Synthesis, characterization and antimicrobial activities of novel silver(I) complexes with coumarin substituted N-heterocyclic carbene ligands

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

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Synthesis, characterization and antimicrobial activities of novel silver(I) complexes with coumarin substituted N-heterocyclic carbene ligands _ Mert Olgun Karatasß a,⇑, Begüm Olgundeniz b, Selami Günal c, Ilknur Özdemir a, Bülent Alıcı a, b Engin Çetinkaya a

Inonu University, Faculty of Science, Department of Chemistry, 44280 Malatya, Turkey Ege University Faculty of Science, Department of Chemistry, 35100 Izmir, Turkey c Inonu University, Faculty of Medicine, Department of Microbiology, 44280 Malatya, Turkey b

a r t i c l e

i n f o

Article history: Received 20 September 2015 Revised 15 December 2015 Accepted 17 December 2015 Available online xxxx Keywords: Antibacterial Antifungal Coumarin Carbene Silver

a b s t r a c t Eight new coumarin substituted silver(I) N-heterocyclic carbene (NHC) complexes were synthesized by the interaction of the corresponding imidazolium or benzimidazolium chlorides and Ag2O in dichloromethane at room temperature. Structures of these complexes were established on the basis of elemental analysis, 1H NMR, 13C NMR, IR and mass spectroscopic techniques. The antimicrobial activities of carbene precursors and silver NHC complexes were tested against standard strains: Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and the fungi Candida albicans and Candida tropicalis. Results showed that all the compounds inhibited the growth of the all bacteria and fungi strains and some complexes performed good activities against different microorganisms. Among all the compounds, the most lipophilic complex bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)3-(naphthalene-2-ylmethyl)benzimidazol-2-ylidene]silver(I) dichloro argentate (5e) was found out as the most active one. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The use of silver as an antimicrobial has been known since early civilizations.1,2 The antimicrobial properties of silver nitrate were known before 1800s and it had been used as an antiseptic in wound care for more than 200 years.3,4 The discovery of penicillin and other new antibiotics limited the use of silver compounds after World War II.4 After a short period of time, resistant organisms such as Pseudomonas aeruginosa, Proteus mirabilis and Proteus morgani surfaced and allowed to the revival of silver nitrate by Moyer in 1965.5 However, the true revival of silver compounds as antimicrobial agents in wound care started by the help of discovery of silver sulfadiazine by Fox.6 Although the silver compounds are well known as antimicrobial agents, the mechanisms of action have not been fully understood yet. In the wound treatment, it is assumed that the slow release of silver cation at the wound sites prevents infection.7 There are three important reasons that limit the usage of silver sulfadiazine and silver nitrate. First of all, these compounds cause the wound site to be reinfected because of losing ⇑ Corresponding author. Tel.: +90 4223773855; fax: +90 4223410037. E-mail address: [email protected] (M.O. Karatasß).

their effects in a short time. Secondly, they may cause discoloration of the skin. Lastly, development of resistance of some organisms to sulphonamides limits these compounds.8,9 Due to these problems, researchers began searching new and more effective antimicrobial compounds and Youngs and co-workers reported silver N-heterocyclic carbenes (NHCs) as a new class of antibiotics in 2004.10 NHCs act as excellent r-donor ligands and they can produce stable metal–NHC complexes11 so, they release silver ions slower than traditional silver-based antimicrobial compounds. Thus, effect of the silver NHC complexes retain over a longer period of time and prevents reinfection in the wound site.10 N-heterocyclic carbene chemistry was first introduced by Wanzlick and Schonherr12 and Öfele13 in 1960s. But, the interest in this area rapidly increased after the isolation of the first stable carbene by Arduengo.14 Since then, various metal–NHC complexes have been prepared and have been used as catalyst in many useful organic transformations.15–19 Moreover, researches on the biological properties of different metal–NHC complexes have attracted much attention, since the first report of the antimicrobial properties of silver–NHCs.20–29 The first silver NHC was prepared by using a free carbene30 and this class of compounds was reported as carbene transfer reagents

