Designing, synthesis, and antimicrobial action of oxazoline and thiazoline derivatives of fatty acid esters

Article abstract of DOI:10.1080/07391102.2016.1255260

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 (PDF) 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 by the broth microdilution method against gram-positive and gram-negative strains of bacteria as well as fungus strains. The investigation into antimicrobial screening revealed that all the compounds were found to be potent antimicrobial agents. After calculating likeness drug properties of the compounds by Prediction of Activity Spectra for Substances 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 PDF of E. coli and CYP 450-14DM of C. 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. Using Bioinformatics approach, we found a correlation between the observed and predicted antimicrobial activities.

Full text of DOI:10.1080/07391102.2016.1255260

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: 09:13

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
1
$#
2$§
3
2
3
Anis Ahmad
Aiman Ahmad , Raja Sudhakar , HimaniVarshney , Naidu Subbarao , Saba
,
4
2*
1*
Ansari , Abdul Rauf and Asad U. Khan
1
2
3
4
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, 202 002, India
Department of Chemistry, Aligarh Muslim University, Aligarh, 202 002, India
School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India
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
0
02, India
$
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: asad.k@rediffmail.com

For Dr. Abdul Rauf, Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India,
Phone: 00919412545345, Email: abduloafchem@gmail.com
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
Br
CH
Br
R2
MeOH, Reflux
N
NH CXNH2
R2
2
X
NH2
X = O/S

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
1
13
characterized by their spectral data (IR, H NMR, C 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 450-
1
4DM 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,
MV,
Molecular Weight
Molecular Volume
PASS,
PSA,
MIC,
SAR,
MRSA,
Sa,
Predicted Activity Spectrum of Substances
Polar Surface Area
Minimal InhibitoryConcentration
Structure-Activity Relationship
Methicillin Resistant S. aureus
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.
015).Nitrogen-containing heterocycles have gained importance because they possess many
2
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.
2
006) 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,
(

1
2R)-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
1
chamber. For column chromatography, silica gel (60-120 mesh) was used. H NMR spectra were recorded
1
3
in CdCl on Bruker DRX-400 spectrometer at 400 MHz, and C NMR was recorded at 100 MHz in
3
CdCl . The chemical shifts (δ) were measuredabout TMS as an internal standard in ppm. Coupling
3
constants (J) are expressed in Hz. Mass spectra were obtained on Jeol SX-102 (FAB) spectrometer. IR
-1
spectra were obtained on Shimadzu 8201 PC spectrophotometer using KBr pellet with absorption in cm .
Products purification was done by column chromatography and the synthesized compounds were
1
13
identified by IR, H NMR, C 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)
-1
Yield: 85%; IR (νmax, cm ) (KBr): 3320 (NH stretching), 2931(C-Hstretching), 1739 (C=O in
2
1
est1437(C=N ring), 1362(C-O-C). H NMR (CDCl , δ ): 4.16 (tdd, 1H,
J
= 6.6 Hz, JH HZ = 10.2
3
H
9
H  CH
2
Hz, JH HE = 17.1 Hz, CH -CH-), 3.85 (dd, 1H, JH = 10.2 Hz, JH = 1.2 Hz, H C-CH), 3.66 (s, 3H, -
2
Z
Z
H
Z HE
OCH ), 3.64 (dd, 1H, JHE H = 17.1 Hz, JHE HZ = 3.6 Hz, H C-CH), 2.32 (s, 2H, NH ), 2.30 (t, J = 7.52
3
E
2
Hz, 2H, -CH COOCH ), 2.12 (m, 2H, -CH CH COOCH ), 1.76 (m, 2H, -CH (CH ) -), 1.31 (br.s, 10H,
2
3
2
2
3
2
2 5
1
3
(
CH ) ). C NMR (CDCl δ ): 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,
2 5 3, C
+
2
4
4.9. MS (ESI): m/z = 279.1512 [M+Na] , Calculated = 279.3300.
.2.2. 2-Amino-4-octyl-5-(carbomethoxyheptyl)-1,3-oxazoline (2b)
-1
Yield: 80%; IR (νmax, cm ) (KBr): 3322 (NH stretching), 2927 (C-H stretching), 1740 (C=O in ester),
2
1
1
3
461 (C=N ring), 1366 (C-O-C stretching). H NMR (CDCl , δ ):4.20 (m, 2H, CH-CH ring), 3.65 (s,
3
H
H, -OCH ), 2.31 (s, 2H, NH ), 2.24 (t, 2H, J = 7.75 Hz, CH COOCH ), 1.56 (m, 4H, CH -CHCH-
3
2
2
3
2
1
3
CH ), 1.44 (m, 2H, CH CH COOCH ), 1.30 (br.s, 18H, (CH ) ), 0.87 (dis.t, 3H, CH ). C NMR (CDCl
3,
2
2
2
3
2 9
3
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,
+
2
4.1, 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
-
1
(
(
(
νmax, cm ) (KBr): 3461-3315 (OH, NH stretching), 2928 (C-H stretching), 1740 (C=O in ester), 1459
2
1
C=N ring), 1363 (C-O-C); HNMR (CDCl , δ ):4.05 (m, 1H, CH-OH), 3.98 (m, 2H, CH-CH ring), 3.66
3
H
s, 3H, -OCH ), 2.32 (s, 2H, NH ), 2.20 (t, 2H, J = 7.14, CH COOCH ), 2.06 (m, 1H, CH-OH), 1.59 (m,
3
2
2
3

