Synthesis, biological evaluations and computational studies of N-(3-(-2-(7-Chloroquinolin-2-yl)vinyl) benzylidene)anilines as fungal biofilm inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.12.046

In the present investigation, new chloroquinoline derivatives bearing vinyl benzylidene aniline substituents at 2nd position were synthesized and screed for biofilm inhibitory, antifungal and antibacterial activity. The result of biofilm inhibition of C.

Full text of DOI:10.1016/j.bmcl.2018.12.046

Accepted Manuscript
Synthesis, biological evaluations and computational studies of N-(3-(-2-(7-
Chloroquinolin-2-yl)vinyl) benzylidene)anilines as fungal biofilm inhibitors
Firoz A. Kalam Khan, Rashmi N. Kaduskar, Rajesh Patil, Rajendra H. Patil,
Siddique Akber Ansari, Hamad M. Alkahtani, Abdulrahman A. Almehizia,
Devanand B. Shinde, Jaiprakash N. Sangshetti
PII:
S0960-894X(18)30993-4
https://doi.org/10.1016/j.bmcl.2018.12.046
BMCL 26215
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 October 2018
17 December 2018
20 December 2018
Please cite this article as: Khan, A.K., Kaduskar, R.N., Patil, R., Patil, R.H., Ansari, S.A., Alkahtani, H.M.,
Almehizia, A.A., Shinde, D.B., Sangshetti, J.N., Synthesis, biological evaluations and computational studies of N-
(
3-(-2-(7-Chloroquinolin-2-yl)vinyl) benzylidene)anilines as fungal biofilm inhibitors, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.12.046
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Synthesis, biological evaluations and computational studies of N-(3-(-2-(7-
Chloroquinolin-2-yl)vinyl) benzylidene)anilines as fungal biofilm inhibitors
a
b
c*
d
Firoz A. Kalam Khan, Rashmi N. Kaduskar, Rajesh Patil , Rajendra H. Patil , Siddique
e
e
e
f
Akber Ansari , Hamad M. Alkahtani , Abdulrahman A. Almehizia , Devanand B. Shinde ,
Jaiprakash N. Sangshetti b*
a
Anjuman-I-Islam’s Kalsekar Technical Campus, School of Pharmacy, Navi Mumbai 410206 (M.S.),
India.
b
Y.B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Aurangabad 431 001 (M.S.), India.
c
Sinhgad Technical Education Society's, Smt. Kashibai Navale College of Pharmacy, Kondhwa
Bk), Pune, India
Department of Biotechnology, Savitribai Phule Pune University, Pune 411007 (M.S.), India.
(
d
e
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia. Po Box 2454, Riyadh 11451.
f
Shivaji University, Vidyanagar, Kolhapur 416004, MS, India
*
Corresponding author. Tel. No.: +91 240-2381129.
E-mail address: rajshama1@yahoo.com (Rajesh Patil)
jnsangshetti@rediffmail.com (J.N. Sangshetti).
ABSTRACT
In the present investigation, new chloroquinoline derivatives bearing vinyl benzylidene aniline
nd
substituents at 2 position were synthesized and screed for biofilm inhibitory, antifungal and
antibacterial activity. The result of biofilm inhibition of C. albicans suggested that compounds 5j
(
IC value= 51.2 μM) and 5a (IC value= 66.2 μM) possess promising antibiofilm inhibition
50 50
when compared with the standard antifungal drug fluconazole (IC = 40.0 μM). Two compounds
5
0
5
a (MIC= 94.2 μg/mL) and 5f (MIC= 98.8 μg/mL) also exhibited good antifungal activity
comparable to standard drug fluconazole (MIC= 50.0 μg/mL). The antibacterial screening
against four strains of bacteria viz. E. coli, P. aeruginosa, B. subtilis, and S. aureus suggested
their potential antibacterial activity and especially all the compounds except 5g were found more
active than the standard drug ciprofloxacin against B. subtilis. To further gain insights into the
possible mechanism of these compounds in biofilm inhibition through the agglutinin like protein
(Als), molecular docking and molecular dynamics simulation studies were carried out. Molecular
1

