Bioorganic & Medicinal Chemistry Letters xxx (2011) xxxxxx

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Bioorganic & Medicinal Chemistry Letters

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Molecular properties prediction and synthesis of novel 1,3,4-oxadiazole analogues as potent antimicrobial and antitubercular agents
Mohamed Jawed Ahsan a,b,, Jeyabalan Govinda Samy a, Habibullah Khalilullah a, Md. Shivli Nomani a, Pankaj Saraswat a, Ramakant Gaur a, Abhimanyu Singh a
a b

New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy College, Alwar, Rajasthan 301 030, India Department of Pharmaceutical Sciences, National Institute of Medical Sciences University, Jaipur 303 121, India

a r t i c l e

i n f o

a b s t r a c t
In the present investigation, a series of 1,5-dimethyl-2-phenyl-4-{[(5-aryl-1,3,4-oxadiazol-2-yl)methyl]amino}-1,2-dihydro-3H-pyrazol-3-one were subjected to molecular properties prediction, druglikeness by Molinspiration (Molinspiration, 2008) and MolSoft (MolSoft, 2007) software, lipophilicity and solubility parameters using ALOGPS 2.1 program. The compounds followed the Lipinski Rule of ve were synthesized for antimicrobial and antitubercular screening as oral bioavailable drugs/leads. Maximum drug-likeness model score (0.95) was found for compound, 4a. All the synthesized compounds were characterized by IR, NMR and mass spectral analysis followed by antimicrobial and antimycobacterial screening. Among the title compounds, compound 4d showed pronounced activity against Mycobacterium tuberculosis H37Rv and isoniazid resistant M. tuberculosis (INHR-TB) with minimum inhibitory concentrations (MICs) 0.78 lM and 1.52 lM, respectively. The compound, 4a showed maximum activity against all bacterial strains with MIC 48 lg/mL comparable to standard drug ciprooxacin, while the compounds, 4e and 4k showed maximum antifungal activity with MIC 816 lg/mL less active than standard drug uconazole. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 10 July 2011 Revised 14 September 2011 Accepted 17 October 2011 Available online xxxx Keywords: Oxadiazoles Antimicrobial agents Antitubercular agents Molecular properties prediction Lipinski Rule of ve

The pathogenic agent of tuberculosis (TB), Mycobacterium tuberculosis (MTB) is responsible for death of 23 million people annually.1 There are estimated 1.3 million multi/extensively drug resistant TB (M/XDR-TB) cases will need to be treated between 2010 and 2015.2 Antibacterial resistance is a cause of increased mortality.3 Fungal infections like Candidiasis, Crytococcosis and Aspergillosis are more common in immuno-compromised patients.4 Life threatening infectious disease caused by multidrug-resistant pathogenic bacteria (Gram-positive/Gram-negative) increased an alarming level around the world. Owing to this increased microbial resistance, new classes of antimicrobial agents with novel mechanisms are todays need to ght against the multidrug-resistant infections. Oxadiazoles are important classes of compounds which have long attracted attention, owing to their remarkable biological and pharmacological properties, such as antitubercular, antibacterial, antiviral, anti-inammatory, antifungal and insecticidal activities.513 Also the azole group of heterocyclic compounds possess signicant pharmacokinetic property, lipophilicity that inuence the ability of drug to reach the target by transmembrane diffusion and show promising activity against resistant TB by inhibiting the

Corresponding author. Tel.: +91 9694087786; fax: +91 144 5121120.

