Biofilm inhibition of linezolid-like Schiff bases: Synthesis, biological activity, molecular docking and in silico ADME prediction

Article abstract of DOI:10.1016/j.bmcl.2014.12.063

Herein, we report the synthesis and screening of linezolid-like Schiff bases as inhibitors of biofilm formation. The result of biofilm inhibition of Pseudomonas aeruginosa suggested that compounds 5h (IC₅₀ value = 12.97 ± 0.33 μM) and 5i (IC₅₀ value = 15.63 ± 0.20 μM) had more inhibitory activity when compared with standard linezolid (IC₅₀ = 15.93 ± 0.18 μM) without affecting the growth of cells (and thus behave as anti-quorum sensing agents). The compounds 5h (MIC range = 2.5-10 μg/mL) and 5i (MIC range = 3.5-10 μg/mL) with 2-chloroquinolinyl and 2-chloro-8-methylquinolinyl motif, respectively, showed antibacterial activity in comparable range of linezolid (MIC range = 2-3 μg/mL) and were more potent when compared with ciprofloxacin (MIC range = 25-50 μg/mL). Thus, the active derivatives were not only potent inhibitors of P. aeruginosa biofilm growth but also efficient antibacterial agents. The docking study of most active compounds 5h and 5i against PqsD enzyme of P. aeruginosa exhibited good binding properties. In silico ADME properties of synthesized compounds were also analyzed and showed potential to develop as good oral drug candidates.

Full text of DOI:10.1016/j.bmcl.2014.12.063

Accepted Manuscript
Biofilm inhibition of linezolid-like Schiff bases: Synthesis, biological activity,
molecular docking and in silico ADME prediction
Jaiprakash N. Sangshetti, Firoz A. Kalam Khan, Rajendra H. Patil, Sayali D.
Marathe, Wasudev N. Gade, Devanand B. Shinde
PII:
S0960-894X(14)01371-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.12.063
BMCL 22308
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
24 September 2014
5 December 2014
19 December 2014
Please cite this article as: Sangshetti, J.N., Kalam Khan, F.A., Patil, R.H., Marathe, S.D., Gade, W.N., Shinde, D.B.,
Biofilm inhibition of linezolid-like Schiff bases: Synthesis, biological activity, molecular docking and in silico
ADME prediction, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2
014.12.063
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Biofilm inhibition of linezolid-like Schiff bases: Synthesis, biological activity,
molecular docking and in silico ADME prediction
a,
a
b
b
Jaiprakash N. Sangshetti *, Firoz A. Kalam Khan , Rajendra H. Patil , Sayali D. Marathe ,
b
c
Wasudev N. Gade , Devanand B. Shinde
a
Dr. Rafiq Zakaria Campus, Y.B. Chavan College of Pharmacy, Aurangabad 431001 (M.S.),
India
b
Department of Biotechnology, University of Pune, Pune 411007 (M.S.), India
c
Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad 431004 (M.S.), India
Corresponding author. Tel.: +91 240-2381129.
*
E-mail address: jnsangshetti@rediffmail.com (J. N. Sangshetti).
ABSTRACT
Herein, we report the synthesis and screening of linezolid-like Schiff bases as inhibitors of
biofilm formation. The result of biofilm inhibition of P. aeruginosa suggested that compounds
5
h (IC50 value= 12.97± 0.33 µM) and 5i (IC50 value= 15.63± 0.20 µM) had more inhibitory
activity when compared with standard linezolid (IC50= 15.93± 0.18 µM) without affecting the
growth of cells (and thus behave as anti-quorum sensing agents). The compounds 5h (MIC
range= 2.5-10 µg/mL) and 5i (MIC range= 3.5-10 µg/mL) with 2-chloroquinolinyl and 2-chloro-
8
-methylquinolinyl motif, respectively, showed antibacterial activity in comparable range of
linezolid (MIC range= 2-3 µg/mL) and were more potent when compared with ciprofloxacin
MIC range= 25-50 µg/mL). Thus, the active derivatives were not only potent inhibitors of P.
(
aeruginosa biofilm growth but also efficient antibacterial agents. The docking study of most
active compounds 5h and 5i against PqsD enzyme of P. aeruginosa exhibited good binding
properties. In silico ADME properties of synthesized compounds were also analyzed and showed
potential to develop as good oral drug candidates.
Keywords: Linezolid-like Schiff bases; biofilm inhibition; antibacterial activity; molecular
docking; ADME properties.
1

