Synthesis of new triazole fused imidazo[2,1-b]thiazole hybrids with emphasis on Staphylococcus aureus virulence factors

Article abstract of DOI:10.1016/j.bmcl.2019.08.025

A series of new triazole fused imidazo[2,1-b]thiazole hybrids (9a–u) were designed, synthesized and evaluated as antimicrobial agents. Compounds 9c, 9d, 9e, 9j and 9l showed promising broad spectrum antimicrobial activity. Further, compound 9c exhibited s

Full text of DOI:10.1016/j.bmcl.2019.08.025

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of new triazole fused imidazo[2,1-b]thiazole hybrids with
emphasis on Staphylococcus aureus virulence factors
a,b
b,c
b,c
b,c
Mohd Adil Shareef , Kanugala Sirisha , Ibrahim Bin Sayeed , Irfan Khan ,
d
a
b,c
e,⁎
Thipparapu Ganapathi , Syed Akbar , C. Ganesh Kumar , Ahmed Kamal ,
a,b,⁎
Bathini Nagendra Babu
a
b
c
d
Centre for Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Tarnaka, 500007 Hyderabad, India
Academy of Scientific and Innovative Research, Ghaziabad 201002, India
Organic Synthesis and Process Chemistry Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
Stem Cell Research Division, National Institute of Nutrition (NIN), Indian Council of Medical Research (ICMR), Hyderabad 500007, Telangana, India
e
School of Pharmaceutical Education and Research (SPER), Jamia Hamdard, New Delhi 110062, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of new triazole fused imidazo[2,1-b]thiazole hybrids (9a–u) were designed, synthesized and evaluated
as antimicrobial agents. Compounds 9c, 9d, 9e, 9j and 9l showed promising broad spectrum antimicrobial
activity. Further, compound 9c exhibited significant anti-biofilm activity with single and mixed biofilm dis-
ruption demonstrated by Field Emission Scanning Electron Microscope (FE-SEM). Furthermore, molecular
docking studies revealed that they interact with the virulence factor, Staphylococcus aureus dehydrosqualene
synthase (CrtM) (PDB ID: 2ZCS). Overall, the findings suggest compound 9c as potential lead for further de-
velopment of novel antibacterial and anti-biofilm agents.
Triazole
Fused
Imidazo[2,1-b]thiazole
Antimicrobial
Staphylococcus aureus
Virulence factors
7
The development of new antibacterial compounds to combat human
bacterial infections has never been more alarming and challenging,
mainly because of the explicit use of antibiotics and the appearance of
defence by neutrophils and reactive oxygen species (ROS). Similarly,
persistent biofilms are a key virulence factor, which are recognized as the
principal cause of chronic and frequent bacterial infections ensuing an
estimated 17 million fresh biofilm infections and more than five hundred
1
bacterial resistance pattern towards a range of antimicrobial agents.
8
The evolution of drug-resistant strains viz., methicillin-resistant Sta-
phylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) has
unfortunately augmented nosocomial and community acquired infec-
tions and the inherent mortality rates, which poses an imperative threat
thousand deaths each year. Biofilms turn the bacteria resistant to con-
9
ventional antibiotics by around 1000-fold. Consequently, there is an
urgent need to counter biofilm formation and develop new antibacterial
agents that can exert anti-biofilm activities.
2
to human health. Only four new classes of antibiotics have been ap-
Imidazo[2,1-b]thiazole scaffold has gained renewed interest in view
1
0–13
proved by FDA over the past 17 years, whilst the majority of current
of their extensive pharmacological activities.
Levamisole, the well
3
,4
drugs have the same well-understood target. In view of these con-
siderations, there is a pressing demand to develop new arsenal of
chemotherapeutic agents, and to investigate newer targets or mechan-
known antihelminthic agent (I, Fig. 1) is one of the orally active imi-
dazo[2,1-b]thiazole derivatives. Besides their clinically proven anti-
helmintic and immunomodulating actions, various imidazo[2,1-b]
thiazole derivatives (II and III, Fig. 1) have exhibited promising anti-
bacterial, antifungal, and anti-tumour activities which has been the
5
isms to explore the antimicrobial activity.
