Imidazole clubbed 1,3,4-oxadiazole derivatives as potential antifungal agents Mohmmad Younus Wani a,,⇑ , Aijaz Ahmad b,, Rayees Ahmad Shiekh c , Khalaf J. Al-Ghamdi c , Abilio J. F. N. Sobral a,⇑ a Departmento de Quimica, FCTUC, Universidade de Coimbra, Rua Larga, Coimbra 3004-535, Portugal b Department of Oral Biological Sciences, University of Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africa c Department of Chemistry, Faculty of Science, Taibah University, PO Box 30002, Al Madinah Al Munawarrah, Saudi Arabia article info Article history: Received 18 March 2015 Revised 18 June 2015 Accepted 20 June 2015 Available online xxxx Keywords: Imidazole 1,3,4-Oxadiazole Pyridine Antifungal activity Docking abstract A series of compounds in which 2-(4-ethyl-2-pyridyl)-1H-imidazole was clubbed with substituted 1,3,4- oxadiazole was synthesized and subjected to antifungal activity evaluation. In vitro assays indicated that several clubbed derivatives had excellent antifungal activity against different strains of laboratory and clinically isolated Candida species. Structural Activity Relationship (SAR) studies revealed that the pres- ence and position of substituents on the phenyl ring of the 1,3,4-oxadiazole unit, guides the antifungal potential of the compounds, where compound 4b, 4c and 4g were found to be active against all the tested fungal strains. Impairment of ergosterol biosynthesis upon the concomitant treatment of 4b, 4c and 4g, revealed the possible mechanisms of antifungal action of these compounds. Inhibitors snugly fitting the active site of the target enzyme, as revealed by molecular docking studies, may well explain their excel- lent inhibitory activity. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Azoles, five-membered heterocyclic compounds with two, three or four nitrogen atoms, constitute a large group of organic substances exhibiting a wide range of biological activities. 1,2 The development of azole based drugs represented a major advance in medical mycology and since then azoles have been serving mankind by fighting infections and deadly microbes. 3,4 Azoles are currently the most popular class of antifungals used in medi- cine. These compounds bind as the sixth ligand to the heme iron in CYP51 by N-heterocycle nitrogen, thereby altering the structure of the active site and acting as non-competitive inhibitors. 5 The effectiveness of azoles as inhibitors of the 14 a-demethylase has been confirmed through several experiments. Some studies test for changes in the production of important downstream ergosterol intermediates in the presence of these compounds. 5–7 However prolonged use of some azoles as antifungal agents has resulted in the emergence of drug resistance among certain fungal strains. 8–10 Consequently, the focus of azole research is beginning to shift toward identifying new efficient drug molecules to circum- vent this major obstacle. A novel strategy of generating a new class of azole based antifungal agents is clubbing together of two or more biologically important azole scaffolds, to achieve a target compound with potentiation of activities. To this end we choose two biologically important scaffolds; imidazole and 1,3,4-oxadia- zole and clubbed them together into one molecule (Fig. 1). Imidazole ring is a part of many existing drugs and same is true for 1,3,4-oxadiazole ring. 11–13 The vital role played by small heterocyclic molecules in drug design cannot be denied. These molecules act as highly function- alized scaffolds. In this context, pyridyl ring, a prominent scaffold present in numerous bioactive molecules, has played a vital role in the development of different medicinal agents. 14 On the other hand, the interesting and versatile biological activities of imida- zoles and 1,3,4-oxadiazoles, including anti-bacterial, anti-fungal, anti-cancer, anti-viral, and antiprotozoal, established them as important pharmacophores. 11,12,15 Following our search for the synthesis of novel azole based antimicrobial agents, 16–18 we syn- thesized novel imidazole clubbed 1,3,4-oxadiazole derivatives bearing a pyridyl moiety on the imidazole ring and substituted phenyl ring on the 1,3,4-oxadiazole unit, to optimize the overall structure for better and promising antifungal efficacy, which could be used as lead structures for further optimization. http://dx.doi.org/10.1016/j.bmc.2015.06.053 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Tel.: +351 923207796; fax: +351 239827703. E-mail addresses: [email protected], [email protected](M.Y. Wani), asobral@ci. uc.pt (A.J.F.N. Sobral). Authors contributed equally. Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc Please cite this article in press as: Wani, M. Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.053