http://dx.doi.org/10.1016/j.bmc.2015.12.032 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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M. O. Karatasß et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

in transmetallation reactions to prepare other metal–NHC complexes.31 After this invention, silver NHC complexes played an important role in the development of the field of NHC complexes and today NHCs have an important place in organometallic chemistry as ligand. Coumarins which belong to a class of compounds, are known as benzopyrones. Benzopyrones are heterocyclic and dicyclic compounds which consist of the fusion of pyron and benzene rings. Coumarin derivatives show various biological and pharmacological properties such as anticoagulant,32 anticancer,33–35 anti-HIV,36–38 anti-inflammatory.39 In addition, coumarin derivatives were also reported as antibacterial and antifungal agents. The antibacterial potential of coumarin derivatives may extend over 1945; Goth reported that dicoumarol inhibited the growth of several strains of bacteria.40 Some coumarin derivatives such as Novobiocin and Chlorobiocin, which occurred naturally were reported as highly active new class of antibiotics.41,42 In recent years antimicrobial properties of coumarin derivatives have drawn considerable interest.43–52 Moreover, in a study, coumarin substituted gold(I)-NHC complexes were synthesized and these complexes were reported as anticancer agents.53 In the light of these knowledge, we aimed to synthesize new series of silver(I) complexes stabilized by NHC ligands which have coumarin group in order to evaluate the role of the combination of coumarin with silver carbene in one structure. Coumarin substituted azolium salts were used as carbene precursors and the silver NHC complexes were synthesized by the reaction of corresponding carbene precursor and Ag2O. The antimicrobial activities of carbene precursors and silver NHC complexes were tested against Gram-negative, Gram-positive bacteria and fungi Candida albicans and Candida tropicalis. 2. Results and discussion 2.1. Chemistry Silver–NHC complexes are commonly prepared according to three different methods: (i) reaction of free NHC with silver salts (but this technique was applied only in a few instances); (ii) reaction of azolium salts with alkaline silver compounds such as Ag2O, Ag2CO3, AgOAc; (iii) reaction of azolium salts with silver salts under basic phase transfer conditions.54 Deprotonation of azolium salts by use of alkaline silver compounds (route ii) is the most commonly used method for the synthesis of silver–NHC complexes. Ag2O is the most widely used compound in this procedure. The first synthesis of a silver NHC complex by using Ag2O was reported by Lin and Wang.31 The use of Ag2O in the synthesis of silver NHCs provides some advantages such as: (i) the reactions can be carried out at room temperature; (ii) air does not have to be excluded; (iii) solvent pretreatments and strong bases are not required.31,55 Therefore route (ii) with Ag2O was used in this work for the preparation of silver NHC complexes. The ligand precursors coumarin substituted azolium salts were ready from our previous study.56 The silver NHC complexes were prepared by treatment of the imidazolium or benzimidazolium chlorides with 0.5 equiv Ag2O in dichloromethane after 24 h in the absence of the light and 20–53% yields were obtained. All complexes were stored in the dark. The synthetic route for silver NHC complexes was given in Scheme 1. The structures of complexes were established by 1H NMR, 13C NMR, mass, IR spectroscopic methods and elemental analyses. We could not achieve a single crystal for any of the eight complexes despite all efforts. Silver NHC complexes show different structural motifs in the solid state depending on the ratio of silver reagent to the azolium salts used in the synthesis. Nature of ligand, temperature, solvent