In this context, efforts were made to explore the dehydrosqualene
synthase enzyme which plays a pivotal role in the biosynthesis of golden
carotenoid pigment, staphyloxanthin produced by Staphylococcus
1
4–17
primary reason for their inclusion in the hybrid framework.
Mo-
lecular hybridization of imidazo[2,1-b]thiazole scaffold with other
pharmacophores for the synthesis of newer chemotherapeutics has
signified a promising and ongoing field of research in the last few years.
We have been exploring the 1,2,3-triazole motif in our laboratory
6
aureus. It is evident that this pigment acts as a virulence factor. It also
acts as an antioxidant by shielding the bacterium to endure within the
host cell against oxidative stress generated because of host immune
⁎
Corresponding authors at: Centre for Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Tarnaka, 500007 Hyderabad, India (B. Nagendra
Babu).
E-mail addresses: pvc@jamiahamdard.ac.in (A. Kamal), bathini@iict.res.in (B. Nagendra Babu).
https://doi.org/10.1016/j.bmcl.2019.08.025
Received 1 March 2019; Received in revised form 8 August 2019; Accepted 11 August 2019
0960-894X/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Mohd Adil Shareef, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.08.025

M.A. Shareef, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
phenyl azides (8a–g) were prepared in one pot from the corresponding
2
1
anilines (7a–g) as per the literature protocol. Finally, the title com-
pounds (9a–u) were synthesized by reaction of corresponding pre-
cursors (6a–c) with phenyl azides (8a–g) in presence of catalytic
amount of CuSO
4
·5H O and sodium ascorbate in tertiary butanol/water
2
22
mixture as click chemistry protocol in good to excellent yields. All the
1
13
synthesized hybrids were confirmed by H NMR, C NMR and HRMS
spectral data.
All the synthesized triazole fused imidazo[2,1-b]thiazole hybrids
2
3
(
9a–u) were screened for their antimicrobial activity and the results
are represented in Table 1. Among them, compounds 9c, 9d, 9e, 9j and
9
l demonstrated promising broad spectrum antibacterial activity
against all the bacterial strains with MIC values ranging between 1.9
and 7.8 µg/mL and also displayed moderate antifungal activity with
MIC values ranging between 7.8 and 15.6 µg/mL.
Fig. 1. Structures of compounds containing imidazo[2,1-b]thiazole and 1,2,3-
triazole motifs.
Structure–activity relationship (SAR) study revealed that that
amongst the synthesized triazole fused imidazo[2,1-b]thiazole hybrids,
compounds (9o–u) with electron donating substituent such as 4-
for the development of newer chemotherapeutic agents.18 In addition,
when pooled with other heterocycles, it contributes in enhancing the
biological activity, as it efficiently binds with various biological targets
via hydrogen bonds, pi-stacking interactions and dipole-dipole inter-
methoxy group at R position exhibited diminished antibacterial ac-
1
tivity, while the compounds (9h–n) with 4-chloro substitution dis-
1
9
actions. Moreover, 1,2,3-triazole motif can be easily synthesized and
is an integral structural motif of various marketed antibacterial agents
such as Tazobactam (IV) and Cefatrizine (V) (Fig. 1). Despite significant
investigations on 1,2,3-triazoles, continuous efforts are still being made
to integrate them in exploring newer agents with potent broad spectrum
antibacterial and anti-biofilm activities.
played better antibacterial activity whereas compounds (9a–g) without
any substitution (R ) demonstrated promising antibacterial activity. On
1
the contrary, compounds 9c, 9d, 9e, 9j, 9k and 9l with substituents like
4
-chloro, 2-bromo 4-fluoro and 4-bromo groups on the phenyl ring of
triazole exhibited pronounced antibacterial activity in comparison with
the compounds 9a, 9b, 9h, 9i, 9o and 9p bearing substituents such as
In view of the above consideration and in furtherance to our on-
going efforts towards the discovery of new potent heterocyclic che-
motherapeutic agents, new triazole fused imidazo[2,1-b]thiazole hy-
brids were designed via molecular hybridization by incorporating
3
,4,5-trimethoxy and 4-methoxy groups (R ). Moreover, compounds
2
(
9f, 9m and 9t) with trifluoromethyl group were found to be having
moderate antibacterial activity against gram positive bacteria and di-
minished activity against gram negative bacteria.
