1-Butylimidazole-derived ionic liquids: synthesis, characterisation and evaluation of their...

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PAPER

1-Butylimidazole

School of Chemical Sciences, Central Univ

India. E-mail: [email protected];

232-60210

† Electronic supplementary informationNMR spectra. See DOI: 10.1039/c4ra08370

Cite this: RSC Adv., 2014, 4, 53634

Received 8th August 2014Accepted 8th October 2014

DOI: 10.1039/c4ra08370a

www.rsc.org/advances

53634 | RSC Adv., 2014, 4, 53634–536

-derived ionic liquids: synthesis,characterisation and evaluation of theirantibacterial, antifungal and anticancer activities†

Prabodh Ranjan, Bheru Singh Kitawat and Man Singh*

A series of new 1-butylimidazole-based ionic liquids (3a–h) have been synthesised by the quaternisation

reaction of 1-butylimidazole with different alkyl- and alkoxy-substituted aryl halides using a microwave

solvent-free approach with z82–95% yield. The solvent-free approach allows the preparation of a

variety of ILs with better yields and purities, making any further purification unnecessary. The structures

were confirmed by FTIR, 1H NMR, 13C NMR and LCMS (Q-TOF). The ILs were screened against Gram-

positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) bacterial strains.

Compounds 3b, 3c, 3e and 3f showed good activities against both S. aureus and B. subtilis strains, while

3a and 3c exhibited good activities against P. aeruginosa strains. The ILs 3a, 3b, 3d and 3e showed better

antifungal activities against the C. albicans strain. Additionally, the compounds were tested for their in

vitro anticancer activities against the MCF-7 and MDA-MB-435 cell lines using an SRB assay protocol to

estimate cell growth. Compounds 3a, 3d and 3e demonstrated 50–60% activity against the MDA-MB-

435 cell line with GI50 values of 67.2, 52.5 and 57.9 mM, respectively, as compared to standard adriamycin

(GI50 24.4 mM).

Introduction

Ionic liquids (ILs) are fused salts constituting cation and anionmoieties.1 They possess some fundamental properties such asnegligible vapour pressure, thermal stability up to 300 �C, non-ammability, high ionic conductivity,2–4 wide electrochemicalstability window2,3 and high polarity, enabling wide kineticcontrol and immiscibility with numerous organic solvents.5 Forthe last few decades, ILs have been recognised as “designersolvents”6–8 because their physical, chemical and biologicalproperties can be tuned to obtain desired task-specic ionicliquids (TILs) for a multitude of applications.9–12 These uniqueproperties of ILs make them widely applicable in differentdomains of chemistry; particularly, they are excellent candi-dates for use in “green synthesis” as green solvents.13–15

Despite the promising results evidenced by the many studiesin which they have been used as solvents or catalysts forchemical synthesis,16,17 as electrolytes for electrochemicaldevices,18 as super capacitors19,20 and in engineering and phys-ical chemistry,21,22 their widespread application is stillhampered by doubts related to some practical drawbacks: (i)cost and possible toxicological concerns; (ii) problems related to

ersity of Gujarat, Gandhinagar-382030,

Fax: +91-079-232-60076; Tel: +91-079-

(ESI) available: Copies of 1H and 13Ca

44

product isolation; and (iii) catalyst recovery. Recently, to over-come at least some of these drawbacks, ether-functionalisedILs, the so-called TSILs, have been synthesised.23,24 For thepast decade, numerous green routes including reaction undersolvent-free conditions25 using non-classical techniques such asultrasonication,26 microwaves and others27,28 have been adoptedfor the synthesis of ILs. Microwave (MW)-assisted synthesis hasbeen used to develop a rapid green synthetic route, resulting inlarge reductions in reaction time (from hours to minutes),uniform temperature and pressure during the course of thereaction and better yields.29 Additionally, the biological activi-ties of several ILs have been investigated. The literature revealsthat many synthetic ILs and their analogues display a widespectrum of biological activities including antibacterial,30,31

anticancer,32,33 antifungal and others.34–36 For this reason, theyare an object of continuously growing interest in academia aswell as in industry. ILs in the environment may also harmaquatic ecosystems and living organisms due to the lack oftoxicity data for designed ILs or ILs that have yet to bedesigned.37 Thus, the toxicity data of ILs must be investigated todetermine their biological and environmental impacts.38,39

In general, earlier studies reveal that the biological activity ofan IL depends on the alkyl chain length of its cationic headgroup, the functional groups present in their chain andelements (nitrogen, sulphur, and oxygen) present in thering.40,41 Thus, structural modication can enhance theirpotency and SAR (structural activity relationship).42

This journal is © The Royal Society of Chemistry 2014

Table 1 Optimisation of reaction conditions under MW solvent-freeconditions

Entry Power (W) Temp. (�C) Time (min) Yield (%)

1 200 100 25 772 250 100 25 793 300 100 25 844 350 100 25 875 400 100 25 876 350 120 25 887 350 140 25 908 350 160 25 91

Table 2 Optimisation of reaction conditions under MW with solvents

Paper RSC Advances

Considering the advantages of theMW solvent-free approachand continuing our investigations on new methodologies forthe synthesis of new heterocyclic ring-bearing compounds,43 wereport herein an efficient and practical procedure for thesynthesis of a series of 1-butylimidazole-based ILs. Additionally,the potential biological activities (i.e., antibacterial, antifungaland anticancer) of ILs against a panel of microorganisms andcell lines have been investigated.

Results and discussionSynthesis and analytical characterisations

The aim of our study is to synthesise novel 1-butylimidazole-based ILs (3a–h) by altering the alkyl- or alkoxy-substitutedaryl bromide thorough MW dielectric heating as well as aconventional heating method. 1-Butylimidazole reacts withdifferent alkyl or alkoxy-substituted aryl bromides to deliver the1-butyl-3-(n-aryl alkyl)-1H-imidazole-3-ium bromide (3a–h)according to Scheme 1. The optimised conditions for ILsynthesis under MW solvent-free, MW with solvent and heatingmethod (or conventional heating method) are summarised inTables 1–3, respectively. The comparisons among the MW-assisted methods and the heating method are summarised inTable 4. The solvent-free MW assisted-method produced goodyield compared to the heating and MW with solvent methods(Table 4).

Optimisation of reaction conditions

Initially, the reaction between equimolar amounts of 1-butyli-midazole (1a) and (2-bromoethoxy)benzene (2c) to deliverproduct (3c) was considered as a model reaction to screen thereaction conditions for methods A–C, which have been dis-cussed below.

Method A: MW solvent-free approach

Initially, the model reaction was conducted under MW solvent-free conditions at 100 �C and a power of 200 W. The reactionwas completed in 25 min and produced a 77% yield of 3c(Table 1, entry 1). To further optimise the MW power, thereaction was performed at 250, 300, 350 and 400 W for 25 min,resulting in 3c with yields of 79, 84, 87 and 87%, respectively(Table 1, entries 2–5).

