Designing, synthesis and antimicrobial action of

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Journal of Biomolecular Structure and Dynamics

ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20

Designing, synthesis and antimicrobial action of oxazoline and thiazoline derivatives of fatty acid esters Anis Ahmad, Aiman Ahmad, Raja Sudhakar, Himani Varshney, Naidu Subbarao, Saba Ansari, Abdul Rauf & Asad U. Khan To cite this article: Anis Ahmad, Aiman Ahmad, Raja Sudhakar, Himani Varshney, Naidu Subbarao, Saba Ansari, Abdul Rauf & Asad U. Khan (2016): Designing, synthesis and antimicrobial action of oxazoline and thiazoline derivatives of fatty acid esters, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2016.1255260 To link to this article: http://dx.doi.org/10.1080/07391102.2016.1255260

Accepted author version posted online: 01 Nov 2016.

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Date: 02 November 2016, At: 21:44

Publisher: Taylor & Francis Journal: Journal of Biomolecular Structure and Dynamics DOI: http://dx.doi.org/10.1080/07391102.2016.1255260

Designing, synthesis and antimicrobial action of oxazoline and thiazoline derivatives of fatty acid esters Anis Ahmad 1$#, Aiman Ahmad2$§, Raja Sudhakar3, HimaniVarshney2, Naidu Subbarao3 , Saba Ansari4, Abdul Rauf 2*and Asad U. Khan1* 1

Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, 202 002, India Department of Chemistry, Aligarh Muslim University, Aligarh, 202 002, India 3 School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India 4 Department of Radiation Oncology, Miller school of Medicine/Sylvester Comprehensive Cancer Center University of Miami, Miami, Florida, 33136, USA # Current address: Department of Radiation Oncology, Miller school of Medicine/Sylvester Comprehensive Cancer Center University of Miami, Miami, Florida, 33136, USA § Current address: Department of Applied Chemistry, Aligarh Muslim University, Aligarh, 202 002, India 2

$ Both authors have equal contribution Running title: Designing and synthesis of antimicrobial agents

* Corresponding Author: Dr.Asad U Khan, Professor, Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP, India-202002. PH: 00919837021912, Fax: 0091-571-2721776; Email: [email protected]

For Dr. Abdul Rauf, Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India, Phone: 00919412545345, Email: [email protected]

GRAPHICAL ABSTRACT: A series of novel oxazoline and thiazoline derivatives of long chain unsaturated fatty acid esters were synthesized from urea and thiourea respectively and screened for their antibacterial activity against Escherichia coli, Methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Klebsiella pneumonia and antifungal activity against Candida albicans, Aspergillus fumigatus, Penicillium marneffei, Trichophyton mentagrophytes.

R1 R1

CH

CH

Br

Br

R2

MeOH, Reflux

N

NH2CXNH2

X NH2

X = O/S

R2

ABSTRACT In this study, a novel series of oxazoline and thiazoline were designed as inhibitors of cytochrome P450 14 alpha-sterol demethylase (CYP51) from Candida albicans and peptide deformylase of Escherichia coli. The long chain dibromo derivative of fatty acid esters on reaction with urea and thiourea gave their corresponding oxazolines and thiazolines respectively. All the compounds were characterized by their spectral data (IR, 1H NMR, 13C NMR and MS) and tested for antibacterial and antifungal activity by disk diffusion assay and minimum inhibitory concentration (MIC) by the broth microdilution method against Gram-positive and Gram-negative strains of bacteria as well as fungus strains. The investigation of antimicrobial screening revealed that all the compounds were found to be potent antimicrobial agents. After calculating likeness drug properties of the compounds by PASS software, ADMET-related descriptors were computed to predict the pharmacokinetic properties for the active and bioavailable compounds by discovery studio 2.5. Molecular docking studies have been performed on Peptide deformylase (PDF) of Escherichia coli and CYP 45014DM of Candida albicans to understand the mode of binding of the molecules in the active site of the receptor. Compounds (2-amino-5-(carbomethoxyoctyl)-1,3-oxazoline , 2-amino-5(carbomethoxyoctyl)-1,3-thiazoline and 2-amino-4-pentyl-5-[(8’R)-8’ hydroxy (carbomethoxydecyl)-1,3-oxazoline ) showed excellent antimicrobial activity nearly equivalent to the control compounds and compounds,2-amino-4-octyl-5-(carbomethoxyheptyl)-1,3-oxazolin, 2-

amino-4-(2'R)(2’-hydroxy octyl)-5-(carbomethoxyheptyl)-1,3-oxazoline and 2-amino-4-pentyl-5[(8’R)-8’-hydroxy(carbomethoxy decyl)-1,3-oxazolineshowed vasodilation and antihypertensive properties. Furthermore, a computational analysis of physicochemical parameters revealed that the most of the compounds possessed drug-like attributes. By using Bioinformatics approach, we found a correlation between the observed and predicted antimicrobial activities. Keywords: oxazoline , thiazoline, drug resistance, antimicrobial agent, docking