and counter anions can also be factors in determining structure. Structures of silver NHC complexes were divided into two main classes in a comprehensive review in 2005: (i) mono NHC complexes and (ii) multi NHC complexes of silver. In the same review, mono NHC complexes were divided into six sub-headings by authors: (i) C2-Ag, (ii) C-Ag-X, (iii) C-Ag-X2, (iv) C-Ag-X3, (v) (C2-Ag-AgX2) and (vi) [Ag2X4]2 (C2-Ag-Ag(X/Y)3) types.57 Thus, it is clear that NMR and IR spectroscopy is not sufficient to clarify the structures of silver carbene complexes, however mass spectroscopy can be used in order to clarify the structures of silver NHC complexes in the solution in the case of the absence of crystallographic data. Mass spectra of complexes showed that maximal peak intensities for each complexes attributable to [AgL2]+ (see Section 4). Mass spectrum of 5f was given in Figure 1. Elemental analyses give molar ratio among silver, ligand and chloride of 1:1:1 for all the complexes. Combination of mass spectra and elemental analyses data suggest that all of eight complexes have a structure such as [Ag(L)2]+[AgCl2] . In literature, Wang and Lin reported structure of [Ag(Et2-Bimy)2]+[AgBr2] and pointed out the existence of these ions in solution31 similar to our complexes which have structures as shown in Scheme 1. In the 1H NMR spectra of carbene precursors, signal of acidic protons (NCHN) were located in the range of 9.41–10.24 ppm (see Ref. 56) and the absence of these downfield signals of acidic protons in the 1H NMR spectra of the silver NHC complexes indicated the formation of silver NHC complexes.57 Olefinic protons are important for characterization of coumarin derivatives and signals of these olefinic protons are numbered as 4 in Scheme 1 and located in the range of 5.42–6.03 ppm as a singlets. 1H NMR spectrum of compounds 2b and 4b were given in Figure 2. In the 13C NMR spectra of carbene precursors, the imino carbons are singlets in the range of 137.7–144.8 ppm (see Ref. 56). In the 13C NMR spectra of the silver NHC complexes, disappearances of the signals of the imino carbons supported the proposed structures. The resonance of carbene carbon was detected at 192.0 ppm for complex 5a, but in the other complexes the signals of carbene carbons were not detected due to labile nature of Ag–Ccarbene bond which was also mentioned in the literature.54,55,57 13C NMR spectrum of compounds 3a and 5a were given in Figure 3. In addition, for further characterization of complexes, 5c and 3c were evaluated by HSQC and COSY NMR techniques. HSQC spectra of 3c was given in Figure 4 as representative. As seen from the spectra, the carbon signal which located in 143.5 ppm overlaps with NCHN proton of 3c (9.95 ppm). This signal showed that imino carbon (NCHN) of 3c is located in 143.5 ppm. HSQC spectra of corresponding complex 5c was given in Figure 5. The disappearance of the signal of imino carbon in the HSQC spectra of 5c indicated the formation of silver– carbene complex. Moreover, COSY spectra of 5c confirms the 1 H–1H interactions (see Supplementary data). According to COSY spectra of 5c, protons of ArCH3 interact with protons of benzene ring of coumarin. Interestingly, COSY spectra also showed that, –NCH2 coumarin protons and olefinic proton interact weakly. The IR data of complexes indicate the presence of –C@O and –C–N– groups and vibrations signals were observed in the range of 1714–1722 cm 1 and 1584–1594 cm 1, respectively. 2.2. Antimicrobial activity Minimal inhibitory concentrations (MICs) of carbene precursors and silver NHC complexes were determined against Staphylococcus aureus, Enterococcus faecalis (Gram-positive), Escherichia coli, Pseudomonas aeruginosa (Gram-negative) bacterial strains and fungal strains Candida albicans and Candida tropicalis. The MICs of synthesized compounds were given in Table 1 and ampicillin, ciprofloxacin and fluconazole were used as standard drugs for comparison.