4
H, CH -CHCH-CH ), 1.49 (m, 2H, CH CH COOCH ), 1.43 (br.s, 18H, (CH ) ), 0.88 (dist.t, 3H,
2 2 2 2 3 2 9
13
CH ). C NMR (CDCl δ ): 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,
3
3, C
+
2
9.4, 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)
-
1
Yield: 65%; IR (νmaxcm ) (KBr): 3475-3314(OH, NH stretching), 2925 (CH chain), 1739 (C=O in
2
2
1
ester), 1455 (C=N ring), 1362 (C-O-C). H NMR (CDCl , δ ):4.12 (m, 1H, CH-OH), 3.85 (m, 2H, CH-
3
H
CH ring), 3.65 (s, 3H, -OCH ), 2.33 (s, 2H, NH ), 2.18 (t, 2H, J = 7.32 Hz, CH COOCH ), 2.02 (m, 1H,
3
2
2
3
CH-OH), 1.60 (m, 4H, CH -CHCH-CH ), 1.53 (m, 2H, CH CH COOCH ), 1.38 (br.s, 18H, (CH ) ),
2
2
2
2
3
2 9
1
3
0
.84 (dist.t, 3H, CH ). C NMR (CDCl δ ): 178.8, 164.7, 78.4, 73.2, 63.6, 52.9, 41.8, 37.3, 34.7, 33.4,
3 3, C
+
3
1.3, 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.
.2.2.3. 2-Amino-5-(carbomethoxyoctyl)-1,3-thiazoline (3a)
=
4
-1
Yield: 83%; IR (νmax, cm ) (KBr): 3324 (NH stretching), 2929 (C-H stretching), 1738 (C=O in ester),
2
1
1
435 (C=N ring), 1359 (C-S-C). HNMR (CDCl , δ ) : 4.16 (tdd, 1H,
J
= 6.6 Hz, JH HZ = 10.2 Hz,
3
H
9
H  CH
2
JH HE = 17.1 Hz, CH
-CH-), 3.85 (dd, 1H, JH = 10.2 Hz, JH = 1.2 Hz, H C-CH), 3.66 (s, 3H, -
Z
H
2
Z
Z HE
OCH ), 3.64 (dd, 1H, JHE H = 17.1 Hz, JHE HZ = 3.6 Hz, H C-CH), 2.32 (s, 2H, NH ), 2.30 (t, J = 7.52
3
E
2
Hz, 2H, CH COOCH ), 2.12 (m, 2H, CH CH COOCH ), 1.76 (m, 2H, CH (CH ) ), 1.31 (br.s, 10H,
2
3
2
2
3
2
2 5
1
3
(
CH ) ). C NMR (CDCl δ ): 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,
2 5 3, C
+
2
4
4.2. MS (ESI): m/z = 294.9914 [M+Na] , Calculated = 295.3956.
.2.2.4. 2-Amino-4-octyl-5-(carbomethoxyheptyl)-1,3-thiazoline (3b)
-1
Yield: 80%; IR (νmax, cm ) (KBr): 3323 (NH stretching), 2926 (C-H stretching), 1740 (C=O
2
1
stretching), 1461(C=N ring), 1355 (C-S-C stretching). H NMR (CDCl , δ ):4.20 (m, 2H, CH-CH ring),
3
H
3
.66 (s, 3H, -OCH ), 2.32 (s, 2H, NH ), 2.30 (t, 2H, J = 7.45 Hz, CH COOCH ), 1.57 (m, 4H, CH -
3 2 2 3 2