ABBREVIATIONS MR,

Moler Refractivity

MW,

Molecular Weight

MV,

Molecular Volume

PASS,

Predicted Activity Spectrum of Substances

PSA,

Polar Surface Area

MIC,

Minimal InhibitoryConcentration

SAR,

Structure-Activity Relationship

MRSA,

Methicillin Resistant S. aureus

Sa,

Staphylococcus aureus

1. INTRODUCTION In most of the healthcare settings, Methicillin-resistant Staphylococcus aureus is the leading cause of infection (DeLeo, Otto, Kreiswirth & Chambers 2010). Candidiasis, Trichomoniasis, and bacterial vaginosis are responsible for more than 90 % cases of vaginitis (Ahmad & Khan 2009). Globally, there is increasing morbidity and mortality annually due to Pneumonia, enteric infections(Bergstrom, Sham, Zarepour & Vallance 2012). Despite the side effects of Amoxicillin, norfloxacin, and ciprofloxacin, they are used to treat the infections of the E.coli like diarrhea(McIsaac & Hunchak 2011). Several factors like theevolution of the new resistant mechanism (production of carbapenemases or extended-spectrum beta-lactamases), toxicity and mobile element (Butcu et al. 2011). The role of ADMET and biopharmaceutical properties of compounds including bioavailability are critical in drug discovery, and drug dosage designing (Leucuta 2014). There is an urgent need for new drugs against biological warfare agents (BWA) and genetic modified strains. Fast in silico techniques like docking and molecular dynamics simulations may be very helpful to provide structures of several new lead compounds quickly for further experimental work against the BWA Bacillus anthracis and Yersinia pestis (Franca, Guimaraes, Cortopassi, Oliveira & Ramalho 2013).There are suitable software like Molegro(®) and Spartan(®)for the prediction of kinetic and thermodynamic parameters of lead compounds, which

may be useful in the design and selection of new and more effective compounds (Giacoppo et al. 2015).Nitrogen-containing heterocycles have gained importance because they possess many biological activities and pharmaceutical properties. Fatty acid substrates due to its own pesticidal, antimicrobial, antidepressant, antitumor activities are being used extensively as a starting material (Farshori, Ahmad, Khan & Rauf 2011; Jubie et al. 2012). Significant biological activities exhibited by oxazolines and their corresponding oxidized derivatives i.e. oxazoles and thiazoles that are commonly found in marine sources(Davyt & Serra 2010). Derivatives of oxazoline and thiazoline have been used as therapeutic agents and have biological properties such as antihypertensive (Remko, Swart & Bickelhaupt 2006), antidiabetic, antitumor(Vassilev et al. 2004; Crane et al. 2006) activities. The oxazoline derivatives also play a significant role in pharmaceutical drug discovery(Wipf, Reeves, Balachandran & Day 2002). Numerous methods had been explored for the synthesis of oxazoline and thiazoline derivatives (Kempe, Lobert, Hoogenboom & Schubert 2009; Padmavathi, Venkatesh, Muralikrishna & Padmaja 2012). Many of these methods involve expensive reagents, longer reaction time, low yields, strong acidic conditions and the use of toxic organic solvents. Due to above limitations, the discovery of new methods with short reaction time and high yields for the preparation of these compounds is of prime importance. We now apply urea and thiourea with dibromo fatty acid esters for the very first time in a mild reaction condition for the direct synthesis of 2-amino-5-substituted and 2-amino-4,5-disubstituted oxazoline and thiazoline derivatives. 2

EXPERIMENTAL PROTOCOL

2.1 Physical and Spectroscopic Measurements All reagents and solvents were received from commercial suppliers. Undec-10-enoic (purity 98%) and (9Z)-octadec-9-enoic (97%) acids were obtained commercially from Fluka Chemicals (Switzerland). (9Z,

12R)-12-Hydroxyoctadec-9-enoic (ricinolic, 98%) acid and (isoricinolic, 98%) acid were isolated from natural sources i.e. Ricinuscommunis and Wrightiatinctoria seed oils, respectively following Gunstone’s partition (Gunstone 1954). The esters of fatty acids (long chain alkenoates) were prepared by refluxing the fatty acid in methanol in the presence of a catalytic volume of concentrated sulfuric acid. The dibromo derivatives of fatty acid esters were synthesized following the literature method (Gunstone 1972). Reactions were monitored by thin-layer chromatography on a glass plate (20×5cm) with a layer of silica gel G (Merck, Mumbai, India, thickness 0.5 mm). A mixture of petroleum ether-diethyl ether was used as developing solvents in different proportions for various compounds and was visualized in an iodine chamber. For column chromatography, silica gel (60-120 mesh) was used.1H NMR spectra were recorded in CdCl3 on Bruker DRX-400 spectrometer at 400 MHz, and