modeling studies suggested the clear role in inhibition of this protein and the resulting biofilm
inhibitory activity.
Keywords: Quinoline; Schiff bases; fungal biofilm inhibition; antibacterial activity; molecular
docking; molecular dynamics
Nitrogen bearing heterocyclic compounds has been the largest class of organic
heterocyclic compounds possessing diverse biological properties and thus continually being
exploited in unmet challenges in drug discovery and design. Quinoline is an important nitrogen
containing heterocyclic ring and its derivatives possess diverse bioactivities such as
1
2
3
4
5
antimicrobial , anticancer, antileishmania . Antimycobacterial , Anticonvulsant , anti-
6
7
8
9
10
inflammatory and analgesic , antiallergenic , anti-alzheimer , antihypertensive , antifungal
1
1
and antidiabetic activities . Some of the quinoline ring based antimicrobial agents currently
being used as therapeutic agents are presented in Figure 1.
CH3
O
N
O
CH3
O
N
NH2 O
F
COOH
F
F
COOH
H C
F
COOH
H C
F
COOH
3
N
3
N
N
N
N
N
F
H
HO
H
HN
HN
HN
F
N
CH3
Grepafloxacin
H2N
Delafloxacin
Ciprofloxacin
Sparfloxacin
F
Figure 1. Currently used quinoline derivatives as antimicrobial agents
Microorganisms are continually evolving with newer means to cope up with antibiotics,
1
2
antibacterials and toxins . The process of cell to cell communication, called Quorum Sensing
QS) in bacteria, results in altered gene expression and subsequent survival and pathogenesis of
(
bacteria 13-14. Quorum sensing mediates formation of biofilm which is an association of micro-
organisms where cells adhere to each other within matrix of extracellular polymeric substances
15.
Thus the QS results in release of variety of chemical signals, formation of pores and formation
of water channels conferring the resistance in microbial colonies 16-17. Among the different
microbes, the Gram negative bacterium Pseudomonas aeruginosa is responsible for biofilm
growth and has gained much attention, since they can form biofilms in urinary tract, kidney,
lungs, leading to inflammations and septic shock. Thus, to study bacterial biofilm inhibition, P.
2

aeruginosa can be used as model organism 18. In immune-compromised patients Candida
albicans can cause superficial and systemic infections 19-20. Further, in hospitalized patients, it is
2
1
the major cause of morbidity and mortality . The QS support the formation of complex biofilm
from the budding state and subsequent colonization, entry in blood stream and virulence. Thus,
2
2
the biofilm formation has aggrieved the problem of resistance in C. albicans infections . All
these issues suggest that there is strong need to develop more effective antimicrobial treatment
mainly against infections associated with biofilm formation. At molecular level it has been
reported that the cell surface glycoproteins from Agglutinin-like protein family (Als1–Als7 and
Als9) found in the fungi like C. albicans, are involved in adhesion of the fungus to host cells and
2
3
biofilm formation . A recent report suggests that the inhibitors of Als proteins can be potential
2
4
biofilm inhibitors . It has been also established that the signaling molecules like 2-n-heptyl-3-
hydroxy-4(1H)-quinolone, called Pseudomonas Quinolone Signal (PQS) and 2-n-heptyl-4-
hydroxyquinoline (HHQ) derived from AnthraniloylCoA (ACoA) are responsible for biofilm
2
5
formation . Considering all above facts and in continuation of our ongoing efforts for synthesis
of antimicrobial compounds acting on different targets 26-29, we are herewith reporting the
synthesis of some new quinoline analogues which have been evaluated for their inhibitory
activity against biofilm from C.albicans. These new quinoline analogues were designed on the
basis of key signaling molecules, PQS and HHQ as shown in Figure 2. The possible binding
mode and mechanism of inhibition of the Als protein, molecular docking and dynamics was
carried out and reported to augment the biofilm inhibitory potential of these molecules.
3