Microorganisms are present almost everywhere in nature and infections caused by
pathogenic microorganisms could lead to illnesses even a fatal one. Although several classes of
antibacterial agents are presently available and used for clinical treatment, however, their
advances in medical care are threatened by a natural phenomenon known as “antimicrobial
1
, 2
resistance”. A number of life threatening infections caused by multi-drug resistant microbial
3
pathogens have reached an alarming level in the hospitals and the community. Treatment is
4
further hampered when the bacterium is growing in biofilms. Biofilms are defined as
conglomerations of bacterial cells protected by a self-synthesized extrapolymeric substance
5
(
EPS). By forming biofilm, microorganisms are protected inside, and show an increased
resistance to antimicrobial agents including antibiotics. As a consequence infections caused by
6
bacterial biofilms are generally chronic and very difficult to eradicate. Due to the rising levels of
resistance to conventional antibiotics; there is an urgent need for new therapeutic options.
The formation of biofilm is quorum sensing (QS) mediated phenomenon. The QS
is an inter-cell communication system aided by released chemical signals when cell density
7
reaches a critical concentration. In particular, P. aeruginosa is the Gram-negative bacterium
responsible for biofilm growth has attracted considerable attentions, as this pathogen are able to
form biofilms in lungs, kidney, urinary tract, causing inflammations and septic shock in patients.
8
Thus, P. aeruginosa is used as model organism to study biofilm inhibition. Three different QS
systems are known for P. aeruginosa i.e. las, rhl, and pqs. The las and rhl are both LuxR-type
transcriptional activators that use N-(3-oxododecanoyl)homoserine lactone and N-
(
butanoyl)homoserine lactone, respectively, as signal molecules, whereas the pqs system utilizes
the 2-heptyl-3-hydroxy-4(1H)-quinolone, called Pseudomonas Quinolone Signal (PQS) and its
9
immediate precursor of 2-heptyl-4-quinolone (HHQ). Biosynthesis of PQS and HHQ signal
10
molecules in P. aeruginosa is presented in Figure 1. Both PQS and HHQ bind to the
transcriptional regulator PqsR, the receptor of the pqs system which regulates the production of
virulence determinants including biofilm development. As HHQ and PQS activate PqsR,
inhibition of their synthesis should lead to a decrease of virulence factor production without
1
1
interfering with bacterial growth. PqsD is an essential enzyme in the biosynthesis of HHQ and
PQS as it catalyzes the last step in HHQ formation, the condensation of anthraniloyl-CoA
(
ACoA) with β-ketodecanoic acid. Therefore PqsD is considered as an attractive therapeutic
1
2
target for drug development and treatment of P. aeruginosa infections. It is believed that
2