13

C NMR was recorded at 100 MHz in

CdCl3. The chemical shifts (δ) were measuredabout TMS as an internal standard in ppm. Coupling constants (J) are expressed in Hz. Mass spectra were obtained on Jeol SX-102 (FAB) spectrometer. IR spectra were obtained on Shimadzu 8201 PC spectrophotometer using KBr pellet with absorption in cm-1. Products purification was done by column chromatography and the synthesized compounds were identified by IR, 1H NMR, 13C NMRand mass spectra. 2.2 Synthesis 2.2.1 General procedure for preparation of dibromo derivative of fatty acid esters (1a-d). Fatty acid ester (0.1 mole) was dissolved in carbon tetrachloride and an equimolar amount of bromine (added dropwise to the reaction mixture) at 0˚C. The reaction was stirred until all the fatty acid ester was used. 2.2.2 General procedure for synthesis of oxazoline and thiazoline derivatives of fatty acid esters: 0.1 Mole of dibromo derivative of fatty acid ester 1a-d dissolved in methanol and equimolar amount of urea/thiourea was added and then the mixture of the reactant were refluxed on a paraffin bath. The

reaction was continued till all reactants were consumed. After completion solvent was evaporated on a water bath and then worked up with diethyl ether-water. Product was purified by column chromatography. All these novel compounds were yellow oily liquid and were characterized from their spectral data. 4.2.1.2. 2-Amino-5-(carbomethoxyoctyl)-1,3-oxazoline (2a) Yield: 85%; IR (νmax, cm-1) (KBr): 3320 (NH2 stretching), 2931(C-Hstretching), 1739 (C=O in est1437(C=N ring), 1362(C-O-C). 1H NMR (CDCl3, δH): 4.16 (tdd, 1H, J H  CH = 6.6 Hz, 9

Hz, J H  H = 17.1 Hz, CH2-CH-), 3.85 (dd, 1H, E

OCH3), 3.64 (dd, 1H, J H

E H

= 17.1 Hz, J H

E

HZ

JHZ H =

10.2 Hz,

J HZ H E =

2

JH HZ

= 10.2

1.2 Hz, HZC-CH), 3.66 (s, 3H, -

= 3.6 Hz, HEC-CH), 2.32 (s, 2H, NH2), 2.30 (t, J = 7.52

Hz, 2H, -CH2COOCH3), 2.12 (m, 2H, -CH2CH2COOCH3), 1.76 (m, 2H, -CH2(CH2 )5-), 1.31 (br.s, 10H, (CH2)5). 13C NMR (CDCl3, δC): δ 179.8, 163.4, 79.2, 64.7, 51.4, 36.3, 35.9, 34.0, 29.8, 29.1, 28.7, 26.7, 24.9. MS (ESI): m/z = 279.1512 [M+Na]+ , Calculated = 279.3300. 4.2.2. 2-Amino-4-octyl-5-(carbomethoxyheptyl)-1,3-oxazoline (2b) Yield: 80%; IR (νmax, cm-1) (KBr): 3322 (NH2 stretching), 2927 (C-H stretching), 1740 (C=O in ester), 1461 (C=N ring), 1366 (C-O-C stretching). 1H NMR (CDCl3, δH):4.20 (m, 2H, CH-CH ring), 3.65 (s, 3H, -OCH3), 2.31 (s, 2H, NH2), 2.24 (t, 2H, J = 7.75 Hz, CH2COOCH3), 1.56 (m, 4H, CH2-CHCHCH2), 1.44 (m, 2H, CH2CH2COOCH3), 1.30 (br.s, 18H, (CH2)9), 0.87 (dis.t, 3H, CH3).13C NMR (CDCl3, δC): 178.7, 164.2, 79.0, 63.8, 51.3, 37.3, 34.0, 31.9, 30.7, 29.9, 29.7, 29.3, 28.7, 28.3, 26.5, 25.8, 24.5, 24.1, 22.7, 14.1. MS (ESI): m/z = 377.3010 [M+Na]+, calculated = 377.5155. 4.2.2.1. 2-Amino-4-(2'R)(2’-hydroxy octyl)-5-(carbomethoxyheptyl)-1,3-oxazoline (2c)Yield: 82%; IR (νmax, cm-1) (KBr): 3461-3315 (OH, NH2 stretching), 2928 (C-H stretching), 1740 (C=O in ester), 1459 (C=N ring), 1363 (C-O-C); 1HNMR (CDCl3, δH):4.05 (m, 1H, CH-OH), 3.98 (m, 2H, CH-CH ring), 3.66 (s, 3H, -OCH3), 2.32 (s, 2H, NH2), 2.20 (t, 2H, J = 7.14, CH2COOCH3), 2.06 (m, 1H, CH-OH), 1.59 (m,

