1-Butylimidazole-derived ionic liquids: synthesis, characterisation and evaluation of their antibacterial, antifungal and anticancer activities

Abstract

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 ≈82–95% 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 μM, respectively, as compared to standard adriamycin (GI₅₀ 24.4 μM).

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Introduction

Ionic liquids (ILs) are fused salts constituting cation and anion moieties.¹ They possess some fundamental properties such as negligible vapour pressure, thermal stability up to 300 °C, non-flammability, high ionic conductivity,^2–4 wide electrochemical stability window^2,3 and high polarity, enabling wide kinetic control and immiscibility with numerous organic solvents.⁵ For the last few decades, ILs have been recognised as “designer solvents”^6–8 because their physical, chemical and biological properties can be tuned to obtain desired task-specific ionic liquids (TILs) for a multitude of applications.^9–12 These unique properties of ILs make them widely applicable in different domains of chemistry; particularly, they are excellent candidates for use in “green synthesis” as green solvents.^13–15

Despite the promising results evidenced by the many studies in which they have been used as solvents or catalysts for chemical synthesis,^16,17 as electrolytes for electrochemical devices,¹⁸ as super capacitors^19,20 and in engineering and physical chemistry,^21,22 their widespread application is still hampered by doubts related to some practical drawbacks: (i) cost and possible toxicological concerns; (ii) problems related to product isolation; and (iii) catalyst recovery. Recently, to overcome at least some of these drawbacks, ether-functionalised ILs, the so-called TSILs, have been synthesised.^23,24 For the past decade, numerous green routes including reaction under solvent-free conditions²⁵ using non-classical techniques such as ultrasonication,²⁶ microwaves and others^27,28 have been adopted for the synthesis of ILs. Microwave (MW)-assisted synthesis has been used to develop a rapid green synthetic route, resulting in large reductions in reaction time (from hours to minutes), uniform temperature and pressure during the course of the reaction and better yields.²⁹ Additionally, the biological activities of several ILs have been investigated. The literature reveals that many synthetic ILs and their analogues display a wide spectrum of biological activities including antibacterial,^30,31 anticancer,^32,33 antifungal and others.^34–36 For this reason, they are an object of continuously growing interest in academia as well as in industry. ILs in the environment may also harm aquatic ecosystems and living organisms due to the lack of toxicity data for designed ILs or ILs that have yet to be designed.³⁷ Thus, the toxicity data of ILs must be investigated to determine their biological and environmental impacts.^38,39

In general, earlier studies reveal that the biological activity of an IL depends on the alkyl chain length of its cationic head group, the functional groups present in their chain and elements (nitrogen, sulphur, and oxygen) present in the ring.^40,41 Thus, structural modification can enhance their potency and SAR (structural activity relationship).⁴²

Considering the advantages of the MW solvent-free approach and continuing our investigations on new methodologies for the synthesis of new heterocyclic ring-bearing compounds,⁴³ we report herein an efficient and practical procedure for the synthesis of a series of 1-butylimidazole-based ILs. Additionally, the potential biological activities (i.e., antibacterial, antifungal and anticancer) of ILs against a panel of microorganisms and cell lines have been investigated.

Results and discussion

Synthesis and analytical characterisations

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

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

Table 1 Optimisation of reaction conditions under MW solvent-free conditions

Entry	Power (W)	Temp. (°C)	Time (min)	Yield (%)
1	200	100	25	77
2	250	100	25	79
3	300	100	25	84
4	350	100	25	87
5	400	100	25	87
6	350	120	25	88
7	350	140	25	90
8	350	160	25	91

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Table 2 Optimisation of reaction conditions under MW with solvents

Entry	Solvent	Power (W)	Temp (°C)	Time (min)	Yields (%)
1	ACN	300	100	25	76
2	Toluene	300	100	25	80
3	EtOH	300	100	25	75
4	MeOH	300	100	25	70
5	DCM	300	100	25	—
6	Toluene	350	100	25	81
7	Toluene	300	120	25	84
8	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	Reflux	—	5	70
2	100 °C	Reflux	—	4	69
3	100 °C	Reflux	—	3	68
4	100 °C	Reflux	—	2	62
5	120 °C	Reflux	—	4	73
6	120 °C	Reflux	—	3	72
7	120 °C	Reflux	—	2	69
8	140 °C	Reflux	—	3	84
10	160 °C	Reflux	—	3	85

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Table 4 Comparison between MW methods and the heating method

ILs	MW solvent-free		MW with solvent		Heating method (solvent free)
	Time (min)	Yield (%)	Time (min)	Yield (%)	Time (h)	Yield (%)
3a	25	92	30	85	3	82
3b	25	85	30	84	3	79
3c	25	90	30	85	3	84
3d	25	84	30	74	3	81
3e	25	93	30	79	3	85
3f	25	86	30	82	3	83
3g	25	82	30	79	3	81
3h	25	95	30	78	3	87

