Synthesis and antifungal activity investigation of a novel clotrimazole derivative

Azole antifungal agents disrupt fungal ergosterol synthesis that is essential for the formation of fungal cell membrane by preventing 14-α-demethylase enzyme from binding to its substrate. Clotrimazole is one of the first generations of azole antifungal agents. To discover a novel azole antifungal agent, biphenyl derivative was synthesised together with clotrimazole by multistep linear synthesis. Structures of synthesised azole agents have been validated by spectral analysis and potential antifungal activity of both compounds was determined on an yeast, E.coli and M.luteus by using a disk diffusion method. Clotrimazole and its biphenyl derivative were active against yeast but a novel compound resulted less activity than clotrimazole. Antibacterial effect was not observed for either azole agents.


INTRODUCTION
Azole antifungals, including clotrimazole, act as an inhibitor to the fungal ergosterol synthetic pathway.Ergosterol is an essential compound for fungal cell wall integrity and requires that the C-14 methyl group of sterol to be removed if it is to maintain its integrity and normal functionality.Azole antifungals target the haeme protein, which is an active catalytic site of 14α-demethylase (CYP51) enzyme that catalyses 14-αdemethylation of lanosterol in the ergosterol synthetic route.The inhibition of 14-α-demethylase enzyme leads to the accumulation of demethylated compounds, hence results in the death of fungal cell [1 -3].Clotrimazole is, one of the first generations of azole drugs, was first introduced by Bayer Ag (Germany) in 1967.Since that time this compound has been extensively studied.Clotrimazole has a broad spectrum of activity and is effective against Candida and Aspergillus species [4,5].As antifungal resistance develops in fungi and other factors occur that affect the result of fungal infection treatment, scientists invent new antifungals.A number of structure-activity relationship studies of antifungal agents have been conducted over the past years.The main target of azole antifungals is CYP51 (14-α-demethylase) of p450 enzymes and many studies focused on the computer aided structure modelling of this enzyme in relation to the activity of antifungals.Some structure-activity relationship studies of azole derivatives with the aid of molecular modelling of the CYP51 enzyme active site and the substrate binding mode suggest that conjugated aromatic moieties, including biphenyl and pyrrolylphenyl residues, confer better antifungal activity due to the favourable steric interaction within the enzyme's active site cavity.Also, if an electron withdrawing group is placed in the para position of the phenyl group, then antifungal activity was increased [6].In this study we aimed to design novel azole antifungal agents based on clotrimazole core structure.

EXPERIMENTAL
General: All reactions were conducted under nitrogen.Solvent concentration was performed on a Buchi rotary evaporator R-210.Flash column chromatography was conducted on silica 40-60 µ, 60 Е with ethyl acetate and petrol as eluent unless specified otherwise.Melting points were determined by a Stuart SMP11 apparatus.IR spectra were obtained on a Varian 800 FT-IR. 1 H and 13 C NMR spectra were obtained on a BRUKER AVANCE-300 and a JEOL ECS-400.Mass spectra were recorded on a Waters LCT-Premier ESI high resolution mass spectrometer.

Preparation of 1-((2-chlorophenyl)diphenylmethyl)-1H-imidazole (4):
Thionyl chloride (0.82 g, 6.9 mmol) was added into a solution of alcohol (2) (0.69 g, 2.3 mmol) and the mixture stirred at 0 0 C for 1 hour.The reaction mixture was left at reflux stirring for overnight.The organic solvent was evaporated and the residue washed with acetonitrile (2 x 20 mL) to afford compound (3) as an yellow oily mass that was submitted for the further reaction directly without purification and structure elucidation.Imidazole (0.3 g, 4.4 mmol) with Et 3 N (0.6 g, 6.6 mmol) was added to the solution of compound (3) (0.7 g, 2.2 mmol).The reaction mixture was left at room temperature at reflux stirring for 72 hours.The organic solvent was removed and EtOAc (20 mL) with water (20 mL) was added to the residue.The aqueous layer was extracted with EtOAc (2 x 20 mL).Combined organic layers were washed with water and dried over MgSO 4 prior to evaporation.The crude product was purified by flash chromatography to afford 0.69 g of compound (4) as a white crystal (yield 91%  7): Solution of alcohol ( 6) (1 g, 3.8 mmol) was added to pyridinium chlorochromate (1.2 g, 5.7 mmol) and celite (4 g) and the reaction mixture stirred at reflux for 3 hours at room temperature.The reaction mixture was filtered and the collected organic solution was dried over MgSO 4 and concentrated to afford 1.11 g (100% yield) of ketone (7) as a white crystal.mp 87-90 0 C (lit 99-100 0 C) [9], R f = 0.47; IR (neat) ν max /cm -