4H, CH2-CHCH-CH2), 1.49 (m, 2H, CH2CH2COOCH3), 1.43 (br.s, 18H, (CH2)9), 0.88 (dist.t, 3H, CH3).13C NMR (CDCl3, δC): 179.2, 162.9, 79.1, 72.4, 65.1, 52.9, 42.3, 36.1, 35.2, 34.6, 33.9, 31.7, 29.9, 29.4, 29.0, 28.8, 28.6, 27.7, 25.8, 14.1.MS (ESI): m/z = 393.3526 [M+Na]+, calculated = 393.5149. 4.2.2.2. 2-Amino-4-pentyl-5-[(8’R)-8’-hydroxy(carbomethoxy decyl)-1,3-oxazoline (2d) Yield: 65%; IR (νmaxcm-1) (KBr): 3475-3314(OH, NH2 stretching), 2925 (CH2 chain), 1739 (C=O in ester), 1455 (C=N ring), 1362 (C-O-C). 1H NMR (CDCl3, δH):4.12 (m, 1H, CH-OH), 3.85 (m, 2H, CHCH ring), 3.65 (s, 3H, -OCH3), 2.33 (s, 2H, NH2), 2.18 (t, 2H, J = 7.32 Hz, CH2COOCH3), 2.02 (m, 1H, CH-OH), 1.60 (m, 4H, CH2-CHCH-CH2), 1.53 (m, 2H, CH2CH2COOCH3), 1.38 (br.s, 18H, (CH2)9), 0.84 (dist.t, 3H, CH3). 13C NMR (CDCl3, δC): 178.8, 164.7, 78.4, 73.2, 63.6, 52.9, 41.8, 37.3, 34.7, 33.4, 31.3, 29.8, 29.0, 28.7, 28.5, 28.4, 28.3, 26.9, 25.8, 14.0. MS (ESI): m/z = 393.3894 [M+Na]+, calculated = 393.5149. 4.2.2.3. 2-Amino-5-(carbomethoxyoctyl)-1,3-thiazoline (3a) Yield: 83%; IR (νmax, cm-1) (KBr): 3324 (NH2 stretching), 2929 (C-H stretching), 1738 (C=O in ester), 1435 (C=N ring), 1359 (C-S-C). 1HNMR (CDCl3, δH) : 4.16 (tdd, 1H, J H  CH = 6.6 Hz, 9

J H  H E = 17.1 Hz, CH2-CH-), 3.85 (dd, 1H, J H

OCH3), 3.64 (dd, 1H, J H

E H

= 17.1 Hz, J H

E

HZ

Z

H

= 10.2 Hz,

J HZ H E =

2

JH HZ

= 10.2 Hz,

1.2 Hz, HZC-CH), 3.66 (s, 3H, -

= 3.6 Hz, HEC-CH), 2.32 (s, 2H, NH2), 2.30 (t, J = 7.52

Hz, 2H, CH2COOCH3), 2.12 (m, 2H, CH2CH2COOCH3), 1.76 (m, 2H, CH2(CH2 )5), 1.31 (br.s, 10H, (CH2)5).13C NMR (CDCl3, δC): δ 177.2, 165.9, 79.5, 62.2, 50.8, 35.8, 34.3, 33.8, 28.8, 28.6, 28.3, 25.8, 24.2. MS (ESI): m/z = 294.9914 [M+Na]+ , Calculated = 295.3956. 4.2.2.4. 2-Amino-4-octyl-5-(carbomethoxyheptyl)-1,3-thiazoline (3b) Yield: 80%; IR (νmax, cm-1) (KBr): 3323 (NH2 stretching), 2926 (C-H stretching), 1740 (C=O stretching), 1461(C=N ring), 1355 (C-S-C stretching). 1H NMR (CDCl3, δH):4.20 (m, 2H, CH-CH ring), 3.66 (s, 3H, -OCH3), 2.32 (s, 2H, NH2), 2.30 (t, 2H, J = 7.45 Hz, CH2COOCH3), 1.57 (m, 4H, CH2-

CHCH-CH2), 1.43 (m, 2H, CH2CH2COOCH3), 1.29 (br.s, 18H, (CH2)9), 0.89 (dist.t, 3H, CH3).