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Optimisation of reaction conditions

Initially, the reaction between equimolar amounts of 1-butylimidazole (1a) and (2-bromoethoxy)benzene (2c) to deliver product (3c) was considered as a model reaction to screen the reaction conditions for methods A–C, which have been discussed 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 reaction was completed in 25 min and produced a 77% yield of 3c (Table 1, entry 1). To further optimise the MW power, the reaction 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, no improvement in yield was found when power was increased from 350 to 400 W. Thus, 350 W was chosen as the optimum MW power. Next, temperature was optimised by conducting the model reaction at 120 °C, 140 °C and 160 °C; the quaternisation yield was increased to 88%, 90% and 91%, respectively (Table 1, entries 6–8). On increasing the temperature from 140 °C to 160 °C, no better yield was noted. Thus, the suitable reaction conditions for the model reaction were a MW power of 350 W and a temperature of 140 °C. The scope and versatility of the optimised conditions under the MW solvent-free approach were studied by preparing a series of eight new ILs (3a–h) using differently substituted alkyl and alkoxy bromide (2a–h). It was found that ILs having oxygen in their alkyl chain produced better 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 phenyl ring. The structures of ILs were confirmed using various spectroscopic techniques.

Method B: MW with solvent approach

In this method, to study the effect of solvent on reaction conditions such as MW power, temperature and yield, the model reaction was conducted using acetonitrile (ACN), toluene, ethanol (EtOH), methanol (MeOH) and dichloromethane (DCM) as solvents at 100 °C and an MW power of 300 W. After 25 min of reaction, 3c was produced with yields of 76, 80, 75 and 70% for ACN, toluene, EtOH and MeOH, respectively (Table 2, entries 1–4). The reaction did not proceed when DCM was used as a solvent (Table 2, entry 5).

We found that 300 W and 100 °C using toluene as a solvent produced a higher yield of 3c (80%; Table 2, entry 2) compared to other solvents. Thus, further reaction conditions were optimised considering toluene as the solvent. The reaction carried out 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, entry 7). The reaction conducted at 250 W and 120 °C gave a 77% yield (Table 2, entry 8), which was lower than that for a power of 300 W. Considering these results, the optimised conditions used to synthesise a series of ILs (3a–h) were as follows: toluene as 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 refluxed at temperatures of 100 °C, 120 °C, 140 °C and 160 °C with constant stirring at times varying from 2 to 5 h. To optimise the reaction duration, the reaction mixture was refluxed at 100 °C for 5, 4, 3 and 2 h, resulting in 70, 69, 68, and 62% yields, respectively (Table 3, entries 1–4). Furthermore, the reaction mixture was refluxed 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 were observed to increase the yield of 3c, whereas the yield of 3c was decreased by shortening the reaction duration. Interestingly, the reactions carried out at temperatures of 100 °C and 120 °C for 3 h had good yields compared to other reaction times; increases in yield of about 1% or 2% with respect to the other reaction times at the same temperatures (100 °C or 120 °C) were found. Thus, a reaction duration of 3 h was chosen as optimal for this method. After optimising the reaction duration, the reaction temperature was optimised by performing the reaction at 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 for the same period of time. Thus, the optimal reaction conditions for the model reaction using the solvent free heating method were a temperature of 140 °C and a reaction time of 3 h. The scope and versatility of the solvent-free heating method under the optimised conditions were studied by preparing a series of eight new ILs (3a–h) using differently substituted alkyl bromides (2a–h).

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

It was found that higher MW irradiation power did not increase the product yield. Furthermore, an improvement in yield was observed when the reaction took place in the absence of solvent under MW. Therefore, the MW solvent-free method has been standardised and offers a simplification of laboratory techniques as it does not require the use of stirrers, reflux condensers, water separators or noxious solvents. The identities of the ILs synthesised by the heating and MW-induced methods were established by their co-TLC and superimposable IR spectra. For each method, the IL (3c) was confirmed using proton NMR, LCMS, and FTIR data.