OH
N
O
OH
N
H
HHQ
PQS
R
Cl
N
N
Figure 2. Strategy of design of fungal biofilm inhibitors.
The 2-substituted quninoline derivatives 5(a-j) were synthesized as per the scheme
outlined in Scheme 1. Sigma or Avra grade of compounds and solvents were used in the
synthesis of titled compounds and used without further purification.
4

Cl
N
CH3
OHC
CHO
+
1
2
a
NH2
+
Cl
N
CHO
R
(a-j)
3
4
b
Cl
N
N
R
5(a-j)
Scheme 1: Synthesis of N-(3-(-2-(7-Chloroquinolin-2-yl)vinyl) benzylidene)anilines 5(a-j).
nd
Iron-catalyzed C(sp3)–H functionalization of methyl group substituted at 2 position of
3
0
7
-chloroquinoline was achieved through a analogues reported method . Thus, the intermediate
3-(2-(7-Chloroquinolin-2-yl)-vinyl)benzaldehyde was obtained by stirring a solution of 7-
3
chloro-2-methylquinoline 1 (2.0 g, 11.26 mmol), isophthalaldehyde 2 (1.80 g, 13.4 mmol) and
Fe(OAc) (98 mg, 0.56 mmol) in AcOH (65 µL, 1.13 mmol) and toluene (12 mL) at 110 °C for
2
3
0 h. The progress of reaction was monitored by TLC. When the mixture was cooled to room
temperature, it afforded a solid precipitate which was recovered by filtration and recrystallised
by using EtOAc (25 mL) as a light-yellow solid (Yield: 60%). The mixture of compound 3 (1.0
mmol) and different aromatic amines 4(a-j) (1.0 mmol) in absolute ethanol (15 mL) with
catalytic amount of glacial acetic acid (3.0 mmol) was refluxed to obtain the titled compounds
5
(a-j). The completion of reaction was monitored by TLC and the mixture was kept overnight.
The crude solid was collected and recrystallized from ethanol. The physical data of the
5

synthesized compounds 5(a-j) is given in Table 1. The purity of the synthesized compounds was
determined by preparative TLC (Merck) with iodine vapor visualization technique. The melting
points were measured in open capillary tubes and are uncorrected. 13C NMR and H NMR
spectra were recorded on 400MHz BRUKER spectrometer. Chemical shifts for NMR studies are
reported in parts per million (ppm), using TMS as an internal standard. Agilent technology 1200
series HPLC paired to a 6130 mass spectrometer with electron spray ionization (ESI) was used to
record mass spectrum. The characterization data (Supplementary data) suggested the synthesis of
proposed compounds.
1
Table 1 Physical data for N-(3-(-2-(7-Chloroquinolin-2-yl)vinyl)benzylidene)anilines 5(a-j).
0
Entry
R
Mol. Formula
C H Cl N
Yield (%)
Rf value
0.71
0.68
0.65
0.60
0.70
0.76
0.65
0.68
0.72
0.70
Mp ( C)
110
5
a
87
82
90
85
83
84
84
84
84
85
p-Cl
p-NO2
m-NO2
p-OCH3
p-CH3
2
4
16
2
2
5
b
90-92
C H ClN O
24
16
3
2
5
c
220
C H ClN O
24
16
3
2
5
d
110-112
80
C H ClN O
25
19
2
2
2
2
5
e
C H ClN
25
19
5
f
140-144
138-140
110-114
130-134
140-142
-H
C H ClN
24 17
5
g
o-CH3
o-CF3
m-CF3
p-CF3
C H ClN
25 19
5
h
C H ClF N
2
25
16
3
5
i
C H ClF N
25
16
3
2
2
5
j
C H ClF N
25
16
3
The synthesized compounds 5(a-j) were evaluated for in vitro biofilm inhibitory activity
in C. albican strain. The reported method 31-32 of in vitro biofilm inhibitory activity was
followed. The N-(3-(-2-(7-Chloroquinolin-2-yl)vinyl)benzylidene)anilines 5(a-j) were evaluated
for biofilm inhibition by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT)
5
assay which is measure of metabolic activity of cells. C. albicans cells (10 cells/ well) were
inoculated in RPMI medium and exposed to various concentrations (0-100 µM) of compounds
o
5
(a-j) and standard drug fluconazole and further incubated for 24 h at 30 C. The media was
removed and the biofilm formed was washed with phosphate buffer. Each well was further
treated with 100 µL (1µg/µL) of MTT and the culture was further incubated for 3 h in dark. The
6

