Biofilm inhibition and DNA binding studies of isoxazole-triazole conjugates in the development of effective anti-bacterial agents

Article abstract of DOI:10.1016/j.molstruc.2019.127144

Isoxazole-triazole conjugates (8a-q) were synthesized using click chemistry approach and their biological activities were explored to develop novel antibacterial agents. In vitro antibacterial screening against Gram-positive as well as Gram-negative bacterial strains identified compounds 8b and 8m with potent inhibitory potential against selective bacterial cells. 8b showed IC₅₀ value of 67.6 μg/mL against P. aeruginosa while 8m exhibited better activity against Gram-positive bacteria S. pneumoniae and E. faecalis having IC₅₀ values 74.13 and 44.7 μg/mL, respectively. Effect on growth kinetics of the bacterial cells as well as cytotoxicity studies on human embryonic kidney cells (HEK293) further supports their biological potential. Compound 8m significantly inhibited biofilm formation of E. coli cells visualized by scanning electron microscopy (SEM) analysis. The interaction of these compounds with ctDNA, as their possible mode of action, was studied using multi-spectroscopic techniques and molecular docking. The data suggested that compound 8m intercalate in the minor groove of DNA.

Full text of DOI:10.1016/j.molstruc.2019.127144

Journal Pre-proof
Biofilm inhibition and DNA binding studies of isoxazole-triazole conjugates in the
development of effective anti-bacterial agents
Farhat Habib, Shadab Alam, Afzal Hussain, Babita Aneja, Mohammad Irfan,
Mohamed F. Alajmi, Phool Hasan, Parvez Khan, Md Tabish Rehman, Omar
Mohammed Noman, Amir Azam, Mohammad Abid
PII:
S0022-2860(19)31253-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.127144
MOLSTR 127144
Reference:
To appear in: Journal of Molecular Structure
Received Date: 27 June 2019
Revised Date: 16 September 2019
Accepted Date: 27 September 2019
Please cite this article as: F. Habib, S. Alam, A. Hussain, B. Aneja, M. Irfan, M.F. Alajmi, P. Hasan, P.
Khan, M.T. Rehman, O.M. Noman, A. Azam, M. Abid, Biofilm inhibition and DNA binding studies of
isoxazole-triazole conjugates in the development of effective anti-bacterial agents, Journal of Molecular
Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.127144.
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HEK293 Cells
100
50
0
8m
Strain
IC50 in µg/mL
E. coli
83.17
74.13
44.66
0
5
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
1
2
4
8
6
0
1
2
4
8
6
1
2
1
2
S. pneumoniae
E. faecalis
Concentration(µM)
Cytotoxicity
Docking studies
Biofilm inhibition

Biofilm inhibition and DNA binding studies of isoxazole-
triazole conjugates in the development of effective anti-
bacterial agents
Farhat Habiba,b, Shadab Alama, Afzal Hussainc, Babita Anejaa,b, Mohammad Irfana,
Mohamed F. Alajmic, Phool Hasana, Parvez Khand, Md Tabish Rehmanc, Omar
Mohammed Nomane, Amir Azamb*, Mohammad Abida
aMedicinal Chemistry Laboratory, Department of Biosciences, Jamia Millia Islamia, New
Delhi-110025, India.
bDepartment of Chemistry, Jamia Millia Islamia, New Delhi-110025, India.
cDepartment of Pharmacognosy College of Pharmacy, King Saud University, Riyadh, 11451,
Kingdom of Saudi Arabia
dCentre for Interdisciplinary Research in Basic Science, Jamia Millia Islamia, New Delhi-
110025, India.
eMedicinal, Aromatic and Poisonous Plants Research Center, College of Pharmacy, King
Saud University, Riyadh-11451, Kingdom of Saudi Arabia.
Corresponding author
M. Abid: mabid@jmi.ac.in; Tel: +91-8750295095
Co-corresponding author
A. Azam: amir_sumbul@yahoo.co.in; Tel: +91-9818963639

Abstract
Isoxazole-triazole conjugates (8a-q) were synthesized using click chemistry approach and
their biological activities were explored to develop novel antibacterial agents. In vitro
antibacterial screening against Gram-positive as well as Gram-negative bacterial strains
identified compounds 8b and 8m with potent inhibitory potential against selective bacterial
cells. 8b showed IC50 value of 67.6 µg/mL against P. aeruginosa while 8m exhibited better
activity against Gram-positive bacteria S. pneumoniae and E. faecalis having IC50 values
74.13 and 44.7 µg/mL, respectively. Effect on growth kinetics of the bacterial cells as well as
cytotoxicity studies on human embryonic kidney cells (HEK293) further supports their
biological potential. Compound 8m significantly inhibited biofilm formation of E. coli cells
visualized by scanning electron microscopy (SEM) analysis. The interaction of these
compounds with ctDNA, as their possible mode of action, was studied using multi-
spectroscopic techniques and molecular docking. The data suggested that compound 8m
intercalate in the minor groove of DNA.
Keywords: Isoxazole-1,2,3-triazole, Antibacterial agents, Biofilm inhibition, DNA binding,
Cytotoxicity, Anti-bacterial.

1. Introduction
Bacterial infections are posing a serious threat to human health and are the leading
cause of chronic diseases worldwide. The situation is becoming worst due to the emergence
of resistant and new pathogenic bacterial strains [1]. The tremendous increase has been
observed in bacterial resistance due to the greater access to antibiotic drugs in developing
countries. According to a recent review on antimicrobial resistance; annual mortality
associated with antimicrobial resistance (AMR) is approximately 700,000 [2]. Inaction in
containing this menace is likely to cause more than 10 million deaths every year by 2050,
which accounts for more deaths than that caused by cancers and road accidents. Most of these
deaths are expected in developing countries of Asia and Africa [3]. The severity of the global
health crisis has propelled the Infectious Disease Society of America (IDSA) to issue the
challenge of developing ten new antibiotics by 2020 [4]. Therefore, to confront the global
burden of antibacterial resistance, it is necessary to develop new antibiotics which can disrupt
the processes necessary for bacterial cell growth and proliferation to fight against bacterial
infections.
There are many factors called virulence attributes which help pathogens to survive,
multiply and pathogenesis within the host. These factors include adherence, colonisations,
and invasive factors, capsule formation, secretion of endotoxins and exotoxins, etc [5].
Instead of the planktonic state, bacterial cells exist in a sessile state within extracellular
matrix called a biofilm. The formation of biofilm in pathogens plays an important role in
adherence, colonization, defense mechanism and ultimately helps in pathogenesis [6]. Many
reports established the direct connection of biofilm formation with bacterial infections such
as chronic lung infections, skin infections, periodontitis, endocarditis and nosocomial
infections [7-9]. Thus targeting biofilm formation can be helpful to stop the colonization or
adhesion of bacterial population at the very initial level and eliminate the infection.
Literature gives strong pieces of evidence regarding heterocyclic scaffolds containing
nitrogen and oxygen having potent biological applications [10]. Over the last decades,
progress on the pharmacological activity and synthesis of variously substituted
triazole/isoxazole derivatives has been added to the scientific literature [11]. Compounds
containing a triazole ring have shown a broad spectrum of activities, including anti-
inflammatory [12], analgesic [13], antitumor [14], anti-diabetic [15] and anticonvulsive [16].
Additionally, several reports highlight the importance of triazole motif against microbes:
bacteria [17], fungi [18], protozoa [19], and viruses [20]. Similarly, isoxazoles are commonly

encountered in a variety of natural products and drugs with a diverse range of
pharmacological activities [21]. Isoxazoles have also been previously reported as an
antidiabetic, analgesic, antiarrhythmic, anti-inflammatory, antifungal, antibacterial,
anticancer, anti-HIV, anti-Alzheimer’s disease, etc. [22-23]. Recently, the strategy of
generating a new class of azole based antibacterial agents by clubbing together of two or
more biologically important azole scaffolds showed the significant potential of inhibiting
bacterial growth [24,25]. Some commonly available drugs which contain 1,2,3-triazole and
isoxazole unit are shown in Fig. 1. Moreover, reports suggest that the central structural motifs
of biological interest, for instance, triazole, when coupled to another heterocyclic ring have
manifested therapeutic potential in different domains including against microbial pathogens
[26].
Incited by promising results of these scaffolds, herein, we report a series of isoxazole-
triazole conjugates and explored their antibacterial potential against two Gram-positive
(Streptococcus pneumoniae MTCC 655, Enterococcus faecalis MTCC 439) and four Gram-
negative (Pseudomonas aeruginosa ATCC 2453, Salmonella typhimurium MTCC 3224,
Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922) bacterial strains.
Toxicity studies using human RBCs and HEK293 (human embryonic kidney cells) confirmed
their non-toxic nature, thus supports further pharmacological investigations. To elucidate
their mode of action, DNA binding studies were performed using multi-spectroscopic
techniques, further supported by molecular docking studies.