By comparing the yields of 3c at different powers, noimprovement in yield was found when power was increasedfrom 350 to 400 W. Thus, 350 W was chosen as the optimum

Scheme 1 General scheme for the synthesis of 1-butyl-3-(n-arylalkyl)-1H-imidazole-3-ium bromide (3a–h).


MW power. Next, temperature was optimised by conducting themodel reaction at 120 �C, 140 �C and 160 �C; the quaternisationyield was increased to 88%, 90% and 91%, respectively (Table 1,entries 6–8). On increasing the temperature from 140 �C to160 �C, no better yield was noted. Thus, the suitable reactionconditions for the model reaction were a MW power of 350 Wand a temperature of 140 �C. The scope and versatility of theoptimised conditions under the MW solvent-free approach werestudied by preparing a series of eight new ILs (3a–h) usingdifferently substituted alkyl and alkoxy bromide (2a–h). It wasfound that ILs having oxygen in their alkyl chain producedbetter yields compared to ILs with oxygen in their alkyl chain.The overall yield of ILs was found to be in the range of 82–90%depending on the substitution of the alkyl chain on the phenylring. The structures of ILs were conrmed using various spec-troscopic techniques.

Method B: MW with solvent approach

In this method, to study the effect of solvent on reactionconditions such as MW power, temperature and yield, themodel reaction was conducted using acetonitrile (ACN),

Entry Solvent Power (W) Temp (�C) Time (min) Yields (%)

1 ACN 300 100 25 762 Toluene 300 100 25 803 EtOH 300 100 25 754 MeOH 300 100 25 705 DCM 300 100 25 —6 Toluene 350 100 25 817 Toluene 300 120 25 848 Toluene 250 120 25 77

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Table 3 Optimisation of reaction conditions for the heating method

Entry Temp. Method Solvents Time (h) Yield (%)

1 100 �C Reux — 5 702 100 �C Reux — 4 693 100 �C Reux — 3 684 100 �C Reux — 2 625 120 �C Reux — 4 736 120 �C Reux — 3 727 120 �C Reux — 2 698 140 �C Reux — 3 8410 160 �C Reux — 3 85

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toluene, ethanol (EtOH), methanol (MeOH) and dichloro-methane (DCM) as solvents at 100 �C and an MW power of300 W. Aer 25 min of reaction, 3c was produced with yields of76, 80, 75 and 70% for ACN, toluene, EtOH and MeOH,respectively (Table 2, entries 1–4). The reaction did not proceedwhen DCM was used as a solvent (Table 2, entry 5).

We found that 300 W and 100 �C using toluene as a solventproduced a higher yield of 3c (80%; Table 2, entry 2) comparedto other solvents. Thus, further reaction conditions were opti-mised considering toluene as the solvent. The reaction carriedout at 350 W and 100 �C gave an 81% yield (Table 2, entry 6),representing a 1% improvement compared to 300 W (Table 2,entry 2). At 300 W and 120 �C, the yield was 84% (Table 2, entry7). The reaction conducted at 250W and 120 �C gave a 77% yield(Table 2, entry 8), which was lower than that for a power of300 W. Considering these results, the optimised conditionsused to synthesise a series of ILs (3a–h) were as follows: tolueneas solvent, MW power of 300 W, and temperature of 120 �C.

Method C: heating method (solvent-free)

Equimolar amount of reactants (1a and 2c) were reuxed attemperatures of 100 �C, 120 �C, 140 �C and 160 �C with constantstirring at times varying from 2 to 5 h. To optimise the reactionduration, the reaction mixture was reuxed at 100 �C for 5, 4, 3and 2 h, resulting in 70, 69, 68, and 62% yields, respectively

Table 4 Comparison between MW methods and the heating method

ILs

MW solvent-free MW with

Time (min) Yield (%) Time (m

3a 25 92 303b 25 85 303c 25 90 303d 25 84 303e 25 93 303f 25 86 303g 25 82 303h 25 95 30

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(Table 3, entries 1–4). Furthermore, the reaction mixture wasreuxed at 120 �C for 4, 3 and 2 h, giving 73, 72 and 69% yields,respectively (Table 3, entries 5–7).

Increases in temperature and reaction duration wereobserved to increase the yield of 3c, whereas the yield of 3c wasdecreased by shortening the reaction duration. Interestingly,the reactions carried out at temperatures of 100 �C and 120 �Cfor 3 h had good yields compared to other reaction times;increases in yield of about 1% or 2% with respect to the otherreaction times at the same temperatures (100 �C or 120 �C) werefound. Thus, a reaction duration of 3 h was chosen as optimalfor this method. Aer optimising the reaction duration, thereaction temperature was optimised by performing the reactionat 140 �C and 160 �C for 3 h, resulting in yields of 84 and 85%,respectively.

Interestingly, the reaction performed at 160 �C gave 1%better yields compared to the reaction performed at 140 �C forthe same period of time. Thus, the optimal reaction conditionsfor the model reaction using the solvent free heating methodwere a temperature of 140 �C and a reaction time of 3 h. Thescope and versatility of the solvent-free heating method underthe optimised conditions were studied by preparing a series ofeight new ILs (3a–h) using differently substituted alkylbromides (2a–h).

Thus, considering these optimised conditions, a series of ILshave been synthesised. It could be concluded that in the non-conventional methods A and B under MW irradiation, thereaction rate is considerably enhanced, reducing the reactiontime from hours to minutes, and the yield and purity of theproducts are enhanced compared to the heating method. Thecomparisons of reaction time and yield among all methods aregiven in Table 4.

It was found that higher MW irradiation power did notincrease the product yield. Furthermore, an improvement inyield was observed when the reaction took place in the absenceof solvent under MW. Therefore, the MW solvent-free methodhas been standardised and offers a simplication of laboratorytechniques as it does not require the use of stirrers, reuxcondensers, water separators or noxious solvents. The identitiesof the ILs synthesised by the heating and MW-induced methodswere established by their co-TLC and superimposable IRspectra. For each method, the IL (3c) was conrmed usingproton NMR, LCMS, and FTIR data.

solvent Heating method (solvent free)

in) Yield (%) Time (h) Yield (%)

85 3 8284 3 7985 3 8474 3 8179 3 8582 3 8379 3 8178 3 87


Paper RSC Advances

ILs derived from imidazole may have varied structuresdepending on the nature of the cationic and anionic parts. Inthe present work, the cationic parts of the ILs consist of1-butylimidazole and were kept xed, while the anionic partswere varied by using alkyl bromide chains of different lengths.The purpose behind the synthesis of imidazole-based ILs withvarying structures is to understand the nature of these novelsolvents. The prevailing methods of IL preparation with imid-azole compounds such as 1,3-disubstituted imidazoliumhalides generally require longer reaction times and result inconsiderable contamination with halide ions (X ¼ Br�, Cl�, I�,F�). Additional steps are also required to carry out the quater-nisation that converts the halide-containing precursors into ILs.The purication of ILs is a matter of foremost concern asimpurities have been found to signicantly affect the biologicaland physicochemical properties of these ILs, which in turnregulate their domain of application.23,24