E-mail address: [email protected] (M.J. Ahsan). 0960-894X/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.10.057

biosynthesis of lipids.14,15 Most of the antitubercular drugs (isoniazid, pyrazinamide, etc.) are inhibitors of mycobacterial cell wall synthesis by inhibiting fatty acid synthetase. In the present investigation it has been tried to design and synthesized such novel compounds which include both the advantage of pyrazole and oxadiazole nucleus in the single molecule. All the title compounds (4a4n) were subjected to molecular properties prediction by Molinspiration and MolSoft (MolSoft, 2007) software in order to lter the drugs for synthesis and biological screening and to reduce enormous wastage of expensive chemicals and precious time. Earlier we have reported the antimycobacterial activity of diketones and pyrazoline.16,17 A good bioavailability can be achieved with an appropriate balance between solubility and partitioning properties. The computed log P values (P is the partition coefcient of the molecule in the water/octanol system) are shown in Table 1. The ALOGPS method is part of the ALOGPS 2.1 program used to predict lipophilicity and aqueous solubility of compounds. The lipophilicity calculations within this program are based on the associative neural network approach and the efcient partition algorithm. The Log Kow (Kow-WIN) program estimates the log octanol/water partition coefcient (log P) of organic chemicals and drugs using an atom/ fragment contribution method developed at Syracuse Research Corporation. The XLOGP2 is an atom additive method applying corrections. Computed partition coefcients for drugs studied varied

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Table 1 Physical constants and calculated partition coefcients, solubilities and drug likeness model score of the synthesized compounds

N N O

NH

N O

Ar

4a-n
Compound 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n Ar 2-Chlorophenyl4-Chlorophenyl4-Aminophenyl4-Pyridinyl2-Hydroxyphenyl2-PhenylacetatePhenyl4-Methoxyphenyl2-Methylphenyl3-Methylphenyl4-MethylphenylBenzylPhenoxymethyl2-ThiophenylYield (%) 65 78 82 78 67 62 82 85 89 69 77 80 84 89 Mp (C) 154 132 124 186 114 108 98 118 164 198 212 112 116 102 ALOGPS 4.48 4.48 3.82 3.08 3.03 4.05 3.75 3.77 4.09 4.09 4.09 3.31 3.22 3.67 (12.99 mg/L) (12.99 mg/L) (56.46 mg/L) (0.30 g/L) (0.35 g/L) (37.56 mg/L) (64.57 mg/L) (67.10 mg/L) (30.38 mg/L) (30.38 mg/L) (30.38 mg/L) (0.19 g/L) (0.22 g/L) (78.74 mg/L) KoW-WIN 1.09 1.09 0.47 0.74 0.03 0.05 0.45 0.53 1.00 1.00 1.00 0.94 0.47 0.27 XLOP2 4.70 4.70 3.26 2.83 3.68 3.87 4.08 4.00 4.52 4.52 4.52 3.89 3.76 3.11 Drug-likeness model score 0.95 0.79 0.61 0.60 0.47 0.17 0.44 0.37 0.93 0.82 0.69 0.56 0.85 0.86

between 2.83 and 4.70 (XLOGP2 method) and between 0.74 and 1.09 (KoW-WIN method) is presented in Table 1. Both the XLOGP2 method and KoW-WIN best supported for most of the compounds on the basis of lipophilicity (65) to consider an oral drug/lead. Also the absorption of a drug is usually very low if the calculated solubility is <0.0001 mg/L.18 As per the criterion compounds of the series (4a4n) almost full the requirement of solubility (ALOGPS). Good intestinal absorption, reduced molecular exibility (measured by the number of rotatable bonds), low polar surface area or total hydrogen bond count (sum of donors and acceptors), are important predictors of good oral bioavailability.19,20 Membrane permeability and bioavailability is always associated with some basic molecular descriptors such as log P (partition coefcient), molecular weight (MW), or hydrogen bond acceptors and donors counts in a molecule. Number of rotatable bonds is important for conformational changes of molecules under study and ultimately for the binding with receptors or channels. It is revealed that for passing oral bioavailability criteria, number of rotatable bond should be 610.19 The compounds in this series (4a4n) in general possess high number of rotatable bonds (57) and therefore, exhibit large conformational exibility. Drug-likeness model score (a combined effect of physico-chemical properties, pharmacokinetics and pharmacodynamics of a compound and is represented by a numerical value) was computed by MolSoft (MolSoft, 2007) software for the entire compounds under study. Maximum drug-likeness score was found out to be 0.95 for compound, 4a. Molecular polar surface area (TPSA) is a very useful parameter for the prediction of drug transport properties. TPSA is a sum of surfaces of polar atoms (usually oxygen, nitrogen and attached hydrogen) in a molecule. TPSA and volume is inversely proportional to %ABS. Topological polar surface area (TPSA), that is, surface belonging to polar atoms, is a descriptor that was shown to correlate well with passive molecular transport through membranes and, therefore, allows prediction of transport properties of drugs in the intestines and bloodbrain barrier crossing.18 TPSA was used to calculate the percentage of absorption (%ABS) according to the equation: %ABS = 109 0.345 TPSA, as reported.21 From all these parameters, it can be observed that all the title compounds exhibited a great %ABS ranging from 80.43 to 87.71% and that of standard drug isoniazid has %ABS of 88.78 calculated by MolSoft software. A com-

