41 (3H, m, Ar-H), 7.44 (4H, m, Ar-H), 7.57 (2H, d, J = 8.0 Hz, Ar-H), 7.71 (4H, m, Ar-H), 7.76 (3H, m, Ar-H). 13

C{1H} NMR (125 MHz, d6-DMSO): δ 19.8 (CH3-Ar), 48.1 (CH2-methylbenzyl), 49.9 9

(CH2-benzonitrile), 117.9 (nitrile-C≡N), 110.9, 111.4, 112.8, 115.9, 123.0, 125.7, 127.8, 127.9, 128.7, 128.8, 129.9, 130.1, 130.4, 131.9, 132.0, 132.5, 133.2, 135.7, 136.8, 137.9 (ArC), 189.0 (benzimidazol-2-ylidene C2-Ag). FTIR (KBr disk) cm-1: 2923, ~3045 ν(C-H, aliphatic and aromatic), 2232 ν(C≡N, benzonitrile), 1604 ν(C=N, benzimidazole), 1015 ν(CN, benzimidazole). Anal. Calcd for C46H38Ag1N6F6P1: C 59.6, H 4.1, N 9.1. Found: C 59.9, H 4.3, N 9.6. 2.4. Biological Activity 2.4.1. Solubility studies 2.4.1.1. Aqueous solubility test: Azolium salts and NHC complexes were separately added in excess to 5 mL of distilled water in volumetric flasks. The flasks were vortex-mixed and agitated at 120 RPM in a water bath maintained at 25±2 °C for 72 h. Samples were filtered through a 0.45 µm nylon membrane filter syringe. The amount of compounds dissolved was measured at 260 nm spectrophotometrically using a calibration curve obtained from the respective compound dissolved in acetonitrile. Analysis of solubility for each compound was conducted in triplicate and the data is presented as mean ± SD of mg/mL. 2.4.1.2. DMSO solubility test: Test solutions (strength of 0.5 mM) of all the compounds were prepared with different percentages of DMSO in distilled water (100, 50, 25, 12, 6, 3, 1.5, 0.75, 0.3, 0.1, 0.05, 0.02, 0.01 and 0.005%). Turbidity of all the samples was measured by portable turbidity meter. Analysis for each sample was repeated three times and the data is presented as mean ± SD of Nephelometric Turbidity Units (NTU). 2.4.2. Determination of antimicrobial activity The antimicrobial potentials of the benzimidazolium salts 2 and their Ag-NHC complexes 3 were determined using the agar dilution method. Minimum inhibitory concentration (MIC) was determined against E. coli and B. subtilis using ampicillin as a standard drug by disc diffusion method. The stock solutions of all complexes were prepared using DMSO as a solvent at four different concentrations (3-6 µM). For the disc diffusion method, a loop of the bacterial strain was inoculated into the nutrient broth and was incubated for 16 h at 37 °C. Then, 50 mL of the suspension was applied uniformly on the surface of the nutrient agar 10

plate before placing the antimicrobial assay discs on the plate (four per plate). Test samples of different concentrations of 5 µL were loaded on the discs, and the plates were incubated at 37 °C for 24 h. Later, the average zone of inhibition was measured with a ruler with a resolution of up to 1 mm. The MIC of the compounds was determined based on the lowest concentration of the compound, which inhibited the growth of the bacterial strain using the broth dilution method. 2.4.3. Determination of anticancer activity 2.4.3.1. Cell culture. Initially, HCT 116 cells were allowed to grow under optimal incubator conditions. Cells that had reached a confluency of 70-80% were chosen for cell plating purposes. Old medium was aspirated out of the plate. Later, cells were washed using sterile phosphate-buffered saline (PBS) (pH 7.4), two to three times. PBS was completely discarded after washing. Following this, trypsin was added and distributed evenly on to cell surfaces. Cells were incubated at 37 °C in 5% CO2 for 1 min. Then, the flasks containing the cells were gently tapped to aid cell segregation and observed under inverted microscope (if cell segregation was not satisfactory, cells were incubated for a further minute). Trypsin activity was inhibited by adding 5 mL fresh complete media (10% FBS (Fetal Bovine Serum)). Cells were counted and diluted to obtain a final concentration of 2.5 x 105 cells mL-1, and inoculated into wells (100 µL cells per well). Finally, plates containing the cells were incubated at 37 °C with an internal atmosphere of 5% CO2. 2.4.3.2. Test sample preparation. The compounds were dissolved in DMSO to obtain 10 mM stock solutions and stored at 4 °C. For treatment, the compounds were serially diluted using cultured media to obtain various concentrations (3, 6, 12, 25, 50 and 100 µM) of test samples. As DMSO is used as a vehicle to dissolve the compounds, 0.1% DMSO was used as negative control. 2.4.3.3. MTT assay. Cancer cells (100 µL cells per well, 1.5 x 105 cells mL-1) were inoculated into wells of a microtiter plate. The plate was incubated in a CO2 incubator overnight in order to allow cell attachment. Test substance was diluted with media to the desired concentrations from the stock. Various concentrations (3 to 100 µM) of test substance was added to each well (100 µL/well) containing the cells. The plates were incubated at 37 °C 11