ILs derived from imidazole may have varied structures depending on the nature of the cationic and anionic parts. In the present work, the cationic parts of the ILs consist of 1-butylimidazole and were kept fixed, while the anionic parts were varied by using alkyl bromide chains of different lengths. The purpose behind the synthesis of imidazole-based ILs with varying structures is to understand the nature of these novel solvents. The prevailing methods of IL preparation with imidazole compounds such as 1,3-disubstituted imidazolium halides generally require longer reaction times and result in considerable contamination with halide ions (X = Br⁻, Cl⁻, I⁻, F⁻). Additional steps are also required to carry out the quaternisation that converts the halide-containing precursors into ILs. The purification of ILs is a matter of foremost concern as impurities have been found to significantly affect the biological and physicochemical properties of these ILs, which in turn regulate their domain of application.^23,24

The structures of the synthesised ILs (3a–h) were confirmed with ¹H NMR, ¹³C NMR, FTIR and LCMS (see ESI†). The quaternisation reactions of 1-butylimidazole with different alkyl halides were established by their FTIR spectra; the presence of –C–N bond stretching in region of 1120 to 1290 cm⁻¹ is due to the formation of the quaternary nitrogen of the imidazole ring and oxygen in the aliphatic chain, while peaks at 1630, 1587, 1560 cm⁻¹ are due to the –C=N bond of the five-membered imidazolium ring. The C–N is experiencing an addition that requires an electron of oxygen, thus affecting the stretching of the –C=N bond. Additionally, the peaks at 1497, 1457, 1406 cm⁻¹ are due to the –C=C– bond of phenyl and imidazolium rings. 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 and 3070 to 3134 cm⁻¹ regions, respectively, of all ILs. Positive mode ESI-MS spectra of the synthesised ILs (3a–h) exhibited [M − Br]⁺ molecular ion peaks, confirming their molecular masses. In the ¹H NMR spectra of ILs (3a, 3b, 3d, 3e and 3g), two methylenic protons of the imidazole ring appeared as a doublet from δ 7.70 to 7.92 ppm with a coupling constant of J = 9–12 Hz, while in the ILs (3c, 3f and 3h), the methylenic protons appeared as a singlet. The single proton of the imidazole ring appeared as a singlet in the downfield in the range of δ 9.0 to 9.9 ppm. The differences in the proton shifting of the imidazole ring could be attributed to the alkyl chain lengths in the ILs. For ILs (3a–h), the five aromatic protons appeared as a multiplet in the desired aromatic range. The 9H protons of the butyl chain in all ILs (3a–h) as –CH₃CH₂CH₂CH₂– attached to the imidazole ring were observed in the upper field, where the terminal 3H proton of –CH₃ appeared as a triplet in the region of δ 0.90 ppm with coupling constant J = 7.5 Hz. The 2H of –CH₃CH₂CH₂CH₂– appeared as a sextet at δ 1.10 to 1.30 ppm (J = 7.5 Hz), the 2H of CH₃CH₂CH₂CH₂– as a quintet at δ 1.52 to 1.84 ppm (J = 7.5 Hz) and the 2H of CH₃CH₂CH₂CH₂– as a triplet at δ 3.93 to 4.30 ppm (J = 7.5 Hz) respectively. The –CH₂ protons of alkyl chains attached to quaternary nitrogen atoms of imidazole rings appeared in the range of δ 1.0 to 4.9 ppm as per their chemical shift in the standard splitting pattern as triplet and quintet.

The ¹³C NMR spectra provided final structural elucidation of ILs (3a–h). Signals due to the formation of quaternary nitrogen atoms in 1-butylimidazolium-based ILs were found in the range of δ 48.50 ppm to 55.96 ppm. Thus, the signals for the ILs (3f) and (3h) appeared in the downfield at δ 55.97 ppm, while those of ILs (3b) and (3e) appeared in the upper field at δ 48.57 ppm and δ 48.54 ppm, respectively. The two carbons (–CH=CH–) of the imidazole ring in ILs were found from δ 119.0 ppm to 125.0 ppm, while the third carbon of –N–C=N– appeared in the range of δ 134.70 ppm to 136.57 ppm. In general, the aromatic phenyl ring carbons appeared in the range of 114.4 ppm to 131.0 ppm, while the alkyl-substituted carbon of the phenyl ring appeared in the downfield from δ 134.0 to 163.0 ppm (Fig. 1).

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

The UV-Vis spectra were exclusively recorded for the eight different ILs using DMSO, THF, ACN, MeOH, EtOH, CHCl₃ as solvents. For each solvent, 1 mM of IL was used to determine the electronic transitions.⁴³ The experiments were carried out at rt for ILs (3a–h) in the range of 200 to 600 nm, and the spectra are illustrated in Fig. 2 (see ESI† for the absorption spectra of 3b, 3c, and 3e–h). The extinction coefficient (ε), which is the characteristic molecular property of ILs and reflects their different electronic transitions, was calculated for each band. The quantitative relationships between absorbance, extinction coefficient, concentration and path length are given by the Beer–Lambert law. To study the absorption spectra of the eight different ILs in six solvents, the IL absorption spectra (3a–h) were recorded using DMSO, EtOH, CHCl₃, MeOH, ACN and THF as solvents.