O
O
S
N
N
N
CF3
O
N
O
O
H2N
OH
F
N
H3C
O
H
Tazobactum
N
N
N
O
F
Dicloxacillin
Rufinamide
O
Cl
O
N
R
O
H2N
Cl
N
N
N
N
N
O
O
N
H
N
Isoxazoleꢀ1,2,3 triazole conjugates
H
N
H2N
N
S
H3C
Cl
Cl
O
O
N
O
O
O
H3C
S
OH
N
O
H
O
Cl
NH2
Carboxyamidotriazole
Sulfamethoxazole
Fig. 1. Rationale design strategy of isoxazole-1,2,3-triazole conjugates. Isoxazole and 1,2,3-
triazole moieties of available drugs matched with red and blue dotted lines, respectively. The
linker in green sphere joined isoxazole and 1,2,3-triazole moieties. Substituted phenyl group
(in pink) is selected for SAR study.
2. Materials and Methods
All the chemicals were purchased from Sigma-Aldrich, USA and used without further
purification. Precoated aluminium sheets (Silica gel 60 F254, Merck Germany) were used for
thin-layer chromatography (TLC) and spots were visualized under UV light. The IR spectra
of compounds were recorded on Agilent Cary 630 FT-IR spectrometer and only major peaks
1
13
are reported in cm−1. H and C NMR spectra were obtained at ambient temperature using
Bruker Spectrospin DPX-300 MHz, 400 MHz, Agilent 500 MHz FT-NMR in DMSO-d6 and
CDCl3 using tetramethylsilane (TMS) as an internal standard. Splitting patterns designated as
follows: s, singlet; d, doublet; t, triplet; m, multiplet. Chemical shift values are given in parts
per million (ppm) and coupling constants (J) in Hertz (Hz). Mass spectra were recorded on an

Agilent Quadrupole-6150 LC−MS spectrometer. Melting points were recorded on a digital
Buchi melting point apparatus (M-560) and were reported uncorrected. Purification of the
compounds was done by column chromatography using silica gel (230–400 mesh size) with
hexane/ethyl acetate as eluent. Purity was determined by an Agilent RRLC MS 6320 ion trap
spectrometer using an XBridge C18 1.7 ꢁm column (50 mm × 2.1 mm). Mobile phase
channel A consisted of 5 mM ammonium acetate in water. Mobile phase B consisted of
acetonitrile with a flow rate = 0.8 mL/min; detection was done by UV at 214 nm.
2.1. Synthesis of diketoester (3)
As reported earlier [27]. Briefly, in a round bottom flask, sodium metal (22.47 mmol) was
o
dissolved in anhydrous ethanol at 0 C to give sodium ethoxide solution. To this freshly
prepared solution, a mixture of diethyl oxalate (20 mmol) and p-chloroacetophenone (21
mmol) was slowly added. Thick precipitate was formed and reaction mixture was stirred for
3–4 h at room temperature. After completion of the reaction, the precipitate obtained was
dissolved in 2 N sulphuric acid (72 ml) and the compound was extracted with diethyl ether,
dried over anhydrous Na2SO4 and concentrated under reduced pressure and purified using
column chromatography.
2.2. Synthesis of isoxazole ethyl ester (4)
As reported earlier [28].Briefly, to the stirred solution of diketo ester 3 (1.0 eq) in anhyd.
ethanol was added hydroxylamine hydrochloride (3.0 eq) and the reaction mixture was
refluxed for 2½ h. After completion of the reaction, the reaction mixture was cooled to room
temperature. The solid precipitate obtained was filtered over the Buckner funnel and washed
with cold ethanol. The solid product was dried under vacuo to afford isoxazole ethyl ester.
2.3. Synthesis of isoxazole carboxylic acid (5)
As reported earlier [29]. Hydrolysis of isoxazole ethyl ester 4 (1.0 eq) was done by
employing freshly prepared LiOH (2M) (4.5 eq.) in THF /H2O mixture (3:1) for 3 hours to
give the corresponding acid in quantitative yield which was used further without purification.
2.4. Synthesis of isoxazole alkyne (6)
In a 50 ml round bottom flask, 5-(3-Chlorophenyl) isoxazole-1-carboxylic acid 5 (4.5 mmol)
was dissolved in acetonitrile (10 ml) and cooled to 0 °C followed by slow addition of
propargyl alcohol (5.37 mmol). To this solution, NMM (5.37 mmol), HOBt (9.0 mmol) and
EDC.HCl (5.4 mmol) were added in small portions. The reaction mixture was brought to

room temperature and the progress of the reaction was checked by TLC. After completion of
the reaction, it was concentrated and water was added to the residue. The compound was
extracted with ethyl acetate, dried over anhydrous sodium sulphate and concentrated under
vacuo.
2.4.1. prop-2-yn-1-yl 5-(4-chlorophenyl)isoxazole-3-carboxylate (6)
White solid, mp: 187-189 °C, Yield: 80%, Rf (hexane: ethyl acetate = 7:3): 0.26, IR (neat):
ѵ(cm-1) 3147 (=CH str.), 1734 (C=O str.), 1611 (C=N), 1238 (C-O-N) cm-1. 1H NMR (300
MHz, CDCl3 (δ ppm): 8.18 (s, 1H, Ar-H), 7.74(d, J = 7.2 Hz, 2H, Ar-H), 7.55(d, J = 6.9 Hz
2H, Ar-H), 5.64 (s, 2H, CH2), 3.54(s, 1H, CH).
2.5. General procedure for the synthesis of azides (7a-q)
To a solution of substituted aniline (3.22 mmol) in ethyl acetate kept at 0 °C was added conc.
hydrochloric acid (1.29 mL) followed by drop wise addition of a solution of NaNO2 (3.87
mmol) in water (4.03 mL) over a period of 10 min with constant stirring. After stirring the
reaction mixture for 1 h at 0 °C, a solution of NaN3 (3.87 mmol) in water was added to this
mixture and allowed to stir at room temperature for 3 h. After completion of the reaction, the
mixture was poured into water, extracted with ethyl acetate, dried over anhyd. sodium sulfate,
and concentrated under vacuo to give azide, which was used further without purification.
2.6. General procedure for the synthesis of isoxazole-triazole conjugates (8a-q)
To a solution of substituted aryl azide (1.0 mmol) and alkyne (1.0 mmol) in a mixed solvent
system, THF/H2O (1:2, 9mL), were added sodium ascorbate (0.55 mmol) and CuSO4·5H2O
(0.18 mmol). The reaction mixture was stirred overnight at room temperature. After
completion of the reaction, the solid obtained was filtered and washed with water. The crude
product was purified by silica gel chromatography using ethyl acetate/hexane (3:7) to afford
the title compounds.
2.6.1.
(1-Phenyl-1H-1,2,3-triazol-4-yl)methyl
5-(4-chlorophenyl)isoxazole-3-
carboxylate(8a)
Brown solid, mp: 263-265 °C, Yield: 75%, Rf (hexane: ethyl acetate = 7:3): 0.16, IR(neat):
ѵ(cm-1) 3123 (=CH str., triazole, 1746 (C=O str.), 1611 (C=N), 1240 (C-O-N) cm-1, 1H NMR
(300 MHz, DMSO-d6 (δ ppm): 8.99 (s, 1H, Ar-H), 7.96 (d, J = 8.7 Hz, 2H, Ar-H), 7.90 (d, J
= 7.8 Hz, 2H, Ar-H), 7.64-7.58 (m, 5H, Ar-H), 7.53 (d, J = 7.2 Hz, 1H, Ar-H), 5.59 (s, 2H,
CH2); 13CNMR (75 MHz, DMSO-d6) (δ, ppm): 170.62, 159.42, 157.12, 142.71, 136.95,