with an internal atmosphere of 5% CO2 for 72 h. After this treatment period, 20 µL (5 mg/mL) MTT reagent was added to each well and incubated again for 4 h. After this incubation period, 50 µL MTT lysis solution (DMSO) was added to the wells. The plates were incubated for a further 5 min in a CO2 incubator. Finally, the plates were read at 570 and 620 nm wavelengths using a standard enzyme-linked immunosorbent assay microplate reader. Data were recorded and analyzed for assessment of the effects of test substance on cell viability and growth inhibition. The percentage of growth inhibition was calculated from the optical density (OD) obtained from MTT assay, i.e. hundredth multiple of subtracted OD value of control and surviving cells over OD of control cells. 3. Results and discussion For a detailed screening of the influence of electronic and steric factors originating from NHC backbones on antibacterial and anticancer studies, a series of nitrilefunctionalized benzimidazolium salts and their bis-NHC silver(I) complexes having the general formula [NHC(CN)-Ag-NHC(CN)]+ PF6- was synthesized and successfully characterized. Although, the similar hybrid NHC complexes used for anticancer studies have some consequences in the improvement of their anticancer potential [13], we thought that the presence of the nitrile functionality at 2- or 3- position of the benzonitrile group and a methyl substitution at 2- or 3- position of the benzyl group may also play a significant role in the enhancement of the biological activities. Also, due to its excellent donor properties, nitrile module is the functional group of choice, thus, its incorporation into benzimidazole-based NHC backbone may add interesting antibacterial and anticancer applications to their derived silver(I)-NHC complexes. The data obtained from the CHN microanalyses are in agreement with the plausible structures of the benzimidazolium salts 1a-d and 2a-d and their respective silver(I)-NHC complexes 3a-d. 3.1. Syntheses of benzimidazolium salts 1 and 2 and silver(I)-NHC complexes 3 Reported benzimidazolium salts were salts 1 and 2 were prepared by the similar procedures based on the method used by Dias and Jin for the preparation of azolium salts [12]. The synthesis of 1-(2’/3’-methyl)benzylbenzimidazoles was achieved by N-alkylation reaction of 2/3-methylbenzyl bromide with 1H-benzimidazole mediated by DMSO in the 12

presence of a mild base such as potassium hydroxide acting as a proton transfer agent. The desired benzimidazolium bromides, 1a-d, were obtained by the reaction of 1-(2’methyl)benzylbenzimidazole or 1-(3’-methyl)benzylbenzimidazole with 2-bromomethyl benzonitrile or 3-bromomethyl benzonitrile, respectively in refluxing 1,4-dioxane for 2 days. Bromide salts 1a-d were treated with potassium hexafluorophosphate in methanol and stirred for 4 h to afford nitrile-functionalized benzimidazolium hexafluorophosphate salts 1a-d, respectively in good yield. Reactions involved in the preparation of benzimidazolium salts 1a-d and 2a-d are depicted in Scheme 1. Both, bromide and hexafluorophosphate, salts were recrystalized from methanol/acetonitrile and diethyl ether mixture, respectively. We used two synthetic strategies for the preparation of desired silver(I)- complexes of nitrile-functionalized NHC ligands 3a-d. The first one, depicted in Scheme 2, combination of a benzimidazolium bromide salt 1 in methanol with silver(I) oxide in a 1:0.5 molar ratio added to approximate classic silver(I) metallation conditions led in each case to complete consumption of the benzimidazolium bromide salt and allowed formation of new bis-NHC silver(I) bromide complexes in methanol. A solution of potassium hexafluorophosphate in methanol was added to the silver(I) bromide complex solution at room temperature over a period of 1 h and allowed to stir for further 4 h resulting in the formation of the desired bisNHC silver(I) hexafluorophosphate complexes 3. In the second approach, the benzimidazolium bromide salts 1 in methanol were treated with a methanolic solution of potassium

hexafluorophosphate

at

room

temperature

yielded

the

corresponding

benzimidazolium hexafluorophosphate salts 2. Subsequent reaction of silver(I) oxide with salts 2 in acetonitrile at approximate classic silver(I) metallation conditions afforded the targeted silver(I)-NHC complexes 3. The final products obtained from both the methods were recrystallized by slow addition of ether to the acetonitrile solution of 3 to give white solids in moderate to high yield (65-73%). All the reported compounds are sparingly soluble in water, insoluble in common organic solvents such as benzene and hexane, while completely soluble in DMSO, DMF and acetonitrile. For bioassay, test solutions of the compounds were prepared in DMSO. 3.2. Spectroscopic characterizations 13