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

Each IL showed two bands in the region of 200 to 300 nm, which are attributed to their n → π* and π → π* transitions. The n → π* transition is due to the lone pair electrons of nitrogen present in the imidazole ring and the oxygen in the aliphatic chain, while the π → π* transition is attributed to the conjugated aromatic phenyl ring and imidazole ring and oxygen in the aliphatic chain. The π → π* transition is attributed to the conjugated aromatic phenyl ring and the imidazole ring. The effects of different solvents on the electronic transitions of ILs can be seen from their electronic transition spectra (Fig. 2). DMSO showed the maximum absorbance or hyperchromic shift as compared to other solvents, while MeOH showed the lowest absorbance. In general, DMSO showed a red shift of ≈30 nm (250 nm) in comparison to MeOH (230 nm). The descending order of the ILs (3b–h) can be arranged on the basis of their absorbances 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 ≈ EtOH; (3d): DMSO > ACN > 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 > THF ≈ MeOH > CHCl₃ > EtOH; and (3h): DMSO > ACN > CHCl₃ > THF > EtOH > MeOH. Thus, the extinction coefficient and lambda value of solute in solvents decrease with decreasing polarity and dielectric constant of the solvents. The differences in electronic transitions might result from the different conjugation degrees and different electronic environments of the ILs. The effects of different solvents on the electronic transitions of ILs can be seen from their electronic transition spectra (Fig. 2); DMSO exhibited the maximum absorbance or hyperchromic shift compared to other solvents, while MeOH showed the lowest absorbance.

Biological activities

Antibacterial. Initially, to investigate the ILs as antibacterial agents due to the presence of the ether-substituted aromatic ring and nitrogen-containing five-membered imidazole ring, the compounds were screened for their antibacterial activity against human pathogenic Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) bacterial strains. The compounds were screened at 100 μg mL⁻¹ concentrations using the Agar disc diffusion method, and the zones of inhibition are illustrated in Table 5.

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

Strain↓	Entry
	Ionic liquids						Standards
	3a	3b	3c	3d	3e	3f	S1	S2	S3
a Data represent the mean of three replicates for each concentration. *Diameter in mm calculated by Vernier caliper. ‘—’ means no zone of inhibition, ‘NA’ means not applicable. S1 = chloramphenicol, S2 = ciprofloxacin and S3 = amphotericin-B.
S. aureus	9	15	21	8	16	13	30	22	NA
B. subtilis	9	14	19	10	12	13	26	21	NA
E. coli	—	8	—	8	9	—	29	21	NA
P. aeruginosa	18	11	17	—	13	—	24	22	NA
C. albicans	12	11	—	13	10	—	NA	NA	15
A. niger	—	—	—	—	—	—	NA^*	NA	14

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It is evident from the zone of inhibition data that ILs 3b, 3c, 3e and 3f showed good activities against both Gram positive strains, with zones of inhibition of 15, 21, 16 and 13 mm against S. aureus and 14, 19, 12 and 13 mm against B. subtilis, respectively (Table 5). The ILs 3a and 3d displayed moderate activities against both Gram-positive strains as compared to the standards chloramphenicol (30 mm) and ciprofloxacin (22 mm). On the other hand, ILs 3b, 3d and 3e showed moderate activities against E. coli in comparison to standard drugs, while 3a, 3c and 3f showed no activity against E. coli. ILs 3a and 3c showed good activities against P. aeruginosa, with zones of inhibition of 18 and 17 mm, respectively. The ILs 3b and 3e showed moderate activities, while 3d and 3f showed no activities against P. aeruginosa (Table 5).

Thus, it is clear that the different alkyl chains of the ILs seem to be responsible for their different antibacterial activities because the rest of the structures (i.e., the imidazole and phenyl ring) 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 chain increased the antibacterial activities in comparison to a normal alkyl chain (3b versus 3c). It was also evident that increasing the alkyl chain length to four –CH₂ groups lowered the activities (3b versus 3f), which was attributed to hydrophobic effects. Thus, the introduction of oxygen in the alkyl chain increased the ability of ILs to irritate cell membranes or inhibit bacterial growth, which might be dependent on the ability of the ILs to cross the outer membrane in Gram-negative bacteria and the cell wall in Gram-positive bacteria.⁴⁴

Antifungal activity. The antifungal activities of ILs were determined using the Agar disc diffusion method against C. albicans and A. niger at 100 μg mL⁻¹ concentrations, and the zones 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. niger strain (Table 5). The zones of inhibition were compared to standard drug amphotericin-B, which has a 15 mm zone of inhibition. From the results of antifungal activity, no significant effect of oxygen was found, whereas it showed good activities against bacterial strains.