1
3
CHCH-CH ), 1.43 (m, 2H, CH CH COOCH ), 1.29 (br.s, 18H, (CH ) ), 0.89 (dist.t, 3H, CH ). C
2
2
2
3
2 9
3
NMR (CDCl δ ): 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,
3
,
C
+
2
8.1, 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)
-
1
Yield: 83%; IR (νmax,cm ) (KBr): 3493-3318 (OH, NH stretching), 2930 (C-H stretching), 1738 (C=O
2
1
in ester), 1461 (C=N ring), 1358 (C-S-C). H NMR (CDCl , δ ):4.02 (m, 1H, CH-OH), 3.95 (m, 2H,
3
H
CH-CH ring), 3.66 (s, 3H, -OCH ), 2.32 (s, 2H, NH ), 2.20 (t, 2H, J = 7.46 Hz, CH COOH), 2.00 (m,
3
2
2
1
H, CH-OH), 1.62 (m, 4H, CH -CHCH-CH ), 1.38 (m, 2H, CH CH COOCH ), 1.36 (br.s, 18H,
2 2 2 2 3
1
3
(
CH ) ), 0.79 (dist.t, 3H, CH ). C NMR (CDCl δ ): 178.8, 164.2, 79.8, 71.8, 62.7, 51.8, 38.8, 35.0,
2 9 3 3, C
+
3
4.8, 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.
.2.2.6. 2-Amino-4-pentyl-5-[(8’R)-8’-hydroxy(carbomethoxydecyl)-1,3-thiazoline (3d)
4
-
1
Yield: 68%; IR (νmax,cm ) (KBr): 3493-3317 (OH, NH stretching), 2927 (C-H stretching), 1712 (C=O
2
1
in ester), 1452 (C=N ring), 1340 (C-S-C). H NMR (CDCl , δ ):4.20 (m, 1H, CH-OH), 3.83 (m, 2H,
3
H
CH-CH ring), 3.66 (s, 3H, -OCH ), 2.33 (s, 2H, NH ), 2.30 (t, 2H, J = 7.32, CH COOCH ), 2.14 (m,
3
2
2
3
1
H, CH-OH), 1.58 (m, 4H, CH -CHCH-CH ), 1.49 (m, 2H, CH CH COOCH ), 1.37 (br.s, 18H,
2 2 2 2 3
1
3
(
CH ) ), 0.89 (dist.t, 3H, CH ). C NMR (CDCl δ ): 178.9, 163.4, 79.1, 70.9, 63.1, 51.4, 38.4, 35.1,
2 9 3 3, C
+
3
4.5, 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
976). The microdilution test was performed to study the MIC of synthesized compounds against
bacterial strains, and the results were observed visually and spectrophotometrically.
1
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 non-
bonded contacts.
Associated Content
Supporting Information. No additional experimental details, spectroscopic data for intermediates,
1
13
NMR ( H and C) 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-5-
substituted 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
1
13
petroleum ether: diethyl ether as eluent. IR, H NMR, 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
-1
-1
revealed 3320 cm (NH stretching), 1739 cm (C=O in ester), 1437 (C=N ring stretching), 1362
2
1
(
C-O-C). In the H NMR, the cyclic protons CH -CH were observed at δ 4.16 (tdd, 1H,
J
= 6.6
2
H
9
H  CH
2
Hz, JH HZ = 10.2 Hz, JH HE = 17.1 Hz, CH -CH-), 3.85 (dd, 1H, JHZ H = 10.2 Hz, JHZ HE = 1.2 Hz, H C-
2
Z
CH), 3.64 (dd, 1H, JHE H = 17.1 Hz, JHE HZ = 3.6 Hz, H C-CH), a sharp singlet at 3.66 was observed
E
for three methyl ester protons and the two amine protons were observed at δ 2.32 as a single. The
H
1
3
structure of compound 2a was further confirmed by C NMR spectral data which show peaks at δ
C
1
79.8, 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, drug-
likeness 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
2
007). 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
2
b, 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
2
010).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
3
a 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
3
a (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
2
b 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
003).Molecular properties like membrane permeability and bioavailability are always linked with
2
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 (<5) and a
considerable number of acceptors (<10) as shown in Table 5. For evaluating drug-likeness,
Lipinski’s rule has initiated many extensions (Ghose, Viswanadhan & Wendoloski 1999).