2
0
phenyl triazoles at the 5th position of imidazo[2,1-b]thiazole scaffold.
Based on the significant antimicrobial activity demonstrated by
these hybrids, they were further screened for minimum bactericidal
concentration (MBC) and minimum fungicidal concentration (MFC)
Further evaluated for their in vitro antimicrobial activity against an
array of microorganisms and the most promising candidates were fur-
ther studied for biofilm inhibition potential.
24
against various microbial strains and the results are represented in
Synthesis of triazole fused imidazo[2,1-b]thiazole hybrids (9a–u) is
represented in Scheme 1. Initially, equimolar quantities of substituted
Table 2. Compounds such as 9c, 9d, 9e, 9j and 9l demonstrated pro-
mising broad spectrum activity against all the tested bacterial strains
with MBC values in the range of 3.9–15.6 μg/mL. Minimum fungicidal
concentration (MFC) values of these promising compounds were found
to be ranging amid 15.6 and 31.2 μg/mL.
2
6
-bromoacetophenones (1a–c) and 2-aminothiazole (2) were reflux for
–8 h followed by addition of 2 N HCl and continued the reflex for
another 1–2 h to obtain imidazo[2,1-b]thiazoles (3a–c). These obtained
intermediates were further subjected to Vilsmeier-Haack reaction con-
ditions to afford the corresponding imidazothiazole aldehydes (4a–c)
and the resulting aldehydes were then reacted with ethynylmagnesium
bromide in dry THF to gain the intermediates (5a–c) which upon oxi-
dation by means of 2-iodoxybenzoic acid (IBX) in DMSO provided the
corresponding terminal alkyne precursors (6a–c). Similarly, substituted
Keeping in view of the significance of biofilms, we tested our most
active hybrids (compounds 9c, 9d, 9e, 9j and 9l) for their biofilm in-
25
hibition property. All the tested hybrids were found to possess pro-
mising biofilm inhibition property. The results are represented in
Table 3. The results revealed that compounds 9c, 9d, 9e, 9j and 9l
displayed promising biofilm inhibition activity in the range of
9
.8–21.1 μg/mL for the tested bacterial biofilms. Fascinatingly, com-
pound 9c remarkably exhibited significant anti-biofilm activity against
the entire tested bacterial biofilms. Biofilm IC50 (Half maximal in-
hibitory concentration) of compound 9c was found to be 9.8 and
1
0.3 μg/mL against S. aureus MTCC 96 and E. coli MTCC 739 strains,
respectively, while the IC50 value of standard ciprofloxacin was found
to be 6.7 and 7.3 μg/mL, respectively.
The effect of compound 9c on biofilm formation was studied by FE-
2
6
SEM. The results to this regard are depicted in Fig. 2(a–h). FE-SEM
micrographs of the control biofilms of S. aureus MTCC 96 (Fig. 2a) with
no treatment depicted an intact biofilm with unaltered cell surface and
morphology, while S. aureus MTCC 96 biofilms treated with compound
9
c at dose of its MBC clearly indicated significant disruption and cell
damage (Fig. 2b). Fig. 2c depicts the control group of E. coli MTCC 739
with intact cell morphology. Whereas, the 9c treated biofilms of E. coli
MTCC 739 showed lysis of cells with a damaged membrane as seen in
the Fig. 2d. The mixed biofilms of S. aureus MTCC 96 and E. coli MTCC
7
39 with normal morphology and secreted biofilm matrix are depicted
in the Fig. 2e and g. The mixed biofilms of S. aureus MTCC 96 and E. coli
MTCC 739 treated with the derivative 9c with the biofilm matrix dis-
rupted and the individual cells lysed are depicted in the Fig. 2f and h.
Scheme 1. Synthesis of triazole fused imidazo[2,1-b]thiazole hybrids (9a–u).
2

M.A. Shareef, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Table 1
Antimicrobial activity of the synthesized triazolo fused imidazo[2,1-b]thiazole hybrids (9a–u).