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

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

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

Results are given in terms of GI₅₀ (concentration of drug that produces 50% inhibition of the cells), TGI (concentration of the drug that produces total inhibition of the cells) and LC₅₀ (concentration of the drug that kills 50% of the cells) values calculated from the mean graphs (Fig. 3 and 4) and are given in Table 6. Adriamycin (ADR), which is a chemotherapy drug often used to kill cancerous cells, was used as the standard anticancer drug. Reported parameters are given in Table 6. The compounds which have GI₅₀ values of ≤0.1 μM and ≤24.4 μM were considered to demonstrate anticancer activity against MCF-7 and MDA-MB-435 cell lines, respectively. The GI₅₀ of each IL against the MCF-7 cell line exceeded 100 μM, hence, these ILs were found to be inactive; in contrast, ADR showed a better result (GI₅₀ < 0.1 μM).

Table 6 In vitro testing expressed as growth inhibition of human cancer cell lines MCF-7 and MDA-MB-435 by ILs^a

ILs	MCF-7			MDA-MB-435
	LC50*	TGI*	GI50*	LC50*	TGI*	GI50*
a Data represent means of three replicates for each concentration DMSO was used as solvent. *LC50 = concentration of drug causing 50% cell kill. *TGI = concentration of drug causing total inhibition of cell growth. *GI50 = concentration of drug causing 50% inhibition of cell growth. *GI50 values of ≤0.1 μM and ≤24.4 μM are considered to demonstrate anticancer activity against MCF-7 and MDA-MB-435 cell lines, respectively.
3a	>100	>100	>100	>100	>100	67.2
3b	>100	>100	>100	>100	>100	96.3
3d	>100	>100	>100	>100	>100	52.5
3e	>100	>100	>100	>100	>100	57.9
3g	>100	>100	>100	>100	>100	>100
ADR	>100	60.3	<0.1	>100	70.8	24.4

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Furthermore, ILs were evaluated against the MDA-MB-435 cell line under the same experimental conditions. The ILs 3a, 3d and 3e demonstrated 50–60% activity with GI₅₀ values of 67.2, 52.5 and 57.5 μM, respectively, compared to the standard ADR (GI₅₀ = 24.4 μM). The GI₅₀ values of 3b and 3g were found to be 96.3 and more than 100 μM, respectively, making them inactive against the MDA-MB-435 cell line. The GI₅₀ values of ILs and ADR against the MDA-MB-435 decreased in the following order: ADR > 3d > 3e > 3a > 3b > 3g.

In general the LC₅₀, which is a parameter of cytotoxicity and reflects the molar concentration needed to kill 50% of the cells, was found to exceed 100 μM for the ILs as well as ADR for both cell lines. Thus, it can be concluded that ILs 3d (with two –CH₂ groups in the alkyl chain) and 3e (with an additional oxygen in the –CH₂ chain) demonstrated 50% activity, while increasing the alkyl chain length to four –CH₂ in IL 3g decreased the activity against the MDA-MB-435 cell line. These differences might result from the hydrophobic effect due to the alkyl chain length.

Conclusion

The present study reports the synthesis of imidazole-based ILs using an MW solvent-free approach, which proved to be a compatible, efficient, eco-friendly and green synthetic route. The solvent-free synthesis is better than the solvent-free heating method, having a shorter reaction time (min vs. h), better yield (82–95% yield of ILs 3a–h) and a simpler work-up. Their characterisation was performed using various spectral techniques. Furthermore, ILs 3b and 3d and ILs 3c and 3e possessed good antibacterial activities against Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. Aeruginosa) strains, respectively. ILs 3a, 3b, 3d and 3e showed good activities against the C. albicans fungal strain, whereas none of the ILs expressed activity against A. niger. Additionally, ILs 3a, 3d and 3e showed ≈50% activity against the MDA-MB-435 cell line based on their GI₅₀ values. The cytotoxicities based on their LC₅₀ values were found to be in the range of the standard drug adriamycin. Thus, the synthesised ILs could be used as catalysts, solvents, electrolytes and other components of chemical and medical sciences to design novel TILs by modulating their anionic and cationic parts.¹¹

Experimental

Material and methods

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

The reactions were carried out using an Anton Paar Synthos 3000 microwave reaction system in closed vessels. Reaction progress was monitored by TLC (Merck, silica GF257) using an EtOH and ethyl acetate (1 : 9 v/v) solvent system, and spots were visualised under UV light (RICO scientific industries, Model RSUV-5). FTIR spectra were recorded with a Perkin Elmer spectrum 65 FTIR spectrometer using KBr plates, and characteristic wave numbers are given in cm⁻¹. Mass spectral analysis was accomplished with an Agilent Technologies G6520B LCMS (Q-TOF) mass spectrometer in +ESI ionisation mode. Trifluoroacetic 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 × 50 mm) with flow rate of 0.5 mL min⁻¹. ¹H and ¹³C NMR spectra were recorded at room temperature (rt) in a 5 mm tube using a Bruker Avance III-500 MHz spectrometer in deuterated or CDCl₃ and DMSO-d₆, with TMS as an internal standard. The chemical shifts are given in δ ppm, and coupling constant (J) is given in Hz. Splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), quintet (q), sextet (s) and multiplet (m). The absorption transition (λ_max) was recorded in EtOH at rt in the range of 200–600 nm using an Analytical UV Spectro 2060 plus. The sample was scanned using a cuvette with a path length of 1 cm.