Partition coefficient cLogP in - 0.4 to + 5.6 range
Molar refractivity from 40 to 130
Molecular weight from 160 to 480
A number of heavy atoms from 20 to 70
Molecular Polar Surface Area, total polar surface (TPSA) are calculated based on the
methodology published by Ertl et al. (Ertl, Rohde & Selzer 2000; Parvez et al. 2010) as a sum of
fragment contributions (O- and N-centered polar fragmentsare considered). PSA is an excellent
descriptor of drug absorption in the intestine, blood brain barrier penetration, Caco-2 permeability,
and bioavailability. The PSA is calculated from the surface areas that are occupied by oxygen and
nitrogen atoms and by hydrogen atoms attached to them. Thus, the PSA is closely related to the
hydrogen bonding potential of a compound (Clark 1999). Molecules with PSA values of 140 Å or
more are expected to show poor intestinal absorption (Clark 1999). Table 5 indicates that all the
compounds are within this limit. There is the probability of problems in bioavailability if a
compound has 2 or more than two violations of the ‘Rule of 5’(Lipinski et al. 2001). Note that all
the compounds except 2b and 3b have only one violation of Lipinski’s “Rule of Five.”Volume and

PSA are inversely proportional to % ABS. The corresponding polar surface area and volume are
least for the compounds 2a and 3a among the series. As shown in Table 5, all parameters of the
compounds satisfy the ‘rule of 5’. It was known that a molecule with 10 or fewer rotatable bonds
2
and PSA less than 140 Å would have good oral bioavailability (Veber et al. 2002) as seen in Table
5
.
3
.2.3.3 The Molar Refractivity (MR)
The coefficient of molar refractivity (MR) is positive in QSAR model developed to describe
the antifungal activity of the synthesized compounds as per the equation is given below:
pMIC = 0.018MR – 0.078
af
2
n = 14 r = 0.88 q =0.533 s = 0.088 f = 22.50
2
2
n – number of data points, r – correlation coefficient, q – cross-validated r were obtained by leave
one out method, s – standard error of the estimate and F – Fischer statistics. The equation indicated
that there is a positive correlation between antimicrobial activity and molar refractivity (MR) i.e.
antimicrobial potential of the synthesized compounds will increase with an increase in their MR
values and vice versa. The steric effects of themoleculeare described by its size polarizability which
was represented by MR. MRexplains the activity behavior of newly synthesized compounds. It is
evident from Table5 that all the compounds had a higher value of molar refractivity which favors
the activity ratio.
3
.2.3.4 Bioactivity Score
The bioactivity was also calculated for GPCR ligand, ion channel modulator, kinase inhibitor
and nuclear receptor ligand. In the case of an average organic molecule, if the bioactivity score is
more than 0.00 then is active, if - 0.50 to 0.00 then moderately active and less than - 0.50 isinactive.
The standard drug scores were compared with calculated scores of the synthesized compounds. To