Please cite this article in press as: Wani, M. Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.053
Mohmmad Younus Wani a,�,⇑, Aijaz Ahmad b,�, Rayees Ahmad Shiekh c, Khalaf J. Al-Ghamdi c,Abilio J. F. N. Sobral a,⇑a Departmento de Quimica, FCTUC, Universidade de Coimbra, Rua Larga, Coimbra 3004-535, Portugalb Department of Oral Biological Sciences, University of Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africac Department of Chemistry, Faculty of Science, Taibah University, PO Box 30002, Al Madinah Al Munawarrah, Saudi Arabia
a r t i c l e i n f o
Article history:Received 18 March 2015Revised 18 June 2015Accepted 20 June 2015Available online xxxx
A series of compounds in which 2-(4-ethyl-2-pyridyl)-1H-imidazole was clubbed with substituted 1,3,4-oxadiazole was synthesized and subjected to antifungal activity evaluation. In vitro assays indicated thatseveral clubbed derivatives had excellent antifungal activity against different strains of laboratory andclinically isolated Candida species. Structural Activity Relationship (SAR) studies revealed that the pres-ence and position of substituents on the phenyl ring of the 1,3,4-oxadiazole unit, guides the antifungalpotential of the compounds, where compound 4b, 4c and 4g were found to be active against all the testedfungal strains. Impairment of ergosterol biosynthesis upon the concomitant treatment of 4b, 4c and 4g,revealed the possible mechanisms of antifungal action of these compounds. Inhibitors snugly fitting theactive site of the target enzyme, as revealed by molecular docking studies, may well explain their excel-lent inhibitory activity.
� 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Azoles, five-membered heterocyclic compounds with two,three or four nitrogen atoms, constitute a large group of organicsubstances exhibiting a wide range of biological activities.1,2 Thedevelopment of azole based drugs represented a major advancein medical mycology and since then azoles have been servingmankind by fighting infections and deadly microbes.3,4 Azolesare currently the most popular class of antifungals used in medi-cine. These compounds bind as the sixth ligand to the heme ironin CYP51 by N-heterocycle nitrogen, thereby altering the structureof the active site and acting as non-competitive inhibitors.5 Theeffectiveness of azoles as inhibitors of the 14 a-demethylase hasbeen confirmed through several experiments. Some studies testfor changes in the production of important downstream ergosterolintermediates in the presence of these compounds.5–7 Howeverprolonged use of some azoles as antifungal agents has resultedin the emergence of drug resistance among certain fungalstrains.8–10 Consequently, the focus of azole research is beginning
to shift toward identifying new efficient drug molecules to circum-vent this major obstacle. A novel strategy of generating a new classof azole based antifungal agents is clubbing together of two ormore biologically important azole scaffolds, to achieve a targetcompound with potentiation of activities. To this end we choosetwo biologically important scaffolds; imidazole and 1,3,4-oxadia-zole and clubbed them together into one molecule (Fig. 1).Imidazole ring is a part of many existing drugs and same is truefor 1,3,4-oxadiazole ring.11–13
The vital role played by small heterocyclic molecules in drugdesign cannot be denied. These molecules act as highly function-alized scaffolds. In this context, pyridyl ring, a prominent scaffoldpresent in numerous bioactive molecules, has played a vital rolein the development of different medicinal agents.14 On the otherhand, the interesting and versatile biological activities of imida-zoles and 1,3,4-oxadiazoles, including anti-bacterial, anti-fungal,anti-cancer, anti-viral, and antiprotozoal, established them asimportant pharmacophores.11,12,15 Following our search for thesynthesis of novel azole based antimicrobial agents,16–18 we syn-thesized novel imidazole clubbed 1,3,4-oxadiazole derivativesbearing a pyridyl moiety on the imidazole ring and substitutedphenyl ring on the 1,3,4-oxadiazole unit, to optimize the overallstructure for better and promising antifungal efficacy, whichcould be used as lead structures for further optimization.
Figure 1. General structure of imidazole clubbed 1,3,4-oxadiazole derivatives.