136.25, 130.42, 129.95, 129.37, 128.14, 127.80, 126.10, 125.33, 124.02, 120.73, 101.99,
59.12. LC-MS: m/z [M + H]+: 381.10.
2.6.2.
4-(4-(((5-(4-Chlorophenyl)isoxazole-3-carbonyl)oxy)methyl)-1H-1,2,3-triazol-1-
yl)benzoic acid(8b)
Yellow solid, mp: 261-263 °C, Yield: 77%, Rf. (hexane: ethyl acetate = 7:3): 0.18, IR (neat):
ѵ(cm-1) 3121 (=CH str., triazole, 1741 (C=O str.), 1609 (C=N), 1240 (C-O-N) cm-1.1H NMR
(300 MHz, DMSO-d6). (δ, ppm): 13.17 (s, 1H,COOH), 9.09 (s, 1H,Ar-H), 8.11 (brs, 5H, Ar-
H), 7.98 (d, J = 8.1 Hz, 2H, Ar-H), 7.62 (d, J = 8.4 Hz, 2H, Ar-H), 7.23 (s, 1H, Ar-H), 5.61
(s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) (δ, ppm): 170.60, 159.38, 157.07, 144.38,
143.06, 139.82, 136.23, 131.94, 129.91, 128.11, 125.29, 124.09, 120.62, 119.74, 101.95,
59.02 LC-MS: m/z [M - H]+: 423.11.
2.6.3. (1-(4-methylphenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8c)
White solid, mp.: 209-211 °C, Yield: 77%, Rf (hexane: ethyl acetate = 7:3) : 0.321, IR (neat):
ѵ(cm-1) 3140 (=CH str., triazole, 1730 (C=O str.), 1613 (C=N), 1233 (C-O-N) cm-1.1H NMR
(300 MHz, CDCl3 (δ, ppm): 8.06 (s, H, Ar-H ), 7.66 (d, J= 8.4 Hz, 2H, Ar-H), 7.54 (d, J= 8.4
Hz, 2H, Ar-H), 7.39 (d, J = 8.7 Hz, 2H, Ar-H), 7.25 (d, J = 8.1 Hz, 2H, Ar-H), 6.86 (s, 1H,Ar-
13
H), 5.56 (s, 2H, CH2), 2.36 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6) (δ, ppm): 170.60,
159.40, 157.11, 142.57, 138.99, 136.24, 135.62, 134.71, 130.72, 129.93, 128.13, 127.79,
126.10, 125.32, 123.85, 120.57, 101.96, 59.11, 21.04. LC-MS: m/z [M + H]+: 395.07.
2.6.4. (1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8d)
Cream solid, mp: 180-182 °C, Yield: 86%, Rf(hexane: ethyl acetate = 7:3): 0.37, IR (neat):
ѵ(cm-1) 3138 (=CH str., triazole, 1730 (C=O str.), 1611 (C=N), 1248 (C-O-N) cm-1.1H NMR
(300 MHz, CDCl3) (δ, ppm): 8.01 (s, 1H, Ar-H), 7.66 (d, J = 8.4 Hz, 3H, Ar-H), 7.51 (d, J =
9 Hz, 2H, Ar-H), 7.39(d, J = 8.4 Hz, 2H, Ar-H), 6.95(d, J = 9.0 Hz, 2H, Ar-H), 5.55 (s, 2H,
13
CH2), 3.80(s, 3H, OCH3); CNMR (75 MHz, DMSO-d6) (δ, ppm): 170.60, 159.87, 159.41,
157.11, 142.43, 136.23, 130.33, 129.93, 128.13, 125.31, 123.96, 122.37, 115.37, 101.97,
59.14, 56.04. LC-MS: m/z [M + H]+: 411.19.
2.6.5 (1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8e)

Light brown solid, mp: 181-183 °C, Yield: 54%, Rf(hexane: ethyl acetate = 7:3): 0.39, IR
(neat): ѵ(cm-1) 3155 (=CH str., triazole, 1737 (C=O str.), 1610 (C=N), 1231 (C-O-N) cm-1.1H
NMR (300 MHz, DMSO-d6). (δ, ppm): 8.96 (s, 1H, Ar-H), 7.99(d, J = 8.4 Hz, 4H, Ar-H),
13
7.64-7.59 (m, 3H, Ar-H), 7.47 (t, J = 8.7 Hz, 2H, Ar-H), 5.59 (s, 2H, CH2); C NMR (75
MHz, DMSO-d6) (δ, ppm): 170.60, 163.85, 160.57, 159.39, 157.09, 142.71, 136.23, 133.51,
129.93, 128.12, 125.29, 124.26, 123.16, 123.04, 117.40, 117.09, 101.97, 59.06. LC-MS: m/z
[M + H]+: 399.18.
2.6.6. (1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8f)
white solid, mp: 204-206 °C Yield: 76%, Rf(hexane: ethyl acetate = 7:3): 0.393 IR (neat):
1
ѵ(cm-1) 3123 (=CH str., triazole, 1737 (C=O str.), 1603 (C=N), 1236 (C-O-N) cm-1.1.. H
NMR (300 MHz, DMSO-d6). (δ, ppm): 8.98 (s, 1H, Ar-H), 7.99-7.96 (m, 4H, Ar-H), 7.69-
13
7.61 (m, 4H, Ar-H), 7.55 (s, 1H, Ar-H), 5.60 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6)
(δ, ppm): 170.61, 159.39, 157.08, 142.87, 136.24, 135.74, 133.65, 130.36, 129.92, 128.12,
125.30, 124.08, 122.39, 101.95, 59.04. LC-MS: m/z [M + H]+: 415.2.
2.6.7. (1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8g)
White solid, mp: 203-204 °C, Yield: 78%, Rf(hexane: ethyl acetate = 7:3): 0.387. IR (neat):
ѵ(cm-1) 3125 (=CH str., triazole, 1734 (C=O str.), 1613 (C=N), 1249 (C-O-N) cm-1.1H NMR
(300 MHz, DMSO-d6). (δ, ppm): 9.18 (s, 1H, Ar-H), 8.46 (d, J = 9.0 Hz, 2H, Ar-H), 8.25 (d,
J = 9.0 Hz, 2H, Ar-H), 7.99 (d, J = 8.4 Hz, 2H, Ar-H), 7.64-7.58 (m, 3H, Ar-H), 5.62 (s, 2H,
CH2); 13C NMR (75 MHz, DMSO-d6) (δ, ppm): 170.61, 162.25, 159.39, 157.06, 147.45,
143.38, 136.29, 133.61, 129.96, 128.15, 126.06, 124.46, 121.32, 101.96, 58.93. LC-MS: m/z
[M + H]+: 408.0.
2.6.8 (1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8h)
Light brown solid, mp: 127-129 °C, Yield: 81%, Rf(hexane: ethyl acetate = 7:3): 0.472, IR
(neat): ѵ(cm-1) 3162 (=CH str., triazole, 1754 (C=O str.), 1609 (C=N), 1249 (C-O-N) cm-1.1H
NMR (300 MHz, DMSO-d6). (δ, ppm): 8.76 (s, 1H, Ar-H), 7.99(d, J = 8.4 Hz, 2H, Ar-H),
7.79(d, J = 7.5 Hz, 2H, Ar-H), 7.73(d, J = 7.8 Hz, 2H, Ar-H), 7.64-7.57 (m, 5H, Ar-H), 5.62
(s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) (δ, ppm): 170.62, 159.42, 157.09, 141.69,