disrupting the cell-to-cell communication instead of killing the bacteria will result in less
selective pressure than with conventional antibacterial agents such as antibiotics and thus will
1
3
circumvent the problem of resistance. Structurally, PqsD most closely resembles FabH (β-
ketoacyl-acyl carrier protein synthase III) having the same catalytic triad consisting of Cys112,
His257 and Asn287. Therefore, inhibitors of FabH (Schiff base derivatives) might also act as
14
PqsD inhibitors.
Linezolid, a synthetic antibacterial was approved in 2000 by the FDA for the treatment of
community-acquired and nosocomial pneumonia, complicated and uncomplicated skin and soft-
tissue infections, and infections caused by methicillin-resistant S. aureus (MRSA) and
1
5
vencomycin-resistant enterococci (VRE). Inspection of the chemical structure of linezolid
revealed that a fluorine atom at the 3′ position practically doubles in vitro and in vivo activity,
and the electron-donating nitrogen atom in the morpholine ring helps maintain high antibiotic
1
6
potency and an acceptable safety profile. Compounds with the structure of -C=N- are known as
Schiff bases, which are usually synthesized by the condensation of primary amines and active
carbonyl groups. Number of researchers studied the synthesis and activity of Schiff bases (e.g.
1
7
YKAs3003) as potent antibacterial agent with FabH inhibitory activity. More et al. have
1
8
reported thiazole Schiff bases as biofilm inhibitors of P. aeruginosa. We, therefore, decided to
couple (Figure 2) these two moieties i.e. 4-(2-fluorophenyl) morpholinyl and -C=N- (Schiff base)
to exploit the synthesis of a series of linezolid-like Schiff bases 5(a-i) as antibacterial agents.
1
9, 20
Here, in continuous of our work on synthesis of bioactive molecules,
we report the
design and synthesis of a series of linezolid-like Schiff bases 5(a-j), and the study of their effects
on inhibition of P. aeruginosa biofilm. The compounds were also evaluated for antibacterial
against B. subtilis, E. coli, and P. aeruginosa. To explore the underlying mechanisms of biofilm
inhibition, we docked active compounds 5h and 5i against P. aeruginosa PqsD enzyme. We have
also assessed the synthesized compounds for in silico ADME prediction and results showed that
compounds could be exploited as oral drug candidate.
The synthetic approaches employed for synthesis of linezolid-like Schiff bases 5(a-i) are
outlined in Scheme 1. The 4-(2-fluoro-4-nitrophenyl) morpholine 3 was synthesized starting
from commercially available morpholine 1 and 3,4-difluoronitrobenzene 2 in good yield (85 %)
according to published procedure. The 3-fluoro-4-morpholinoaniline 4 was obtained by
21
reduction of 4-(2-fluoro-4-nitrophenyl) morpholine 3 using Pd/C and ammonium formate.
3

Further, to expand the series, linezolid-like Schiff bases 5(a-i) were prepared refluxing the
compound 4 (0.03 mol) with various substituted aromatic/heterocyclic aldehydes (0.03 mol) in
ethanol (20 mL) using glacial acetic acid (0.06 mol) as catalyst. After completion of reaction
(
monitored by TLC), mixture was concentrated to obtained solid product. The solid product
formed was filtered, dried and recrystallized from ethanol. All the derivatives 5(a-i) were
prepared similarly by treating with corresponding aldehydes. The physical data of the
synthesized compounds 5(a-i) are presented in Table 1. All the reactions proceeded well in 8-10
h to give products in very good yields (82–94%). The purity of the synthesized compounds was
checked by TLC and melting points were determined in open capillary tubes on a Buchi 530
melting point apparatus and are uncorrected. All synthesized derivatives 5(a–i) were
1
13
characterized using IR, H NMR, C NMR, Mass and elemental analysis (Supplementary data)
and data suggested for proposed structures.
The synthesized linezolid-like Schiff bases 5(a-i) were evaluated for anti-biofilm activity
to explore a possible role of Schiff bases in inhibiting/impeding the formation of biofilm in P.
aeruginosa. Most of the bacteria acquire the resistance to antibiotics by virtue of the synthesis of
an extracellular matrix (biofilm). So, an anti-biofilm approach is the promising approach to
tackle the aforementioned problem. The biofilm inhibition study was performed using crystal
1
8
violet assay retention method using linezolid as standard. This method is based on hypothesis
that higher the biofilm formation greater the extent of absorption of crystal violet and, and thus
less is the effectiveness of the compounds. The IC50 value (concentration that decreased biofilms
by 50 %) of synthesized compounds is presented in Table 2.
The linezolid-like Schiff bases 5(a-i) had shown good biofilm inhibition activity (IC50
range= 12.97-39.39 µM) against P. aeruginosa strain. Compounds 5h (IC50= 12.97 µM) and 5i
(
IC50= 15.63 µM) were more active than standard linezolid (IC50= 15.93 µM). The structure-
activity relationship (SAR) revealed that the compounds 5(a-i) showed varied biofilm inhibition
activity depending upon the various substituents present on aromatic ring (-Ar). Compound 5a
(
IC50= 39.39 µM) with Ar= Phenyl showed lowest biofilm inhibition activity among the
synthesized compounds. Compounds with halogen substituents like 4-fluoro 5b (IC50= 31.09
µM) and 2,6-dichloro 5c (IC50= 27.46 µM) at aromatic ring (-Ar) had shown improved in biofilm
inhibitory activity. Introduction of 3,4-dihdroxy group 5d (IC50= 31.61 µM) and 3,4-dimethoxy
group 5e (IC50= 27.87 µM) on aromatic ring resulted in no significant increase in biofilm
4