NHN
N
NN
NClO
O
O
O
Dry Acetone, K2CO3
NH2NH2.H2O
C2H5OH
NN
N
HN
ONH2
NN
N
O NN
R
RCOOHPOCl3
(1) (2)
(3)(4a-4j)
Cl
ClCl
Cl
NH2
NO2 NH2
O
O
Cl
4a 4b 4c 4d
4e 4f 4g 4h
4i 4j
O
R =
Scheme 1. Synthesis of imidazole clubbed 1,34-oxadiazole derivatives (4a–4j).
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2. Results and discussion
2.1. Chemistry
The starting material 4-ethyl-2-(1H-imidazol-2-yl)pyridine (1)employed in the preparation of hydrazide (3) was obtained fromSigma and used after purification by recrystallization. 2-(2-(4-ethylpyridin-2-yl)-1H-imidazol-1-yl)acetohydrazide (3) wasobtained by refluxing the starting material 1 with ethylchloroac-etate under basic conditions and subsequent treatment withhydrazine hydrate in ethanol (Scheme 1). The target imidazoleclubbed 1,3,4-oxadiazole derivatives (4a–j) were prepared byrefluxing an intimate mixture of hydrazide 3 with appropriate car-boxylic acids in phosphorous oxychloride (Scheme 1). All the com-pounds were obtained in 50–70% yield and recrystallized fromappropriate solvents, and were found to be highly soluble inmethanol, ethanol, DMSO, DMF, acetonitrile, dichloromethane,and chloroform. Structure of all the synthesized compounds wasestablished by FTIR, 1H NMR, 13C NMR and ESI MS spectral analysis.
2.2. Biological evaluation
2.2.1. Minimum inhibitory concentration (MIC)Evaluation of the MIC values by broth microdilution assay
showed that out of the 10 tested compounds (4a–j), 3 compounds(4b, 4c and 4g) showed high antifungal activities against both thetested laboratory Candida strains as well as clinical isolatedCandida strains, with MIC values ranging from 0.002 to0.125 mg/ml (Table 1); while as the other test compounds possessweaker or no antifungal activity (>1 mg/ml). The order of sensitiv-ity on the basis of MIC values is 4g < 4b < 4c. From these results, itcan be observed that the compounds containing nitro and aminogroups attached respectively at position-4 of the phenyl ring ofthe 1,3,4-oxadiazole unit, (4b, 4c) and compound bearing an acetylgroup at position-2 of the phenyl ring (4g) showed high levels ofantifungal activity. Sensitivity to the test compounds to differentpathogens was observed in the order of Candidaalbicans > Candida tropicalis. Compound 4g bearing an acetyl groupat position-2 of the phenyl ring, displayed better antifungal activitycompared to the compounds 4b and 4c, where the phenyl ring issubstituted at position-4 by nitro and amino groups respectively.All the strains have MIC values <0.064 mg/ml for fluconazole(FLC) and therefore all the strains are considered as FLC-suscepti-ble isolates.
2.2.2. Disc diffusion assayIn order to further confirm the antifungal activity of 4b, 4c and
4g, disc diffusion assay was performed against all the fungal strainsat their respective 1/2 �MIC, MIC and MIC � 2 concentrations. Thedata, summarized in Table 2, confirmed that these test compoundspossess significant antifungal activity against the tested pathogens.The highest zone of inhibition (16 mm) was measured in C. albicanswhen treated with MIC � 2 of 4g, followed by 15 mm and 14 mmof ZOI in C. albicans and C. tropicalis when treated with 4b atMIC � 2 concentrations, respectively. In case of clinical strains,
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ZOI ranges from 05 mm to 11 mm, when cells were treated withMIC � 2 of the 4b, 4c and 4g. At sub-MIC values of the test com-pounds the ZOI’s were significantly less. The results summarizedin Table 2 also depicted that 4g is more effective followed by 4cand 4b, which is congruent with the MIC results observed in brothdilution assay. As expected no inhibition zones were observedwhen cells were treated with the negative vehicle control (1%DMSO), which proves the solvent is having no effect on the testedorganisms and all the antimicrobial effect is due to the tested com-pounds. In this assay, for comparison, known antifungal drug FLCwas also included which showed significant zones of inhibition.