136.24, 134.79, 132.26, 131.04, 129.91, 128.96, 128.90, 128.13, 127.87, 125.30, 101.95,
59.0; LC-MS: m/z [M + H]+: 414.9.
2.6.9 (1-(2-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8i)
Brown solid, mp: 151-153 °C, Yield: 87%, Rf(hexane: ethyl acetate = 7:3): 0.147, IR (neat):
1
ѵ(cm-1) 3130 (=CH str., triazole, 1726 (C=O str.), 1611 (C=N), 1238 (C-O-N) cm-1.. H
NMR (300 MHz, DMSO-d6). (δ, ppm): 8.89 (s, 1H, Ar-H), 8.25 (d, J = 7.8 Hz, 1H, Ar-H),
13
8.01-7.84 (m, 5H, Ar-H), 7.63 (d, J = 9.6 Hz, 3H, Ar-H), 5.62 (s, 2H, CH2); C NMR (75
MHz, DMSO-d6) (δ, ppm): 170.65, 159.40, 157.08, 144.49, 142.36, 136.26, 134.94, 131.84,
129.94, 129.43, 128.15, 127.09, 126.05, 125.31, 101.95, 58.91. LC-MS: m/z [M + H]+:
426.07
2.6.10. (1-(2-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8j)
Light brown solid, mp: 150-152 °C, Yield: 68%, Rf(hexane: ethyl acetate = 7:3): 0.454 IR
(neat): ѵ(cm-1) 3173 (=CH str., triazole, 1730 (C=O str.), 1611 (C=N), 1229 (C-O-N) cm-1..
1H NMR (300 MHz, DMSO-d6). (δ, ppm): 8.80 (s, 1H, Ar-H), 8.00-7.97 (m, 2H, Ar-H),
7.89-7.85 (m, 1H, Ar-H), 7.63-7.59 (m, 5H, Ar-H), 7.48-7.44 (m, 1H, Ar-H), 5.61 (s, 2H,
CH2); 13C NMR (75 MHz, DMSO-d6) (δ, ppm): 170.61, 159.41, 157.08, 155.98, 152.66,
142.19, 136.23, 132.00, 131.00, 129.11, 128.12, 127.31, 127.25, 126.48, 125.29, 125.12,
117.75, 101.93, 58.96. LC-MS: m/z [M + H]+: 399.17
2.6.11. (1-(2-Methylphenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8k)
Creamish solid, mp: 153-155 °C, yield: 76%, Rf(hexane: ethyl acetate = 7:3): 0.516, IR
(neat): ѵ(cm-1) 3153 (=CH str., triazole, 1732 (C=O str.), 1611 (C=N), 1251 (C-O-N) cm-1..
1H NMR (300 MHz, DMSO-d6). (δ, ppm): 8.66 (s, 1H, Ar-H), 7.98 (d, J = 8.7 Hz, 2H, Ar-H),
13
7.64-7.59 (m, 3H, Ar-H), 7.49-7.45 (m, 4H, Ar-H), 5.60(s, 2H, CH2), 2.17 (s, 3H, CH3); C
NMR (75 MHz, DMSO-d6) (δ, ppm): 170.59, 159.43, 157.12, 141.66, 136.54, 136.23,
133.52, 131.86, 130.41, 129.92, 128.11, 127.49, 127.26, 126.50, 125.29, 101.95, 59.12,
17.88. LC-MS: m/z [M + H]+: 395.14.
2.6.12. (1-(2-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-
3-carboxylate(8l)

Cream solid, mp: 138-140 °C, Yield: 83%, Rf(hexane: ethyl acetate = 7:3): 0.356. IR (neat):
1
ѵ(cm-1) 3140 (=CH str., triazole, 1737 (C=O str.), 1611 (C=N), 1244 (C-O-N) cm-1.. H
NMR (300 MHz, DMSO-d6). (δ, ppm): 8.64 (s, 1H, Ar-H), 7.99(d, J = 8.7 Hz, 2H, Ar-H),
7.66-7.59 (m, 4H, Ar-H), 7.55 (t, J = 7.8 Hz, 1H, Ar-H), 7.34(d, J = 8.4 Hz, 1H, Ar-H),
13
7.16(t, J = 7.5 Hz, 1H, Ar-H), 5.59 (s, 2H, CH2), 3.87 (s, 3H, OCH3); C NMR (75 MHz,
DMSO-d6) (δ, ppm): 170.61, 159.45, 157.13, 141.34, 136.23, 131.38, 129.92, 128.14, 127.70,
126.28, 125.97, 125.32, 12137, 113.52, 101.94, 59.14, 56.62. LC-MS: m/z: [M + H]+: 330.1.
2.6.13. (1-(3-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8m)
Off-white solid, mp: 167-169 °C, Yield: 73%, Rf(hexane: ethyl acetate = 7:3): 0.548,. IR
(neat): ѵ(cm-1) 3137 (=CH str., triazole, 1721 (C=O str.), 1611 (C=N), 1250 (C-O-N) cm-1..
1H NMR (300 MHz, DMSO-d6). (δ, ppm): 8.74 (s, 1H, Ar-H), 7.98 (d, J = 8.7 Hz, 2H, Ar-H),
7.79-7.58 (m, 7H, Ar-H), 5.62 (s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) (δ, ppm):
170.62, 159.42, 157.08, 141.70, 136.24, 134.78, 132.27, 131.05, 129.92, 128.98, 128.89,
128.13, 127.87, 125.28, 101.93, 59.00. LC-MS: m/z: [M + H]+: 368.29.
2.6.14. (1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8n)
Cream solid, mp: 163-164 °C, Yield: 82%, Rf(hexane: ethyl acetate = 7:3): 0.515. IR (neat):
ѵ(cm-1) 3127 (=CH str., triazole, 1743 (C=O str.), 1612 (C=N), 1238 (C-O-N) cm-1.1H NMR
(300 MHz, DMSO-d6). (δ, ppm): 9.05(s, 1H, Ar-H), 7.99 (d, J = 8.4 Hz, 2H, Ar-H), 7.91.-
7.82 (m, 2H, Ar-H), 7.71-7.61 (m, 4H, Ar-H), 7.38 (dt, J = 1.8, 8.4 Hz, 1H, Ar-H), 5.59 (s,
2H, CH2); 13C NMR (75 MHz, DMSO-d6) (δ, ppm): 170.62, 164.49, 159.38, 157.08, 142.88,
138.04, 136.25, 132.46, 132.34, 129.96, 128.14, 125.28, 124.19, 116.65, 108.39, 101.97. LC-
MS: m/z [M + H]+: 399.4.
2.6.15. (1-(3-methylphenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8o)
Cream solid, mp: 154-156 °C, Yield: 76%, Rf(hexane: ethyl acetate = 7:3): 0.575 , IR (neat):
ѵ(cm-1) 3133 (=CH str., triazole, 1732 (C=O str.), 1612 (C=N), 1237 (C-O-N) cm-1.1H NMR
(300 MHz, DMSO-d6). (δ, ppm): 8.95 (s, 1H, Ar-H), 7.97(d, J = 6.9 Hz, 2H, Ar-H),), 7.56-
7.69 (m, 2H, Ar-H), 7.62-7.57 (m, 3H, Ar-H), 7.48 (t, J = 7.8 Hz, 1H, Ar-H), 7.31 (d, J = 7.5
Hz, 1H, Ar-H), 5.59 (s, 2H, CH2), 2.41 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) (δ,