The structures of the synthesised ILs (3a–h) were conrmedwith 1H NMR, 13C NMR, FTIR and LCMS (see ESI†). The qua-ternisation reactions of 1-butylimidazole with different alkylhalides were established by their FTIR spectra; the presence of–C–N bond stretching in region of 1120 to 1290 cm�1 is due tothe formation of the quaternary nitrogen of the imidazole ringand oxygen in the aliphatic chain, while peaks at 1630, 1587,1560 cm�1 are due to the –C]N bond of the ve-memberedimidazolium ring. The C–N is experiencing an addition thatrequires an electron of oxygen, thus affecting the stretching ofthe –C]N bond. Additionally, the peaks at 1497, 1457, 1406cm�1 are due to the –C]C– bond of phenyl and imidazoliumrings. The oxygen atoms present in a few ILs (3c, 3e, 3g, 3h)showed peaks at 1174 cm�1 due to ether linkages (–C–O–C–).The aliphatic –CH2 and –CH3 appeared in the 2830 to 2970 and3070 to 3134 cm�1 regions, respectively, of all ILs. Positive modeESI-MS spectra of the synthesised ILs (3a–h) exhibited [M� Br]+

molecular ion peaks, conrming their molecular masses. In the1H NMR spectra of ILs (3a, 3b, 3d, 3e and 3g), two methylenicprotons of the imidazole ring appeared as a doublet from d 7.70to 7.92 ppm with a coupling constant of J ¼ 9–12 Hz, while inthe ILs (3c, 3f and 3h), the methylenic protons appeared as asinglet. The single proton of the imidazole ring appeared as asinglet in the downeld in the range of d 9.0 to 9.9 ppm. Thedifferences in the proton shiing of the imidazole ring could beattributed to the alkyl chain lengths in the ILs. For ILs (3a–h),the ve aromatic protons appeared as a multiplet in the desiredaromatic range. The 9H protons of the butyl chain in all ILs (3a–h) as –CH3CH2CH2CH2– attached to the imidazole ring wereobserved in the upper eld, where the terminal 3H proton of–CH3 appeared as a triplet in the region of d 0.90 ppm withcoupling constant J ¼ 7.5 Hz. The 2H of –CH3CH2CH2CH2–

appeared as a sextet at d 1.10 to 1.30 ppm (J ¼ 7.5 Hz), the 2H ofCH3CH2CH2CH2– as a quintet at d 1.52 to 1.84 ppm (J ¼ 7.5 Hz)and the 2H of CH3CH2CH2CH2– as a triplet at d 3.93 to 4.30 ppm(J ¼ 7.5 Hz) respectively. The –CH2 protons of alkyl chainsattached to quaternary nitrogen atoms of imidazole ringsappeared in the range of d 1.0 to 4.9 ppm as per their chemicalshi in the standard splitting pattern as triplet and quintet.


The 13C NMR spectra provided nal structural elucidation ofILs (3a–h). Signals due to the formation of quaternary nitrogenatoms in 1-butylimidazolium-based ILs were found in the rangeof d 48.50 ppm to 55.96 ppm. Thus, the signals for the ILs (3f)and (3h) appeared in the downeld at d 55.97 ppm, while thoseof ILs (3b) and (3e) appeared in the upper eld at d 48.57 ppmand d 48.54 ppm, respectively. The two carbons (–CH]CH–) ofthe imidazole ring in ILs were found from d 119.0 ppm to 125.0ppm, while the third carbon of –N–C]N– appeared in the rangeof d 134.70 ppm to 136.57 ppm. In general, the aromatic phenylring carbons appeared in the range of 114.4 ppm to 131.0 ppm,while the alkyl-substituted carbon of the phenyl ring appearedin the downeld from d 134.0 to 163.0 ppm (Fig. 1).

The UV-Vis spectra were exclusively recorded for the eightdifferent ILs using DMSO, THF, ACN, MeOH, EtOH, CHCl3 assolvents. For each solvent, 1 mM of IL was used to determine theelectronic transitions.43 The experiments were carried out at rtfor ILs (3a–h) in the range of 200 to 600 nm, and the spectra areillustrated in Fig. 2 (see ESI† for the absorption spectra of 3b, 3c,and 3e–h). The extinction coefficient (3), which is the charac-teristic molecular property of ILs and reects their differentelectronic transitions, was calculated for each band. Thequantitative relationships between absorbance, extinctioncoefficient, concentration and path length are given by theBeer–Lambert law. To study the absorption spectra of the eightdifferent ILs in six solvents, the IL absorption spectra (3a–h)were recorded using DMSO, EtOH, CHCl3, MeOH, ACN and THFas solvents.

Each IL showed two bands in the region of 200 to 300 nm,which are attributed to their n / p* and p / p* transitions.The n / p* transition is due to the lone pair electrons ofnitrogen present in the imidazole ring and the oxygen in thealiphatic chain, while the p/ p* transition is attributed to theconjugated aromatic phenyl ring and imidazole ring and oxygenin the aliphatic chain. The p / p* transition is attributed tothe conjugated aromatic phenyl ring and the imidazole ring.The effects of different solvents on the electronic transitions ofILs can be seen from their electronic transition spectra (Fig. 2).DMSO showed the maximum absorbance or hyperchromic shias compared to other solvents, while MeOH showed the lowestabsorbance. In general, DMSO showed a red shi of z30 nm(250 nm) in comparison to MeOH (230 nm). The descendingorder of the ILs (3b–h) can be arranged on the basis of theirabsorbances as (3a): DMSO > ACN > THF > CHCl3 > EtOH >MeOH. In order of their decreasing solvent polarity, the ILs are(3b): DMSO > THF > ACN >MeOH > CHCl3 > EtOH; (3c): DMSO >ACN > CHCl3 > THF > MeOH z EtOH; (3d): DMSO > ACN >

Fig. 1 Structure of reactants (2a–h) used in the IL syntheses.

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Fig. 2 UV-Vis absorption spectra of ILs 3a and 3d.

Table 5 Antimicrobial and antifungal activities of ILs in terms of zoneof inhibition (mm)a

StrainY

Entry

Ionic liquids Standards

3a 3b 3c 3d 3e 3f S1 S2 S3

S. aureus 9 15 21 8 16 13 30 22 NAB. subtilis 9 14 19 10 12 13 26 21 NAE. coli — 8 — 8 9 — 29 21 NAP. aeruginosa 18 11 17 — 13 — 24 22 NAC. albicans 12 11 — 13 10 — NA NA 15A. niger — — — — — — NA* NA 14

a Data represent the mean of three replicates for each concentration.*Diameter in mm calculated by Vernier caliper. ‘—’ means no zone ofinhibition, ‘NA’ means not applicable. S1 ¼ chloramphenicol, S2 ¼ciprooxacin and S3 ¼ amphotericin-B.