clear solution from each well was removed and dimethylsulfoxide (100 µL) was added. The
optical density was recorded at 575 nm. From the growth inhibition curve, the concentration
required to inhibit biofilms by 50 % (IC ) was calculated 33-34. The results of biofilm inbibition
5
0
are given in Table 2.
Table 2 In vitro Biofilm inhibition, antibacterial and antifungal activities of N-(3-(-2-(7-
Chloroquinolin-2-yl)vinyl)benzylidene)anilines 5(a-j).
Anti-
Antifungal
activity (MIC
Values in
biofilm
activity
IC μM)
(
μg/mL)
Antibacterial activity (MIC in μg/mL)
5
0
Entry
P.
C. albicans C. albicans
E. coli
aeruginosa B. subtilis
S. aureus
(
NCIM-
(NCIM-
3471)
(NCIM-
2256)
(NCIM-
2036)
(NCIM-
2063)
(NCIM-
2901)
3
471)
5
a
66.2
92.1
83.6
97.5
96.2
199.1
165.5
94.3
87.2
51.2
40
94.2
121.2
106
45
150
185
120
205
110
68.5
67.5
102
84.5
-
228.5
200.5
190
167
177
102
185
250
142
91.5
-
31.6
15.7
17.4
17.6
16.98
32.35
136.22
36.22
33.44
37.66
-
170.2
165.5
104.2
94.8
98.5
91.6
85.3
55.3
71.3
89.7
-
5
b
5
c
5
d
105
5
5
e
f
164.1
98.8
104.4
282
191
174.4
50
5
5
g
h
5
5
FA
CP
i
j
-
-
50
50
50
25
The title quinoline derivatives 5(a-j) exhibited good to moderate biofilm inhibition
activity (IC in the range 51.2-199.1 μM) against C. albicans strain. Compounds 5j and 5a with
5
0
IC values 51.2 and 66.2 μM respectively, were most active identified compounds in this series.
5
0
The preliminary structure activity relationship (SAR) suggested that the compound 5j (IC =
5
0
5
1.2 μM) with p-trifluoromethyl substituent on the phenyl ring improves biofilm inhibitory
activity as compared to the compound 5f (IC = 199.1 μM) which has unsubstituted phenyl ring.
5
0
But this trifluoromethyl substituent at ortho and meta position as in compounds 5h (IC = 94.3
5
0
μM) and 5i (IC = 87.2 μM) found decreasing the biofilm inhibitory activity. This suggests that
5
0
the small electronegative substituent like trifluoromethyl is more favaorable at para position of
7