ppm): 170.59, 159.40, 157.09, 142.62, 140.16, 136.89, 136.23, 130.16, 129.91, 128.10,
125.29, 123.89, 123.53, 121.09, 117.76, 101.92, 59.11, 21.34. LC-MS: m/z [M + H]+: 395.0
2.6.16. (1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-
3-carboxylate(8p)
Pink solid, mp: 150-152 °C, Yield: 78%, Rf(hexane: ethyl acetate = 7:3): 0.516, IR (neat):
ѵ(cm-1) 3142 (=CH str., triazole, 1730 (C=O str.), 1613 (C=N), 1231 (C-O-N) cm-1. 1H NMR
(300 MHz, DMSO-d6). (δ, ppm): 8.99 (s, 1H, Ar-H), 7.99 (d, J = 7.2 Hz, 2H, Ar-H), 7.63 (d,
J = 7.2 Hz, 3H, Ar-H), 7.58-7.51 (m, 3H, Ar-H), 7.09-7.07 (m, 1H, Ar-H), 5.59 (s, 1H, CH2),,
3.87 (s, 3H, OCH3), 13C NMR (75 MHz, DMSO-d6) (δ, ppm): 170.62, 160.67, 159.39, 157.11
142.64, 137.97, 136.25, 131.37, 129.95, 128.15, 125.31, 124.12, 115.12, 112.67, 106.31,
101.96, 59.8, 56.11. LC-MS: m/z [M + H]+: 410.8.
2.6.17. (1-(3-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl 5-(4-chlorophenyl)isoxazole-3-
carboxylate(8q) Dark brown solid, mp: 160-162 °C, Yield: 87%, Rf(hexane: ethyl acetate =
7:3): 0.375, IR (neat): ѵ(cm-1) 3155 (=CH str., triazole, 1739 (C=O str.), 1611 (C=N), 1235
(C-O-N) cm-1.1H NMR (300 MHz, DMSO-d6). (δ, ppm): 9.23 (s, 1H, Ar-H), 8.76 (s, 1H, Ar-
H), 8.44 (d, J = 8.1 Hz, 1H, Ar-H), 8.35 (d, J = 8.1 Hz, 1H, Ar-H), 7.99 (d, J = 8.7 Hz, 2H,
Ar-H), 7.92 (t, J = 8.1 Hz, 1H, Ar-H), 7.65-7.61 (m, 2H, Ar-H), 5.62 (s, 2H, CH2); 13C NMR
(75 MHz, DMSO-d6) (δ, ppm): 170.61, 159.38, 157.06, 148.98, 143.17, 137.49, 136.24,
132.03, 129.91, 128.10, 126.71, 125.28, 124.47, 123.77, 115.42, 101.93, 58.96. LC-MS: m/z:
[M + H]+:380.2.
2.7. In vitro antimicrobial activity
All the compounds (8a-8q) were screened for their in vitro antibacterial activity
against two Gram-positive (S. pneumoniae, E. faecalis) and four Gram-negative strains (P.
aeruginosa, S. typhimurium, K. pneumoniae, E. coli) bacterialstrains using broth dilution
assay according to the standard protocol for antibacterial assessment by NCCLS.
Ciprofloxacin (CIP) was used as positive control for the study. The compounds were
dissolved in DMSO and serially diluted in nutrient broth medium to achieve the final
concentration of DMSO less than 4%. Varying concentrations (1000 to 7.8 µg/mL) of
compounds were dispensed into a 96-well plate in nutrient broth medium in a final volume of
100 µL. Then, 100 µL of bacterial cells (approximate 2.5×105 cells/mL) were dispensed into
each well of 96-well plate (Tarson) and incubated at 37 °C for 24 h. After incubation period
each well was analyzed for the presence or absence of visual growth of bacterial cells. The

lowest concentration of the test compound at which no visible growth occurs represents its
MIC value. Moreover, after incubation, the growth was measured turbidometrically at 585
nm using a Thermo MultiskanGo spectrophotometer. The IC50 value was defined as the
concentration of the test compound that causes 50% decrease in absorbance compared with
that of the control (no test compound). Log10 concentration versus % growth inhibition
scattered graph was plotted to calculate the IC50 values of the test compounds.
2.8. Growth curve studies
The bacterial cells were freshly revived by subculture on the nutrient agar plate. A
loopful of inoculums was introduced into the nutrient broth and cells were grown for 16 h at
37 °C before use. Approximately 2×105 cells/mL was then inoculated into the freshly
prepared 50 mL sterile nutrient broth medium. Different concentrations, equivalent to 2MIC
(250 ꢁg/mL), MIC (125 ꢁg/mL), MIC/2 (62.5 ꢁg/mL), of compounds were added separately
into the conical flasks containing inoculated medium and incubated at 37 °C and 160 rpm. 7.5
ꢁg/mL concentration of CIP was used as positive control. At predetermined time periods (0,
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 after incubation with agitation at 37 °C), 1 mL
aliquot to each sample was removed from the conical flask and growth was measured
turbidometrically at 585 nm using Thermo Multiskan spectrophotometer. Optical density was
recorded for each concentration against time (h).
2.9. In vitro cytotoxicity
HEK293 cells were cultured and maintained as a monolayer in Dulbecco's Modified
Eagle's Medium (DMEM, Sigma) supplemented with 10% fetal calf serum (Gibco-life
technologies, Thermo Fisher Scientific (USA) and antibiotics (100 IU ml-1 of penicillin and
100 mg mL-1 of streptomycin, Sigma). The cells were cultured at 37 °C in a humidified
atmosphere and 5% CO2. Exponentially growing cells were plated at 1.2×104 cells per well
into 96-well plates and incubated for 48
h
before the addition of the
compounds/metronidazole. Stock solutions of compounds were initially dissolved in 20%
(v/v) DMSO and further diluted with fresh complete medium. The growth-inhibitory effects
of the compounds were measured using standard tetrazolium MTT assay. After 48 h of
incubation at 37 °C, the medium was removed and 25 µL of MTT (5 mg ml-1) in 100 µL of
serum free medium was added to each well. The plates were incubated at 37 °C for 4 h. At
the end of the incubation period, the medium was removed and 100 µL DMSO was added to
each well. The metabolized MTT product was dissolved in DMSO and quantified by reading

the absorbance at 570 nm using an ELISA plate reader. All assays were performed in
triplicate. Percent viability was defined as the relative absorbance of treated versus untreated
control cells.
2.10. Biofilm inhibition assay
Biofilm formation in E. coli and S. pneumoniae was determined by semi-quantitative
method using XTT (2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-
2H-tetrazolium-hydroxide) reduction assay with slight modification in previously known
method [30]. Briefly, aliquots of 100 ꢁL of standardized cell suspension test organisms
prepared in sterile nutrient broth medium containing 0.5% additional glucose and poured into
the wells of micro-titre plate. The plate was incubated at 37 °C for 24 h in static condition to
establish biofilm. After incubation period, the medium was discarded and the plate was
gently washed with PBS to remove non-adherent cells. Further, 100 ꢁL of freshly prepared
medium containing various concentrations of compound 8m was added to the wells. The
plate was again incubated at 37 °C for 24 h under static condition to determine the metabolic
activity of biofilm. The medium was discarded and washed with PBS to remove the non-
adherent cells followed by the addition of 100 ꢁL of prepared XTT salt solution (HiMedia,
India). The plate was incubated at 37 °C in dark for 90 min. Bacterial dehydrogenase activity
reduces XTT tetrazolium salt to XTT formazan, resulting in colorimetric change (turns to
orange) that was correlated with cell viability. The colorimetric changes were measured
spectrophotometrically at 490 nm. The % inhibition data were interpreted from dose-response
curves.
2.11. Biofilm formation assessment by SEM analysis
Biofilm inhibition property of compound 8m was also determined by scanning
electron microscopy (SEM) in E. coli. The sophisticated analytical instrumentation facility
(SAIF) of All India Institute of Medical Sciences, New Delhi was availed for instrumentation
support. 3 mL of sterile nutrient broth containing 0.5% additional glucose was poured into the
6 well plate (Tarson) and incubated with freshly prepared primary culture of E. coli. 10 mm2
of glass pieces were sterilized and dispensed into the medium for biofilm formation on the
surface. The plate was incubated at 37 °C for 24 h. After incubation, the glass pieces were
gently washed with sterile PBS and aseptically transferred into fresh medium followed by
addition of test compound at 250 µg/mL and 500 µg/mL concentrations. The plate was then
incubated at 37 °C for next 24 h. After incubation, the glass pieces was removed and gently