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M. O. Karatasß et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

3

OH

O O

+

O Cl H2SO4(70%) r.t., 24 h

X N

Cl

N

O

O

N DMF, 80 o C, 24 h

O

N

Y

Y

X N

N

N

Cl

O

N

DMF, 80 oC, 24 h

O

O

2a,b

1

Cl

3a-f

0.5 Ag2O, DCM, r.t., 24 h

2a, 4a; X= -H

0.5 Ag2O, DCM, r.t., 24 h

2b, 4b; X= -CH 2CH 2CH3 3a, 5a; Y= -H 3b, 5b; Y= -CH=CH2

O O

X

AgCl2

N

Ag

6

N O

X

3

3d, 5d; Y=

2 1

1

AgCl2

N

N Ag

2

N 3e, 5e; Y=

3f, 5f; Y=

O O

4a,b

Y

3

O 1

N

5

4

O

O

O

O

Y

N 5

4

N

O

3c, 5c; Y= -CH2CH 2CH3

2

1

5a-f

Scheme 1. Synthesis of imidazolium (2a,b), benzimidazolium (3a–f) chlorides and silver–NHC complexes (4a,b and 5a–f) with NMR numbering scheme. Compound 1, imidazolium and benzimidazolium chlorides were ready from our previous study.56

Figure 1. LC–MS spectrum of complex 5f.

As shown in Table 1, antimicrobial activities against bacteria and fungi were observed in 800–25 lg/mL concentrations. As it is seen from the results, all the compounds inhibited the growth of all bacteria and fungi strains. The silver NHC complexes are more active than corresponding imidazolium or benzimidazolium chlorides as expected. If we compare the effects of alkyl or aryl groups which substituted on the nitrogen atom for carbene precursors, there are some minor differences, except from naphthalene group. However, according to these results, we cannot reach any definite opinion about the effects of these alkyl or aryl groups. Among the carbene precursors, naphthalene substituted benzimidazolium chloride 3e is much more active than others and this compound is more lipophilic than other carbene precursors. Interestingly, 3e showed higher inhibitory activity (25 lg/mL) than seven silver

NHC complexes except from 5e against Gram-positive bacteria strains. In addition, compound 3e showed good inhibitory activities (25 lg/mL) against fungi strains. Complex 5e which is corresponding complex of 3e showed good activities (25 lg/mL) against Gram-positive bacteria and fungi strains. The microorganisms cell wall is surrounded by a lipid membrane which favours the passage of lipid soluble materials. So, it is assumed that, lipophilicity of NHCs and their metal complexes is important in contributing their antimicrobial effects22,58 and high activities of 3e and 5e against bacteria and fungi strains is supportive for this hypothesis. Among the other carbene precursors and silver NHC complexes, 5d also showed good inhibitory activities (25 lg/mL) against fungi strains. Furthermore it must be noted that among all of the compounds, complex 5d showed the highest inhibitory

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M. O. Karatasß et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

Figure 2. 1H NMR spectra of compounds 2b and 4b.

activity (50 lg/mL) against Gram-negative bacteria Escherichia coli. It is known that Gram-negative bacteria have an outer cell membrane in addition to cytoplasmic membrane in Gram-positive bacteria. This cell membrane provides an additional protective barrier compared with Gram-positive bacteria so, it is more difficult to kill Gram-negative bacteria.59 In literature, some similar N-benzylated silver NHC complexes and their antimicrobial activities against organisms used in this study were reported. The observed antimicrobial activities of compounds synthesized in this work are comparable with these similar complexes.60–63 3. Conclusion In this paper, eight new coumarin substituted silver NHC complexes were synthesized and characterized by 1H NMR, 13C NMR, mass, IR spectroscopic methods and elemental analyses. Combination of MS and elemental analyses data suggest that complexes have a structure such as [AgL2]+[AgCl2] . Antimicrobial properties of eight carbene precursors and their corresponding silver NHC complexes were tested against Staphylococcus aureus, Enterococcus faecalis (Gram-positive), Escherichia coli, Pseudomonas aeruginosa (Gram-neg-