phenyl ring. Hydrophobic substituent like o-methyl is less favorable as in compound 5g (IC =
5
0
1
65.5 μM). A halogen group like chloro at para position on pheyl ring as in compound 5a (IC50
66.2 μM) was also found increasing the biofilm inhibitory activity than the unsubstituted
=
compound. The substituents like p-methoxy and p-nitro shows moderate antibiofilm activity in
compounds 5b (IC50 = 92.1 μM) and 5d (IC50 = 97.5 μM). But the m-nitro substitution in
compound 5c (IC = 83.6 μM) was found better than the p-nitro substitution. The structure
5
0
activity relationship suggests that the trifluoromethyl group with significant electronegative
characteristic exhibit the highest antibiofilm activity than the corresponding chloro substituted
compound. The hydrophobic substituent’s shows decrease in antibiofilm activity as compared to
such electronegative substituents. The reference compound Fluconazole exhibited slightly better
antibiofilm inhibition activity (IC = 40.0 μM) than the series under investigation.
5
0
The synthesized compounds were also evaluated for in vitro antifungal activity. Standard
agar method 35-36 as per the guidelines of The Clinical and Laboratory Standards Institute (CLSI)
was employed. The solutions of title compounds and standard fluconazole were prepared in
DMSO. The C. albicans was subcultured on the Sabouraud dextrose agar (SDA) and incubated
0
at 25 C for 72 h. The resultant C. albicans cells were suspended in sterile distilled water to
5
achieve 10 cells/mL dilution. This standardized C. albicans suspension was inoculated on
control plates (plates without standard drug, fluconazole and plates for solvent control, DMSO)
0
and test plates with title compounds. The plates were incubated at 25 C for 48 h. The lowest
concentration of compounds (MIC) required to prevent growth of macroscopically visible
colonies of C. albicans in control plates and test plates were measured by taking readings at 48 h
and 72 h. The synthesized compounds exhibited good to moderate antifungal activity (IC50
range = 94.2 – 282.0 μM). Compounds 5a and 5f exhibited good antifungal activity with IC50
values of 94.2 and 98.8 μM, which are comparable to the reference compound Fluconazole with
IC value of 50 μM. All other compounds exhibited moderate antifungal activity as their IC
5
0
50
value is above 100 μM and below 300 μM. Interestingly the compound 5f which is unsubstituted
exhibited better antifungal activity than its antibiofilm inhibitory activity. In case of compound
5
j though the antibiofilm activity is higher the corresponding antifungal activity is moderate.
Further, the substituent p-chloro improves the antifungal activity considerably suggesting that
only electronegative substituent is favorable for antifungal activity.
8

The synthesized compounds were also evaluated for their in vitro antibacterial activity.
The antibacterial potency in terms of MIC values was determined as per the guidelines of CLSI
35-36.
Nutrient Broth containing microbial spores was prepared from 24 h old bacterial cultures on
ο
-4
-5
nutrient agar (Hi-media) at 37±1 C. A concentration of 1× 10 -10 colony forming unit (CFU)
in the bacterial suspension was adjusted with sterile saline solution. Two fold serial dilutions of
the synthesized compounds and standard drug ciprofloxacin were prepared to obtain the
concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, and 3.13 μg/mL. The control and test
ο
samples were incubated in BOD incubators at 37± 1 C for bacteria. The MICs were measured
after 24 h of incubation.
The title compounds 5(a-j) showed good to moderate antibacterial activities against
different bacterial strains. All the compounds 5(a-j) (MIC range = 15.7 – 37.6 μg/mL) were
found possessing better antibacterial activity than ciprofloxacin (MIC= 50 μg/mL) except
compound 5g (MIC= 136.2 μg/mL) against B. subtilis strain. This suggests that the hydrophobic
methyl substituent decreases antibacterial activity against B. subtilis. The compound 5a (MIC=
4
5 μg/mL) was found more active than the reference ciprofloxacin (MIC= 50 μg/mL) against E.
coli. Compound 5g (MIC= 68.5 μg/mL), 5h (MIC= 67.5 μg/mL) and 5j (MIC= 84.5 μg/mL)
exhibited moderate antibacterial activity against E. coli. The MIC value for other compounds
(5b-5f and 5i) was found above 100 μg/mL, suggesting their decreased antibacterial activity
against E. coli. All the compounds were found exhibiting less antimicrobial activity against P.
aeruginosa, except compound 5j (MIC= 91.5 μg/mL) as compared to standard drug
ciprofloxacin (MIC= 50.0 μg/mL). This suggests that the synthesized compounds are less
effective on gram negative organisms. The synthesized compounds exhibited moderate
antimicrobial activity against S. aureus (MIC range= 55.3 – 170.2 μg/mL). Compound 5h was
found moderately active against S. aureus (MIC= 55.3 μg/mL), and compounds 5d-5j have MIC
value below 100.0 μg/mL, suggesting their moderate potency against the S. aureus. The results
of antimicrobial activity also suggest that the compound 5j is almost effective against all test
organisms.
9