putational study for prediction of ADME properties of all molecules performed is presented in Table 2. The number of rotatable bonds (NROTB) and Lipinskis rule of ve were also calculated.22 The rule states that most molecules with good membrane permeability have log P 6 5, molecular weight 6500, number of hydrogen bond acceptors 610, and number of hydrogen bond donors 65. This rule is widely used as a lter for drug-like properties. Furthermore, none of the compounds violated Lipinskis parameters, making them potentially promising agents for antitubercular and antimicrobial therapy. The 1,5-dimethyl-2-phenyl-4-{[(5-aryl-1,3,4-oxadiazol-2-yl)methyl]amino}-1,2-dihydro-3H-pyrazol-3-one analogues (4an) described in this study are shown in Table 1 and the reaction sequence for the synthesis is summarised in Scheme 1. In the initial step 4-amino-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3one (1) (0.1 mol) and chloroacetic acid (2) (0.1 mol) in dry acetone and potassium carbonate reuxed for 8 h giving [(1,5-dimethyl-3oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)amino]acetic acid (3). In the subsequent step [(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)amino]acetic acid (3) was treated with appropriate hydrazides in 5 ml phosphorus oxychloride furnished the titled compounds (4a4n). The yields of the title compounds were ranging from 62% to 89% after recrystallization with absolute ethanol. The purity of the compounds was checked by TLC using eluants toluene/ethyl acetate/formic acid (5:4:1) and petroleum ether/toluene/ethyl acetate (5:4:1) and elemental analyses. The spots were located under iodine vapour/UV light. Both the analytical and spectral data (IR, 1H NMR) of all the synthesized compounds were in full harmony with the proposed structures. In general, the IR spectra of the compounds afforded absorption at 15311567 cm1 due to C@N group, band at 11531174 cm1 due to stretching of oxadiazole ring. In the Nuclear Magnetic Resonance spectra (1H NMR) the signals of the respective protons of the synthesized titled compounds were veried on the basis of their chemical shift, multiplicities and coupling constants in DMSO-d6. The spectra showed a singlet at d 1.912.23 ppm corresponding to CH3 group (pyrazolone ring); a singlet at d 2.272.39 ppm corresponding to CH3 (aromatic); a singlet at d 2.492.61 ppm corresponding to CH3 group (NCH3); a doublet at d 3.934.07 ppm corresponding to CH2; a singlet at d