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CHCl3 > THF >MeOH > EtOH; (3d): DMSO > ACN > CHCl3 > THF> MeOH > EtOH; (3e): CHCl3 > DMSO > ACN > EtOH > THF >MeOH; (3f): DMSO > ACN > CHCl3 > THF > MeOH > EtOH; (3g):DMSO > ACN > THFzMeOH > CHCl3 > EtOH; and (3h): DMSO> ACN > CHCl3 > THF > EtOH > MeOH. Thus, the extinctioncoefficient and lambda value of solute in solvents decrease withdecreasing polarity and dielectric constant of the solvents. Thedifferences in electronic transitions might result from thedifferent conjugation degrees and different electronic environ-ments of the ILs. The effects of different solvents on the elec-tronic transitions of ILs can be seen from their electronictransition spectra (Fig. 2); DMSO exhibited the maximumabsorbance or hyperchromic shi compared to other solvents,while MeOH showed the lowest absorbance.

Biological activities

Antibacterial. Initially, to investigate the ILs as antibacterialagents due to the presence of the ether-substituted aromaticring and nitrogen-containing ve-membered imidazole ring,the compounds were screened for their antibacterial activityagainst human pathogenic Gram-positive (S. aureus and B.subtilis) and Gram-negative (E. coli and P. aeruginosa) bacterialstrains. The compounds were screened at 100 mg mL�1

concentrations using the Agar disc diffusion method, and thezones of inhibition are illustrated in Table 5.

It is evident from the zone of inhibition data that ILs 3b, 3c,3e and 3f showed good activities against both Gram positivestrains, with zones of inhibition of 15, 21, 16 and 13mm against

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S. aureus and 14, 19, 12 and 13 mm against B. subtilis, respec-tively (Table 5). The ILs 3a and 3d displayed moderate activitiesagainst both Gram-positive strains as compared to the stan-dards chloramphenicol (30 mm) and ciprooxacin (22 mm). Onthe other hand, ILs 3b, 3d and 3e showed moderate activitiesagainst E. coli in comparison to standard drugs, while 3a, 3c and3f showed no activity against E. coli. ILs 3a and 3c showed goodactivities against P. aeruginosa, with zones of inhibition of 18and 17 mm, respectively. The ILs 3b and 3e showed moderateactivities, while 3d and 3f showed no activities against P. aeru-ginosa (Table 5).

Thus, it is clear that the different alkyl chains of the ILs seemto be responsible for their different antibacterial activitiesbecause the rest of the structures (i.e., the imidazole and phenylring) were kept constant. The ILs with two or three –CH2 chains(3b and 3d) and –CH2–O– (3c and 3e) showed good activities.The introduction of oxygen as an ether linkage in the alkyl chainincreased the antibacterial activities in comparison to a normalalkyl chain (3b versus 3c). It was also evident that increasing thealkyl chain length to four –CH2 groups lowered the activities (3bversus 3f), which was attributed to hydrophobic effects. Thus,the introduction of oxygen in the alkyl chain increased theability of ILs to irritate cell membranes or inhibit bacterialgrowth, which might be dependent on the ability of the ILs tocross the outer membrane in Gram-negative bacteria and thecell wall in Gram-positive bacteria.44

Antifungal activity. The antifungal activities of ILs weredetermined using the Agar disc diffusion method against C.albicans and A. niger at 100 mg mL�1 concentrations, and thezones of inhibition are summarised in Table 5. Compounds 3a,3b, 3d and 3e had zones of inhibition of 12, 11, 13 and 10 mm,respectively, and showed good activities against C. albicans,while all the ILs (3a–f) showed no activity against the A. nigerstrain (Table 5). The zones of inhibition were compared tostandard drug amphotericin-B, which has a 15 mm zone ofinhibition. From the results of antifungal activity, no signicanteffect of oxygen was found, whereas it showed good activitiesagainst bacterial strains.


Fig. 3 MCF-7 cell line growth as a percentage of the control versusdrug (ILs and ADR) concentration.

Table 6 In vitro testing expressed as growth inhibition of humancancer cell lines MCF-7 and MDA-MB-435 by ILsa

ILs

MCF-7 MDA-MB-435

LC50* TGI* GI50* LC50* TGI* GI50*

3a >100 >100 >100 >100 >100 67.23b >100 >100 >100 >100 >100 96.33d >100 >100 >100 >100 >100 52.53e >100 >100 >100 >100 >100 57.93g >100 >100 >100 >100 >100 >100ADR >100 60.3 <0.1 >100 70.8 24.4

a Data represent means of three replicates for each concentration DMSOwas used as solvent. *LC50¼ concentration of drug causing 50% cell kill.*TGI ¼ concentration of drug causing total inhibition of cell growth.*GI50 ¼ concentration of drug causing 50% inhibition of cell growth.*GI50 values of #0.1 mM and #24.4 mM are considered to demonstrateanticancer activity against MCF-7 and MDA-MB-435 cell lines,respectively.

Paper RSC Advances

Anticancer activity. The in vitro anticancer activities of veILs 3a, 3b, 3d, 3e and 3g were determined against the MCF-7and MDA-MB-435 cell lines at four dosage levels of 0.1, 1.0, 10and 100 mM in DMSO. A test consisted of a 48 h continuous drugexposure protocol using SRB assay to estimate cell growth.Suitable positive controls were run in every experiment. Eachexperiment was repeated in triplicate, and growth relative to thecontrol was plotted as a function of drug concentration (Fig. 3and 4) to calculate numerous parameters.

Results are given in terms of GI50 (concentration of drug thatproduces 50% inhibition of the cells), TGI (concentration of thedrug that produces total inhibition of the cells) and LC50

(concentration of the drug that kills 50% of the cells) valuescalculated from the mean graphs (Fig. 3 and 4) and are given inTable 6. Adriamycin (ADR), which is a chemotherapy drug oenused to kill cancerous cells, was used as the standard anticancerdrug. Reported parameters are given in Table 6. Thecompounds which have GI50 values of #0.1 mM and #24.4 mM

Fig. 4 MDA-MB-435 cell line growth as a percentage of the controlversus drug (ILs and ADR) concentration.


were considered to demonstrate anticancer activity againstMCF-7 and MDA-MB-435 cell lines, respectively. The GI50 ofeach IL against the MCF-7 cell line exceeded 100 mM, hence,these ILs were found to be inactive; in contrast, ADR showed abetter result (GI50 < 0.1 mM).

Furthermore, ILs were evaluated against the MDA-MB-435cell line under the same experimental conditions. The ILs 3a,3d and 3e demonstrated 50–60% activity with GI50 values of67.2, 52.5 and 57.5 mM, respectively, compared to the standardADR (GI50 ¼ 24.4 mM). The GI50 values of 3b and 3g were foundto be 96.3 and more than 100 mM, respectively, making theminactive against theMDA-MB-435 cell line. The GI50 values of ILsand ADR against the MDA-MB-435 decreased in the followingorder: ADR > 3d > 3e > 3a > 3b > 3g.