Synthesis of ILs

A series of eight novel ILs (3a–h) were synthesised according to Scheme 1 using 1-butylimidazole (1a) as a cationic moiety and substituted 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-assisted method using solvent (method B); and a solvent-free heating method (method C).

Method (A): MW assisted solvent-free approach

An equimolar ratio of the reactants 1-butylimidazole (1a, 0.04 mol) and aryl alkyl bromide (2a–h, 0.04 mol) were placed into Teflon reaction vials and reacted for 25 min at a microwave power of 350 W, a pressure of 20 bar and a temperature of 140 °C. The reaction was continuously monitored with TLC every 60 s until its completion. After completion, the ILs were washed 4–6 times with 10 mL of EA and then dried at 60 °C for 1 h on a rotary evaporator under reduced pressure. The ILs were kept under vacuum overnight to ensure the complete removal of moisture and EA before taking the spectroscopic measurements. All ILs were viscous with a light yellowish colour. The yields 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 aryl alkyl bromide (2a–h, 0.04 mol) were placed into Teflon reaction vials. 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 with TLC until completion. After completion, toluene was removed by rotary evaporation, and the ILs were washed 4–6 times with 10 mL of ethyl acetate. The ILs were then dried at 60 °C for 1 h on a rotary evaporator under reduced pressure. The ILs was further kept in a vacuum oven overnight to ensure the complete removal of moisture and EA before taking the spectroscopic measurements. The obtained ILs were viscous and light yellowish in colour. The yields and reaction times are given in Table 4.