13

C

NMR (CDCl3, δC): δ 177.5, 164.9, 79.1, 63.9, 52.0, 36.3, 33.8, 31.5, 30.2, 29.9, 29.7, 29.5, 28.8, 28.7, 28.1, 28.0, 25.1, 24.6, 22.6, 14.0. MS (ESI): m/z = 393.3214 [M+Na]+, calculated = 393.5811. 4.2.2.5. 2-Amino-4-(2'R)(2’-hydroxy octyl)-5-(carbomethoxy heptyl)-1,3-thiazoline (3c) Yield: 83%; IR (νmax,cm-1) (KBr): 3493-3318 (OH, NH2 stretching), 2930 (C-H stretching), 1738 (C=O in ester), 1461 (C=N ring), 1358 (C-S-C). 1H NMR (CDCl3, δH):4.02 (m, 1H, CH-OH), 3.95 (m, 2H, CH-CH ring), 3.66 (s, 3H, -OCH3), 2.32 (s, 2H, NH2), 2.20 (t, 2H, J = 7.46 Hz, CH2COOH), 2.00 (m, 1H, CH-OH), 1.62 (m, 4H, CH2-CHCH-CH2), 1.38 (m, 2H, CH2CH2COOCH3), 1.36 (br.s, 18H, (CH2)9), 0.79 (dist.t, 3H, CH3). 13C NMR (CDCl3, δC): 178.8, 164.2, 79.8, 71.8, 62.7, 51.8, 38.8, 35.0, 34.8, 33.5, 31.4, 29.9, 29.5, 28.8, 28.7, 28.5, 28.0, 27.7, 25.8, 14.0. MS (ESI): m/z = 409.0123 [M+Na]+, calculated = 409.5805. 4.2.2.6. 2-Amino-4-pentyl-5-[(8’R)-8’-hydroxy(carbomethoxydecyl)-1,3-thiazoline (3d) Yield: 68%; IR (νmax,cm-1 ) (KBr): 3493-3317 (OH, NH2 stretching), 2927 (C-H stretching), 1712 (C=O in ester), 1452 (C=N ring), 1340 (C-S-C). 1H NMR (CDCl3, δH):4.20 (m, 1H, CH-OH), 3.83 (m, 2H, CH-CH ring), 3.66 (s, 3H, -OCH3), 2.33 (s, 2H, NH2), 2.30 (t, 2H, J = 7.32, CH2COOCH3), 2.14 (m, 1H, CH-OH), 1.58 (m, 4H, CH2-CHCH-CH2), 1.49 (m, 2H, CH2CH2COOCH3), 1.37 (br.s, 18H, (CH2)9), 0.89 (dist.t, 3H, CH3). 13C NMR (CDCl3, δC): 178.9, 163.4, 79.1, 70.9, 63.1, 51.4, 38.4, 35.1, 34.5, 34.2, 33.4, 31.3, 29.0, 28.9, 28.6, 28.5, 28.3, 26.9, 25.7, 14.0. MS (ESI): m/z = 409.1391 [M+Na]+, calculated = 409.5805. 2.3 Prediction of Hidden Biological Potential of the Synthesized Compounds The activity of the molecules was predicted, using PASS (Prediction of Activity Spectra for Substances) which estimates the probable biological activity spectra for compounds under study

based on their structural formulae presented in MOL file or SDF file format using Marvin applet (Pospieszny, Koenig, Kowalczyk & Brycki 2014). 2.4 ADME & Toxicity Studies In silico ADME studies were performed by using ADME Descriptors algorithm of Accelrys Discovery Studio 2.5 in which various pharmacokinetic parameters like Aqueous Solubility (Cheng & Merz 2003), Human Intestinal Absorption (Egan, Merz & Baldwin 2000), plasma protein binding (PPB) (Colmenarejo, Alvarez-Pedraglio & Lavandera 2001), blood-brain-barrier (BBB) penetration (Kelder, Grootenhuis, Bayada, Delbressine & Ploemen 1999), Cytochrome P450-14DMinhibition (Susnow & Dixon 2003) and hepatotoxicity levels (Cheng & Dixon 2003) were estimated for 5 ligands. Obtained results were cross-checked with the standard levels listed in (Table 2). Toxicity profiling of all the five ligands was performed by employing Toxicity prediction –extensible protocol of Accelrys DiscoveryStudio 2.5. Toxicity profile includes screening for carcinogenicity, aerobic biodegradability, developmental toxicity potentials, AMES mutagenicity, and ocular and skin irritancy (Xia, Maliski, Gallant & Rogers 2004). OSIRIS property explorer an online tool was used to study teratogenicity effects of the ligands (Sravani, N Duganath, Gade & Sandeep Reddy 2012). The structures of all the novel compounds are shown in Scheme 1 and 2. 2.5 Prediction of Physicochemical Properties The