In general the LC50, which is a parameter of cytotoxicity andreects the molar concentration needed to kill 50% of the cells,was found to exceed 100 mM for the ILs as well as ADR for bothcell lines. Thus, it can be concluded that ILs 3d (with two –CH2

groups in the alkyl chain) and 3e (with an additional oxygen inthe –CH2 chain) demonstrated 50% activity, while increasingthe alkyl chain length to four –CH2 in IL 3g decreased theactivity against the MDA-MB-435 cell line. These differencesmight result from the hydrophobic effect due to the alkyl chainlength.

Conclusion

The present study reports the synthesis of imidazole-based ILsusing an MW solvent-free approach, which proved to be acompatible, efficient, eco-friendly and green synthetic route.The solvent-free synthesis is better than the solvent-free heatingmethod, having a shorter reaction time (min vs. h), better yield(82–95% yield of ILs 3a–h) and a simpler work-up. Their char-acterisation was performed using various spectral techniques.Furthermore, ILs 3b and 3d and ILs 3c and 3e possessed goodantibacterial activities against Gram-positive (S. aureus and B.subtilis) and Gram-negative (E. coli and P. Aeruginosa) strains,

RSC Adv., 2014, 4, 53634–53644 | 53639

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respectively. ILs 3a, 3b, 3d and 3e showed good activities againstthe C. albicans fungal strain, whereas none of the ILs expressedactivity against A. niger. Additionally, ILs 3a, 3d and 3e showedz50% activity against the MDA-MB-435 cell line based on theirGI50 values. The cytotoxicities based on their LC50 values werefound to be in the range of the standard drug adriamycin. Thus,the synthesised ILs could be used as catalysts, solvents, elec-trolytes and other components of chemical and medicalsciences to design novel TILs by modulating their anionic andcationic parts.11

ExperimentalMaterial and methods

Chemicals used were of analytical reagent grade and procuredfrom Sigma-Aldrich. Ethyl acetate (EA), DCM, ACN, toluene,EtOH, DMSO, cyclohexane and EtOH solvents were from Ran-kem, India and used without further purication.

The reactions were carried out using an Anton Paar Synthos3000 microwave reaction system in closed vessels. Reactionprogress was monitored by TLC (Merck, silica GF257) using anEtOH and ethyl acetate (1 : 9 v/v) solvent system, and spots werevisualised under UV light (RICO scientic industries, ModelRSUV-5). FTIR spectra were recorded with a Perkin Elmerspectrum 65 FTIR spectrometer using KBr plates, and charac-teristic wave numbers are given in cm�1. Mass spectral analysiswas accomplished with an Agilent Technologies G6520B LCMS(Q-TOF) mass spectrometer in +ESI ionisation mode. Tri-uoroacetic acid (0.02%) in water and acetonitrile–EtOH(60 : 40 v/v) was run as the mobile phase at a ratio of 30 : 70% v/v on an Agilent zorbax 300 SB-C18 column (3.5 mm, 4.6 � 50mm) with ow rate of 0.5 mL min�1. 1H and 13C NMR spectrawere recorded at room temperature (rt) in a 5 mm tube using aBruker Avance III-500 MHz spectrometer in deuterated or CDCl3and DMSO-d6, with TMS as an internal standard. The chemicalshis are given in d ppm, and coupling constant (J) is given inHz. Splitting patterns are described as singlet (s), doublet (d),triplet (t), quartet (q), quintet (q), sextet (s) and multiplet (m).The absorption transition (lmax) was recorded in EtOH at rt inthe range of 200–600 nm using an Analytical UV Spectro 2060plus. The sample was scanned using a cuvette with a pathlength of 1 cm.

Synthesis of ILs

A series of eight novel ILs (3a–h) were synthesised according toScheme 1 using 1-butylimidazole (1a) as a cationic moiety andsubstituted aryl alkyl and alkoxy bromide as anionic moieties(2a–h). Three different methods were employed: an MW-assisted solvent-free method (method A); an MW-assistedmethod using solvent (method B); and a solvent-free heatingmethod (method C).

Method (A): MW assisted solvent-free approach

An equimolar ratio of the reactants 1-butylimidazole (1a, 0.04mol) and aryl alkyl bromide (2a–h, 0.04 mol) were placed intoTeon reaction vials and reacted for 25 min at a microwave

53640 | RSC Adv., 2014, 4, 53634–53644

power of 350 W, a pressure of 20 bar and a temperature of140 �C. The reaction was continuously monitored with TLCevery 60 s until its completion. Aer completion, the ILs werewashed 4–6 times with 10 mL of EA and then dried at 60 �C for1 h on a rotary evaporator under reduced pressure. The ILs werekept under vacuum overnight to ensure the complete removal ofmoisture and EA before taking the spectroscopic measure-ments. All ILs were viscous with a light yellowish colour. Theyields and reaction times are given in Table 4.

Method (B): MW assisted synthesis with solvent approach

An equimolar ratio of 1-butylimidazole (1a, 0.04 mol) and arylalkyl bromide (2a–h, 0.04 mol) were placed into Teon reactionvials. Subsequently, 10 mL of toluene was added to each vial,and the reactions were carried out for 30 min at 300 W, 20 bar,and 120 �C. The reactions were continuously monitored withTLC until completion. Aer completion, toluene was removedby rotary evaporation, and the ILs were washed 4–6 times with10 mL of ethyl acetate. The ILs were then dried at 60 �C for 1 hon a rotary evaporator under reduced pressure. The ILs wasfurther kept in a vacuum oven overnight to ensure the completeremoval of moisture and EA before taking the spectroscopicmeasurements. The obtained ILs were viscous and lightyellowish in colour. The yields and reaction times are given inTable 4.

Method (C): heating method (solvent-free)

To a 50 mL round-bottom ask, an equimolar ratio of 1-buty-limidazole (1a, 0.04 mol) and aryl alkyl bromide (2a–h, 0.04 mol)was added, and the reaction system was reuxed for 3 h at140 �C with constant stirring. The reaction progress wascontinuously monitored at regular intervals with TLC untilcompletion. Aer completion, the newly synthesised ILs werewashed 4–6 times with 10 mL of EA. The ILs were dried at 60 �Cfor 1 h on a rotary evaporator under reduced pressure and keptin a vacuum oven overnight to ensure the complete removal ofmoisture and EA. All ILs were viscous and light yellowish incolour. The reaction times and yields of the novel ILs (3a–h)synthesised by all three methods are compared in Table 4.