identify an active moiety, proper bioactivity, suitable modifications in the structure were made. The
bioactivity score for synthesized derivatives is shown in Table 6which hadexcellent bioactivity
scores. In the series, 2b, 2c, 3c, and 2d showed better GPCR ligand scores than the FDA approved
drugs ciprofloxacin and fluconazole (0.12 and 0.12 respectively).
3
.3 In vitro antimicrobial activity evaluation
3
.3.1 Antibacterial Studies
The antimicrobial activity of the newly synthesized compound was carried out by disc
diffusion method (Espinel-Ingroff, Boyle & Sheehan 2001; Balikci, Acikgoz, Guvenman, Celikbilek
Ozdem 2014) against Escherichia coli (ATCC-25922), Staphylococcus aureus, Pseudomonas
&
aeruginosa (ATCC-27853), Streptococcus pyogenes and Klebsiella pneumoniae (Clinical isolates).
7
Standard inoculums (1-2 X10 c.f.u. /ml 0.5 McFarland standards) were spread onto the
surface of sterile agar plates. Whatman no. 1 filter paper was used to make the discs of 6 mm
diameter. For sterilization, the discs were kept at 140 °C for one hr. These sterile discs were soaked
into the known concentration of the synthesized compounds and were placedonto the culture plates.
For positive control, Ciprofloxacin (30 µg) discwas put on to the culture plates. The inverted culture
plates were incubated at 37 °C for 24 hrs. The susceptibility was evaluatedby the diameter of the
zone of inhibition against Gram-negative and Gram-positive bacteria and compared with the
controls (Table 7).
Minimum inhibitory concentrations (MICs) were found by the broth microdilution method.
The nutrient broth, which contained a logarithmic serially two-fold diluted amount of the newly
5
synthesized compound and controls were inoculated with approximately 5 X10 c.f.u. /ml of actively
dividing bacteria cells. The cultures of the bacterial strains were incubated for 24 hours at 37 °C and
the growth were monitored visually and spectrophotometrically. The lowest concentration of the

compound required to inhibitbacterial growth was regarded as minimum inhibitory concentration
MIC). For obtaining the minimum bacterial concentration (MBC), 0.1 ml volume was taken from
each tube and spread on agar plates.The number of colonies forming units (CFU) was counted after
4 hrs. of incubation, at 35°C. The lowest concentration of the compound in which 99.9% of the
(
2
inoculums were killed was defined as MBC, and the results are given (Table 8).
Antibacterial screening data revealed that all the tested compounds showed moderate to good
antibacterial activity. Among all the synthesized compounds, compound 2a (12.5 µg/ml), and 3a (25
µg/ml) showed highest antibacterial activity against E. coli. These results are almost equivalent to
approved drug ciprofloxacin. The newly synthesized compounds 2a and 3a showed good
antimicrobial activities against Gram-positive bacteria like S. pyogenes, S. aureus,and Gram-
negative bacteria E. coli. All the compounds were more effective against Gram-positive bacteria as
compared to Gram-negative bacteria.
3
.3.2 Antifungal Assay
The stock solutions of the newly synthesized compounds were prepared in DMSO at a
concentration of 200 mg/ml. Agar dilution and microdilution method were used to perform the
minimum inhibitory concentration (MIC) as well as minimum fungicidal concentration (MFC) of
synthetic derivatives (Yoshida, Jono & Okonogi 1997; Chowdhary et al. 2011). The compounds
were diluted in solid and broth media to obtain a final concentration from 0.0312 to 256 μg/ml,
using PDA and RPMI 1640 media. The inocula of the yeasts were prepared from 2-7 days mature
colonies grown. Fluconazole and itraconazole or griseofulvin, depending on the kind of fungus were
used as positive controls, and the solvents of the compounds were usedas negative blanks, (Tables 9
and 10). All the compounds had good antifungal effects against tested clinical species of Candidaas
shown in Table 9. Among all the synthesized compounds, 2a and 3a showed higher antifungal