inhibition activity and showed activity equal to compounds 5b and 5c with halogen substituents.
The replacement of phenyl/substituted phenyl rings by heterocyclic rings like indolyl 5f (IC50
9.06 µM) and furanyl 5g (IC50= 35.87 µM) showed no improvement in inhibitory activity. The
=
2
2
2
quinoline and its derivatives have been reported as potent antibacterial activity. In order to
explore the effect of quinoline nucleus, we studied the biofilm inhibition by replacing the
phenyl/substituted phenyl rings (-Ar) with quinoline ring. The introduction of 2-chloro-3-
quinolinyl ring 5h (IC50= 12.97 µM) and 5i (IC50= 15.63 µM) led to the increase in inhibitory
activity by 2-3 folds and resulted in lead compounds of the synthesized library. The substitution
th
of methyl group at 8 position of 2-chloro-3-quinolinyl ring 5i resulted in slight decrease in
activity than compound 5h with 2-chloro-3-quinolinyl ring.
Having identified the leads with potent anti-biofilm activity and to better understand the
biofilm inhibition by compounds, we performed the field emission scanning electron microscopy
(
FESEM) analysis of compounds 5h and 5i (Figure 3). In absence of compounds, cells of P.
aeruginosa were seen as enmeshed-covered structures, and cells were enclosed, a typical of
biofilm structures. When the cells of P. aeruginosa were subjected to inhibitory concentration of
compounds 5h and 5i, there was a prominent decrease in the biofilm formation and cells were
seen as spatially distributed. More importantly, the numbers of the planktonic cells (cells in
suspension) were not affected, suggesting that the inhibition of the biofilm in P. aeruginosa is
1
8
QS mediated.
The main problem associated to biofilm infections is the dissemination of biofilm cells
into the systemic circulation. In order to colonize new surfaces and to prevent density-mediated
2
3
starvation within the mature biofilm, the cells detach and disseminate. Dispersal is
accomplished by shedding, detachment, or shearing. This may result in bloodstream infection,
depending on the host immune system and bioburden of cells released. Single cells released by
shedding are susceptible to antibiotics and can be controlled by antimicrobial therapy and/or the
host’s immune system. However, those released in clumps retain antibiotic resistance and may
embolize at a distant anatomic site to develop metastatic infections such as endocarditis or
2
4
osteomyelitis. Therefore, we have evaluated these compounds for antibacterial activity against
one Gram-positive bacterium, namely B. subtilis (NCIM-2063) and two Gram-negative bacteria,
namely E. coli (NCIM-2256), and P. aeruginosa (NCIM-2036). Interestingly, our results
5