3-Benzyl-1-butyl-1H-imidazol-3-ium bromide (3a)

Chemical formula: C14H19BrN2; colour: light yellowish; state:liquid; FTIR (KBr, cm�1): nmax 3133, 3070 (–CH str., –CH3); 2968,2936, 2877 (–CH str., –CH2); 1629, 1562 (–C]N, imidazole);1499, 1460, 1408 (–C]C); 1365, 1322, 1278, 1211 (C–C); 1160(C–N); 1108, 1049, 1029 (–C–H, bending); 820, 757, 714 (str.,–CH2). UV-Vis [l in nm (3 in M�1 L�1)]: DMSO [250 (3000)]; EtOH[220, 260 (2108, 190)]; THF [235, 280 (2770, 2046)]; EtOH [220(2151)]; CHCl3 [240 (2745)]; ACN [240 nm (2796)]. 1H NMR(500 MHz, DMSO-d6): d 0.89 (t, 3H, J ¼ 7.5 Hz, –CH3); 1.25 (sex,2H, J ¼ 7.5 Hz, –CH2); 1.79 (q, 2H, J ¼7.5 Hz, –CH2); 4.23 (t, 2H,J ¼ 7.0 Hz, –CH2); 5.53 (s, 2H, –CH2); 7.39–7.50 (m, 5H, Ar ring);7.92 (d, 2H, J ¼ 10.5 Hz, imidazole ring); 9.60 (s, 1H, imidazolering). 13C NMR (125 MHz, DMSO-d6): d 13.25 (–CH3), 18.75,31.26, 48.55, 55.95 (–CH2), 122.44, 122.78, (C2, C3 imidazolering), 127.99, 128.32, 128.40, 128.93, 128.95 (C2, C6, Ar ring),


Paper RSC Advances

134.95 (C1, Ar ring), 136.09 (C1, imidazole ring). +ESI-MS (m/z):calculated for C14H19N2

+ (M � Br)+ 215.1543, found 215.1515.

1-Butyl-3-phenethyl-1H-imidazol-3-ium bromide (3b)

Chemical formula: C15H21BrN2; colour: light yellowish; state:liquid. FTIR (KBr, cm�1): nmax 3137, 3062 (–CH str., –CH3); 2964,2932, 2873 (–CH str., –CH2); 1629, 1606 (–C]N, imidazole),1562, 1495, 1456 (–C]C); 1361, 1333 (C–C); 1164 (–C–N); 1085,1033, 860 (–C–H, bending); 753, 706 (C–H, –CH2). UV-Vis[l in nm (3 in M�1 L�1)]: DMSO [280, 290 (3046, 2959)]; EtOH[280 (252)]; THF [280 (2018)]; EtOH [280 (319)]; CHCl3[280 (257)]; ACN [280 (437)]. 1H NMR (500 MHz, DMSO-d6): d0.90 (t, 3H, J ¼ 7.5, –CH3); 1.26 (sex, 2H, J ¼ 7.5 Hz, –CH2); 1.77(q, 2H, J ¼ 7.5 Hz, –CH2); 2.59 (t, 2H, J ¼ 7.5 Hz, –CH2); 4.16(t, 2H, J¼ 7.0 Hz, –CH2); 4.20 (t, 2H, J¼ 7.5 Hz, –CH2); 7.31–7.19(m, 5H, Ar ring); 7.83 (d, 2H, J ¼ 12.0, imidazole ring); 9.27(s, 1H, imidazole ring). 13C NMR (125 MHz, DMSO-d6): d 13.26(–CH3); 18.76, 30.70, 31.44, 48.57 (5, –CH2); 122.42, 122.43(C2, C3, imidazole ring); 126.07 (C2, C4, C6 Ar ring); 128.30(C3, C5, Ar ring); 136.01 (C1, imidazole ring); 140.43 (C1, Ar ring).+ESI-MS (m/z): calculated for C15H21N2

+ (M � Br)+ 229.1699,found 229.1661.

1-Butyl-3-(2-phenoxyethyl)-1H-imidazol-3-ium bromide (3c)

Chemical formula: C15H21BrN2O; colour: light yellowish; state:liquid. FTIR (KBr, cm�1): nmax 3143, 3084 (–CH str., –CH3); 2958,2934, 2871 (–CH str., –CH2); 1599, 1587, 1564 (–C]N, imid-azole); 1493, 1469 (–C]C); 1387, 1359 (C–C); 1296, 1237 (–C–N);1174 (–C–O); 1083, 1056 (–C–H, bending); 914, 760, 697 (str.,–CH2). UV-Vis [l in nm (3 in M�1 L�1)]: DMSO [250, 280 (3222,2699)]; EtOH [230, 275 (2538, 2036)]; THF [235, 275 (2770,2678)]; EtOH [230, 275 (2538, 1896)]; CHCl3 [240, 275 (2796,1682)]; ACN [245, 275 (3046, 2244)]. 1H NMR (500 MHz, CDCl3):d 0.90 (t, 3H, J¼ 7.5, –CH3); 1.30 (sex, 2H, J¼ 7.5 Hz, –CH2); 1.84(q, 2H, J ¼ 7.5 Hz, –CH2); 4.85–4.30 (m, 6H, –CH2); 6.90 (d, 2H, J¼ 8.0, C2, C6, Ar ring); 6.94 (d, 2H, J¼ 7.0, C3, C5, Ar ring); 7.23 (t,1H, J ¼ 7.5, C4 Ar ring); 7.80, 7.56 (s, 2H, C2, C3 imidazole ring);9.93 (s, 1H C1, imidazole ring). 13C NMR (125 MHz, DMSO-d6): d13.32 (–CH3); 19.24, 31.86, 49.24, 49.63, 66.04 (–CH2); 114.41(C2, C6, Ar ring); 121.51 (C4, Ar ring); 122.03, 123.20 (C2, C3

imidazole ring); 129.49 (C3, C5, Ar ring); 136.52 (C1, imidazolering); 157.43 (C1, Ar ring). +ESI-MS (m/z): calculated forC15H21N2O

+ (M � Br)+ 245.1648, found 245.1618.

1-Butyl-3-(3-phenylpropyl)-1H-imidazol-3-ium bromide (3d)

Chemical formula: C16H23BrN2; colour: light yellowish; state:liquid. FTIR (KBr, cm�1): nmax 3139, 3072 (–CH str., –CH3); 2962,2934, 2871 (–CH str., –CH2); 1627, 1603, 1564 (–C]N, imidazole);1497, 1457 (–C]C); 1410, 1363, 1335 (C–C); 1166 (C–N); 1083,1032 (–C–H, bending); 855, 752, 705 (str., –CH2). UV-Vis[l in nm (3 in M�1 L�1)]: DMSO [250, 275 (3000, 1625)]; EtOH[225, 280 (1767, 998)]; THF [235, 280 (2538, 2444)]; EtOH[225, 280 (2131, 1226)]; CHCl3 [240, 275 (2745, 1470)]; ACN [240,275 (2770, 1149)]. 1H NMR (500 MHz, DMSO-d6): d 0.86 (t, 3H,–CH3); 1.10 (sex, 2H, J ¼ 7.5 Hz, –CH2); 1.68 (q, 2H, J ¼ 7.0 Hz,–CH2); 3.14 (t, 2H, J ¼ 7.0 Hz, –CH2); 4.11 (t, 2H, J ¼ 7.0, –CH2);


4.45 (t, 2H, J ¼ 7.0, –CH2); 7.16 (d, 2H, J ¼ 7.5 Hz, C2, C6 Ar ring);7.23 (t, 2H, J¼ 7.0, C3, C5 Ar ring); 7.29 (t, 1H, J¼ 7.5 Hz, Ar ring);7.78 (s, 2H, C2, C3, imidazole ring); 9.08 (s, 1H, imidazole ring).13C NMR (125 MHz, DMSO-d6): d 13.21 (–CH3); 18.54, 30.68,31.23, 35.21 (–CH2); 48.44, 49.83 (–CH2); 122.41 (C2, C3 imidazolering); 126.81 (C4, Ar ring); 128.56 (C2, C3, C5, C6, Ar ring); 135.9(C1, imidazole ring); 136.7 (C1, Ar ring). +ESI-MS (m/z): calculatedfor C16H23N2

+; (M � Br)+ 243.1831, found 243.1832.