With the aid of NMR spectroscopic handles of both the benzimidazolium salts 1 and 2 forms in hand the respective silver(I)-NHC complexes 3 in CD3CN/d6-DMSO was investigated. In the case of the hexafluorophosphate salts 2a, 2b and 2c, 2d the 1H NMR spectra were quasi-identical with those resonance signals shown by the bromide salts 1a, 1b and 1c, 1d, respectively. Analysis of the 1H NMR spectra of these salts revealed the presence of a characteristic C2 proton resonance in the range δ 9.87-9.99, indicating the formation of desired benzimidazolium salts [14]. Spectra also evidenced the peaks in the range δ 7.218.04, δ 5.84-6.04, δ 5.74-5.89 and δ 2.31-2.37, attributed to the resonance of aromatic, benzonitrilic, benzylic and methyl protons, respectively. Similarly, in the case of 13C NMR analysis, spectra of compounds having same substitutions at 1- and 3- position of the heterocyclic ring, but different counterions evidenced the presence of quasi-identical resonance peaks. Spectra revealed the presence of a characteristic C2 carbon resonance in the downfield region in the range δ 143.0-144.3, indicating the successful formation of salts. Finally, a set of aromatic, nitrilic and aliphatic carbon resonances were appeared in the range δ 113-137, δ 110-111.5 and δ 18.7-51.2, respectively. All these spectral observations are consistent with the literature [15]. Analysis of the complex 1H NMR spectra recorded in CD3CN/d6-DMSO revealed the complete disappearance of signal corresponding to C2 proton of the respective salt. This observation confirmed the formation of the desired bis-NHC complexes 3 via C2 proton abstraction. However, all other proton resonances corresponding to the respective salt appeared in the complex spectra with negligible differences in the peak positions. On the other hand, in the case of 13C NMR spectra of complexes 3c and 3d, the resonance signals due to the silver(I) metalated carbene carbon appeared at δ 166.2 and 189.0 indicating the formation of Ag-C bonds. Interestingly, the complexes 3a and 3b displayed two doublets centered at δ 190.2, 191.8 and δ 190.0, 191.6 attributed to the resonance of metalated carbene carbons bound to

107

Ag and

109

Ag with the coupling constant of 200 Hz in both the cases.

The presence of carbene 13C-107/109Ag coupling is feasible for silver-NHC complexes having non-coordinating counterions, and has been attributed to the non-labile nature of carbene CAg bond. Conversely, this phenomenon is not found in the cases of 3c and 3d complexes, indicating that the corresponding C-Ag bonds are labile in nature. These observations are in 14

line with the similar silver(I)-NHC complexes previously reported by us [16] and many others [17]. Apart from this major observation, spectra evidenced almost no/negligible changes in the resonance of other carbon nuclei compared to their respective salts. Furthermore, the identity of the new silver(I)-NHC complexes was confirmed as predicted on the basis of a comparison of the 1H and

13

C NMR spectral resonances with independently

prepared complexes. Considering that the present work involved the nitrile-functionalities, FTIR spectroscopic characterization was followed to obtain the insights on the ligational behavior of nitrile group or the possibility of a weak interaction with the metal centre. FTIR spectra of silver(I)-NHC complexes 3 in comparison with benzimidazolium salts 1 and 2 were measured using KBr disc method over 4000-400 cm-1 range. Salt spectra displayed a characteristic medium intensity band at around 2230 cm-1 attributed to the stretching vibrations of C≡N module [18]. This band remains unaltered in the complex spectra, indicating its noninvolvement in the coordination. This could be attributed to the stronger ligation effect offered by the carbene carbon than nitrile nitrogen atom. Apart from this, salt spectra revealed the presence of medium intensity strong bands at around 1610 and 1110 cm1