Test Compds
R
1
R
2
Minimum inhibitory concentration (µg/mL)
Gram positive bacteria
Gram negative bacteria
Fungus
B. s a
S. a b
M. l c
S. a d
E. c e
P.a f
K. p g
C .a h
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
H
3,4,5-Tome
3.9
3.9
3.9
3.9
3.9
3.9
7.8
3.9
3.9
3.9
3.9
3.9
15.6
3.9
15.6
7.8
3.9
7.8
7.8
15.6
3.9
0.9
7.8
7.8
1.9
1.9
3.9
7.8
> 125
7.8
7.8
7.8
7.8
7.8
> 125
7.8
7.8
7.8
7.8
7.8
7.8
15.6
7.8
0.9
–
3.9
3.9
> 125
> 125
1.9
> 125
> 125
3.9
31.2
15.6
3.9
15.6
15.6
7.8
H
4-OCH
4-Cl
3
3.9
7.8
H
3.9
1.9
H
2-Br 4-F
4- Br
3.9
1.9
1.9
3.9
3.9
7.8
H
7.8
3.9
3.9
3.9
3.9
7.8
H
4-CF
3
3.9
3.9
> 125
> 125
> 125
> 125
3.9
> 125
> 125
> 125
> 125
3.9
> 125
> 125
> 125
3.9
15.6
15.6
15.6
7.8
g
h
i
H
4-F
> 125
15.6
3.9
> 125
7.8
Cl
3,4,5-Tome
Cl
4-OCH
4-Cl
3
3.9
j
Cl
3.9
3.9
3.9
7.8
k
l
Cl
2-Br 4-F
4- Br
3.9
3.9
> 125
3.9
> 125
3.9
3.9
7.8
Cl
3.9
3.9
3.9
7.8
m
n
o
p
q
r
Cl
4-CF
3
> 125
3.9
> 125
3.9
> 125
> 125
> 125
> 125
> 125
> 125
> 125
> 125
> 125
0.9
> 125
> 125
> 125
> 125
> 125
> 125
> 125
> 125
> 125
0.9
> 125
7.8
15.6
15.6
7.8
Cl
4-F
OCH
OCH
OCH
OCH
OCH
OCH
OCH
–
3
3
3
3
3
3
3
3,4,5-Tome
15.6
15.6
3.9
15.6
15.6
3.9
> 125
7.8
4-OCH
4-Cl
3
15.6
15.6
7.8
> 125
15.6
15.6
> 125
> 125
0.9
2-Br 4-F
4- Br
3.9
3.9
s
7.8
3.9
7.8
t
4-CF
4-F
–
3
7.8
15.6
3.9
7.8
u
3.9
15.6
a
CPF
0.9
0.9
–
a
MCZ
–
–
–
–
–
–
–
–
3.9
a
–
No activity.
CPF, Ciprofloxacin and MCZ, Miconazole as reference drugs (Controls). The tests were done in triplicates and represented as mean values for [a] Bacillus subtilis
MTCC 121; [b] Staphylococcus aureus MLS-16 MTCC 2940; [c] Micrococcus luteus MTCC 2470; [d] Staphylococcus aureus MTCC 96; [e] Escherichia coli MTCC 739; [f]
Pseudomonas aeruginosa MTCC 2453; [g] Klebsiella planticola MTCC 530 and [h] Candida albicans MTCC 3017.
Table 2
Minimum bactericidal/fungicidal concentration (µg/mL) of the synthesized triazolo fused imidazo[2,1-b]thiazole hybrids (9a–u).