washed with PBS and kept in fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1
M phosphate buffer, pH 7.4) overnight. Samples were again washed with PBS and dried to
examine under scanning electron microscope.
2.12. DNA binding studies
ctDNA was purchased from Sigma Aldrich (St. Louis, USA) and was used without
further purification. The solutions were prepared in tris-HCl buffer (pH 7.2). The
concentration of ctDNA was determined from absorption spectroscopy by using the average
extinction coefficient value of 6600 M-1cm-1 of a single nucleotide at 260 nm, which was
further validated by Nano Drop. The purity of DNA solution was checked by taking
absorbance ratio A260nm/A280nm [31].
2.12.1. UV-Vis spectroscopy
Absorption spectra were recorded on Analytik Jena Specord-250 spectrophotometer
(Konrad-Zuse-Strasse Jena, Germany). Quartz cuvettes of 1.0 cm were used. Absorbance
titrations were performed by maintaining fixed concentration of 8m (5 ꢁM) and varying the
concentrations of ctDNA (0-60 ꢁM) [32]..
2.12.2. Steady state fluorescence
Fluorescence emission spectra were recorded using a Cary Eclipse spectrofluorometer
(Varian, USA) equipped with a 150 W xenon lamp in a 1.0 cm quartz cell (Santa Clara, CA)
and a thermostated water bath, in an emission wavelength range of 450-600 nm upon
excitation at 420 nm. Fluorescence spectra were acquired by maintaining the concentration
8m constant (5 ꢁM) and varying the concentrations of ctDNA (0-80 ꢁM) at 298K [33].
2.12.3. CD Spectroscopy
Jasco-715 spectropolarimeter, equipped with a microcomputer, and 1.0 cm quartz cell
was used to record the CD spectra. All the CD measurements were carried out at 298K using
a thermostatically controlled cell holder attached to a Neslab RTE-110 water bath with an
accuracy of ±0.1 K. All the CD spectra were recorded in the range of 200-350 nm at a scan
speed of 50 nm/min [34].
2.13. Docking studies
Docking of compound 8m with the DNA dodecamer duplex of sequence
d(CpGpCpGpApApTpTpCpGpCpG) (PDB ID: 8BNA) was performed using AutoDock Vina
4.2. The structure of compound 8m was drawn using ChemDraw and converted into PDB

format using online free web server of National Cancer Institute (NCI) named SMILES
translator tool (http://cactus.nci.nih.gov/translate/). The desired format of docking i.e.
PDBQT was obtained using ADL tools. The complete DNA dodecamer was covered within
grid box and used for docking process. The interactions between compound 8m and DNA
was visualized by using Pymol [35]. The validity of the docking protocol adopted in this
study was performed by re-docking the ligand (HOECHST-33258) present in the X-ray
crystal structure of DNA (PDB ID: 8BNA) and comparing the RMSD between the docked
and crystal structure poses as described previously [36]. The binding affinity of ligands
towards DNA was estimated using the following relation, as described earlier [37].
∆G = −RT ln Ka
where, R and T are the universal gas constant (=1.987 cal/mol/K), and temperature (=298K)
respectively
3. Results and discussion
3.1. Chemistry
A series of novel isoxazole-1,2,3-triazole conjugates (8a-q) was synthesized as shown
in Scheme 1. Commercially available p-chloroacetophenone (1) and diethyl oxalate (2) were
converted into diketoester (3) via oxalylation process in the presence of freshly prepared
sodium ethoxide. Further, the diketoester (3) on reaction with hydroxylamine hydrochloride
in absolute ethanol at reflux condition underwent [3+2] cyclo-condensation reaction to give
isoxazole ethyl ester (4). Basic hydrolysis of isoxazole ethyl ester by employing freshly
prepared LiOH (2M) in THF/H2O mixture for 2½ h gave the corresponding acid in
quantitative yield. Further 5-(4-chlorophenyl) isoxazole-3-carboxylic acid (5) was
propargylated in the presence of hydroxyl benzotriazole (HOBt) and 1-(3-
dimethylaminopropyl)-3 ethylcarbodiimide (EDC.HCl) in acetonitrile to yield corresponding
alkyne (6). All the intermediates were purified by column chromatography to give desired
compounds in moderate to high yields as confirmed by spectroscopic analysis. In another set
of reaction, variously substituted aniline containing either electron-withdrawing or electron-
donating groups were converted into their corresponding azides (7a-q) via diazotization using
sodium nitrite and hydrochloric acid followed by sodium azide treatment. Finally, azide and
alkyne underwent Cu(I)-catalyzed [3+2] cycloaddition reaction in the presence of a catalytic
amount of CuSO4 and sodium ascorbate (NaAsc) in THF/H2O (1:2) mixture to yield the title
compounds (8a-q) in good to excellent yield (Scheme 1). The proposed structures were

confirmed by elemental analysis, FT-IR, 1H, 13C NMR & Mass spectral data, and purity was
checked by an Agilent RRLC MS 6320 ion trap spectrometer.
In the IR spectra of all the compounds (8a-q), the main evidence for their formation
came from the disappearance of absorption band at 2090 cm-1 corresponding to azide
functionality indicating that azide had been completely consumed in the reaction and the
peaks corresponding to alkyne ≡CH and C≡C which appeared in the range 3250-3376 cm-1
and 2112-2137 cm-1 respectively were also absent from their spectra. All the synthesized
compounds (8a-q) gave the characteristic peak of triazole ring in the region 3065-3189 cm-1
as broad absorption band. All the other absorption bands also appeared in the expected
regions.
Scheme 1: Synthesis of Isoxazole-1,2,3-triazole conjugate: Reagents and conditions: (a) Na
metal, C2H5OH, 4 h, r.t., 70% (b) C2H5OH, NH2OH.HCl, 2½ h, 78% (c) THF:H2O, LiOH,

2½ h, r.t., 95% (d) EDC.HCl, HOBt, acetonitrile, Propargyl alcohol, r.t., 80% (e) NaNO2,
HCl, NaN3, 92-98% (f) CuSO4.5H2O, NaASc, THF:H2O, overnight, r.t., 54-87%.
1
In the H NMR spectra, these compounds showed a sharp singlet in the region
7.18-7.67 ppm assigned to triazole ring, confirming its formation. The characteristic
singlets at 5.54-4.66 ppm and 5.50-4.36 ppm were also observed for OCH2 and C6H5CH2
protons of triazole derivatives 8a-q respectively. While a sharp singlet corresponding to
≡CH group which appeared in the region 2.49-2.57 ppm was absent from the spectra.
Besides, all the aromatic and aliphatic protons appeared at their expected chemical shifts
and integral values.
The formation of the triazole ring was further confirmed by 13C NMR spectral data in
which both the carbons of triazole ring appeared in the range 120.23-127.34 ppm and 138.32-
145.92 ppm respectively. The carbon signals of OCH2 and C6H5CH2 groups were resonated
at 62.16- 65.23 ppm and 54.25-55.99 ppm respectively, while all the other carbons gave
peaks at their expected values.
The mass spectra of most of the compounds showed [M+H]+ and [M-H]- peak which
was per their molecular formula thus further confirming their formation. The isoxazole
alkyne (6) showed [M+Na]+peak. In the mass spectra of compound 8m, fragmentation was
observed and no peak corresponding to molecular ion peak was observed.
3.2. In vitro antimicrobial activity
Antimicrobial screening against two Gram-positive (S. pneumoniae, E. faecalis) and
four Gram-negative strains (P. aeruginosa, S. typhimurium, K. pneumoniae, E. coli) bacterial
strains was done using microdilution method [38]. CIP was used as a reference drug and the
results are presented as IC50 in µg/mL (Table 1). In the preliminary in vitro antibacterial
screening, compounds showed selective inhibition of bacterial strains across the screening
panel. We synthesized 17 compounds with different substitution on benzene attached to the
triazole moiety. Methyl, methoxy, halides (-Cl and -F), carboxylic and nitro group were
substituted at different positions. Although no strong structure-activity relationship could be
established, but it was observed based on substitution of various groups at different position
of phenyl group, compounds became selective inhibitor against the respective bacterial strain.
Compounds 8a-b, 8d-f, 8m, 8o and 8q showed good to moderate activity against selective
bacterial strains. The Compound 8a showed moderate activity against S. pneumoniae and K.
pneumoniae with IC50 values of 295.8 and 274.4 µg/mL, respectively. Compound 8b with p-