, ascribable to the stretching vibrations of C=N and C-N modules of heterocyclic ring

system. These bands, however, shifted to lower energy region in the complex spectra indicating the coordination of the carbene carbon to silver centre. Finally, both the spectra evidenced the presence of aliphatic and aromatic C-H stretching vibrational bands at around 2940 and 3140 cm-1, respectively. These IR spectral observations are in line with the literature [19]. 3.3. Single crystal X-ray diffraction studies The molecular structure of silver(I)-NHC complexes 3a and 3c were unambiguously confirmed by single crystal X-ray diffraction studies. The crystal data and structure refinement details for complexes 3a and 3c are tabulated in Table 1. Single crystals suitable for X-ray diffraction analysis were grown from slow diffusion of diethyl ether into an acetonitrile solution of the complex at room temperature. Attempts to crystallize other complexes led to the formation of either multiple crystals or amorphous solids, which did not 15

give single crystals suitable for X-ray diffraction studies. As anticipated from the spectral analysis, the benzimidazol-2-ylidene carbene carbon is coordinated to the silver(I) centre in both the cases. Complex 3a

crystallizes in

the monoclinic space group

P2

with

two

crystallographically independent structural units (unit A and B) having one half-molecules of both the complex cations and two half-molecules of the hexafluorophosphate anions in the asymmetric unit lies about a twofold symmetry axis. Both the crystallographically independent structural units, A and B, are well ordered structures having no co-crystalized solvent or water molecules. Perspective views of both the units of complex 3a are shown in Figures 1 and 2, respectively. Bond parameters of these two units differ by a small extent [20], and are tabulated in Table 2 and 3, respectively. X-ray diffraction analysis of units A and B of complex 3a revealed monomeric species with the silver(I) centre is in a dicoordinate environment, which is best described as distorted linear coordination by the two carbene carbon atoms of two benzimidazol-2-ylidene ligands with the coordination bond angle of 175.0(2)° for C1A-Ag1A-C1AA and 173.8(1)° for C1B-Ag1B-C1B. The internal ring angles at the carbene carbon center (N-C-N) of benzimidazole ring are 105.6(3) and 105.6(3)° for N1A-C1A-N2A and N1B-C1B-N2B, respectively are a bit wider than the typical bond angle which is around 104o [21]. The silver-carbene bond distances, Ag1A-C1A and Ag1B-C1B are found to be 2.080(3) and 2.084(4) Å which compare well with those of the similar silver(I)-NHC complexes [22]. As evidenced by FTIR analysis, nitrile nitrogen atoms directing away from the metal centre without involving in the coordination with silver(I). Particularly in this case, a weak π-π stacking interaction is observed between two benzene units of benzimidazole rings of units A and B with an interaction distance of 3.890(6) Å (centroid to centroid). In the extended crystal structure of complex 3a, the PF6 anions link the silver(I)-complex cations into three-dimensional networks by C-H---F hydrogen bonds with distances ranging from 2.382-3.101 Å [23]. Complex 3c is an elegant structure crystalizes in the monoclinic space group C2/c, in which, one half formula unit in the asymmetric unit of the structure occupied an asymmetric unit and lies on a twofold symmetry axis. Selected bond distances and angles for the 16

aforementioned complex are tabulated in Table 4. The molecular structure of complex 3c, depicted in Figure 3, reveals the expected monomeric species having distorted-linear coordination geometry around the silver(I) centre with a coordination angle of 170.3(1)° for C1-Ag1-C1A. This bond angle is somewhat less for a linear Ag complex, showing the operation of two significant C16HA…Ag and C16HB…Ag (benzonitrilic protons to silver) preagostic interactions with the bond distance of 2.861(4) [24]. These interactions perhaps, influence the deviation in coordination geometry from 180o, implicitly manifest as an preagostic interaction effect. The silver-carbene carbon bond distances for Ag1-C1 and Ag1A-C1A are the same i.e., 2.080(3) Å [25], suggesting that in solution this compound is not undergoing reversible NHC dissociation in a fashion analogous to that for 3a. Unlike complex 3a, the NHC ligands in complex 3c adopt a syn-arrangement at the silver(I) centre, probably due to the sterically demanding nature of both, the benzyl and benzonitrile, substituents. The internal ring angle at the carbene center (N-C-N) is 105.6(2)°, is in line with the similar reported complexes [26]. Interestingly, the planes defined by the NHC rings are twisted by a dihedral angle of 120.45° with respect to each other. Similarly, terminal nitrile groups displayed a bond angle of 169.6(9)° at nitrile carbon atom, which is much deviated from the linearity might be due to its involvement in the strong hydrogen bonding (2.869(6) Å) with C12H of neighboring unit. In the extended structure, the complex cations and hexafluorophosphate anions are connected via C-H---F and C-H---N hydrogen bonds (2.465-3.761 Å) along with weak π-π stacking interactions between two neighboring benzimidazole rings (centroid to centroid distance of 3.588(7) Å), forming a threedimensional array. 3.4. Antimicrobial studies The ability of NHCs to form stable transition metal complexes, especially with silver(I) makes them versatile pharmacophores, which depends on the nature of the carbene carbon and metal bond strength. The antimicrobial activity of silver(I)-NHC complexes has been reported a decade ago by Youngs et al [27] and developments in this field are still in progress. Currently, the most promising antimicrobials among all investigated silver(I) carbene complexes are derived from benzimidazole-based NHC ligands [28]. Our research 17