activity (64and 128 µg/ml respectively) against standard species of Candida and clinical resistant
isolates of Candida tropicalis and Candida albicans (Table 9 and 10). Molecular weight has a
significant role in bioactivity. The antimicrobial activity of the series (3a-3d) was slightly lower
than the series (2a-2d). Consequently, it was advocated that bioactivity was decreased due to the
generation of steric hindrance by high molecular weight compounds at the binding sites.
3
.4 Molecular docking studies
Synthesized compounds were docked against the protein (PDB ID: 1E9X & 1G2A) using GOLD
software, and were ranked based on the GOLD fitness score. Xscore is performed to find out the
predicted binding affinity of each protein-ligand complex and LIGPLOT is shown to represent the
protein-ligand interactions in 2D with detailed information of the residues which are involved in
bonded and non-bonded interactions.
3
.4.1 Molecular docking with peptide deformylase of E.coli
Results were analyzed after docking of protein peptide deformylase of E.coli (PDB ID:1G2A) with
each ligand in which, GOLD fitness score ranges between 65.85 to 56.78 whereas for known
inhibitors it ranges from 56.00 to 47.87. For all compounds, Xscore is varying between -8.09 to -
6
.92 kcal/mol (Table 11). After analysis, it was found that the compounds have equal contribution in
hydrophobic interactions and hydrogen bond interactions with the targeted protein. Especially,
compounds 2a, 3a, and 3d have better interactions than other molecules. Glutamic acids (Glu) 133
alone forms ahydrogen bond with all the synthesized molecules and other binding site residues are
involved in hydrophobic and non-bonded interactions (Figure 4a and b). The LIGPLOT and docking
figures of the rest of the other molecules have been shown in SupplementaryFiguresS1a, b to Figure
S7a, b. All the synthesized compounds having interactions and GOLD fitness score better than
ciprofloxacin were treated as control (Table 11)

3
.4.2 Molecular docking with cytochrome P450 14 alpha-sterol demethylase (CYP51)
After docked with CYP51 protein, results were analyzed with each ligand in which GOLD fitness
score ranges between 58.9 to 51.69 whereas for known inhibitors it ranges from 51.31 to 36.71. For
synthesized compounds, Xscore is ranging between -8.10 to -7.69 kcal/mol whereas for known
inhibitors, ranges from -8.06 to 7.35 kcal/mol. After analysis, it was found that the compounds had
equal contribution in hydrophobic interactions and hydrogen bond interactions with the targeted
protein. Especially, Compounds 2a, 3c, 2d, 3a, 3b, 3d, and 2b has better interactions than standards
drugs. Lysine 97 alone forms maximum hydrogen bonds with (2a, 3a, 2b, 3b, 2c, 3c, 2d, and 3d) of
the synthesized molecules and other binding site residues are involved in hydrophobic and non-
bonded interactions (Figure 5a and b). The LIGPLOT and docking figures of rest of the other
molecules have been shown(Supplementary Figures S8a, b to figure S14a, b). All the synthesized
compounds have at least one hydrogen bonded interaction whereas the control does not have bonded
interaction. But all the compounds including control have more non-bonded interactions with the
protein IE9X (Table 12).
3
.5 Structure Activity Relationship (SAR)
Chemical structure and antimicrobial activity of substituted phenol compounds have been reported
previously against Gram-negative and Gram-positive bacteria (Suter 1941; Daoud, Dickinson &
Gilbert 1983).
Structure–activity relationship of antimicrobial agents was summarized by means of
equations
(
Lien, Hansch & Anderson 1968) based on a multiple regression analysis methods(Hansch & Fujita
964) to establishing the correlation between structural properties and activities (Lien et al. 1968;
Kubinyi 1993). These authors suggested that the correlations varied with bacteria and type of
1