In order to gain insights of possible mode of inhibition of Als protein and mechanism of
biofilm inhibition, molecular docking studies were performed. The crystal structure of Als-3
adhesin from C. albicans in complex with hepta-threonine (PDB ID: 5LEB) was retrieved from
www.rcsb.org and used for docking with Autodock Vina 37 program. The 2D structures of
compounds were drawn with Marvin Sketch 5.6.0.0, which were transformed to the 3D geometry
and subsequently energy minimized in UCSF Chimera 1.8 38 interface using steepest descent and
conjugate gradient search criteria. The protein structure was refined by removing water and
nonstandard residues and energy minimization with Amber ff12SB force field. Polar hydrogen
were added in the protein structure and docking was carried out using the grid box of size 22 ×
2
0 × 20 along x, y and z axis with 1 A° spacing. The key interactions of ligands with residues at
active site and estimation of binding free energy in kcal/mol was considered for the analysis of
results of docking studies.
The active site of Als-3, where hepta-threonine binds, has the surrounding residues
Val172, Ser170, Gly297, Thr296, Arg294, Lys59, Gly27 and Tyr21. The interaction between the
best docked conformer of ligands with these surrounding residues at this active site was
analyzed. These results are shown in Table 3. The docking results and experimental results are
concordant. The docking results suggest that the hydrogen bond interaction with the residue
Tyr21 or Tyr226 is most crucial in deciding the potency of these compounds. The best docked
conformers of compounds 5a, 5b, 5d and 5j produce such hydrogen bond interaction. The
estimated binding free energy for these compounds is also lower (-7.8 to -8.6 Kcal/mol) as
compared to other compounds suggesting their favorable interactions at the active site. The
naphthyl ring forms hydrophobic π–π stacking interaction with residues like Leu293, Val161,
Trp295, Tyr166 or Val172.
1
0

Table 3: Results of docking studies
Compound Estimated binding free
Interactions with key residues
ID
energy in Kcal/mol
Hydrogen bond
Hydrophobic
5
a
-8.5
-8.1
-8.2
-7.8
-7.9
-7.4
-7.6
-8.0
Tyr21
Tyr21
--
Val172, Leu193
Val172, Val162, Lys59, Trp295
Val161, Tyr166, Ser170
Val172, Trp295, Leu167, Val161
Pro29, Tyr226
5
b
5
c
5
d
Tyr21
--
5
e
5
f
--
Pro29, Tyr226
5
5
g
h
--
Ala19, Ala18, Pro29, Tyr226
Val161, Thr168, Val172
--
Val161, Ser170, Thr296, Trp295,
Val268, Tyr271
Val161, Leu293, Arg171, Val172,
Pro29, Lys59, Gly27
5
5
i
j
-8.3
-8.6
--
Tyr226
The extent and pattern of substitution on iminophenyl ring was found crucial in favorable
binding at active site of Als-3. The chloro substitution in compound 5a forms halogen bond with
Val172. The trifluoromethyl substitution at para position in compound 5j forms hydrophobic
halogen bonds with Val172, Arg171, Pro29 and Gly27. Such hydrophobic halogen bond
interactions in compound 5j may be responsible for the highest biofilm inhibitory activity of this
compound. The unsubstituted compound 5f was not fond making any key interactions and was
least active. The hydrophobic methyl substitution at ortho position in compound 5g was also
found unable to make above key interactions and also less active. The trifluoro methyl
substitution at ortho position could not produce the key interactions in compound 5h and this
compound was found less active than compound 5j. The central phenyl ring in all the compounds
except compound 5j could not form any hydrophobic π–π stacking interaction. In compound 5j
the central phenyl ring in a conformer was found oriented favaorably to form π–π. stacking
interaction with Val161. Figure 3 shows the key interactions at the active site for all the
compounds.
1
1