Test Compds
Minimum bactericidal/fungicidal concentration (µg/mL)
Gram positive bacteria
Gram negative bacteria
Fungus
B. s a
S. a b
M. l c
S. a d
E. c e
P. a f
K. p g
C .a h
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
7.8
15.6
15.6
3.9
7.8
7.8
> 125
> 125
3.9
> 125
> 125
7.8
62.5
31.2
7.8
31.2
31.2
15.6
15.6
15.6
31.2
31.2
31.2
15.6
15.6
15.6
15.6
15.6
> 125
15.6
31.2
31.2
15.6
15.6
15.6
31.2
7.8
7.8
15.6
3.9
7.8
7.8
7.8
3.9
7.8
3.9
3.9
7.8
7.8
7.8
7.8
15.6
7.8
7.8
7.8
7.8
7.8
7.8
15.6
> 125
15.6
15.6
15.6
15.6
15.6
> 125
15.6
15.6
15.6
15.6
15.6
15.6
31.2
15.6
1.9
7.8
> 125
> 125
> 125
> 125
7.8
> 125
> 125
> 125
> 125
7.8
> 125
> 125
> 125
7.8
g
h
i
15.6
7.8
> 125
31.2
7.8
> 125
15.6
7.8
7.8
j
7.8
7.8
7.8
7.8
k
l
7.8
7.8
7.8
> 125
7.8
> 125
7.8
7.8
7.8
7.8
7.8
15.6
> 125
15.6
> 125
15.6
> 125
15.6
31.2
> 125
> 125
1.9
m
n
o
p
q
r
31.2
7.8
> 125
7.8
> 125
7.8
> 125
> 125
> 125
> 125
> 125
> 125
> 125
> 125
> 125
1.9
> 125
> 125
> 125
> 125
> 125
> 125
> 125
> 125
> 125
1.9
31.2
15.6
7.8
31.2
31.2
7.8
31.2
31.2
7.8
15.6
15.6
31.2
7.8
7.8
7.8
s
15.6
15.6
7.8
7.8
t
31.2
7.8
u
a
CPF
1.9
1.9
1.9
–
a
MCZ
–
–
–
–
–
–
–
3.9
a
–
No activity; CPF, Ciprofloxacin and MCZ, Miconazole as reference drugs (Controls). The tests were done in triplicates and represented as mean values for [a]
Bacillus subtilis MTCC 121; [b] Staphylococcus aureus MLS-16 MTCC 2940; [c] Micrococcus luteus MTCC 2470; [d] Staphylococcus aureus MTCC 96; [e] Escherichia coli
MTCC 739; [f] Pseudomonas aeruginosa MTCC 2453; [g] Klebsiella planticola MTCC 530; and [h] Candida albicans MTCC 3017.
3

M.A. Shareef, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Table 3
Biofilm inhibitory concentration (µg/mL) of the synthesized triazolo fused imidazo[2,1-b]thiazole hybrids (9a–u).
Compound
Biofilm inhibitory concentration (µg/mL)
B. s a
S. a b
M. l c
S. a d
E. c e
P. a f
K. p g
9
9
9
9
9
c
d
e
j
11.4 ± 0.22
13.5 ± 0.09
16.0 ± 0.26
13.2 ± 0.06
13.5 ± 0.08
6.6 ± 0.06
10.63 ± 0.03
12.8 ± 0.08
14.4 ± 0.07
11.9 ± 0.08
14.0 ± 0.06
6.6 ± 0.06
11.23 ± 0.20
12.3 ± 0.06
13.9 ± 0.09
13.7 ± 0.08
16.0 ± 0.04
6.8 ± 0.05
9.8 ± 0.08
13.5 ± 0.15
21.8 ± 0.07
18.4 ± 0.03
21.3 ± 0.06
6.7 ± 0.05
10.3 ± 0.20
18.2 ± 0.20
20.2 ± 0.20
14.0 ± 0.24
16.2 ± 0.8
7.3 ± 0.04
14.2 ± 0.8
18.2 ± 0.8
20.2 ± 0.07
19.3 ± 0.06
17.5 ± 0.05
7.4 ± 0.04
14.6 ± 0.09
17.3 ± 0.08
21.1 ± 0.08
16.8 ± 0.18
20.5 ± 0.06
8.6 ± 0.04
l
CPF
CPF, Ciprofloxacin, as reference drug (Control). The tests were carried out in triplicates and represented as mean values for [a] Bacillus subtilis MTCC 121. [b]
Staphylococcus aureus MLS-16 MTCC 2940. [c] Micrococcus luteus MTCC 2470. [d] Staphylococcus aureus MTCC 96. [e] Escherichia coli MTCC 739. [f] Pseudomonas
aeruginosa MTCC 2453. [g] Klebsiella planticola MTCC 530. Bold signifies promising values.