1-Butyl-3-(3-phenoxypropyl)-1H-imidazol-3-ium bromide (3e)

Chemical formula: C16H23BrN2O; colour: light yellowish; state:liquid. FTIR (KBr, cm�1): nmax 3139, 3072 (–CH str., –CH3); 2962,2934, 2871 (–CH str., –CH2); 1631, 1599 (–C]N, imidazole); 1591,1568, 1493, 1473 (–C]C); 1390, 1335 (C–C); 1237 (C–N); 1174(–C–O, C–O–C); 1115, 1083, 1044 (–C–H, bending); 953, 823, 882,756, 693 (C–H, –CH2). UV-Vis [l in nm (3 inM�1 L�1)]: DMSO [245,275 (3000, 1442)]; EtOH [230, 275 (2409, 1216)]; THF [235, 280(2377, 2208)]; EtOH [230, 275 (2201, 1224)]; CHCl3 [245, 255(3046, 3000)]; ACN [240, 275 (2569, 1227)]. 1H NMR (500 MHz,DMSO-d6): d 0.87 (t, 3H, J ¼ 7.5 Hz, –CH3); 1.22 (sex, 2H, J ¼ 7.5Hz, –CH2); 1.74 (q, 2H, J ¼ 7.5 Hz, –CH2); 2.29 (q, 2H, J ¼ 6.5 Hz,–CH2); 4.01 (t, 2H, J¼ 6.0 Hz, –CH2); 4.16 (t, 2H, J¼ 7.0 Hz, –CH2);4.37 (t, 2H, J ¼ 7.0 Hz, –CH2); 6.89 (d, 2H, J ¼ 8.0 Hz, C2, C6 Arring); 6.94 (t, 1H, J ¼ 7.5 Hz, C4); 7.28 (t, 2H, J ¼ 7.5 Hz, C3, C5 Arring); 7.85 (d, 2H, J¼ 18.0 Hz, C2, C3, imidazole ring); 9.31 (s, 1H,imidazole ring). 13C NMR (125 MHz, DMSO-d6): d 13.24 (–CH3);18.75, 28.89, 30.96, 46.53, 48.54, 64.39 (–CH2); 114.34 (C2, C6, Arring); 120.75 (C4, Ar ring); 122.50 (C2, C3, imidazole ring); 129.47(C3, C5, Ar ring);136.16 (C1, imidazole ring); 158.11 (C1, Ar ring). +ESI-MS (m/z): -calculated for C16H23N2O

+ (M � Br)+ 259.1805, found 259.1774.

1-Butyl-3-(4-phenylbutyl)-1H-imidazol-3-ium (3f)

Chemical formula: C17H25BrN2; colour: light yellowish; state:liquid. FTIR (KBr, cm�1): nmax 3133, 3066, 3027 (–CH str., –CH3);2964, 2936, 2865 (–CH str., –CH2); 1606, 1566 (–C]N, imid-azole), 1495, 1456 (–C]C–); 1369, 1329 (C–C); 1164 (C–N); 1116,1029 (–C–H, bending); 860, 753, 702 (C–H, –CH2). UV-Vis [l innm (3 in M�1 L�1)]: DMSO [250, 275 (3000, 1625)]; EtOH [225,280 (2081, 998)]; THF [235, 280 (2538, 2444)]; EtOH [225, 270,280 (2131, 1232, 1226)]; CHCl3 [240, 275 (2745, 1470)]; ACN [240,275 (2770, 1149)]. 1H NMR (500 MHz, DMSO-d6): d 0.88 (t, 3H, J¼ 7.5 Hz, –CH3); 1.23 (sex, 2H, J¼ 7.5 Hz, –CH2); 1.52 (q, 2H, J¼7.5 Hz, –CH2); 1.858–1.746 (m, 4H, –CH2); 2.60 (t, 2H, J¼ 7.5 Hz,–CH2); 4.25–4.18 (m, 4H, –CH2); 7.28–7.15 (m, 5H, C2–C6 Arring); 7.87 (s, 2H, C2, C3, imidazole ring); 9.41 (s, 1H, imidazolering). 13C NMR (125 MHz, DMSO-d6): d 13.24 (–CH3); 18.74,27.41, 28.99, 31.25, 34.29, 48.54, 55.97 (–CH2); 119.36, 122.44(C2, C3, imidazole ring); 125.78 (C4, Ar ring); 128.24 (C2, C3, C5,C6, Ar ring); 135.96 (C1, imidazole ring); 141.57 (C1, Ar ring).+ESI-MS (m/z): calculated for C17H25N2

+ (M � Br)+ 257.2012,found 257.1996.

1-Butyl-3-(4-phenoxybutyl)-1H-imidazol-3-ium bromide (3g)

Chemical formula: C17H25BrN2O; colour: light yellowish; state:liquid. FTIR (KBr, cm�1): nmax 3139, 3076 (–CH str., –CH3); 2962,

RSC Adv., 2014, 4, 53634–53644 | 53641

RSC Advances Paper

2934, 2875 (–CH str., –CH2); 1627, 1599 (–C]N, imidazole);1587, 1564, 1493, 1473 (–C]C); 1390, 1335 (C–C); 1296 (–C–N);1245, 1166 (–C–O, C–O–C); 1119, 1083, 1032 (–C–H, bending);760, 693 (C–H, –CH2). UV-Vis [l in nm (3 in M�1 L�1)]: DMSO[250 (3000)]; EtOH [225 (2032)]; THF [235, 280 (2721, 2569)];EtOH [240 (2721)]; CHCl3 [225 (2092)]; ACN [240 (2770)]. 1HNMR (500 MHz, CDCl3): d 0.9 (t, 3H, J¼ 7.5 Hz, –CH3); 1.25 (sex,2H, J¼ 7.5 Hz, –CH2); 1.70 (q, 2H, J¼ 6.5 Hz, –CH2); 1.80 (q, 2H,J ¼ 7.5 Hz, –CH2); 1.97 (q, 2H, J ¼ Hz, –CH2); 3.99 (t, 2H, J ¼ 6.0,–CH2); 4.17 (t, 2H, J¼ 7.0, –CH2); 4.26 (t, 2H, J¼ 7.0, –CH2); 6.93(t, 3H, J¼ 7.5 Hz, C2, C4, C6 Ar ring); 7.29 (t, 2H, J¼ 8.0, C3, C5 Arring); 7.84 (d, 2H, J ¼ 10.0, imidazole ring); 9.28 (s, 1H, imid-azole ring). 13C NMR (125 MHz, DMSo-d6): d 18.50 (–CH3); 24.02,30.59, 31.55, 35.93, 36.49, 53.81, 71.76 (–CH2); 119.61 (C2, C6, Arring); 125.77 (C2, C3, imidazole ring and C4, Ar ring); 127.64 (C3,C5, Ar ring); 134.70 (C1, imidazole ring); 163.63 (C1, Ar ring).+ESI-MS (m/z): calculated for C17H25N2O

+ (M � Br)+ 273.1961,found 273. 1943.