demonstrated that most potent biofilm inhibitors 5h (MIC= 2.5 µg/mL) and 5i (MIC= 3.5
µg/mL) showed also a significantly potent antibacterial activity against P. aeruginosa (Table 2).
Minimum inhibitory concentration (MIC) values for antibacterial activity were
2
5
determined using standard agar method using linezolid and ciprofloxacin as standards.
Dimethyl sulfoxide was used as solvent control. From antibacterial data (Table 2), the
synthesized compounds 5(a-i) had exhibited good to excellent antibacterial activity. All the
synthesized compounds 5(a-i) (MIC range= 5-40 µg/mL) showed better activity than
ciprofloxacin (MIC= 50 µg/mL) but less active than linezolid (MIC= 3 µg/mL) against B. subtilis
strain. The compounds 5e (MIC= 20 µg/mL), 5h (MIC= 10 µg/mL) and 5i (MIC= 10 µg/mL)
were most active against E. coli when compared with that ciprofloxacin (MIC= 25 µg/mL). The
Compound 5c (MIC= 25 µg/mL) was equipotent with that of ciprofloxacin against E. coli strain.
However, all the compounds 5(a-i) (MIC range= 10-40 µg/mL) were less active than linezolid
(
(
(
MIC= 2 µg/mL) against E. coli strain. Most of the compounds i.e., 5a (MIC= 20 µg/mL), 5b
MIC= 20 µg/mL), 5d (MIC= 20 µg/mL), 5e (MIC= 20 µg/mL), 5h (MIC= 2.5 µg/mL) and 5i
MIC= 7.5 µg/mL) showed potent antibacterial activity against P. aeruginosa when compared
with ciprofloxacin (MIC= 25 µg/mL). The compound 5f (MIC= 25 µg/mL) was equipotent to
ciprofloxacin against P. aeruginosa strain. Further, the compound 5h (MIC= 2.5 µg/mL) was
equipotent and compound 5i (MIC= 3.5 µg/mL) showed comparable activity with linezolid
(
MIC= 2.5 µg/mL) against P. aeruginosa strain. Compounds 5e, 5h and 5i showed broad
spectrum activity active against both Gram-positive and Gram-negative bacteria. Inspection of
the chemical structure of linezolid-like Schiff bases 5(a-i) revealed that morpholinyl, 3-
2
6
fluorophenyl and Schiff base groups (FabH inhibition) could be responsible for potent
antibacterial activity (Figure 4).
The structure-activity relationship (SAR) studies for antibacterial activity revealed that
replacement of H atom of phenyl ring 5a by halogens like, 4-fluoro 5b or 2,6-dichloro 5c atoms
resulted in the improving of their antibacterial activity. Introduction of 3,4-dihdroxy group 5d
resulted in improved antibacterial activity than compounds with halogen substituents 5b and 5c.
Replacement of 3,4-dihydroxy group 5d with 3,4-dimethoxy group 5e resulted in better activity.
This suggested that compounds with electron-donating groups on aromatic ring (-Ar) showed
better activity. When phenyl/substituted phenyl ring was replaced by heterocyclic rings like
indolyl 5f and furanyl 5g, the antibacterial activity was altered and exhibited less antibacterial
6