Fig. 2. (a–h) Effect of compound 9c on the dual
species biofilms formed by S. aureus MTCC 96 and E.
coli MTCC 739; a. S. aureus biofilm untreated; b. S.
aureus biofilm treated with 9c depicting the lysis of
S. aureus MTCC 96; c. E. coli biofilm untreated; d. E.
coli biofilm treated with 9c depicting the lysis of E.
coli MTCC 739; e. Dual species biofilm of S. aureus
and E. coli, untreated (4000×); f. Dual species bio-
film of S. aureus and E. coli, treated with 9c
(
4000×); g. Dual species biofilm of S. aureus and E.
coli, untreated (17000×); h. Dual species biofilm of
S. aureus and E. coli, treated with 9c (17000×).
In the current study, the dual species biofilm inhibition was assessed
(PDB ID: 2ZCS) was obtained from protein data bank.31 Docking studies
were carried out to identify the Protein–Ligand interactions of the most
active compounds namely 9c, 9d, 9e, 9j and 9l using Molegro Virtual
for the compound 9c by testing against Staphylococcus aureus MTCC 96
2
7
and Escherichia coli MTCC 739 dual species biofilms. The results to
this context are represented in Table 4. The results revealed that mixed
biofilm IC50 of compound 9c was found to be 14.3 μg/mL as compared
to ciprofloxacin which exhibited an IC50 of 11.6 μg/mL.
3
2
Docker.
Investigation of the receptor ligand complex models was carried out
based on the parameters such as MolDock score, H–Bond energy,
Protein–Ligand interactions and Water–Ligand interactions of the
docked compound inside the active site. The results are represented in
Table 6 and the Protein–Ligand interactions were represented in Fig. 3.
The amino acid residues present in the active binding site of the protein
2ZCS can interact with these compounds which evidently reveal the
binding positions of ligands with the protein.
Furthermore, the most potential compounds (9c, 9d, 9e, 9j and 9l)
were evaluated for their in vitro cytotoxicity against normal human lung
2
8
cell line MRC5 (ATCC No. CCL 171) using MTT assay and the results
are represented in Table 5. In this assay, ciprofloxacin was used as
positive control along with DMSO as negative control. IC50 values are
represented in µg/mL as mean ± S.D. The results revealed that the
cytotoxicity of these compounds was found to be in the range of
Interestingly, compounds 9c, 9d, 9e, 9j and 9l were found to be
binding in a similar orientation and with the same binding mode when
superimposed as shown in Supplementary Fig. S3. The key ligand in-
teractions (steric) with the amino acid residues of the target include
Arg45, Phe22, Gln165, Gly161, Leu164, Leu141, Tyr41, Val137,
Ala134, His18 and Asn168. Almost, all the docked poses interact with
these residues, which indicate that this active site is the most favorable
binding site for the compounds. None of the compounds showed H-
2
4–37 µg/mL while their antibacterial activity (MIC) was found to be in
the range of 1.9–7.8 µg/mL. These findings suggests that compound 9c,
9
d, 9e, 9j and 9l exhibited lower cytotoxicity to normal cell lines
compared to the antibacterial activity.
The promising antibacterial results encouraged us to further in-
vestigate the possible mode of action. In view of the importance of
2
9,30
dehydrosqualene synthase (CrtM) as a virulence factor,
the mole-
cular docking studies were carried out on this enzyme. The three di-
mensional prote in crystal structure of the S. aureus C(30) carotenoid
dehydrosqualene synthase complexed with bisphosphonate BPH-700
Table 5
Cytotoxicity assay of the most active derivatives (9c, 9d, 9e, 9j
and 9l) on MRC5 normal cell line.
Table 4
Compound code
IC50 values (μg/mL)
Mixed biofilm inhibitory concentration (µg/mL) of compound 9c.
9
c
36.1 ± 0.5
48.2 ± 0.9
28.1 ± 0.2
24.2 ± 0.2
37.3 ± 0.4
53.4 ± 0.3
ⱡ
Test Compd
Dual biofilm inhibitory concentration (µg/mL)
9d
9
e
9
c
14.3 ± 0.26
11.6 ± 0.19
9j
Ciprofloxacin
9l
Ciprofloxacin
ⱡ S. aureus MTCC 96 + E. coli MTCC 739.