octanol/water

partition

coefficient

ClogP

being

a

measure

of

hydrophobicity/lipophilicity was calculated using ChemDraw Ultra 11.0 software (Cambridge Soft Corporation) (Lipinski, Lombardo, Dominy & Feeney 2001). The physicochemical parameters including octanol partition coefficients (miCLog P), MW, HBD, HBA, TPSA, and Rotatable bonds

were calculated using a molinspiration server (http://www.molinspiration.com/cgi-bin/properties) and ChemAxon (chemicalize.org) (Parvez, Jyotsna & Taibi Ben 2010; Parvez et al. 2010). 2.6 Bioactivity Score The drugs were also checked for the bioactivity by calculating the activity score. All the bioactivity parameters were checked with the help of software ChemSketch 11 and Molinspiration drug-likeness score online (www.molinspiration.com). A calculateddrug-likeness score of each newly synthesized compound was compared with standard drugs. 2.7 Antibacterial Assay The antibacterial activity of the synthesized compounds was completed by the disk diffusion method and measured by Halo Zone Test (Cruickshank, Duguid, Marmion & Swain 1975; Collins 1976). The microdilution test was performed to study the MIC of synthesized compounds against bacterial strains, and the results were observed visually and spectrophotometrically.

2.8 Antifungal Activity Clinical samples were obtained from the Gynaecology OPD and NICU (Neonatal Intensive Care Unit) of Jawaharlal Nehru Medical College (JNMC), Aligarh Muslim University, Aligarh, India. Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), oatmeal, and RPMI 1640 were used for agar dilution and macrodilution methods. The clinical isolates of fungi including Candida albicans, Candida tropicalis, and Candida parapeilosis were purified and subcultures on SC, SCC, and PDA media before testing. To obtain the stock solutions of the compounds, 200 mg/ml of the compound was dissolved in DMSO. The compounds were diluted in liquid broth and a solid medium to obtain a final concentration from 0.0312 to 256 mg/ml, using PDA and RPMI 1640 media. The inocula of the yeasts were prepared from 1-10 days mature colonies grown. Fluconazole

and itraconazole or griseofulvins were used as positive and the solvents of the compounds as negative blanks. 2.9 Molecular Docking Crystal structures of protein (PDB ID: 1G2A & 1E9X) were downloaded from PDB. They were prepared with default parameters and energy minimized up to 1000 cycles by using Protein preparation wizard module- Schrodinger 9.1. Synthesized ligands were drawn by ChemDraw 11.1. Then the drawn molecules along with their known inhibitors were prepared and converted to 3D structures by Ligprep module-Schrodinger 9.1. Binding site residues like TYR76 PHE78 MET79 PHE83 PHE89 LYS97 MET99 HIS101 SER252 PHE255 HIS259 THR260 LEU321 ILE323 MET433 VAL434 for 1E9X and GLU42 GLY43 ILE44 GLY45 GLN50 GLU87 GLU88 GLY89 CYS90 LEU91 CYS129 HIS132 GLU133 HIS136 were predicted by Pocket-Finder software, and they were confirmed by our previous data. Protein-ligand docking was performed by GOLD v5.0.1 software, which uses a genetic algorithm for docking and produces the result based on GOLD fitness score. By using default parameters except for the number of runs per ligand which was changed to 50 instead ten runs. Binding affinity was calculated for the protein-ligand interaction complex by Xscorev1.2.1. The post-docking analysis was performed by using LIGPLOT for each protein-ligand interactions to know the residues involved in a hydrogen bond, lipophilic and nonbonded contacts. Associated Content Supporting Information. No additional experimental details, spectroscopic data for intermediates, NMR (1H and 13C) spectra of compounds. 3 RESULTS AND DISCUSSION 3.1 Chemistry

Derivatives of fatty acid have already been reported as an antimicrobial agent in our laboratory (Farshori, Banday, Ahmad, Khan & Rauf 2010; Farshori et al. 2011),and it has been suggested that the antimicrobial activity of the organic compounds can be enhanced by incorporating fatty acid side chain. In the present study, we have discussed the synthesis, characterization and antimicrobial activities of oxazoline and thiazoline derivatives of fatty acid esters. The synthesis of 2-amino-5substituted and 2-amino-4, 5-disubstituted oxazolines 2a-d and thiazolines3a-d was carried out by refluxing urea and thiourea, with various dibromo derivatives of olefinic and hydroxyl olefinic fatty acid esters 1a-d in methanol (Scheme 1). All the reactions were observed by thin-layer chromatography (TLC). The nucleophilic substitution of urea and thiourea to dibromo derivatives of olefinic and hydroxyl olefinic fatty acid esters gives an inseparable isomeric mixture of oxazolines and thiazolines, respectively. The products 2a-d and 3a-d were purified on silica gel column with petroleum ether: diethyl ether as eluent. IR, 1H NMR,