Fig. 3. Key interactions observed for all compounds in docking studies.
1
2

The docking simulation does not address the dynamic interaction between protein-ligand
complexes. Further, the protein structure is kept rigid during the docking simulation, whereas
proteins can undergo significant conformational changes while performing their function. Thus,
molecular dynamics (MD) simulation was performed on Gromacs 5.1.4 39 to augment the
docking results and to gain deeper insights of the mode of action of ligands. The complex of Als-
3
with best docked conformer of ligand was used during MD simulation. The complexes of two
less active compounds 5f and 5g and two most active compounds 5a and 5j were selected for 1
nanoseconds MD simulation. The topology of protein was generated using CHARMM36 all-
4
0
atom force field , where as ligand topology was generated using CHARMM General Force
Field (CGenFF) 41 from CGenFF server available at https://cgenff.paramchem.org.
With topologies of protein and ligand the topology of complex was created. The
complexes were solvated using simple point charge solvent model (SPC) 216 in a dodecahedral
unit cell with minimum distance of 1 A° between the protein’s outermost atoms and walls of the
unit cell. Subsequently the systems were then neutralized by addition of counter ions. In order to
remove the steric clashes the energy minimization was performed until the maximum force value
on any atom is below 10.0 kJ/mol. The system was equilibrated with position restraint NVT and
NPT at 300K for 100 ps each. Linear Constraint Solver (LINCS) algorithm was exploited to fix
4
2
the covalent bonds of the system . Long range electrostatic interactions were measured with
4
3
Particle Mesh Ewald (PME) scheme . The van der waals and coulomb interaction cutoff’s
were set with 12.0 A°. Different modules of GROMACS package were employed for the
analysis of the results.
MD simulations of 1 ns each were performed for complexes of two less active
compounds (compound 5f and compound 5g) and two more active compounds (compound 5a
and 5j). MD simulation gave an impetus of the overall stability of these complexes. The overall
stability in terms of root-mean-square-deviation (RMSD), Root-mean square fluctuation
(RMSF), number of H-bonds and energy evaluation was evaluated.
Root mean square deviation (RMSD), which is the measure of the deviation of main
chain atoms of the protein, help determines the dynamic stability of the system during the course
of simulation. The complexes of more active compounds (compound 5a and 5j) were found to be
stabilized during 1 ns simulation with average RMSD of 0.113 and 0.126. The complex of
1
3

compound 5g stabilize with RMSD of 0.175 where as the complex of compound 5f was found
stabilize with RMSD of 0.693. As the RMSD values for these two complexes were found
deviating these compounds require longer simulations and probably producing unfavorable
interactions with the protein. Figure 4 shows the RMSD of the backbone atoms during MD
simulation.
Fig 4. The RMSD of the backbone atoms of all complexes during MD simulations.
Thus the low RMSD of complexes of compound 5a and 5j shows the substitutions at
para position of phenyl ring in these compounds was better stabilized whereas the unsubstituted
compound 5f display larger RMSD. Further the hydrophobic methyl substitution at ortho
position in compound 5g also shows slightly larger RMSD. This suggest that the importance of
halogen substitution at para position of phenyl ring.
Root mean square fluctuation (RMSF) is characteristic to the elasticity of the native
protein structure which is investigated through the fluctuations in backbone atoms and atoms of
ligand. The RMSF evaluation helps in investigating the accuracy and stability of a system during
simulation. As shown in Figure 5, the fluctuations in atom 2000 onwards were observed for
complexes of compound 5f and 5g, whereas such fluctuations are minimal for the complexes of
1
4

compounds 5a and 5j. The least fluctuations in compound 5a and 5j could be due to the most
favorable substitution at para position of phenyl group which allows the ligand to properly orient
within the active site.
Fig 5. The RMSF of all complexes back bone atoms during MD simulations.
The binding affinity of ligands depends on capacity to form hydrogen bonding with key
residues. The stability of system increases as the number of hydrogen bond interaction increases.
Figure 6 shows number of hydrogen bonds formed between ligands and active site residues
during entire simulation.
1
5