group recently reported a series of mono- and binuclear silver(I) complexes derived from nitrile-functionalized NHCs shown marked nuclease activities on pUC18 plasmid DNA using gel electrophoresis technique in the presence and absence of an external oxidizing agent such as H2O2 [29]. In line with this profile which is an indication that these complexes can be used as effective antimicrobial agents, the reported complexes 3 are evaluated for antimicrobial activity. To gauge the antibacterial performances of the reported benzimidazolium salts 2 and silver(I)-NHC complexes 3, the effect of these compounds at different concentrations (3-6 µM) against E. coli, S. aereus and B. subtilis bacteria was studied by disc diffusion method using 5 µL of each test compound. Results of the antimicrobial performances of salts 2 and complexes 3 at different concentrations against both the aforementioned bacteria are summarized in Table 5. All the benzimidazolium salts displayed almost no activity against both the tested bacteria. Overall, the present silver(I)-NHC systems displayed good efficiency toward E. coli bacterial strains, especially complexes 3b, 3c and 3d displayed higher activity of about 21±1 (entry 6), 23±0 (entry 7) and 22±1 (entry 8) mm diameter of zone of inhibition, respectively at 6 µM concentration, which are comparable with the activity of standard used. In the latter two entries, it is noteworthy that in addition to the nitrile substitution at 3- position of the benzonitrile unit, a methyl substitution adding steric hindrance leads to increase of activity. This observation is further supported by the decreased activity (16±0 mm diameter of zone of inhibition, entry 5) of complex 3a, which is a structural analogue of complex 3b. On the other hand, S. aereus bacterial strain found little resistant toward complexes 3a-d compared to E. coli. Complexes 3a-c displayed moderate activity of about 12-13 mm diameter of zone of inhibition, while complex 3d showed the best activity in the tested series with 15±1 mm diameter of zone of inhibition, which can be compared with the activity of the standard drug ampicillin (15±1 mm diameter of zone of inhibition) used for the bioassay. Interestingly, a dibenzyl-substituted benzimidazol-2-ylidene silver acetate complex [30] displayed lesser antibacterial activity while present benzonitrile derivatives evinced relatively better potential against the same bacterial strains. Surprisingly, against B. subtilis 18

bacterial strains, complexes 3b and 3c displayed improved activity compared to other family members with 20±1 and 17±0 mm diameter of zone of inhibition, respectively at maximum concentration tested. It has been proposed from these results that the complexes having (3’nitrile)benzyl substitution would account for the superior antibacterial performances of the complexes 3c and 3d in comparison to the analogous complexes 3a and 3b against E. coli. These activities of the test compounds against both the bacteria are comparable with the activity of standard used. Further studies into this proposed concept for the complexes are however, necessary to better appreciate the significance of the structure activity relationships responsible for relative activities of the complexes. 3.5. Solubility studies Among the compounds tested, all the azolium salts displayed more pronounced solubility in the water compared with the complexes as depicted in Table 6. However, the complexes demonstrated considerably low aqueous solubility. Therefore, in the present study DMSO was used as a solubilizer to dissolve the compounds. Table 6 depicts minimum percentage of DMSO required to solubilise the compounds. In addition, turbidity of the compounds prepared with different percentages of DMSO was measured and the data is presented in Table 6. 3.5. Anticancer studies Since pioneering works, notably by the research groups of Youngs [31], Tacke [30], Huynh [32] and Ghosh [33], on anticancer potentials of mono- and bis-NHC silver(I) complexes, this field has gathered increasing interest, especially because these compounds have filled the deficiencies of nonplatinum-based metallopharmaceuticals in this area. Therefore, a large number of synthetic pathways leading to biologically active mono-NHC silver(I) acetate as well as bis-NHC silver(I) halide complexes have now been employed, as recently highlighted in review articles [34]. Encouraged by these promising results, we examined the anticancer potential of both, benzimidazolium salts 2 and silver(I)-NHC complexes 3, against the human colon cancer (HCT 116) cell line by an MTT-based assay, in culture. Half maximal inhibitory concentration (IC50) values against the HCT 116 cell line