13

C NMR and mass spectra were used to

characterize the newly synthesized compounds. The structure of compound 2ais outlined in Scheme 2. A detailed spectral description for compound 2ais discussed below. IR spectrum of compound 2a revealed 3320 cm

-1

(NH2 stretching), 1739 cm-1 (C=O in ester), 1437 (C=N ring stretching), 1362

(C-O-C). In the 1H NMR, the cyclic protons CH2-CH were observed at δH4.16 (tdd, 1H, J H  CH = 6.6 9

Hz,

JH HZ

= 10.2 Hz, J H  H = 17.1 Hz, CH2-CH-), 3.85 (dd, 1H,

CH), 3.64 (dd, 1H, J H

E

E H

= 17.1 Hz, J H

E

HZ

JHZ H =

10.2 Hz,

J HZ H E =

2

1.2 Hz, HZC-

= 3.6 Hz, HEC-CH), a sharp singlet at 3.66 was observed

for three methyl ester protons and the two amine protons were observed at δH 2.32 as a single. The structure of compound 2a was further confirmed by 13C NMR spectral data which show peaks at δC 179.8, 163.4, 79.2, 64.7, 51.4, 36.3, 35.9, 34.0, 29.8, 29.1, 28.7, 26.7, 24.9. The mass spectra showed characteristic molecular ion peak in accord with the molecular formula. In the same way, all other synthesized compounds were also characterized by their spectral data

3.2 In Silico Study 3.2.1 Prediction Activity Spectra for Substances (PASS) Drug-likeness score of the compounds are presented in (Table 1) and was calculated using PASS software. Based on the various molecular properties and structural features of the compound, druglikeness score determines the similarity of the synthesized compound to the known drug. The synthesized compound to be a drug, it should not have zero or negative drug-likeness score. The higher drug-likeness 0.984 and 0.964 were observed for the compounds 3c and 2a respectively which proved their probability to use as a future therapeutic agent. The compound 2a having oxazole heterocyclic ring have higher drug-likeness score compared to 2b having thiazole heterocyclic ring. Further substitution of heterocyclic rings of 2a and 2b with long alkoxy side chain, decreased the drug-likeness score. The optimum length of alkoxy side chain is favorable for ADMET profile of the synthesized compounds(Williams, Sugandhi, Macri, Falkinham & Gandour 2007). However, further substitution of 8-(2-amino-4,5-dihydro-thiazol-5-yl)-octanoic acid methyl ester with 2-hydroxy-octyl side chain enhanced the drug-likeness score of the compounds 2c-3d because it favors the ADMET profile of the compounds as also reported in our previous study. The most common cause of glaucoma is ischemic injury due to pro-inflammatory cytokines glutamate, oxygen free-radical, and nitric oxide (Husain, Abdul, Singh, Ahmad & Husain 2014). Compound 2b, 2c, and 2d have validation and antihypertensive properties apart from antimicrobial properties and hence can play a significant role in neuroprotection against glaucoma and heart disease. The algorithm used in PASS successfully discriminated the drug-like compounds from drug-unlike compounds and similar studies have also been carried out earlier(Lagunin, Filimonov & Poroikov 2010).Compound 2b, 2c, and 2d have validation and antihypertensive properties apart from antimicrobial properties and hence can play a significant role in neuroprotection against glaucoma