ative) bacterial strains and fungal strains Candida albicans and Candida tropicalis. Effects of the addition of coumarin group to silver NHC complexes were investigated in this study. The results showed that addition of coumarin to the NHC scaffold did not increase antibacterial and antifungal activities significantly, while the most lipophilic complexes 5d and 5e showed good activities. They were found out as promising candidates to be used as antimicrobial agents. Finally, these observations suggest that silver carbene framework and lipophilicity of complexes are responsible for antimicrobial activity. Detailed investigations on synthesis and evaluation of the biological activities of novel silver NHC complexes and coumarin substituted other metal NHC complexes are in progress. 4. Experimental All reactions for the preparation of silver NHC complexes were carried out in standard schlenk type flasks under an atmosphere of dry argon in the absence of light. Chemicals and solvents were purchased from Sigma Aldrich. DCM was used for synthesis of silver complexes as solvent and was dried over P2O5. 4-Chloromethyl-6, 8-dimethylcoumarin, imidazolium and benzimidazolium chlorides

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M. O. Karatasß et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

Figure 3.

5

13

C NMR spectra of compounds 3a and 5a.

Figure 4. HSQC spectra of 3c.

were ready from our previous study.56 Melting points were determined in open capillary tubes by Electrothermal-9200 melting point apparatus. FT-IR spectra were recorded on ATR unit in the range of 400–4000 cm 1 with Perkin Elmer Spectrum 100 Spectro-

fotometer. 1H NMR and 13C NMR spectra were recorded using a Bruker FT spectrometers operating at 300.13 MHz (1H), 75.47 MHz (13C). Chemical shifts are given in ppm relative to tetramethylsilane (TMS). NMR multiplicities are abbreviated as

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Figure 5. HSQC spectra of 5c.

Table 1 Minimal inhibitory concentrations (lg/mL) of imidazolium (2a,b) and benzimidazolium chlorides (3a–f) and silver NHC complexes (4a,b and 5a–f) tested against bacteria and fungi Compound

Bacteria

Fungi

Gram-positive

1 2a 2b 3a 3b 3c 3d 3e 3f 4a 4b 5a 5b 5c 5d 5e 5f Ampicillin Ciprofloxacin Fluconazole

Gram-negative

Staphylococcus aureus

Enterococcus faecalis

Escherichia coli

Pseudomonas aeruginosa

Candida albicans

Candida tropicalis

400 800 800 400 400 200 400 25 200 200 200 200 400 100 50 25 100 3.12 0.39 —

400 800 800 400 400 800 400 25 200 200 200 200 400 100 100 25 100 1.56 0.78 —

800 800 800 800 400 800 800 400 400 100 100 100 200 200 50 200 200 3.12 1.56 —

800 800 800 800 400 800 800 400 400 100 100 100 200 200 100 200 200 — 3.12 —

400 400 800 400 200 200 200 25 200 50 50 100 200 50 25 25 200 — — 3.12

200 400 800 400 200 200 200 25 200 50 50 100 200 50 25 25 200 — — 3.12

follows: s = singlet, d = doublet, t = triplet, quin = quintet, sex = sextet, m = multiplet signal. Elemental analyses were performed by LECO CHNS-932 elemental analyzer at IBTAM (Inonu University Scientific and Technological Research Central). LC–MS spectra were performed on an Agilent 1100 LC/MSD SL mass spectrometer equipped with an electrospray ion source.

4.1. General procedure for preparation of silver NHC complexes A solution of 0.5 mmol (115 mg) of Ag2O, 1 mmol of corresponding imidazolium (2a,b) or benzimidazolium (3a–f) chloride and activated 4 Å molecular sieves in dichloromethane (25 mL) was stirred at room temperature for 24 h. After this period, the