COOH substitution increased its activity particularly against P. aeruginosa with IC50 value of
67.6 µg/mL. This compound showed significant inhibition of Gram-negative as well as
Gram-positive bacteria. Compound 8d with p-methoxy substitution selectively inhibited S.
typhimurium, S. pneumoniae and E. coli with IC50 values of 244.9, 325.1 and 239.8 µg/mL,
respectively. Similarly, the substitution of –F at para position enabled compounds 8e as a
potent antibacterial agent which selectively inhibited K. pneumoniae, P. aeruginosa and S.
pneumoniae with IC50 values of 157.4, 212.3 and 228.0 µg/mL, respectively. Compound 8f
with substitution of –Cl at para position showed increased activity against all the bacterial
strains. After changing the position of chlorine on meta-position as in compound 8m, it
showed good to moderate activity against all the bacterial strains with IC50 values less than
100 µg/mL except K. pneumonia (IC50 value = 105.9 µg/mL). Moreover, the substitution
with p–COOH and –Cl is responsible for antibacterial activity of compounds which also
depicted in Table 1. It showed good activity against Gram-positive bacteria S. pneumoniae
and E. faecalis with potent antibacterial potential having IC50 values 74.13 and 44.7 µg/mL,
respectively. Thus, based on the above observations, we selected compound 8b and 8m, as it
showed good to moderate activity against all the strains used in the study, for further
biological evaluation.
Table 1. In vitro antibacterial activity of compounds (8a-q) against Gram-positive and Gram-
negative strains in µg/mL.
Compound
IC50 in µg/mL
P.
S.
S.
E.
K.
E. coli
aeruginosa typhimurium pneumoniae faecalis
pneumoniae
544.50
67.60
903.64
199.06
601.17
244.90
413.23
270.39
>1000
428.54
295.80
153.81
699.84
325.08
228.03
167.10
331.13
805.37
482.21
114.29
812.83
519.99
790.67
171.00
>1000
>1000
274.78
136.77
>1000
496.59
157.39
183.23
>1000
767.36
332.12
154.88
281.83
239.80
457.08
213.79
831.76
363.07
8a
8b
8c
8d
8e
8f
676.08
950.60
212.32
153.81
948.90
448.74
8g
8h

628.05
397.19
322.84
110.92
92.25
400.49
564.93
>1000
737.90
165.19
512.86
393.55
712.85
292.41
538.26
639.73
707.94
>1000
74.13
788.86
383.7
>1000
>1000
812.83
>1000
105.92
346.73
>1000
287.07
454.98
832.88
891.22
891.25
>1000
83.17
8i
8j
>1000
578.09
44.66
8k
8l
8m
8n
8o
8p
8q
948.41
197.69
315.50
252.34
301.99
360.57
634.16
194.28
201.37
542.00
271.64
234.42
154.88
1095.52
524.80
776.24
3.3. Cytotoxicity assessment of lead inhibitors by hemolysis and MTT assays
3.3.1. Hemolytic assay
Based on preliminary antibacterial results, compounds 8b and 8m were selected to
evaluate their cytotoxicity on human red blood cells as well as cell line. The hemolytic assay
was performed to check the toxicity of the selected compounds (8b and 8m) on human red
blood cells (hRBCs) [39]. At 1 mg/mL concentration, 8b and 8m exhibited moderate toxicity
causing only 7% and 30% hemolysis, respectively compared to the standard drug CIP with
35% hemolysis. However, at 100 µg/mL concentration these compounds exhibited fairly less
toxicity causing only 0.6% and 4% cell lysis, respectively. At 25 µg/mL concentration, these
compounds were completely safe causing only 0.05% and 1% cell lysis, respectively (Fig. 2).

Hemolysis assay
40
30
20
10
0
8b
8m
Cip
0.025
0.05
0.1
0.2
0.4
0.6
0.8
1
Concentration in microgram/mL
Fig. 2. Hemolysis assay of compounds 8b and 8m showing % lysis of human RBCs.
3.3.2. Inhibition of cell proliferation by MTT assay
To evaluate the cytotoxicity effect of selected compounds, human embryonic kidney
cells (HEK293) were used. The compounds were screened with the help of a standard MTT
assay in the concentration range of 0-200 µM, for 48 h [40]. Interestingly, the results show
that the treatment of these compounds doesn’t affect the viability of HEK293 cells even at
200 µM concentration. These results suggested that the selected compounds 8b and 8m were
non-cytotoxic to HEK293 cells in the tested concentration range as shown in Fig. 3. From
these cell viability results, we can speculate that these molecules may be taken as promising
lead molecules against bacterial infection, as shown in sub-micromolar concentration ranges
they do not possess toxicity towards normal embryonic human kidney cells, but for bacterial
cells.

HEK293 Cells
100
50
0
Concentration(µM)
Fig. 3. MTT assay results at concentration range of 0-200 µM of 8b (left) and; 8m (right).
3.4. Growth curve
To investigate the effect of lead compounds, growth curve studies were performed
against specific susceptible bacterial strains. Compound 8m was selected as a lead inhibitor
to perform further biological studies. We also performed growth curve study for 8b against P.
aeruginosa. Growth curve study for compound 8m was performed against S. pneumoniae, E.
coli and E. faecalis strains. CIP was used as a standard drug while untreated cells were used
as positive control. The results showed that significant growth inhibition of P. aeruginosa
and S. pneumoniae occurred due to the presence of compounds 8b and 8m, respectively. At
higher concentrations i.e. 2MIC and MIC of both compounds, an extending lag phase up to
24 h were observed in P. aeruginosa as well as S. pneumoniae (Fig. 4). Moreover, at low
concentration of compound 8b, the lag phase ended in between 4-6 h in P. aeruginosa.
Similarly, at the sub-MIC concentration of the compound, the cells of S. pneumoniae enter
into log phase between 2-4 h. Further, compound 8m showed moderate growth inhibition
against E. coli and E. faecalis strains. Although, no measurable growth occurred in presence
of 2MIC concentration, the cells were grown with very less growth rate at MIC concentration
of compound 8m. There was not any significant change in growth in the presence of sub-MIC
concentration against E. coli and E. faecalis. Thus the above observations indicate the
bactericidal nature of compounds 8b and 8m against P. aeruginosa and S. pneumoniae,
respectively. Compound 8m was found more effective towards specific Gram-positive
bacterium S. pneumoniae.

Fig. 4. Dose dependent growth curve of E. coli (a); E. faecalis (b); S. pneumoniae (c) in
presence of 8m; and (d) P. aeruginosa in presence of compound 8m. No growth occurs in the
presence of high concentration i.e. 2MIC and MIC concentration of compounds 8b and 8m.
3.5. Assessment of biofilm formation
On the basis of better antibacterial potential against most of the bacterial strains used
in the study as well as toxicity, compound 8m was selected for further biological assays. The
attribute of biofilm formation in pathogenic microorganism plays an important role in
virulence. The biofilm inhibition assessment using XTT was performed against E. coli, S.
pneumoniae and E. faecalis bacterial strains. The concentrations equal to 8MIC, 4MIC, 2MIC
and MIC were used to explore it biofilm inhibition activity. The results indicated that
compound 8m significantly inhibit biofilm formation in S. pneumoniae and E. faecalis
bacterial strains. Although, the effect of compound 8m against biofilm formation by E. coli
was comparatively low at lower concentrations it significantly inhibit biofilm formation at
8MIC concentration (Fig. 5).