and heart disease. The algorithm used in PASS successfully discriminated the drug-like compounds from drug-unlike compounds and similar studies have also been carried out earlier. 3.2.2 Absorption, distribution, metabolism, and excretion (ADME) and Toxicity Researchers are involved in synthesizing newly substituted derivatives to enhance the antibacterial potential of already known chemicals and in predicting physicochemical properties of hypothetical antibacterial pharmacophores (Parvez et al. 2010). Standard levels of ADMET descriptors are calculated using Discovery Studio 2.5 software (Table 2).Table 3 depicts calculated ADMET descriptors for all the synthesized novel compounds. Data illustrates that synthesized compounds have no or very low aqueous solubility. These can act as non-inhibitors of CYP2D6 and can assimilate in Phase-I metabolism. Binding of drugs to plasma proteins may reduce their bioavailability (Pellegatti 2011). Most of the compounds synthesized in this work show less plasma protein binding parameters. Results of ADMET test indicate that these compounds can reach the desired targets. These compounds also passed ocular irritancy, mutagenicity, and skin sensitization tests and were found to be aerobically biodegradable. Taken together, it can be concluded that all the newly synthesized compounds have desired properties to act as drugs. Using ADMET_PSA_2D and ADMET_A logp98 properties, Blood Brain Barrier (BBB) and Human Intestinal Absorption (HIA) ADME plots for all the synthesized compounds, are shown in Figure 1. As seen in the figure, values for all compounds except 2a and 3a fall outside the 99% ellipse (undefined) in BBB-PLOT, indicating that excluded compounds (all except 2a and 3a)cannot penetrate BBB and hence there are fewer chances for nervous system toxicity. Compounds 2a and 3a fall inside the 99% ellipse and hence can cross BBB. Whether a drug crosses, BBB can be

beneficial or harmful depending on where it has to be targeted and its toxicity profile. Strategies are available to make non-penetrating drugs reach the brain (Gabathuler 2010). Also can be inferred from figure 1, all compounds can be properly absorbed by intestines upon ingestion, as their values have fallen inside 99% ellipse. Although oral route is the most shared and easy way of administering drugs in the body, due to low intestinal absorption, bioavailability decreases. Different strategies have been developed to overcome demerits of oral delivery (Xu, Ling & Zhang 2013). 3.2.3 Virtual screenings and molecular properties calculations When a new molecule is designed to use as a drug, it has to be checked on ADMET and “Lipinski’s Rule of Five” to determine whether the molecule has drug-like properties or not. Desirable properties of a molecule to act as the drug may include better solubility, plasma stability, favorable absorption, good bioavailability, pharmacology binding efficiency, and safety. In silico methods developed for the efficient screening of synthesized compounds, helps to save resources and speed up the drug discovery process [24, 25]. In the present work, we have performed a similar study for synthesized compounds and compared them with the values obtained for standard approved drugs Ciprofloxacin and Fluconazole. 3.2.3.1 pKa and cLog P Determining pKa and cLogP parameters is essential in drug testing [26].Molecules possessing ionizable group at physiological pH possess biological activity and hence are preferred to be used as drugs. The pKa is the pH at which ionizable group of the molecule is 50% protonated.

It

affects pharmacokinetic parameters; like Solubility, permeability, metabolism, bioavailability and excretion of the compound. For a compound existing as a tautomer, the most stable one is used

for calculating pKa values. The presence of particularsubstituent which results in resonance stabilization/charge

delocalization

also

has

a

very major

effect

on

pKa

of

the

compounds(Settimo, Bellman & Knegtel 2013). The pKa values of the tested compounds are plottedvs. MIC, (Figure 2a). As shown in Figure2a, the pKa values can be correlated with the activity of the compounds under study. The highest pKa value was observed for the compound 3a (Table 5). Calculated cLogP value (partition coefficient) is a measure of the lipophilicity of a compound. Lipophilicity acts as a determinant of pharmacokinetic behavior of drugs [28]. It can influence the distribution of drug into tissues, binding and absorption of the drug and also determines the solubility of the compound. For Achievingproper absorption as well as cell permeability, cLogP value between 2 and 3 is considered as optimal in an oral drug program and must never be above 5.0 for a compound to be considered as a drug. On this basis, all the compounds (2a to 3d) except 2b and 3b are having log P values under the acceptable criteria (Table 5). Compounds 2a and 3a having lower log P values as compared to other compounds in the series have also shown good antibacterial activity (figure 2b). Compounds that have low cLogP, as well as low MIC values, can be active as anti-microbial agents [29].Distribution of cLogP versus MW has been shown in Figure 3. 3.2.3.2 “Rule of Five” Properties the compound should have high intestinal absorption, and good oral bioavailability (Muegge 2003).Molecular properties like membrane permeability and bioavailability are always linked with some basic molecular descriptors such as cLogP (partition coefficient), molecular weight (MW), hydrogen bond acceptors and donors count in a molecule. According to “Lipinski Rule of Five” for

goodmembrane permeability molecules should have molecular weight ≤ 500, cLog P ≤ 5, the number of hydrogen bond acceptors ≤ 10, and the number of hydrogen bond donors ≤ 5. This rule is widely used as a filter for drug-like properties(Lipinski et al. 2001). A compound that fulfills at least three out of the four criteria is said to Lipinski’s “Rule of Five.” There will be poor permeation or absorption if a compound has more than 5 H-bond donors or 10 H-bond acceptors. The series (2a-3d) under investigation have less number of hydrogen bond donors (