2.2.3. WST1 cytotoxicity assayHigh antifungal activities of the test compound 4b, 4c and 4g
were further confirmed by the WST1 cytotoxicity assay. Percentcytotoxicity of the test compounds at different concentrationsagainst all the tested pathogens are calculated as:
Figure 2 represents the % cytotoxicity of compounds 4b, 4c and4g, in different strains of C. albicans and C. tropicalis, at MIC andsub-MIC values. In case of laboratory strains, at MIC value of 4b,the % cytotoxicity was 57.94 while as at 1/2 MIC and 1/4 MIC val-ues of 4b the % cytotoxicity was 35.57 and 16.88, respectively. Thehighest % cytotoxicity for laboratory strains was observed in C. albi-cans ATCC10261 (87.27) and C. tropicalis ATCC750 (80.81) with thetreatment of MIC value of 4c while as the lowest % cytotoxicity for
Figure 2. % Cytotoxicity in presence of 4b, 4c and 4g against different fungi strains. Different concentrations of the test compounds (MIC and sub-MIC values) are representedby different color bars.
Table 1Minimum inhibitory concentrations (mg/ml) of imidazole clubbed 1,3,4-oxadiazole derivatives (4a–4j) for different fungal strains
Compounds Laboratory strains Clinical Strains
C. albicans ATCC10261 C. tropicalis ATCC750 C. albicans 2323 C. albicans 2367 C. albicans 2779 C. tropicalis 2029 C. tropicalis 2356
C+ is standard drug (Fluconazole)C� is solvent neative control (1% DMSO)
* 1/2 �MIC.** MIC.
*** MIC � 2
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laboratory strains was observed when C. tropicalis ATCC750 cellswere treated with 1/4 MIC value of 4b (15.6%). For clinical strains,the highest % cytotoxicity was observed in C. albicans 2323 (60.62)and C. tropicalis (2029) with the MIC treatment of 4g (Fig. 2). Inclinical isolates, least % cytotoxicity was observed as 7.31 and8.02 in C. albicans 2779 with the treatment of 1/4 MIC values of4g and 4b, respectively (Fig. 2). From these result it can be con-cluded that the higher concentration of the test compounds is inhi-bitory for cell proliferation and drastically reduces biomass ofmicrobial cell culture.
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2.2.4. Sterol quantitationSterol quantification in absence and presence of the test com-
pounds revealed the possible mechanisms of action for the antifun-gal activity of 4b, 4c and 4g. The effect of 4b, 4c and 4g onergosterol biosynthesis in different Candida strains is summarizedin Table 3 and represented in Figure 3. A dose dependent decreasein ergosterol biosynthesis was observed when isolates were grownwith 1/2 MIC values of the test compounds and a flat line wasobserved at MIC values, which is a representation of completeergosterol biosynthesis inhibition (Fig. 3). As shown in Table 3,
Table 3Ergosterol biosynthesis inhibition with respect to control in different Candida isolates upon treatment with the active compounds (4b, 4c and 4g)
Fungal isolates Mean ergosterolcontent of cellsgrown with 4b
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the decrease in the total cellular ergosterol content for standardlaboratory strains ranged from 41% to 100% with respect tountreated control cells, while as the total cellular ergosterol con-tent decreases for clinical isolates ranged from 31% to 93%. The per-centage inhibition of ergosterol biosynthesis in clinical Candidastrains at MIC and 1/2 MIC values was less in comparison to stan-dard Candida cells. Fluconazole, as expected, inhibits 100% ofergosterol biosynthesis in all the tested strains of Candida.
Among all the tested compounds against pathogenic fungalstrains, 4g possess low MIC values inhibiting the growth ofCandida cells more efficiently followed by 4b and 4c. Disc diffusionstudies as a function of varied concentration also follow the samepattern of effective input, thereby indicating differences in sensi-tivity of microbial cells to these bioactive compounds. When cellcytotoxicity assay was performed, only a marginal decline in cyto-toxicity was observed at lower concentrations, while at higher con-centrations of test compounds significant levels of cell cytotoxicitywere observed. Tetrazolium salts are widely used as an assay forfungal cell viability and are widely used as indicators of cellularproliferation and biomass for eukaryotic cells. Live cells reducethe tetrazole ring and a colored formazan product is formed, whichcan be assessed visually and quantified spectrophotometrically.There are reports that such assays are used to test the cell viabilityof Candida and other fungal and bacterial cells.19,20 Assay resultindicates that higher concentration is inhibitory for cell prolifera-tion and drastically reduces biomass of microbial cell culture. Inan attempt to understand the mechanism of antifungal action of4b, 4c and 4g, we quantified the ergosterol content in C. albicansATCC10261, C. tropicalis ATCC750 and one clinical strain C. albicans2323, after treating these cells with sub-MIC and MIC values of theactive compounds 4b, 4c and 4g. The mechanism of action of allthe azole drugs including fluconazole and ketoconazole, whichare the most widely used antifungal drugs to treat candidiasis, is
Figure 3. UV spectrophotometric sterol profiles of standard C. albicans ATCC10261 (A),respective 1/2 MIC and MIC values of 4b, 4c and 4g. Control (�ive) represent the normal cdrug fluconazole.