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

To a 50 mL round-bottom flask, an equimolar ratio of 1-butylimidazole (1a, 0.04 mol) and aryl alkyl bromide (2a–h, 0.04 mol) was added, and the reaction system was refluxed for 3 h at 140 °C with constant stirring. The reaction progress was continuously monitored at regular intervals with TLC until completion. After completion, the newly synthesised ILs were washed 4–6 times with 10 mL of EA. The ILs were dried at 60 °C for 1 h on a rotary evaporator under reduced pressure and kept in a vacuum oven overnight to ensure the complete removal of moisture and EA. All ILs were viscous and light yellowish in colour. 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): ν_max 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 [λ in nm (ε 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): δ 0.89 (t, 3H, J = 7.5 Hz, –CH₃); 1.25 (sex, 2H, J = 7.5 Hz, –CH₂); 1.79 (q, 2H, J =7.5 Hz, –CH₂); 4.23 (t, 2H, J = 7.0 Hz, –CH₂); 5.53 (s, 2H, –CH₂); 7.39–7.50 (m, 5H, Ar ring); 7.92 (d, 2H, J = 10.5 Hz, imidazole ring); 9.60 (s, 1H, imidazole ring). ¹³C NMR (125 MHz, DMSO-d₆): δ 13.25 (–CH₃), 18.75, 31.26, 48.55, 55.95 (–CH₂), 122.44, 122.78, (C₂, C₃ imidazole ring), 127.99, 128.32, 128.40, 128.93, 128.95 (C₂, C₆, Ar ring), 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): ν_max 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 [λ in nm (ε 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): δ 0.90 (t, 3H, J = 7.5, –CH₃); 1.26 (sex, 2H, J = 7.5 Hz, –CH₂); 1.77 (q, 2H, J = 7.5 Hz, –CH₂); 2.59 (t, 2H, J = 7.5 Hz, –CH₂); 4.16 (t, 2H, J = 7.0 Hz, –CH₂); 4.20 (t, 2H, J = 7.5 Hz, –CH₂); 7.31–7.19 (m, 5H, Ar ring); 7.83 (d, 2H, J = 12.0, imidazole ring); 9.27 (s, 1H, imidazole ring). ¹³C NMR (125 MHz, DMSO-d₆): δ 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): ν_max 3143, 3084 (–CH str., –CH₃); 2958, 2934, 2871 (–CH str., –CH₂); 1599, 1587, 1564 (–C=N, imidazole); 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 [λ in nm (ε 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): δ 0.90 (t, 3H, J = 7.5, –CH₃); 1.30 (sex, 2H, J = 7.5 Hz, –CH₂); 1.84 (q, 2H, J = 7.5 Hz, –CH₂); 4.85–4.30 (m, 6H, –CH₂); 6.90 (d, 2H, J = 8.0, C₂, C₆, Ar ring); 6.94 (d, 2H, J = 7.0, C₃, C₅, Ar ring); 7.23 (t, 1H, J = 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): δ 13.32 (–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, imidazole ring); 157.43 (C₁, Ar ring). +ESI-MS (m/z): calculated for C₁₅H₂₁N₂O⁺ (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): ν_max 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 [λ in nm (ε 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): δ 0.86 (t, 3H, –CH₃); 1.10 (sex, 2H, J = 7.5 Hz, –CH₂); 1.68 (q, 2H, J = 7.0 Hz, –CH₂); 3.14 (t, 2H, J = 7.0 Hz, –CH₂); 4.11 (t, 2H, J = 7.0, –CH₂); 4.45 (t, 2H, J = 7.0, –CH₂); 7.16 (d, 2H, J = 7.5 Hz, C₂, C₆ Ar ring); 7.23 (t, 2H, J = 7.0, C₃, C₅ Ar ring); 7.29 (t, 1H, J = 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): δ 13.21 (–CH₃); 18.54, 30.68, 31.23, 35.21 (–CH₂); 48.44, 49.83 (–CH₂); 122.41 (C₂, C₃ imidazole ring); 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): calculated for 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): ν_max 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 [λ in nm (ε in M⁻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): δ 0.87 (t, 3H, J = 7.5 Hz, –CH₃); 1.22 (sex, 2H, J = 7.5 Hz, –CH₂); 1.74 (q, 2H, J = 7.5 Hz, –CH₂); 2.29 (q, 2H, J = 6.5 Hz, –CH₂); 4.01 (t, 2H, J = 6.0 Hz, –CH₂); 4.16 (t, 2H, J = 7.0 Hz, –CH₂); 4.37 (t, 2H, J = 7.0 Hz, –CH₂); 6.89 (d, 2H, J = 8.0 Hz, C₂, C₆ Ar ring); 6.94 (t, 1H, J = 7.5 Hz, C₄); 7.28 (t, 2H, J = 7.5 Hz, C₃, C₅ Ar ring); 7.85 (d, 2H, J = 18.0 Hz, C₂, C₃, imidazole ring); 9.31 (s, 1H, imidazole ring). ¹³C NMR (125 MHz, DMSO-d₆): δ 13.24 (–CH₃); 18.75, 28.89, 30.96, 46.53, 48.54, 64.39 (–CH₂); 114.34 (C₂, C₆, Ar ring); 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): ν_max 3133, 3066, 3027 (–CH str., –CH₃); 2964, 2936, 2865 (–CH str., –CH₂); 1606, 1566 (–C=N, imidazole), 1495, 1456 (–C=C–); 1369, 1329 (C–C); 1164 (C–N); 1116, 1029 (–C–H, bending); 860, 753, 702 (C–H, –CH₂). UV-Vis [λ in nm (ε 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): δ 0.88 (t, 3H, J = 7.5 Hz, –CH₃); 1.23 (sex, 2H, J = 7.5 Hz, –CH₂); 1.52 (q, 2H, J = 7.5 Hz, –CH₂); 1.858–1.746 (m, 4H, –CH₂); 2.60 (t, 2H, J = 7.5 Hz, –CH₂); 4.25–4.18 (m, 4H, –CH₂); 7.28–7.15 (m, 5H, C₂–C₆ Ar ring); 7.87 (s, 2H, C₂, C₃, imidazole ring); 9.41 (s, 1H, imidazole ring). ¹³C NMR (125 MHz, DMSO-d₆): δ 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): ν_max 3139, 3076 (–CH str., –CH₃); 2962, 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 [λ in nm (ε 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)]. ¹H NMR (500 MHz, CDCl₃): δ 0.9 (t, 3H, J = 7.5 Hz, –CH₃); 1.25 (sex, 2H, J = 7.5 Hz, –CH₂); 1.70 (q, 2H, J = 6.5 Hz, –CH₂); 1.80 (q, 2H, J = 7.5 Hz, –CH₂); 1.97 (q, 2H, J = Hz, –CH₂); 3.99 (t, 2H, J = 6.0, –CH₂); 4.17 (t, 2H, J = 7.0, –CH₂); 4.26 (t, 2H, J = 7.0, –CH₂); 6.93 (t, 3H, J = 7.5 Hz, C₂, C₄, C₆ Ar ring); 7.29 (t, 2H, J = 8.0, C₃, C₅ Ar ring); 7.84 (d, 2H, J = 10.0, imidazole ring); 9.28 (s, 1H, imidazole ring). ¹³C NMR (125 MHz, DMSo-d₆): δ 18.50 (–CH₃); 24.02, 30.59, 31.55, 35.93, 36.49, 53.81, 71.76 (–CH₂); 119.61 (C₂, C₆, Ar ring); 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): ν_max 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 [λ in nm (ε 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): δ 0.9 (t, 3H, J = 7.5 Hz, –CH₃), 1.26 (sex, 4H, J = 7.0 Hz, –CH₂); 1.43 (d, 2H, J = 7.0 Hz, –CH₂); 1.69–1.83 (m, 6H, –CH₂); 3.93 (t, 2H, J = 6.0, –CH₂); 4.20 (d, 4H, J = 7.0 Hz, –CH₂); 6.90 (t, 3H, J = 3.0 Hz, C₂, C₄, C₆ Ar ring); 7.27 (t, 2H, J = 6.0 Hz, C₃, C₅ Ar ring); 7.88 (s, 2H, C₂, C₃, imidazole ring); 9.42 (s, 1H, imidazole ring). ¹³C NMR (125 MHz, DMSO-d₆): δ13.23 (–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, Ar ring); 135.95 (C₁, imidazole ring); 158.55 (C₁, Ar ring). +ESI-MS (m/z): calculated for C₁₉H₂₉N₂O⁺ (M − Br)⁺ 301.2274, found 301.2252.