Fig 6. Hydrogen bond formation for Compounds 5a, 5f, 5g and 5j.
It is clearly evident that in complex of compound 5a, the hydrogen bond interactions
were formed throughout the simulation whereas in complex of compound 5j, the hydrogen bond
formation was observed after 300 ps simulation. Interestingly, in complex of compound 5f no
hydrogen bond formation was observed throughout the simulation and in complex of compound
5
g only one hydrogen bond interaction during initial phase of simulation was observed. This
supports the experimental activities of these compounds against the target protein.
In conclusion, a series of quinoline ring bearing schiff’s bases 5(a-j) were designed and
synthesized as promising fungal biofilm inhibitors and as antifungal and antibacterial agents. The
compounds 5j (IC = 51.2 μM) and 5a (IC = 66.2 μM) with o-hydroxy and p-methoxy
5
0
50
1
6

substituents showed promising C. albicans biofilm inhibition activity in comparison with
standard fluconazole (IC = 40.0 μM). All the compounds except compound 5g, were also found
5
0
possessing promising antimicrobial activity against B. subtilis, and are more potent than the
standard drug ciprofloxacin. The synthesized compounds were found less active on the P.
aeruginosa strain, suggesting the ineffectiveness on Gram-negative organisms. The docking
study supports the experimental biofilm inhibition and suggests that for such activity the
compounds require halogen substitution at para position of the phenyl ring. Hydrophobic
substitutions on phenyl ring are unfavorable for binding of the conformers at the active site of
Als-3 protein. Molecular dynamics also supports these finding suggesting that hydrogen bond
formation is key in inhibition of the Als-3, and substitutions at para position of phenyl ring with
halogens or groups involved in hydrogen bond formation could lead to promising biofilm
inhibitors.
Acknowledgements
The authors are thankful to the Mrs. Fatma Rafiq Zakaria, Chairman, Maulana Azad
Educational Trust and Dr. Zahid Zaheer, Principal, Y.B. Chavan College of Pharmacy, Dr. Rafiq
Zakaria Campus, Aurangabad 431 001 (M.S.), India for providing the laboratory facility. The
authors SAA, HMA and AAA would like to extend their sincere appreciation to the Deanship of
Scientific Research at King Saud University for funding through the Research Group Project No.
RG-1439-010.
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7

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Graphical Abstract
Synthesis, biological evaluations and computational studies of N-(3-(-2-(7-
Chloroquinolin-2-yl)vinyl) benzylidene)anilines as fungal biofilm inhibitors
a
b
c
d
Firoz A. Kalam Khan, Rashmi N. Kaduskar, Rajesh Patil* , Rajendra H. Patil , Siddique
e
e
e
f
Akber Ansari , Hamad M. Alkahtani , Abdulrahman A. Almehizia , Devanand B. Shinde ,
Jaiprakash N. Sangshetti b*
a
Anjuman-I-Islam’s Kalsekar Technical Campus, School of Pharmacy, Navi Mumbai 410206 (M.S.),
India.
b
Y.B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Aurangabad 431 001 (M.S.), India.
c
Sinhgad Technical Education Society's, Smt. Kashibai Navale College of Pharmacy, Kondhwa
Bk), Pune, India
Department of Biotechnology, Savitribai Phule Pune University, Pune 411007 (M.S.), India.
(
d
e
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia. Po Box 2454, Riyadh 11451.
f
Shivaji University, Vidyanagar, Kolhapur 416004, MS, India
*
Corresponding author. Tel. No.: +91 240-2381129.
E-mail address: rajshama1@yahoo.com (Rajesh Patil)
jnsangshetti@rediffmail.com (J.N. Sangshetti).
2
4

4
4.
2
5