1-Butyl-3-(6-phenylhexyl)-1H-imidazol-3-ium bromide (3h)

Chemical formula: C19H29BrN2O; colour: light yellowish; state:liquid. FTIR (KBr, cm�1): nmax 3137, 3073 (–CH str., –CH3); 2940,2864 (–CH str., –CH2); 2469, 2059 (–CH2, bending); 1605, 1601,1585, 1564 (–C]N, imidazole); 1496, 1468 (–C]C); 1387, 1335(C–C); 1299, 1247 (C–N); 1170 (–C–O–C–); 1118, 1082, 1033 (–C–H, bending); 885, 756, 695 (str., –CH2). UV-Vis [l in nm (3 in M�1

L�1)]: DMSO [245, 255, 275 (2959, 2921, 1411)]; EtOH [225, 225(2137, 1031)]; THF [235, 275 (2658, 2469)]; EtOH [225, 275 (2119,1164)]; CHCl3 [240, 275 (2699, 1091)]; ACN [240, 275 (2721,1164)]; 1H NMR (500 MHz, DMSO-d6): d 0.9 (t, 3H, J ¼ 7.5 Hz,–CH3), 1.26 (sex, 4H, J ¼ 7.0 Hz, –CH2); 1.43 (d, 2H, J ¼ 7.0 Hz,–CH2); 1.69–1.83 (m, 6H, –CH2); 3.93 (t, 2H, J ¼ 6.0, –CH2); 4.20(d, 4H, J ¼ 7.0 Hz, –CH2); 6.90 (t, 3H, J ¼ 3.0 Hz, C2, C4, C6 Arring); 7.27 (t, 2H, J ¼ 6.0 Hz, C3, C5 Ar ring); 7.88 (s, 2H, C2, C3,imidazole ring); 9.42 (s, 1H, imidazole ring). 13C NMR (125MHz, DMSO-d6): d13.23 (–CH3); 18.74, 24.86, 25.20, 28.40, 29.22,31.27, 48.61, 55.96, 67.03 (–CH2); 114.32 (C2, C6, Ar ring); 120.31(C4, Ar ring); 122.41 (C2, C3, imidazole ring); 129.41 (C3, C5, Arring); 135.95 (C1, imidazole ring); 158.55 (C1, Ar ring). +ESI-MS(m/z): calculated for C19H29N2O

+ (M � Br)+ 301.2274, found301.2252.

Antibacterial activity

The synthesised ILs 3a–f were evaluated against the humanpathogenic Gram-positive Staphylococcus aureus (NCIM 2079),Bacillus subtilis (NCIM 2250) and Gram-negative Escherichia coli(NCIM 2109) and Pseudomonas aeruginosa (NCIM 2036) bacte-rial strains using the Agar disc diffusion method.45 Stock solu-tions (1000 mg mL�1) of each compound were prepared inDMSO. Assays were carried out with concentrations of 100 mgmL�1 using HiMedia antibiotic disks and chloramphenicol andciprooxacin as reference drugs. The nutrient agar microbio-logical medium used for Gram-positive and Gram-negativestrains was obtained from HiMedia (India) and had thefollowing composition (in grams per litre): sodium chloride,5.0; beef extract, 10.0; and peptone, 10.0 (pH 7.2). The zones of

53642 | RSC Adv., 2014, 4, 53634–53644

inhibition were measured in millimetres (mm) for ILs (3a–f)aer 24 h of incubation at 37 �C and pH 7.2.

Antifungal activity

The synthesised ILs were screened against the human patho-genic fungal strains Candida albicans (NCIM 3471) and Asper-gillus niger (NCIM 545) using the Agar disc diffusion method at100 mg mL�1.45 Potato dextrose agar (HiMedia, India) was usedas the medium for A. niger and had the following composition(grams per liter): potato infusion, 200.0; and dextrose, 20.0 (pH5.2). The microbiological medium for C. albicans was MGYP (allingredients from HiMedia) with the following composition(grams per litre): malt extract, 3.0; glucose, 10.0; yeast extract,3.0; and peptone, 5.0 (pH 6.4). The zones of inhibition weremeasured in millimetres (mm) for 3a–f aer 24 h of incubationat 37 �C. Amphotericin-B was used as a standard drug tocompare the zones of inhibitions. The samples were prepared inDMSO as a solvent.

Anticancer activity

The in vitro anticancer activities of ILs 3a, 3b, 3d, 3e, 3g weredetermined for the human malignant breast cancer cell linesMCF-7 and MDA-MB-435 at four dose levels (0.1, 1.0, 10 and 100mM) in DMSO. The test consisted of a 48 h continuous drugcontact protocol using sulforhodamine B (SRB) assay to esti-mate cell growth. The experimental procedure followed the NCISRB assay protocols.46 Briey, this assay relies on the uptake ofthe negatively charged pink aminoxanthine dye andsulforhodamine-B (SRB) by basic amino acids in the cells. Agreater number of cells corresponds to a greater amount of dyebeing taken up, and aer xing, when the cells are lysed, thereleased dye exhibits greater absorbance. The SRB assay wasfound to be more dependable, sensitive, simple, reproducibleand rapid than the formazan-based assays and gives the bestresults.47 Appropriate positive controls were run in each exper-iment, and each experiment was repeated in triplicate. The cellgrowth as a percentage of the control was plotted against drugconcentration (Fig. 2 and 3) to calculate various parameters. Theresults are given in terms of GI50 (concentration of drug thatproduces 50% inhibition of the cells), TGI (concentration of thedrug that produces total inhibition of the cells) and LC50

(concentration of the drug that kills 50% of the cells) valuescalculated from the plots. The results of anticancer activities aregiven in Table 6.

Acknowledgements

The authors are thankful to the Central University of Gujarat,Gandhinagar, for nancial and infrastructural support andinstrumental facilities. They also thank Dr A. Juvekar, ACTRECKharghar, Navi Mumbai for anticancer activity tests and DrUlhaas K. Patil for antibacterial tests.

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