19

for salts 2 and complexes 3 are summarized in Table 7. All tested compounds were completely soluble in DMSO. In comparison to complexes 3a-d, the benzimidazolium salts 2a-d showed lesser activity with IC50 values of 64.3±2.5, 58.3±4.5, 83.9±1.5 and 72.4±1.0 µM, respectively (entries 1-4). It is difficult to draw a conclusion on the anticancer potential of salts, as the trend observed is irregular. The complex 3d was found to be less active (14.9±0.8 µM, entry 8) than 5-fluorouracil (standard drug, showing IC50 value of 5.2±0.3 µM, entry 9) and in general displayed higher activity as compared to the 2-nitrile/3-nitrilebenzyl substituted complexes 3a-3c (18.8±1.3, 20.6±1.5 and 19.2±1.0 µM, respectively, entries 5-7). This finding agrees well with what has been proposed in the case of antibacterial studies. The observation of higher cytotoxicity in the case of complex 3d containing (3’-nitrile)benzyl (meta-nitrile) substitutions with weak electron-withdrawing abilities (due to decreased negative inductive effect), as compared to the complexes 3a and 3b, having (2’-nitrile)benzyl (ortho-nitrile) substitutions, may be correlated to the relatively more electron-rich silver(I) center in the former. However, detailed mechanistic studies are necessary to draw an appreciate conclusion for relative cytotoxicity of the complexes. Photomicrographs (Figure 4) revealed that the complexes 3a-d caused savior damages to the normal cell morphology as compared to the control cells as well as cells treated with the salts 2a-d. The HCT 116 cells treated with complex 3d displayed marked signs of cytotoxicity caused by the complex, which affected the viable characteristic features of the cells. This is evident from its photomicrograph (J), where only debris can be seen along with few dead cells. While salts have almost similar mode of action on the HCT 116 cell line as all of them affected the cellular morphology of almost all cells. 4. Conclusions In summary, the syntheses, characterizations and biological evaluations of a series of nitrile-functionalized benzimidazolium salts and respective bis-NHC silver(I) complexes have been reported. Salts have been prepared by the successive N-alkylation method, while complexes from the in situ deprotonation of azolium salts at basic reaction conditions. All reported compounds were characterized by 1H and 20

13

C NMR, and FTIR spectral and

elemental analysis techniques. Additionally, molecular structure of complexes 3a and 3c was established using single crystal X-ray diffraction method. Our study on biological evaluations has furnished the following main results. First, a new class of compounds suitable for antimicrobial and anticancer studies has been developed. Secondly, the complexes 3c and 3d having (3’-nitrile)benzyl substitutions have been found to exhibit promising antimicrobial activity against E. coli, while the complex 3d displayed significant anticancer activity with IC50 value in low micromolar concentration. This is because of the presence of nitrile functionality at 3- position of the benzyl group which relatively increased the electron density on the metal centre compared to other series members having nitrile group at 2- position.

Acknowledgements R.A. Haque thanks Universiti Sains Malaysia (USM) for the Research University (RU) grant 1001/PKIMIA/811217. S.Y. Choo thanks USM for the RU Grant 1001/PKIMIA/836013. Appendix A. Supplementary material CCDC 1012974 and 1012975 contain the supplementary crystallographic data for complexes 3a and 3c, respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or Email: [email protected]. Crystal packing diagrams (Figures S1 and S2) of the aforementioned compounds are also available as supplementary material. Figure S3 presents the compiled dose-dependent response curves, while Figure S4 represents effects of increasing amounts of test compounds on the percentage inhibition of HCT 116 cell proliferation. References [1]

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Figure captions Scheme 1. Synthesis of benzimidazolium halide and hexafluorophosphate salts 1a-d and 2ad. Scheme 2. Synthetic route to silver(I) complexes (3a-d) of nitrile-functionalized benzimidazol-2-ylidenes. Figure 1. ORTEP view of single crystal X-ray structure of silver(I)-NHC complex 3a (unit A) with 50% displacement ellipsoids. Hydrogen atoms and counter hexafluorophosphate anion have been omitted for clarity. Figure 2. ORTEP view of single crystal X-ray structure of silver(I)-NHC complex 3a (unit B) with 50% displacement ellipsoids. Hydrogen atoms and counter hexafluorophosphate anion have been omitted for clarity. Figure 3. ORTEP view of single crystal X-ray structure of silver(I)-NHC complex 3c with 50% displacement ellipsoids. Hydrogen atoms and counter hexafluorophosphate anion have been omitted for clarity. Figure 4. Photographs of the control HCT 116 cells (A) and cells treated with 5-fluorouracil (B), benzimidazolium hexafluorophosphate salts 2a-d (C, E, G and I) and silver(I)–NHC complexes 3a-d (D, F, H and J), respectively after 72 hours of incubation.