Biofilm Inhibition
100
80
60
40
20
0
E. faecalis
S. pneumoniae
E. coli
1000
500
250
125
Concentration in micorgram/mL
Fig. 5. Biofilm inhibition by XTT assay. Percentage of biofilm inhibition in E. coli, S.
pneumoniae and E. faecalis in presence of to 8MIC, 4MIC, 2MIC and MIC concentration of
compound 8m.
The quantitative assessment of biofilm formation by XTT assay was further
examined under scanning electron microscopy (SEM). Biofilm formation on the surface of
materials occurs due to the secretion of the extracellular matrix which primarily consists of
polysaccharide. Two concentrations equal to 4MIC and 2MIC values of compound 8m were
taken to compare the efficacy against biofilm formation with untreated cells. The cells in
untreated samples appeared in clusters and embedded in the extracellular matrix (Fig. 6). The
sample treated with 4MIC concentration showed very few scattered bacterial cells. Similarly,
significant damage in biofilm formation showed at 2MIC concentration (Fig. 6). The results
indicated the inhibition of biofilm formation after exposed to compound 8m and strongly
support the data of the previous experiment.
b
a
c
Fig. 6. Scanning electron microscopy of biofilm formation by E. coli. in a) untreated sample;
b) sample exposed to 4MIC; and c) 2MIC concentration of compound 8m.
3.6. DNA Binding Studies

The cleavage of DNA due to the binding of some heterocyclic compounds has been
explored and reported in the literature [41-43]. Some reports mainly emphasize the efficacy
of isoxazole and 1,2,3-triazole containing compounds as potential DNA binding agents which
eventually used as antibacterial [44-46]. Thus, we hypothesized DNA as a primary target for
our designed compounds. All the experiments of DNA binding studies were performed with
compound 8m.
3.6.1. Electronic/UV-visible absorption spectral studies
UV-Visible spectroscopy is the most widely used technique to detect and identify
interactions between small molecules with biomolecules and formation of a complex. The
strength of interaction was correlated with the magnitude of change in absorbance or shift in
the peak position [47]. The ability of ctDNA to interact with small molecules was evaluated
by hypochromism or hyperchromism. The UV absorption spectrum of 8m with increasing
concentrations of ctDNA is shown in Fig. 7. In the absence of DNA, compound 8m showed
maximum absorbance at 420 nm. On addition of first DNA titer (5ꢁM), absorbance is
increased indicating DNA-8m complex formation. On subsequent additions of DNA,
hyperchromism with slight blue shift was observed.
Fig. 7. Absorbance spectra of 8m with increasing concentrations of ct-DNA (0-60 µM)
3.6.2. Fluorimetric Studies
Because of its high sensitivity, selectivity and convenience, fluorescence emission
spectroscopy is widely used to get insights into the binding mode of small molecules with
biomolecular structures [48]. Since the endogenous fluorescence property of DNA is poor,
fluorescence spectra of compound 8m were studied with increasing concentrations of ctDNA

at 298K. Fig. 8 shows the fluorescence spectra of 8m with increasing concentrations of
ctDNA (0-80 ꢁM) at 298 K, with emission maxima at 562 nm upon excitation at 420 nm. On
subsequent addition of ctDNA, the emission intensity increases significantly without any
significant change in emission maxima which is direct evidence for the interaction between
8m and ctDNA. The absence of quenching effect and the subsequent increase in emission
intensity might be attributed to the hydrophobic environment of compound inside the DNA
helix as quenching occurs due to photoelectron transfer from the guanine base of DNA to the
excited states of the molecule. The emission data demonstrate that 8m binds with ctDNA
through intercalative mode.
Fig. 8. Fluorescence emission spectra of compound 8m with increasing concentrations of
ctDNA (0-80 µM) at 298K.
3.6.3. Circular dichroism spectroscopy
CD spectroscopy is a highly sensitive technique employed here to investigate the
conformational changes in secondary structure and molecular orientations relative to DNA
helix. The CD spectrum of ctDNA exhibits a positive band at ~275 nm due to base stacking
and a negative band at ~245 nm due to the right-handed helicity of B-DNA form which was
sensitive to interactions with small molecules. It has been well documented that groove
binding and electrostatic interaction of small molecules show less or no perturbation on the
base stacking and helicity bands, whereas intercalation alters the intensities of both the bands
significantly, thus stabilizing the right-handed B conformation of DNA. The CD spectrum of
ctDNA with the increasing concentrations of 8m was shown in Fig. 9. As it can be observed

that upon addition of compound 8m, CD spectrum was significantly perturbed. The ellipticity
of both positive and negative band changed upon addition of compound 8m to ct-DNA
revealing conformational changes and base stacking within the DNA molecule. Thus the
observed result supported an intercalative mode of binding. All the CD spectra were recorded
in the range of 200-350 nm at a scan speed of 50 nm/min [48].
15
0 µM
10
5
5 µM
10 µM
15 µM
0
-5
-10
-15
-20
-25
220
240
260
280
300
320
Wavelength (nm)
Fig. 9. Circular dichroism spectra of ctDNA with increasing concentrations of 8m.
3.6.5. Molecular docking studies
The validity of docking protocol employed here was confirmed by performing re-
docking of the ligand (HOECHST-33258) present in the X-ray crystal structure and
comparing the RMSD between the docked pose and crystal structure pose. Supplementary
Fig. S40 shows the binding of ligand at the minor groove of DNA as present in the X-ray
structure and as predicted by molecular docking. The RMSD between the two poses was
found to be 0.8356 Å, which is much lower than the allowed limit of 2.0 Å, confirming the
validity of the adopted docking protocol.
To further evaluate the nature of the interaction of the lead compound with DNA,
molecular docking studies were performed with the DNA dodecamer duplex of sequenced
(CpGpCpGpApApTpTpCpGpCpG) (PDB ID: 8BNA). The best docking pose of compound
8m with low energy (-9.9 kcal mol-1), corresponding to a binding affinity of 1.82 × 107 M-1)
is presented in Fig. 11. The compound is interacting within the minor groove of DNA when
combined with additional H-bonding interaction facilitates better DNA binding [49]. For
comparative analysis, we also performed docking of a known minor groove binder

(HOECSHT-33258) to the dodecameric DNA molecule. Supplementary Fig. S41 shows the
binding of HOECSHT-33258 at the minor groove of DNA. The structure was stabilized by
two hydrogen bonds between N1 and N4 of HOECSHT-33258 with DT7 and DT8 of
dodecameric DNA. The docking energy and binding affinity of HOECSHT-33258 towards
DNA was estimated to be -11.6 kcal mol-1 and 3.22 × 108 M-1, respectively. It is worth to
note that the binding affinity of 8m was at least 10-folds higher the known minor groove
binder, HOECSHT-33258.
Fig. 10. In silico studies of lead compound 8m with DNA dodecamer (PDB ID: 8BNA).
Docking pose of compound 8m along with reference compound HOECSHT-33258 showing
interaction with DNA dodecamer in (a) cartoon image; (b) surface image. (c) LigPlot image
showing hydrophobic interactions as well as hydrogen bond interaction with dg-10 and dg16
residue of DNA dodecamer.
4. Conclusion
To summarize, a series of isoxazole-triazole conjugates (8a-q) was synthesized by
1,3-cycloaddition reaction approach. Amongst all, 8b and 8m exhibited better antibacterial
activity towards particular bacterial strains and showed no cytotoxicity against human cells
(HEK293) even at higher concentrations. Compound 8m showed significant inhibition
potential against E. coli biofilm formation. Compound 8m was further evaluated for DNA