antimicrobial agents. Thelipophilicproperty of the molecule plays the most crucial role for the
antimicrobial properties of the compounds apart from a relatively minor contribution from electronic
properties (Lien et al. 1968).
In this study, there are two important parameters which contributesto to the antimicrobial
activity, one is the hydrogen-bonding acidity which play important role in the polar contribution and
the partition coefficient as the dispersive component. The hydrophobicity of materials is an
important parameter because it is directly related to membrane permeation [16]. It has also been
reported that there is direct correlation between the potency of aliphatic alcohols and their lipid
solubility through the hydrophobic interaction between alkyl chains from alcohols and lipid regions
in the membrane (McKarns et al. 1997).We suggested that a similar hydrophobic interaction might
in the lipid like nature of the bacterial membranes. Bacterial membrane loses its permeability due to
hydrophobic interaction which ultimately causing death of the bacteria(Hunt 1975; McKarns et al.
1
997). Some authors have suggested that the antimicrobial effects may be due to a balance between
the hydrophobic and the hydrophilic portions of the molecule (Kubo, Muroi, Himejima & Kubo
993; Kubo, Muroi & Kubo 1993; Kubo, Muroi & Kubo 1995).
The series 1 contains compounds 2a-2d with an oxazoline ring, and series 2 contains compounds 3a-
d with thiazoline ring. The physicochemical properties of the compounds (2a-3d) are presented in
1
3
Table 5. The compounds (2b, 3b, 3a, and 2c) form hydrogen bonds with the target proteins of C.
albicans.Correspondingly, all the compounds also form hydrogen bond interaction with the peptide
deformylase (PDF) protein of E. coli. The structural activity study shows that antimicrobial activity
is dependent on the oxygen atom of non-ionic methyl ester as well as on the nature of the substituent
(hydroxyl group, and anamino group) on heterocyclic ring’s side chains. These compounds in
general exhibited favorable calculated SAR parameters: good octanol-water partition coefficient

(log P) and low values (below 140 Å2) of the polar surface area (PSA) that linked closely to higher
bioavailability. This fact strongly prompted us to study these derivatives as potential new
antimicrobial substances. All the compounds have most favorable values of log P and PSA due to
the presence of heterocyclic ring containing amino group in their structures. Among compounds (2a
and 3a), compound (3a) showed better antibacterial activity compared to compound (2a) due to the
presence of thiazoline ring. Further substitution of oxazoline/thiazoline ring of 2b and 3b with
octane side chain,decrease the antimicrobial and ADMET activities. It has been reported previously
that antimicrobial activity increases as the alkyl chain length increases, but after reaching to an
optimum point, the antimicrobial activity starts decreasing with increasing alkyl chain (Daoud et al.
1
983). Whereas, replacement with ahydroxyl group on octane side chain of 2b and 3b, enhanced the
ADMET and antimicrobial activities of compound 2c and 3c. The hydroxyl group plays
animportanat role in the orientation and localization of the materials near the microbial membrane
by virtue of hydrogen bonding with water molecules and ester linkages of fatty-acylresidues
(Dombek & Ingram 1984). The hydrogen-bonding forces are directly related to the H-bond acidity
which contribute to a better balance through strong hydrogen bonding. (Ornek, Apan, Ogurel &
Ornek 2014).Substitution of the oxazoline ring with pentane and 9-hydroxy-undecanoic acid methyl
ester side chains do not affect much more the ADMET and antimicrobial activities of compound 2d.
Whereas substitution of thiazoline ring with pentane and 9-hydroxy-undecanoic acid methyl ester
side chains enhanced the ADMET and antimicrobial activities of compound 3d.The hydroxyl group
on undecanoic acid methyl ester side chain made an additional hydrogen bond with glutamic acid 42
which enhance the activity of 3d molecule. The topological polar surface area (TPSA) of 3d is lower
than the 2d molecule because 3d has thiazoline heterocyclic ring.