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M. J. Ahsan et al. / Bioorg. Med. Chem. Lett. xxx (2011) xxxxxx Table 2 Pharmacokinetic parameters important for good oral bioavailability of title compounds (4an) Compound Rule 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n %ABS 87.27 87.71 80.43 84.01 81.76 84.96 87.71 84.82 87.71 87.71 87.71 87.92 85.04 87.36 Volume (A3) 381.64 381.18 371.49 361.70 375.09 395.09 363.99 395.84 383.59 385.01 384.93 380.68 394.25 358.87 TPSA (A2) 63.00 61.71 82.80 72.43 78.95 69.68 61.71 70.10 61.71 61.71 61.71 61.11 69.45 62.73 NROTB 5 5 5 5 5 7 5 6 5 5 5 6 7 5 HBA <10 4 4 4 5 5 5 4 5 4 4 4 4 5 5 HBD <5 1 1 3 1 2 1 1 1 1 1 1 1 1 1 Log P 65 2.72 2.70 1.13 0.80 1.71 1.98 2.02 2.07 2.38 2.39 2.48 1.64 0.98 1.90 MW <500 395.11 395.11 376.16 362.15 377.15 391.16 361.15 391.16 375.17 375.17 375.17 375.17 391.16 367.11 Lipinskis violations 61 0 0 0 0 0 0 0 0 0 0 0 0 0 0

%ABS, percentage of absorption; TPSA, topological polar surface area; NROTB, number of rotatable bonds; MW, molecular weight; Log P, logarithm of compound partition coefcient between n-octanol and water; HBA, number of hydrogen bond donors; HBD, number of hydrogen bond acceptors.

N N O
1

NH2

Dry Acetone ClCH2COOH 2 K2CO3

N N O

NH

COOH

3 ArCONHNH 2 POCl3

N N O

NH

N O

Ar

4a-4n
Scheme 1. Protocol for the synthesis of oxadiazoles.

4.95.2 ppm corresponding to NH; a multiplet at d 6.587.89 ppm corresponding to aromatic protons. The elemental analysis results were within 0.4% of the theoretical values. All the synthesized compounds (4ar) were tested for their in vitro antimycobacterial activity against MTB and INHR-MTB by agar dilution method using double dilution technique recommended by the National Committee for Clinical Laboratory Standards.23 The MTB and INHR-TB clinical isolate were obtained from Tuberculosis Research Centre, Alwar, India. The MIC was dened as the minimum concentration of compound required to inhibit 90% of bacterial growth and MIC of the compounds were reported in Table 3 with standard drug isoniazid for comparison. The synthesized title compounds (4a4n) were screened for antibacterial activity against Staphylococcus aureus (NCIM 2079), Bacillus subtilis (NCIM 2439), Escherichia coli (NCIM 5051) and Pseudomonas aeruginosa (ATCC 10145) bacterial strains by disc-diffusion method.24,25 All the bacterial strains were procured from National Chemical Laboratory, Pune and American Type Culture Collection (ATCC) and Gene Bank, Institute of Microbial Technology, Chandigarh, India. Ciprooxacin was used as a standard drug. Minimum inhibitory concentrations (MICs) were determined by broth dilution technique. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as minimum inhibitory concentration (MIC). The synthesized title compounds

(4a4n) were screened for antifungal activity against Aspergillus niger (NCIM 1196) and Candida albicans (NCIM 3471) in DMSO by agar diffusion method26,27 All the fungal strains were procured from National Chemical Laboratory, Pune, India. The lowest concentration (highest dilution) required to arrest the growth of fungus was regarded as minimum inhibitory concentration (MIC). The MIC of compounds was reported in Table 3 with standard drugs ciprooxacin and uconazole for comparison. Among the fourteen synthesized compounds, ve compounds were found to be active with minimum inhibitory concentration of 0.786.81 lM against MTB. The compound, 4d was found to be active against MTB and INHR-TB with MICs of 0.78 lM and 1.52 lM, respectively. The antimycobacterial activity of compound, 4d was comparable to isoniazid (INH) against MTB while 7.6-folds more active than INH against INHR-TB. The compound 4a, 4b, 4c and 4n showed good to moderate inhibitory activity against MTB with MICs 1.976.81 lM. The compound, 4c was found to be 1.4-fold more active than INH against INHR-TB. The N-aryl with electronegative group substitution showed maximum activity, in which 4-pyridinyl group had maximum inhibition when compare to 2-chlorophenyl, 4-chlorophenyl and 4-aminophenyl group substitution. The electron releasing group such as 4-methoxyphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl etc. showed less inhibition. All the compounds showed