4

M.A. Shareef, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Table 6
MolDock score, H–Bond energy, Protein–Ligand interactions and Water–Ligand interactions of the most active compounds (9c, 9d, 9e, 9j and 9l) and antimicrobial
Agent II with the target (PDB: 2ZCS).
Code
MolDock Score
−141.546
−138.455
−139.608
−141.545
−141.81
Protein–Ligand interactions
Hydrogen bonds
Water–Ligand interactions
9
9
9
9
9
c
d
e
j
−126.757
0
0
0
0
0
−14.789
[
Arg45, Phe22, Gln165, Gly161, Leu164, Leu141, Tyr41]
[HOH334, HOH353, HOH568]
−8.092
−130.363
Val137, Leu141, Gly161, Leu164, Ala134, Gln165, Phe22, His18]
−129.461
Leu141, Leu164, Gly161, Phe22 Ala134, Gln165, His18]
−127.338
Arg45, Gln165, Asn168, Ala134, Phe22, Gly161, Leu164, Val137]
−132.742
Leu141, Gly161, Leu164, Ala134, Phe22, Gln165, Asn168]
−137.126
Leu164, Leu141, Gly138, Asp48, Val133, Ala134]
[
[HOH334, HOH353, HOH568]
−10.147
[
[HOH334, HOH353, HOH568]
−14.207
[
[HOH334, HOH353, HOH568]
−9.068
l
[
[HOH334, HOH353, HOH568]
−17.624
Agent II
−157.702
−2.952
[
[Tyr248, Gln165]
[HOH334, HOH568]
bond interactions with amino acid residues. However, they exhibited
strong H–Bond interactions with co-crystal water molecules (HOH334,
HOH353, HOH568) which also play an important role in ligand binding
9l demonstrated promising broad spectrum activity against all the
tested bacterial strains with MBC values ranging between 3.9 and
15.6 μg/mL. Further, biofilm inhibition assay revealed that compounds
9c, 9d, 9e, 9j and 9l displayed promising biofilm inhibition in the
tested bacterial biofilms, especially the compound 9c exhibited sig-
nificant anti-biofilm activity against all the tested bacterial biofilms and
its IC50 was found to be 9.8 and 10.3 μg/mL against S. aureus MTCC 96
and E. coli MTCC 739 strains, respectively. FE-SEM micrographs clearly
indicated that the compound 9c caused biofilm disruption and was
found to be a promising biofilm inhibitor. Furthermore, mixed biofilm
assay revealed that the dual species mixed biofilms were effectively
disrupted by compound and the mixed biofilm IC50 of compound 9c
was found to be 14.3 μg/mL. Further, MTT assay revealed that the most
potential compounds were non toxic to MRC5 normal cell line.
Moreover, docking studies predicted the binding modes of the most
potential compounds with the target enzyme. Therefore, these findings
offer insights to design and develop new antibacterial leads with em-
phasis on the virulence factors such as biofilms and dehydrosqualene
synthase (CrtM) of S. aureus.
3
3,34
with target.
Among the docked compounds, compound 9c and 9j
displayed significant Water–Ligand interactions while the antimicrobial
agent II showed strong H–Bond interactions with amino acid residues
(
Tyr248, Gln165) and co-crystal water molecules (HOH334, HOH568)
apart from steric interactions. Overall, molecular docking studies pro-
vided valuable molecular insights about the interactions and binding
modes of most active compounds with dehydrosqualene synthase en-
zyme.
In conclusion, a series of new triazolo fused Imidazo[2,1-b]thiazole
hybrids (9a–u) were synthesized and evaluated for their in vitro anti-
bacterial, MBC, antifungal, MFC and biofilm inhibition activities.
Among them, the hybrids 9c, 9d, 9e, 9j and 9l demonstrated promising
broad spectrum antibacterial activity against the entire set of tested
pathogens with MIC values ranging between 1.9 and 7.8 μg/mL and
displayed moderate antifungal activity with MIC values ranging be-
tween 7.8 and 15.6 μg/mL. In addition, compounds 9c, 9d, 9e, 9j and
Fig. 3. Docking interactions of compounds 9c, 9d, 9e, 9j and 9l antimicrobial agent (II) with dehydrosqualene synthase (PDB: 2ZCS) of Staphylococcus aureus.
5

M.A. Shareef, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Acknowledgments
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