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known to target the ergosterol biosynthesis pathway. No major dif-ferences were observed in the pattern of sterol contents (four peakcharacteristic curve) of the standard and clinical untreated Candidaisolates. All the cells treated with the three active compounds 4b,4c and 4g showed altered ergosterol biosynthesis in a dose-depen-dent manner. Candida strains have been classified into ergosteroltolerant and ergosterol intolerant strains.21,22 The former Candidastrains are able to grow in absence of the ergosterol while as thelater strains are intolerant to ergosterol absence and therefore theirMIC values coincides with the concentrations at which 100% ergos-terol biosynthesis inhibition is reached. The strains used in the pre-sent study showed significant inhibition of ergosterol biosynthesisat sub-MIC values, which implies that these strains are ergosteroltolerant strains. The dose-dependent antifungal activity of thesecompounds therefore appears to originate from the ergosterolbiosynthesis inhibition in a manner similar to that of thefluconazole.
2.2.5. Molecular docking studiesTo gain insight into the possible mechanism of action and bind-
ing interactions, the most active compounds (4b, 4c and 4g) weredocked into the active site of cytochrome P450 lanosterol 14a-demethylase of C. albicans using autodock 4.2 and autodock vinasoftware package. The active site pocket consists of amino acidresidues LEU139, LYS143, LEU150, LEU204, ILE304, MET306,GLY307, GLY308, GLN309, THR311, SER312, ARG381, HID468,PRO462, PHE463, GLY464, HIS468, ARG469, CYS470, ILE471,GLY472, GLU473, PHE475, and Heme. Binding affinity values ofdocked active compounds 4b, 4c and 4g were found to be in therange of �9.7 to �12.6 kcal mol�1 (Table 4). The more negativethe relative binding energy, the more potent the binding of drugto protein. The docking result indicated that synthesized com-pounds were held in the active pocket by forming combination
C. tropicalis ATCC750 (B) and C. albicans 2323 (C). The cells were treated with theells without any treatment and control (+ive) represent the cells treated with known
Figure 4. Docking of compounds 4b, 4c and 4g into the active site pocket ofcytochrome P450 lanosterol 14a-demethylase of C. albicans.
M. Y. Wani et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx 5
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of hydrogen bonds, hydrophobic bonds and van der Waals interac-tions with the enzyme. The docking study also revealed that theoxadiazole and pyridine ring had formed hydrogen bonding inter-actions with enzyme suggesting that oxadiazole and pyridine ringis important for inhibiting the 14a-demethylase of C. albicans whenin combination with imidazole ring (Fig. 4). The ANO2 group of thephenyl ring and nitrogen of 1,3,4-oxadiazole ring of 4b formedhydrogen bonds with ARG381 amino acid residue and heme groupof the protein respectively. In case of compound 4c the nitrogen of1,3,4-oxadiazole ring formed hydrogen bond with the heme group,where as nitrogen atom of the pyridine ring of 4g coordinated withthe heme group of the modeled protein in a similar way, mimick-ing fluconazole, which coordinates with the heme group throughits triazole nitrogen. Nitrogen of 1,3,4-oxadiazole ring of the samecompound also formed hydrogen bonds with THR311 amino acidresidue of the protein (Fig. 4). On the basis of activity data anddocking results, it was found that all the active compounds hadthe potential to inhibit 14a-demethylase of C. albicans and there-fore can be considered as lead structures for further optimization.