Scheme 1. Synthesis of benzimidazolium halide and hexafluorophosphate salts 1a-d and 2ad. 25

N

N Br

N

N N

+

Dioxane

N

Reflux, 2 d

N

Br

KPF6 MeOH

N

Stir, 4h

N

PF6

R1 1a, 2a: R1= 2-CH3 1b, 2b: R1= 3-CH3

R1

1a, 1b

2a, 2b

N

N

Br

N N

R1

+

Dioxane

N

Reflux, 2 d

N

N

Br

KPF6 MeOH

N

Stir, 4h

N

PF6

R1 1c, 2c: R1= 2-CH3 1d, 2d: R1= 3-CH3

R1

R1

1c, 1d

2c, 2d

Scheme 2. Synthetic route to silver(I) complexes (3a-d) of nitrile-functionalized benzimidazol-2-ylidenes. 26

N 2 N

R1

R2

R2

Br

i) Ag2O, MeOH Stir 2 d, rt

N

ii) KPF6, MeOH Stir 4 h, rt

N

Ag+

N N

R2

Ag2O, MeCN Stir 2 d, rt

N 2 N PF6

PF6 R1 1a-d

R2

R1 1a, 2a, 3a: R1= 2-CH3, R2= 2-CN 3a-d 1b, 2b, 3b: R1= 3-CH3, R2= 2-CN 1c, 2c, 3c: R1= 2-CH3, R2= 3-CN 1d, 2d, 3d: R1= 3-CH3, R2= 3-CN

27

R1 2a-d

Figure 1. ORTEP view of single crystal X-ray structure of silver(I)-NHC complex 3a (unit A) with 50% displacement ellipsoids. Hydrogen atoms and counter hexafluorophosphate anion have been omitted for clarity.

28

Figure 2. ORTEP view of single crystal X-ray structure of silver(I)-NHC complex 3a (unit B) with 50% displacement ellipsoids. Hydrogen atoms and counter hexafluorophosphate anion have been omitted for clarity.

29

Figure 3. ORTEP view of single crystal X-ray structure of silver(I)-NHC complex 3c with 50% displacement ellipsoids. Hydrogen atoms and counter hexafluorophosphate anion have been omitted for clarity.

30

Figure 4. Photographs of the control HCT 116 cells (A) and cells treated with 5-fluorouracil (B), benzimidazolium hexafluorophosphate salts 2a-d (C, E, G and I) and silver(I)–NHC complexes 3a-d (D, F, H and J), respectively after 72 hours of incubation.

31

Table 1. Crystal data and structure refinement details for silver(I)-NHC complexes 3a and 3c .

Formula Formula weight Crystal system Space group Unit cell dimensions a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) Z Density(calcd) (g/cm3) Abs. coeff. (mm-1) F(000) Crystal size (mm) Temperature (K) Radiation (Å) θ Min, Max (°) Data set Total. Uniq. Data R (int) Nref, Npar R, wR2, S

3a C46H38AgF6N6P 927.66 Monoclinic P2

3c C46H38AgF6N6P 927.66 Monoclinic C2/c

14.1781 (3) 8.1798 (2) 18.8919 (3) 90.00 108.5310 (10) 90.00 2077.37 2 1.483 0.592 944 0.52 x 0.28 x 0.13 125 (2) Mo Kα 0.71073 2.16, 30.18 -19:20,-11:11,-26:26 12205 0.06 (3) 8193, 545 0.0893, 0.1114, 0.976

19.9481(6) 13.9681(4) 17.1364(7) 90.00 114.8200(10) 90.00 4333.8(3) 4 1.422 0.568 1888 0.865 x 0.111 x 0.074 294(2) Mo Kα 0.71073 1.841, 30.141 -28:28, -19:19, -24:23 45853 0.0380 6371, 274 0.0662, 0.1701, 1.072

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Table 2. Pertinent bond distances and angles of silver(I)-NHC complex 3a (unit A).

Bond distances (Å) Ag1A-C1A C14A-C15A C8A-C9A N1A-C8A N1A-C1A Bond angles (°)

2.080(3) 1.440(7) 1.515(6) 1.453(6) 1.353(5)

N2A-C1A N2A-C16A C16A-C17A C22A-C23A

1.360(5) 1.469(5) 1.516(6) 1.523(7)

C1A-Ag1A-C1AA C9A-C14A-C15A N1A-C8A-C9A C1A-N1A-C2A N1A-C2A-C7A

175.0(2) 119.7(4) 111.3(3) 111.0(3) 106.1(3)

N1A-C1A-N2A C1A-N2A-C7A N2A-C16A-C17A C17A-C22A-C23A

105.6(3) 111.6(3) 113.8(3) 120.4(4)

33

Table 3. Pertinent bond distances and angles of silver(I)-NHC complex 3a (unit B).