activity. The introduction of 2-chloro-3-quinolinyl ring 5h led to the most active compound of
the synthesized library and showed increase in antibacterial by 2-16 folds. Introduction of methyl
th
group at 8 position of 2-chloro-3-quinolinyl ring 5i led to slight decrease in activity than
compound with 2-chloro-3-quinolinyl ring 5h.
In order to rationalize the mechanism of biofilm inhibition and to establish a basis to
explain the SAR of the PqsD inhibitors, molecular docking study was performed for compounds
5
h and 5i (most active), 5a (least active), and linezolid (standard). Molecular docking study of
2
7
the compounds was performed against PqsD enzyme of P. aeruginosa (PDB ID: 3H76) using
2
8
VLife MDS 4.3 package following standard procedure. The binding interaction of compounds
is shown in Figure 5. Both the active compounds 5h and 5i had shown good binding interaction
with binding energy -65.24 kcal/mol and -62.54 kcal/mol, respectively. The docking interactions
indicated that compounds held deep into the active pocket by combination of various
hydrophobic and van der Waals interactions with the enzyme. Compound 5h held in active site
by forming van der Waals interactions by amino acid residues like Glu140, Leu142, Leu155,
Leu158, Leu159, Gly160, Leu193, Phe218, Met220, Gly222, Met225, Phe126, His257, Pro259,
Asn260, Ile263, Asn287, Met288, and Ala289. The methylene groups (-CH -) of morpholine
2
part of compound 5h had formed major hydrophobic interactions with active site chain of
Glu140, Leu142, Ser143, Leu159, Gly160, Asp161, Asn 287, Met288, and Ala289. The nitrogen
atom of Schiff base (-C=N-) had formed strong hydrogen bond with amino acid Asn287 (1.673
Å).
The docking study of compound 5i revealed that compound is buried deep into the active
site by forming various van der Waals interactions with amino acid residues like, Leu142,
Lys144, Arg145, Met146, Asp147, Leu155, Leu159, Leu193, Gly216, Glu217, Phe218, Met220,
Gly222, Met225, Pro259, and Asn287. The methylene groups (-CH -) of morpholine core had
2
formed various hydrophobic interactions with amino acid residues like, Arg145, Met146,
Asp147, Leu155, Leu193, Gly216, Glu217, Met225, and Pro259. The methyl group of quinoline
ring had also formed the hydrophobic interactions with amino acids like, Leu155, Leu193,
Met225, and Pro259. The amino acids Arg145 (2.558 Å) and Phe218 (1.972 Å) had formed
strong hydrogen bonds with fluorine atom of phenyl ring. The standard linezolid had also shown
good binding interactions with PqsD enzyme with binding energy -60.25 kcal/mol. Linezolid is
held in active site of enzyme by forming various hydrophobic and van der Waals interactions
7

with amino acid residues like, Leu81, Gln111, Lys114, Arg145, Met146, Asp147, Leu193,
Thr195, Gly216, Glu217, Phe218 and Ser317. The fluorine atom of phenyl ring had formed
hydrogen bond with amino acid Phe218 (1.902 Å). Amino acid residue Arg145 had formed
hydrogen bonds with both oxygen atoms of oxazolidinone ring i.e. C=O (2.188 Å) and -O-
(
2.406 Å).
The docking study was also performed for least active compound 5a for better
understanding of mechanism of biofilm inhibition. The docking study revealed the compound 5a
has shown poor binding energy with PqsD enzyme (-35.36 kcal/mol) as compare to active
compounds 5h and 5i. Interestingly, only amino acid residues like, Arg145, Met146 and Glu217
had formed hydrophobic interactions with compound 5a but had shown good van der Waals
interactions with amino acid residues like, Leu81, Gln111, Lys144, Arg145, Met146, Thr195,
Gly216, Glu217, Phe218 and Ser317. The nitrogen atom of morpholine ring and fluorine atom of
phenyl ring had formed hydrogen bonding with amino acids Arg145 (1.919 Å) and Phe218
(
1.938 Å), respectively. The compound 5a had not shown any type of binding interactions with
amino acid residues like, Cys112, His257 and Asn287 (resembling catalytic amino acids between
1
4
FabH and PqsD enzymes) and this may be the reason for its poor biofilm inhibition activity. On
the basis of activity data and docking result, it was found the compounds 5h and 5i had potential
to inhibit PqsD enzyme of P. aeruginosa.
Many potential therapeutic agents fail to reach the clinic because of their unfavorable
absorption, distribution, metabolism, and elimination (ADME) parameters. Therefore, a
computational study of synthesized compounds 5(a-i) was performed for assessment of ADME
properties and value obtained is depicted in Table 3. Polar surface area (TPSA), number of
rotatable bonds (n-ROTB), molecular volume (MV), and Lipinski’s rule of five were calculated
2
9
using Molinspiration online property calculation toolkit. From all these parameters, it can be
observed that all the synthesized compounds exhibited excellent % absorption (86.47–98.56 %).
The most active compounds 5h and 5i showed 95.98 % absorption. Furthermore, none of the
compounds violated Lipinski’s rule of five and thus showing possible utility of series for
developing the compound with good drug like properties. A molecule likely to be developed as
an orally active drug candidate should show no more than one violation of the following four
criteria: logP (octanol–water partition coefficient) ≤5, molecular weight ≤500, number of
3
0
hydrogen bond acceptors ≤10 and number of hydrogen bond donors ≤5. All the synthesized
8