An idea for further optimization of the designed molecules canbe generated from the fact that all the molecules have the samebasic ring skeleton, which differs only in the presence of differentsubstituents at position 5 of the 1,3,4-oxadizole ring. Compoundsbearing a phenyl ring, 2-chloro, 4-chloro or 2,4-dichloro phenylring at position 5 of the 1,3,4-oxadizole ring in the molecules, didnot show any appreciable antifungal activity. Presence of 4-iso-propylphenyl or 4-chlorophenoxy ring at the same position alsoresulted in same. However presence of 4-nitrophenyl, 4 aminophe-nyl or 2-acetoxyphenyl ring at this position brought an appreciablechange in the antifungal properties of the compounds. Substitutionof 4-aminophenyl with a 2-aminophenyl ring resulted in the loss ofthe activity. The structure of the designed compounds needs to befurther optimized by placing different substituents at position-5 ofthe 1,3,4-oxadizole ring or another strategy could be the replace-ment of the 1,3,4-oxadiazole ring with a different azole pharma-cophore to achieve the best molecular scaffold, which can beused as a future drug.
3. Conclusion
A new series of 2-(4-ethyl-2-pyridyl)-1H-imidazole clubbed1,3,4-oxadiazole derivatives (4a–4j) was synthesized.Antimicrobial screening against different fungal strains resultedin finding of three compounds 4b, 4c and 4g as better antifungalagents compared to the other homologs. The dose-dependent anti-fungal activity of these compounds appears to originate from theergosterol biosynthesis inhibition in a manner similar to that ofthe fluconazole. Docking studies revealed that the active com-pounds snugly fit into the active site of the target protein andmay well explain their promising activities. Pyridine and oxadia-zole rings were found to be important for the activity, where asthe presence of different substituents on the phenyl ring of the1,3,4-oxadiazole unit, played a major role in deciding their antifun-gal activities. The structure of the designed compounds can be fur-ther optimized by placing different substituents at position-5 ofthe 1,3,4-oxadiazole ring or replacing the substituted 1,3,4-oxadi-azole ring by other azole pharmacophore for better efficacy.
4. Materials and methods
4.1. Chemistry
All the chemicals were purchased from Aldrich ChemicalCompany (USA). Precoated aluminum sheets (silica gel 60 F254,Merck Germany) were used for thin-layer chromatography (TLC)
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and spots were visualized under UV light. Melting points (mp)were performed using a Mel-temp instrument, and the resultsare uncorrected. FT-IR spectra were recorded using a ThermoNicolet 380 instrument equipped with a Smart Orbit ATR attach-ment. 1H and 13C NMR spectra were recorded at ambient temper-ature on a Bruker AVANCE 400 NMR spectrometer using standardparameters. All chemical shifts are reported in d units with refer-ence to the residual peaks of CDCl3 (d 7.24, 1H NMR; d 77.0, 13CNMR) or DMSO-d6 (d 2.50, 1H NMR; d 39.52, 13C NMR). ESI-MSwas recorded on a MICROMASS QUATTRO II triple quadrupolemass spectrometer.
4.1.1. General procedure for synthesis of imidazole clubbed1,3,4-oxadiazole derivatives (4a–j)4.1.1.1. Ethyl 2-(2-(4-ethylpyridin-2-yl)-1H-imidazol-1-yl)ac-etate (2). A mixture of 4-ethyl-2-(1H-imidazol-2-yl)pyridine (1)(1 mmol), ethylchloroacetate (1 mmol) and potassium carbonate(1.5 mmol) in dry acetone (5–10 ml) was refluxed for 50 h. Thereaction mixture was filtered hot and the solvent was distilledoff from the filtrate. The crude ester thus obtained was purifiedby recrystallization from ethanol.
4.1.1.2. 2-(2-(4-Ethylpyridin-2-yl)-1H-imidazol-1-yl)acetohy-drazide (3). A mixture of 2-methyl-5-nitro-1-imidazo-ethylac-etate 2 (2 mmol) and hydrazine hydrate (2 mmol) in ethanol(10 ml) was refluxed for 10 h. The solution on cooling gave a solidmass of hydrazide 3, which was collected by filtration, and recrys-tallized from ethanol.