Preparation of [1,1'-biphenyl]-4-yl(4-fluorophenyl) (phenyl)methanol (8):
Solution of 4-fluoro-1bromobenzene (0.9 g, 5.4 mmol) was added to magnesium turnings (0.13 g, 5.4 mmol).The reaction mixture was stirred at reflux, then at room temperature for further 6 hours.A solution of ketone (7) (0.7 g, 2.7 mmol) was added and the reaction mixture left stirring at room temperature for overnight.The reaction mixture was quenched with a saturated ammonium chloride solution (30 mL).The aqueous layer was separated and extracted with Et 2 O (2 x 20 mL).The combined organic layers were washed,with water then dried over MgSO 4 and evaporated.Purification of the crude product was conducted by flash chromatography to give 0.73 g of alcohol (8) as a colourless oil (76% yield).R f = 0.35; IR (neat) ν max /cm -

RESULTS AND DISCUSSION
The antifungal agent clotrimazole (4) was synthesised in three steps (Scheme-1).

Scheme 1. Synthesis of clotrimazole
The synthesis started with commercially available ketone (1) and preparation of alcohol (2) was successful (36% yield).Chlorinated compound (3) has been prepared and submitted for further substitution reaction immediately without purification, characterisation and the structure elucidation due to instability of chlorinated compound (3) as this was previously reported [10].Characterisation of clotrimazole (4) was achieved with spectral analysis including 2D ( 1 H-1 H) COSY NMR, MS and IR to confirm the structure.Synthesis of a novel azole biphenyl derivative (10) was carried out in 5 steps linear synthetic route (Scheme 2).The overall yield of the entire synthesis was low at 26 %.There was no previous analytical data of the structure of compound (10), the spectral analysis supported the formation of a new compound.
The antifungal activity of the biphenyl derivative (10, Z-6.1) together with clotrimazole (Z-1.6) was determined using Kirby-Bauer agar diffusion method.Fluconazole -25 µg (FCN) standard test sample and a DMSO dissolved disk were used as control agents.Similar inhibition zones were observed for the biphenyl derivative (10) and DMSO dissolved disks excluding clotrimazole and fluconazole on yeast after growing 24 hours (Figure 1, A).This result was found to be related to solvent we used to dissolve our test samples for the bioassay and therefore we decided to grow yeast further 24 hours.After growing 48 hours, different inhibition zones were observed as indicated in Table 1 (Figure 1, B).The novel azole derivative (10) had the highest activity at 200 µg, where as the clotrimazole showed more activity at twice as less concentration compared to the novel azole.Moreover, the compound (10) was not active against both M.luteus and E.coli after 24 hours of growing.

CONCLUSIONS
We successfully synthesised clotrimazole (4) and a novel biphenyl derivative as 1-([1,1'-biphenyl]-4-yl(4fluorophenyl)(phenyl)methyl)-1H-imidazole (10) through multistep synthesis.The overall yield of novel clotrimazole derivative (10) was comparably low at 26 % We found that the compound (10) was active against an yeast but less than compound (4).DMSO was believed not to be an appropriate solvent for antifungal testing by virtue of its physical property (high boiling point).Therefore, it was impossible to test compounds in higher concentration due to limited absorption of the disks we used.The newly synthesised compound (10) has been proven not to be a potential candidate for further antifungal agent development.

Table 1 .
Inhibition zones on an yeast plate