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Table 3 Antimicrobial and antitubercular screening of the title compounds (4an) Compound Minimum inhibitory concentration (lg/mL) Antibacterial S. aureus 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n Ciprooxacin Fluconazole Isoniazid 4 8 16 16 128 512 512 >512 256 >512 512 256 128 8 4 B. subtilis 4 8 32 16 64 256 512 >512 256 >512 512 256 128 16 4 E. coli 4 4 16 16 128 256 512 256 256 512 256 256 64 8 4 P. aeruginosa 8 8 64 32 128 512 512 >512 512 >512 512 256 128 8 4 A. niger 256 256 128 256 8 512 512 16 8 128 4 512 >512 128 2 Antifugal C. albicans 256 128 128 256 16 512 512 32 16 256 4 512 512 256 1 MIC (lM) Antitubercular MTBa 1.97 3.94 4.15 0.78 >13.25 >12.78 10 >12.78 >13.31 >13.31 10 10 12.78 6.81 0.78 MTBb 12.65 12.65 8.29 1.52 NT NT 13.8 NT NT NT 13.31 >13.31 12.78 13.62 11.58

NT = not tested. a Mycobacterium tuberculosis H37Rv. b INHR-TB.

low to high antibacterial activity. The compound 4a showed good antibacterial activity while compounds 4c, 4d and 4n showed moderate activity with MICs 864 lg/mL against various bacterial strains. The activity of compound, 4a (MIC 4 lg/mL) against S. aureus, B. subtiltis and E. coli and the activity of compound, 4b (MIC 4 lg/mL) against E. coli was found to be comparable with that of the standard drug ciprooxacin. The N-aryl with electronegative group substitution such as 2-chlorophenyl, 4-chlorophenyl and 2-thiophenyl group showed maximum antibacterial activity. The antifungal activity of the title compound was found to be low to moderate. The compounds, 4e, 4i and 4k showed moderate antifungal activity with MICs, 416 lg/mL. The compound, 4k showed maximum antifungal activity, albeit less active than the standard drug uconazole. The N-aryl with 2-hydroxyphenyl, 4-methylphenyl and 2-methylphenyl group substitution showed maximum antifungal activity than electronegative group substitution. When the biological activities were related with computational calculated drug-likeness score model it was found out that the compound, 4a with maximum drug-likeness score model (0.95) showed maximum antibacterial activity. The compounds, 4i and 4k showed maximum antifungal activity have drug-likeness score model of 0.93 and 0.69, respectively. The compound 4a, 4b, 4c and 4d showed maximum antimycobacterial inhibition had drug-likeness score model 0.95, 0.79, 0.61 and 0.60, respectively. All the active compounds were tested for cytotoxicity (IC50) in VERO cells at concentrations of 62.5 lg/mL or 10 times the MIC. After 72 h exposure, viability was assessed on the basis of cellular conversion of MTT into a formazan product using the Promega Cell Titer 96 Non-radioactive Cell proliferation method. Most of the active compounds were found to be non-toxic up to 62.5 lg/mL.28 The molecular docking simulation for possible action on InhA are currently under investigation. Also studies to acquire more information about quantitative structureactivity relationships (QSAR) and MDR are in progress in our laboratory. The oxadiazole derivatives discovered in this study may provide valuable therapeutic intervention for the treatment of tubercular disease. Acknowledgments Authors are thankful to the management people of Alwar Pharmacy College, Alwar, Rajasthan, India for providing research

facilities. The authors are also thankful to Dr. K. P. Singh and Dr. (Mrs.) Shobha Tomar, National Institute of Medical Sciences University, Jaipur, Rajasthan, India for their guidance. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2011.10.057. References and notes
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