4.1.1.3. Imidazole clubbed 1,3,4-oxadiazole derivatives (4a–j). Toa mixture of 2-(2-(4-ethylpyridin-2-yl)-1H-imidazol-1-yl)acetohy-drazide (3) (1 mmol) and respective carboxylic acids (1 mmol), phos-phorous oxychloride (5 ml) was added and the reaction mixture wasrefluxed for 6–8 h. After the completion of the reaction the contentswere cooled to room temperature and poured onto crushed ice.Neutralization by 5% sodium bicarbonate solution resulted in a pre-cipitate, which was collected by filtration and dried. Further purifica-tion was done by recrystallization from a mixture of DCM/Methanol.
4.2.1. Strains, media and growth conditionsTwo standard laboratory strains C. albicans ATCC10261 and C.
tropicalis ATCC750 and five clinical C. albicans and C. tropicalisstrains isolated from the HIV patients were used in this study.The clinical Candida isolates were collected and identified by theDepartment of Microbiology, All India Institute of MedicalSciences, New Delhi, India and the entire patient and sampledetails were noted in the records (as allotted registration num-bers). All stock cultures were initially grown on Yeast-extractPeptone Dextrose Agar (YPDA) (HiMedia, India). To initiate growthfor experimental purposes, cells from agar media were sub-cul-tured in broth media and incubated for 48 h at 37 �C. All otherchemicals and cell cytotoxicity assay kit were of analytical gradeand were procured from Merck Ltd, India. DMSO and Fluconazole(FLC) were purchased from Sigma Fluke.
4.2.2. Antimicrobial susceptibility4.2.2.1. Determination of minimal inhibitory concentration(MIC). The MICs of all the newly synthesized derivatives (4a–j)against all the fungi were determined by the Clinical andLaboratory Standards Institute recommended broth microdilutionmethods M27-A3.23 All the compounds were diluted to yield a con-centration of 1 mg/ml using 1% DMSO. The positive control flu-conazole (1 mg/ml) and the negative vehicle control (1% DMSO)were also included in every set of experiments. Media and culturecontrols were included to confirm the sterility and viability,respectively. All the experiments were carried out in triplicate ateach concentration level and repeated thrice.
4.2.2.2. Disc diffusion assay. On the basis of the promising anti-fungal activity as observed in broth dilution assay, inhibitory activ-ity of 4b, 4c and 4g on solid agar media against both the standardand clinical fungal strains was determined with the help ofdisc diffusion method as described previously.7 Fungal cells(103 cells ml�1) grown in YPD were mixed in molten agar media(�40 �C) and poured into a 100-mm-diameter petriplates. Filterdiscs were kept on solid agar and test compound (10 ll of1/2 �MIC, MIC and MIC � 2) were spotted on the discs.Fluconazole and 1% DMSO as positive and vehicle controls werealso pipetted onto 4-mm-diameter filter disc. All the plates wereincubated at 37 �C for 48 h incubation periods. After incubationperiods, diameters of zone of inhibition were recorded in millime-ters and were compared with that of the controls.
4.2.2.3. WST1 cytotoxicity assay. WST1 cytotoxicity assay wasperformed according to the manufacturer’s instructions (MerckLtd, India) as described previously.24 Briefly, cells were treatedwith MIC and sub-MIC concentrations of the test compounds (4b,4c and 4g) for 24 h in a micro-plate followed by incubation of30 min with 10 ll of WST-1/CEC dye at 37 �C. After 1 min shaking,the reaction was stopped by adding 10 ll of 1%SDS. WST-1 tetra-zolium salt was reduced to formazan by cellular dehydrogenasesleading to the generation of deep red color. The red colored for-mazan was then measured at 450 nm by a BIORAD MicroplateReader, which directly correlates to the cell number. The experi-ment for all the strains in the presence of each concentration of testentity was performed in triplicate and results are shown as Mean ±Standard Deviation.
4.2.2.4. Sterol quantitation method. Total intracellular sterolswere extracted as reported earlier.25 Briefly, single Candida
Figure 5. Flow chart diagram for homology modeling.