Bond distances (Å) Ag1B-C1B C14B-C15B C8B-C9B N1B-C8B N1B-C1B Bond angles (°)

2.084(4) 1.444(7) 1.513(6) 1.446(5) 1.367(5)

N2B-C1B N2B-C16B C16B-C17B C22B-C23B

1.358(4) 1.473(5) 1.516(6) 1.515(7)

C1B-Ag1B-C1B C9B-C14B-C15B N1B-C8B-C9B C1B-N1B-C2B N1B-C2B-C7B

173.8(1) 120.1(4) 111.9(3) 110.5(3) 106.5(3)

N1B-C1B-N2B C1B-N2B-C7B N2B-C16B-C17B C17B-C22B-C23B

105.6(3) 111.9(3) 112.3(3) 120.6(4)

34

Table 4. Pertinent bond distances and angles of silver(I)-NHC complex 3c.

Bond distances (Å) Ag1-C1 2.080(3) Ag1A-C1A 2.080(3) C14-C15 1.489(8) C8-C9 1.504(4) N1-C8 1.446(4) Bond angles (°)

N1-C1 N2-C1 N2-C16 C16-C17 C21-C23

1.352(3) 1.348(4) 1.462(3) 1.505(4) 1.459(6)

C1-Ag1-C1A C9-C14-C15 N1-C8-C9 C1-N1-C2 N2-C1-N1

C1-N2-C7 C2-C7-N2 N2-C16-C17 C22-C21-C23 N3-C23-C21

111.6(2) 105.8(2) 112.7(2) 120.0(4) 169.6(9)

170.3 (1) 122.2(4) 114.6(2) 110.6(2) 105.6(2)

35

Table 5. Antibacterial activity results of salts 2a-d and respective silver(I)-NHC complexesa 3a-d against E. coli, S. aereus and B. subtilis by disc diffusion methodb. Entry Compounds

1 2 3 4 5 6 7 8

2a 2b 2c 2d 3a 3b 3c 3d Ampicillin

9 10 11 12 13 14 15 16

2a 2b 2c 2d 3a 3b 3c 3d Ampicillin

9 10 11 12 13 14 15

2a 2b 2c 2d 3a 3b 3c

Bacteria

E. coli

S. aereus

Zone of inhibition (mm) 3 µM 4 µM 5 µM 6 µM 11±0 13±0 14±0 16±0 16±0 16±1 18±1 21±1 13±0 14±0 19±1 23±0 12±1 14±1 15±1 22±1 13±0 16±1 18±0 19±1 8±0 7±0 8±0 8±0 9±1

B. subtilis 8±0 12±0 9±0 36

9±0 9±0 9±1 10±1 13±0

11±0 10±1 11±0 12±1 15±1

13±1 12±1 13±0 15±1 20±1

11±0 13±1 15±1 15±0 17±1 20±1 12±1 15±0 17±0

16

8±0 16±1

3d Ampicillin

9±0 12±1 14±0 16±1 17±1 18±1

a

: Volume of the test compound used is 5 µL for each concentrations (3, 4, 5 and 6 µM). : Zone of inhibition values were determined on the basis of two independent experiments using disc diffusion method. b

Table 6. Solubility valuesa of the compounds in aqueous and DMSO solution.

Formulation n

a:

Solubility (mg/ml)

Minimum % of DMSO required to prepare 0.5 mM

Turbidity (NTU) in 1% DMSO

2a

5.14±0.004356

0.09±0.00027

<0.01

2b

7.21±0.00175

0.04±0.00014

<0.01

2c

6.36±0.00552

0.06±0.00022

<0.01

2d

2.64±0.00421

0.43±0.00046

3.8±0.02

3a

2.22± 0.00273

0.51±0.00011

4.4±0.01

3b

1.70±0.00067

0.87±0.00075

5.2±0.02

Results are presented as mean ± S.D of three separate experiments (n=3).

37

Table 7. Anticancer potential of salts 2a-d and respective silver(I)-NHC complexes 3a-d against the HCT 116 cell line expressed in terms of IC50 values.

Entry

Compound

IC50 (in µM)

1

2a

64.3±2.5

2

2b

58.3±4.5

3

2c

83.9±1.5

4

2d

72.4±1.0

5

3a

18.8±1.3

6

3b

20.6±1.5

7

3c

19.2±1.0

8

3d

14.9±0.8

9

5-fluorouracil

5.2±0.3

38

Graphical Abstract (Synopsis)

Four nitrile-functionalized imidazolium salts and corresponding silver(I)-NHC complexes have been reported. All compounds were tested for antibacterial and anticancer activity showing all complexes are promising antibacterial and anticancer agents.

39

Graphical Abstract (Pictogram)

40

Research Highlights


A series of nitrile-functionalized benzimidazolium salts are reported
Ag complexes of nitrile-functionalized NHC are prepared
Crystal structure of two complexes are reported
Salts and complexes are evaluated for antibacterial and anticancer activity
Complexes showed promising activities

41