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reaction mixture was filtered through Celite. Solvent and volatile components were removed under reduced pressure. The crude products were recrystallized from dichloromethane/n-hexane at ambient temperature. All manipulations for the synthesis of silver NHC complexes were carried out in the absence of light. 4.1.1. Bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3(methyl)imidazol-2-ylidene]silver(I) dichloro argentate(I) (4a) Color: white solid, yield: 180 mg (44%), mp: 209–211 °C, FT-IR (cm 1): 2917, 1715, 1588, 1H NMR (300 MHz, CDCl3) d/ppm: 7.29 (s, 1H, H2), 7.24 (s, 1H, H2), 7.13 (d, 1H, H6, J = 1.7 Hz), 7.07 (d, 1H, H6, J = 1.7 Hz), 5.68 (s, 1H, H4), 5.57 (s, 2H, H5), 3.94 (s, 3H, HCH3), 2.45 (s, 3H, H1), 2.42 (s, 3H, H1), 13C NMR (75 MHz, CDCl3) d/ppm: 160.3, 150.0, 135.3, 134.1, 126.8, 123.3, 121.9, 120.6, 116.3, 112.8, 51.7, 39.1, 21.0, 15.7, the carbene carbon was not detected. Anal. Calcd for C32H32N4O4Ag2Cl2: C, 46.49; H, 3.92; N, 6.81. Found: C, 46.77; H, 3.99; N, 6.92. LC–MS: 645.0 [AgL2]+. 4.1.2. Bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3(n-butyl)imidazol-2-ylidene]silver(I) dichloro argentate(I) (4b) Color: white solid, yield: 170 mg (37%), mp: 211–212 °C, FT-IR (cm 1): 2915, 1714, 1590, 1H NMR (300 MHz, CDCl3) d/ppm: 7.27 (s, 1H, H2), 7.25 (s, 1H, H2), 7.12 (d, 1H, H6, J = 1.8 Hz), 7.06 (d, 1H, H6, J = 1.8 Hz), 5.67 (s, 1H, H4), 5.59 (s, 2H, H5), 4.18 (t, 2H, HCH2CH2CH2CH3, J = 7.3 Hz), 2.42 (s, 3H, H1), 2.40 (s, 3H, H1), 1.84 (quin, 2H, HCH2CH2CH2CH3, J = 7.7 Hz), 1.38 (sex, 2H, HCH2CH2CH2CH3, J = 7.7 Hz), 0.98 (t, 3H, HCH2CH2CH2CH3, J = 7.3 Hz), 13C NMR (75 MHz, CDCl3) d/ppm: 160.3, 150.1, 149.9, 135.2, 134.1, 126.6, 122.0, 121.8, 120.8, 116.3, 112.8, 52.2, 51.8, 33.4, 21.0, 19.7, 15.6, 13.7, the carbene carbon was not detected. Anal. Calcd for C38H44N4O4Ag2Cl2: C, 50.30; H, 4.89; N, 6.17. Found: C, 50.34; H, 4.92; N, 6.26. LC–MS: 727.0 [AgL2]+. 4.1.3. Bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3(methyl)benzimidazol-2-ylidene]silver(I) dichloro argentate(I) (5a) Color: white solid, yield: 190 mg (41%), mp: 167–168 °C, FT-IR (cm 1): 2918, 1716, 1594, 1H NMR (300 MHz, CDCl3) d/ppm: 7.57– 7.26 (m, 6H, H2), 6.00 (s, 2H, H5), 5.45 (s, 1H, H4), 4.11 (s, 3H, HCH3), 2.44 (s, 3H, H1), 2.43 (s, 3H, H1), 13C NMR (75 MHz, CDCl3) d/ ppm: 192.0 (C-carbene), 160.4, 150.0, 149.5, 135.2, 134.4, 134.1, 133.5, 126.6, 124.9, 124.8, 120.9, 116.5, 111.9, 111.7, 111.3, 49.0, 36.1, 21.0, 15.7. Anal. Calcd for C40H36N4O4Ag2Cl2: C, 52.03; H, 3.93; N, 6.07. Found: C, 52.11; H, 3.98; N, 6.16. LC–MS: 743.0 [AgL2]+. 4.1.4. Bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3(allyl)benzimidazol-2-ylidene]silver(I) dichloro argentate(I) (5b) Color: white solid, yield: 130 mg (27%), mp: 254–256 °C, FT-IR (cm 1): 2929, 1718, 1592, 1477, 1441, 1H NMR (300 MHz, DMSO-d6) d/ppm: 7.84–7.42 (m, 6H, H2), 6.11 (s, 2H, H5), 6.12 (m, 1H, HCH2CH@CH2), 5.42 (s, 1H, H4), 5.29–5.15 (m, 4H, HCH2CH@CH2), 2.39 (s, 3H, H1), 2.34 (s, 3H, H1), 13C NMR (75 MHz, DMSO-d6) d/ppm: 159.8, 151.2, 149.8, 135.1, 133.9, 133.8, 133.6, 125.8, 125.0, 124.9, 122.7, 118.7, 118.2, 117.1, 113.0, 112.7, 111.5, 51.5, 49.1, 20.9, 15.6, the carbene carbon was not detected. Anal. Calcd for C44H40N4O4Ag2Cl2: C, 54.18; H, 4.13; N, 5.76. Found: C, 54.25; H, 4.21; N, 5.87. LC–MS: 795.0 [AgL2]+. 4.1.5. Bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3(n-butyl)benzimidazol-2-ylidene]silver(I) dichloro argentate(I) (5c) Color: white solid, yield: 110 mg (22%), mp: 265–270 °C, FT-IR (cm 1): 2987, 1722, 1591, 1H NMR (300 MHz, CDCl3) d/ppm: 7.58–7.28 (m, 6H, H2), 5.90 (s, 2H, H5), 5.32 (s, 1H, H4), 4.51 (t, 2H, HCH2CH2CH2CH3, J = 7.3 Hz), 2.47 (s, 6H, H1), 1.97 (quin, 2H,