The presence of hydroxyl groups on alkyl/undecanoic acid methyl ester side chains and
theamino group were making the stable complexes through hydrogen bonding and hydrophobic
interactions within the active site of the selected targets in bacteria and fungi. All the compounds
formed better-docked complexes with higher binding energy compared to approved drugs
ciprofloxacin and fluconazole. These compounds (3a, 2a, and 3d) have high drug-likeness score and
optimum octanol-water partition coefficient (log P), indicating these derivatives as better
antimicrobial agents compared to rest of the other derivatives. The Clog P values were influenced
enough by the presence of the hydroxyl group on alkyl/undecanoic acid methyl ester side chains. An
excellentinvitro activity of all the compounds was described to their strong binding energies coupled
with a high lipophilic character which allows an optimal penetration into the cytosol. All the
compounds are endowed with good interaction energies; good hydrophilic nature positively affects
their ability to cut across the membrane of the bacteria and fungi. The lipophilic nature of all the
synthesized compounds combined with very low PSA values may be the main factor ensuring a
sound degree of distribution in vivo. TPSA values were in the range between 38.669 and 94.154
(Table 5).In this study, it has been pointed out above, that a single functional group can affect the
solubility, the overall electronic and steric profile of the lead molecule. It has also been pointed out
that the overall effect of a given functional group is affected by the other functional groups attached
to it or surrounding it.
4
CONCLUSIONS
The proposed method for the synthesis of novel long-chain oxazoline and thiazoline derivatives was
simple, and all the synthesized compounds obtained in good yield. Prediction of the biological
activity of these compounds by using the PASS software was successful to a great extent. The report
of the study confirms traditional usage of these compounds as antimicrobial agents. Nevertheless,

significant data must be brought forth through the pharmacognostic studies before going for
commercialization. The physicochemical parameters suggested that all the compounds possessed
drug-like attributes. In general, the results proved there was a dependency between biological
activity and lipophilicity, where MIC showed a linear relationship with the lipophilicity.The present
work demonstrated that all the title compounds were exhibiting antibacterial activities. All the
compounds showed antimicrobial activity against all tested microorganisms which are nearly
tantamount to the standard drugs ciprofloxacin and fluconazole. Some compounds like 3a, 2a and
3
d had antifungal activity against clinical isolates of Candida resistant to fluconazole and
itraconazole resistance. The consequences of the docking experiments for the newly synthesized
compounds 2a-3d, with the PDF protein of E. coli were found to be in accordance with the in vitro
antimicrobial activity data. The binding sites of the compounds were found to be in close proximity
to the binding site of the standard drug ciprofloxacin. It was interesting to note that even though the
core construction of all the compounds was same, the level of interaction and binding patterns were
set up to be dissimilar. We anticipated that this kind of docking and anti-pathogenic screening
studies would help in designing of novel drugs that targets microbial protein synthesis. Moreover,
the mode of action of the oxazoline and thiazoline derivatives showed that the affinity of the
compounds for Cytochrome P450-14DM was mainly attributed to hydrogen and their non-bonding
interaction with the active site residues of Cytochrome P450-14DM protein and binding to heme.
Our compounds showed better interaction with Cytochrome P450-14DM protein than that of
fluconazole antifungal agent. The works represented here to offer a new structural type for the
development of new antifungal agents.
ACKNOWLEDGEMENTS

The authors wish to thank the Chairman, Department of Chemistry, Aligarh Muslim University,
Aligarh for providing necessary research facilities and SAIF, Panjab University, Chandigarh for
Spectral services.
Appendix A. Supplementary data
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