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colonies were inoculated in 50 mL of broth containing MIC and 1/2MIC concentrations of 4b, 4c, 4g and FLC as positive control. Afterincubation for 16 h at 37 �C, cells were harvested at 2700 rpm(Sigma 3K30) for 5 min and washed once with sterile distilledwater. The net wet weight of the cell pellet was determined, fol-lowed by the addition of 3 mL of 25% alcoholic potassium hydrox-ide solution (25 g of KOH and 35 mL of sterile distilled waterbrought to 100 mL with 100% ethanol) to each pellet. Vortexedand incubated all tubes in water bath at 85 �C for 1 h. Followingincubation, tubes were allowed to cool to room temperature.Sterols were then extracted by addition of a mixture of 1 mL ofsterile distilled water and 3 mL of n-heptane followed by vigorousvortex mixing for 3 min. The heptane layer was transferred to aclean borosilicate glass screw-cap tube and stored at �20 �C for
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24 h. Prior to analysis, a 1 mL aliquot of sterol extract was dilutedfivefold in 100% ethanol and scanned spectrophotometricallybetween 240 and 300 nm using a Labomed, Inc. spectrophotometer(USA). The presence of ergosterol and the late sterol intermediate24(28) DHE in the extracted sample resulted in a characteristicfour-peaked curve. The absence of detectable ergosterol in extractswas indicated by a flat line. Ergosterol content was calculated as apercentage of the wet weight of the cell by the followingequations:
M. Y. Wani et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx 9
where F is the factor for dilution in ethanol and 290 and 518 are theE values (in percentages per centimeter) determined for crystallineergosterol and 24(28) DHE, respectively.
4.2.2.5. Docking. Homology modeling: The 3D model structure ofcytochrome P450 lanosterol 14a-demethylase of C. albicans wasbuilt using homology modeling. A flow chart diagram for homologymodeling is illustrated in Figure 5. Amino acid sequence of enzymewas obtained from the Universal Protein Resource (http://www.uniprot.org) (Accession Code: P10614) and sequence homologouswas obtained from Protein Data Bank (PDB) using Blast search.Based on the result of blast search, we found lanosterol of yeastbearing PDB ID 4K0F with highest homology of 65%. This wasselected as a template for modeling our protein (P10614).P10614 was submitted to I-TASSER,26 an online server for model-ing and the best model was selected on the basis of higher C-scoreand least Z score. In parallel we also modeled it manually byMODELLER.27 We selected the model with least DOPE score andthen we compared the two results with our template. The modelwith least RMSD was selected as the best one and further usedfor docking studies. A Ramachandran diagram as structure valida-tion is shown in Figure S1 (Supporting information). To have theheme group, necessary for the ligand molecules to bind, in ourmodeled protein, we took the homology modeled structure andaligned it to the templates (PDB code: 3Ld6; 4LXJ), containing theheme in its active site using PyMol, then we figured out the atomsthat are interacting with it. Finally we incorporated the co-ordi-nates of heme in our model and also wrote down the interactionsas extracted from the template file.
Active site prediction: The best model thus obtained was submit-ted to DogSite scorer.28 It predicted the existence of 10 differentpockets. The one with highest P score was selected as the reliableactive site pocket and considered to have a potential active siteresidue.
The 3D structure of ligands were created by ChemDraw 8.0 andconverted to the pdb file format after energy minimization(MOPAC). Ligand preparation was conducted by assigningGastegier charges, merging nonpolar hydrogens, and saving it inpdbqt file format using AutoDock Tools (ADT) 4.2. The docking ofthe active molecules, into the lanosterol 14a-demethylase(CYP51) modeled protein was done by positioning with the activesite cavity. Molecular docking calculations were carried out withAutodock vina.29 The conformation with the lowest binding freeenergy was used for analysis. All molecular docked models wereprepared using PyMOL viewer.30
Acknowledgements
This work was supported by Fundo Europeu deDesenvolvimento Regional-QREN-COMPETE through projectPTDC/AAC-CLI/118092/2010 of Fundação para a Ciência e aTecnologia (FCT) and Postdoctoral grant SFRH/BPD/86581/2012(M.Y. Wani). We also thank Centro de Quimica de Coimbra
Please cite this article in press as: Wani, M. Y.; et al. Bioorg. Med. Chem
(CQC), FCTUC for their support. CQC is funded through ProjectsPest-C/QUI/UI0313/2011 and PEst-OE/QUI/UI0313/2014.
Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmc.2015.06.053.
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