7

HCH2CH2CH2CH3, J = 7.7 Hz), 1.47 (sex, 2H, HCH2CH2CH2CH3, J = 7.8 Hz), 1.01 (t, 3H, HCH2CH2CH2CH3, J = 7.4 Hz), 13C NMR (75 MHz, CDCl3) d/ ppm: 160.3, 150.1, 148.9, 135.3, 134.1, 133.7, 133.5, 126.8, 125.0, 124.9, 120.7, 116.4, 112.1, 112.0, 111.6, 49.9, 49.3, 32.4, 21.0, 20.2, 15.7, 13.8, the carbene carbon was not detected. Anal. Calcd for C46H48N4O4Ag2Cl2: C, 54.84; H, 4.80; N, 5.56. Found: C, 54.94; H, 4.89; N, 5.67. LC–MS: 827.0 [AgL2]+. 4.1.6. Bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3(benzyl)benzimidazol-2-ylidene]silver(I) dichloro argentate(I) (5d) Color: white solid, yield: 280 mg (53%), mp: 256–257 °C, FT-IR (cm 1): 2962, 2920, 1717, 1590, 1H NMR (300 MHz, DMSO-d6) d/ ppm: 7.58–7.34 (m, 11H, H2 and HAr), 6.03 (s, 1H, H4), 5.74 (s, 2H, H5), 5.15 (s, 2H, HCH2Ph), 2.44 (s, 3H, H1), 2.41 (s, 3H, H1), 13C NMR spectra could not be recorded due to limited solubility in common organic solvents. Anal. Calcd for C52H44N4O4Ag2Cl2: C, 58.07; H, 4.12; N, 5.21. Found: C, 58.14; H, 4.16; N, 5.29. LC–MS: 895.0 [AgL2]+. 4.1.7. Bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3(naphthalene-2-ylmethyl)benzimidazol-2-ylidene]silver(I) dichloro argentate(I) (5e) Color: white solid, yield: 120 mg (20%), mp: 264–266 °C, FT-IR (cm 1): 2963, 1722, 1584, 1H NMR (300 MHz, DMSO-d6) d/ppm: 7.93–7.38 (m, 13H, H2 and HAr), 6.16 (s, 2H, H5), 5.94 (s, 2H, HCH2Ar), 5.45 (s, 1H, H4), 2.36 (s, 3H, H1), 2.30 (s, 3H, H1), 13C NMR (75 MHz, DMSO-d6) d/ppm: 159.8, 151.8, 149.8, 143.8, 134.9, 134.1, 133.8, 133.1, 133.0, 132.9, 129.0, 128.2, 128.1, 127.1, 126.9, 126.8, 125.8, 125.4, 125.0, 122.5, 117.0, 112.8, 53.9, 49.4, 20.9, 15.6, the carbene carbon was not detected. Anal. Calcd for C60H48N4O4Ag2Cl2: C, 61.30; H, 4.12; N, 4.47. Found: C, 61.40; H, 4.14; N, 4.86. LC–MS: 995.0 [AgL2]+. 4.1.8. Bis[1-(4-methylene-6,8-dimethyl-2H-chromen-2-one)-3(3,4,5,-trimethoxybenzyl)benzimidazol-2-ylidene]silver(I) dichloro argentate(I) (5f) Color: white solid, yield: 140 mg (22%), mp: 236–241 °C, FT-IR (cm 1): 2961, 1722, 1596, 1H NMR (300 MHz, CDCl3) d/ppm: 7.49–7.26 (m, 6H, H2), 6.53 (s, 2H, HAr), 6.06 (s, 2H, H5), 5.57 (s, 2H, HCH2Ar), 5.42 (s, 1H, H4), 3.83 (s, 3H, HArOCH3), 3.79 (s, 6H, HArOCH3), 2.43 (s, 3H, H1), 2.42 (s, 3H, H1), 13C NMR (75 MHz, CDCl3) d/ppm: 160.2, 153.8, 150.0, 149.6, 138.1, 135.2, 134.1, 133.8, 133.7, 130.2, 126.7, 125.0, 124.9, 120.9, 116.4, 112.4, 111.7, 111.4, 104.2, 60.8, 56.3, 53.8, 49.1, 21.0, 15.6, the carbene carbon was not detected. Anal. Calcd for C58H56N4O10Ag2Cl2: C, 55.48; H, 4.49; N, 4.46. Found: C, 55.54; H, 4.53; N, 4.56. LC–MS: 1077.0 [AgL2]+. 