Antibacterial activity

The synthesised ILs 3a–f were evaluated against the human pathogenic Gram-positive Staphylococcus aureus (NCIM 2079), Bacillus subtilis (NCIM 2250) and Gram-negative Escherichia coli (NCIM 2109) and Pseudomonas aeruginosa (NCIM 2036) bacterial strains using the Agar disc diffusion method.⁴⁵ Stock solutions (1000 μg mL⁻¹) of each compound were prepared in DMSO. Assays were carried out with concentrations of 100 μg mL⁻¹ using HiMedia antibiotic disks and chloramphenicol and ciprofloxacin as reference drugs. The nutrient agar microbiological medium used for Gram-positive and Gram-negative strains was obtained from HiMedia (India) and had the following composition (in grams per litre): sodium chloride, 5.0; beef extract, 10.0; and peptone, 10.0 (pH 7.2). The zones of inhibition were measured in millimetres (mm) for ILs (3a–f) after 24 h of incubation at 37 °C and pH 7.2.

Antifungal activity

The synthesised ILs were screened against the human pathogenic fungal strains Candida albicans (NCIM 3471) and Aspergillus niger (NCIM 545) using the Agar disc diffusion method at 100 μg mL⁻¹.⁴⁵ Potato dextrose agar (HiMedia, India) was used as the medium for A. niger and had the following composition (grams per liter): potato infusion, 200.0; and dextrose, 20.0 (pH 5.2). The microbiological medium for C. albicans was MGYP (all ingredients 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 were measured in millimetres (mm) for 3a–f after 24 h of incubation at 37 °C. Amphotericin-B was used as a standard drug to compare the zones of inhibitions. The samples were prepared in DMSO as a solvent.

Anticancer activity

The in vitro anticancer activities of ILs 3a, 3b, 3d, 3e, 3g were determined for the human malignant breast cancer cell lines MCF-7 and MDA-MB-435 at four dose levels (0.1, 1.0, 10 and 100 μM) in DMSO. The test consisted of a 48 h continuous drug contact protocol using sulforhodamine B (SRB) assay to estimate cell growth. The experimental procedure followed the NCI SRB assay protocols.⁴⁶ Briefly, this assay relies on the uptake of the negatively charged pink aminoxanthine dye and sulforhodamine-B (SRB) by basic amino acids in the cells. A greater number of cells corresponds to a greater amount of dye being taken up, and after fixing, when the cells are lysed, the released dye exhibits greater absorbance. The SRB assay was found to be more dependable, sensitive, simple, reproducible and rapid than the formazan-based assays and gives the best results.⁴⁷ Appropriate positive controls were run in each experiment, and each experiment was repeated in triplicate. The cell growth as a percentage of the control was plotted against drug concentration (Fig. 2 and 3) to calculate various parameters. The results are given in terms of GI₅₀ (concentration of drug that produces 50% inhibition of the cells), TGI (concentration of the drug that produces total inhibition of the cells) and LC₅₀ (concentration of the drug that kills 50% of the cells) values calculated from the plots. The results of anticancer activities are given in Table 6.

Acknowledgements

The authors are thankful to the Central University of Gujarat, Gandhinagar, for financial and infrastructural support and instrumental facilities. They also thank Dr A. Juvekar, ACTREC Kharghar, Navi Mumbai for anticancer activity tests and Dr Ulhaas K. Patil for antibacterial tests.

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Footnote

† Electronic supplementary information (ESI) available: Copies of ¹H and ¹³C NMR spectra. See DOI: 10.1039/c4ra08370a

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