4.2. Antimicrobial activities of imidazolium, benzimidazolium chlorides and silver–NHC complexes Antimicrobial activities of the imidazolium chlorides (2a,b), benzimidazolium chlorides (2a–d) and silver–NHC complexes (4a,b and 5a–f) were determined by using agar dilution procedure recommended by the Clinical and Laboratory Standards Institute.64,65 Minimal inhibitory concentrations for each compound were investigated against standard bacterial strains; Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and were obtained from American Type Culture Collection (Rockville, MD.) and the fungal strains Candida albicans ATCC 10231 and Candida tropicalis ATCC 13803 were obtained from American Type Culture Collection (Rockville, MD). Bacterial strains were subcultured on Muller Hinton Broth (HiMedia Laboratories Pvt. Ltd. Mumbai-India) and fungal strains were also on RPMI 1640 Broth (Sigma–Aldrich Chemie GmbH Taufkirchen, Germany). Their

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turbidities matched that of a McFarland no. 0.5 turbidity standard.66 The stock solution of all compounds were prepared in dimethyl sulfoxide (DMSO). All of the dilutions were done with distilled water. The concentrations of the tested compounds were 800, 400, 200, 100, 50, 25, 12.5 and 6.25 lg/mL. Ampicillin and ciprofloxacin were used as antibacterial standard drugs, while fluconazole were used as antifungal standard drug whose minimum inhibitory concentration (MIC) values are provided. A loopful (0.01 mL) of the standardised inoculum of the bacteria and yeasts (106 CFUs/mL) was spread over the surface of agar plates. All the inoculated plates were incubated at 35 °C and results were evaluated after 16–20 h of incubation for bacteria and 48 h for yeasts. The lowest concentration of the compounds that prevented visible growth was considered to be the minimal inhibitory concentration (MIC). Acknowledgements The authors alone are responsible for the content and writing of the paper. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.12.032. These data include MOL files and InChiKeys of the most important compounds described in this article. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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