compounds followed the criteria for orally active drug and therefore, these compounds can be
further developed as oral drug candidates. The results of this in silico ADME prediction analysis
suggest that the synthesized compounds follow the computational assessment and thus represent
a pharmacologically active framework that should be considered on progressing further potential
hits.
In conclusion, a series of linezolid-like Schiff bases 5(a-i) had been designed and
synthesized efficiently in good yields. The synthesized compounds were evaluated for anti-
biofilm and antibacterial activities. The compounds 5h (IC50= 12.97 µM) and 5i (IC50= 15.63
µM) with 2-chloroquinolinyl and 2-chloro-8-methyl-quinolinyl motifs, respectively, showed
promising P. aeruginosa biofilm inhibition activity in comparison with standard linezolid (IC50
=
1
5.93 µM). Also, compounds 5h (MIC range= 2.5-10 µg/mL) and 5i (MIC range= 3.5-10
µg/mL) showed comparable antibacterial activity with standard linezolid (MIC range= 2-3
µg/mL) and potent activity than standard ciprofloxacin (MIC range= 25-50 µg/mL). FESEM
analysis of compounds 5h and 5i revealed that compounds had inhibited the biofilm without
affecting planktonic cells growth. This is important because when the growth is not affected,
there is no selective pressure for the development of resistant bacteria. Further, to understand the
mechanism of biofilm inhibition, we docked the compounds 5a, 5h, 5i and linezolid against
PqsD enzyme of P. aeruginosa and it was found the compounds 5h and 5i had potential to
inhibit PqsD enzyme. In silico ADME prediction of synthesized library indicated that
compounds had potential to develop as good oral drug candidate. These findings provide
important information for the exploration of compounds 5h and 5i as good oral drug-like anti-
biofilm agents.
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.
Authors are also thankful to SAIF, Punjab University, India for providing NMR spectra.
9

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12

Figure captions:
Figure 1. Biosynthesis of signal molecules HHQ and PQS.
Figure 2. Design of linezolid-like Schiff base derivatives 5(a-i).
Figure 3. Inhibition of P. aeruginosa biofilm (FESEM images). FESEM analysis of P.
aeruginosa biofilm (control) shows bunches of cells surrounded by biofilm. However, in
presence of Schiff bases 5h and 5i individual cells were observed, indicating an inhibition of
biofilm formation.
Figure 4. Structural features of linezolid-like Schiff bases 5(a-i) for antibacterial activity.
Figure 5. Molecular docking study of compounds 5a, 5h, 5i and linezolid with PqsD enzyme of P.
aeruginosa (PDB ID: 3H76). Ligands are shown in red color. Hydrogen bonds are shown as green color.
13

Table 1
Physical data for linezolid-like Schiff bases 5(a-i)
ο
Entry Ar
Molecular formula
Yield (%) Rf value Mp ( C)
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
Phenyl
C
17
C
17
C
17
C
17
C
19
C
19
C
15
C
20
C
21
H
H
H
H
H
H
H
H
H
17FN
2
O
94
86
90
89
94
88
82
90
85
0.64
0.52
0.48
0.59
0.55
0.52
0.61
0.46
0.44
194-196
202-204
236-238
220-222
246-248
180-182
172-174
260-262
274-276
4-Flourophenyl
16
F
2
N
2
O
2,6-Dichlorophenyl
3,4-Dihydroxyphenyl
3,4-Dimethoxyphenyl
3-Indolyl
15Cl
2
FN O
2
17FN
21FN
18FN
15FN
2
2
3
2
O
O
O
O
3
3
g
h
i
3-Furanyl
2
2-Chloro-3-quinolinyl
2-chloro-8-methyl-3-quinolinyl
17ClFN
3
O
19ClFN
3
O
14

Table 2
In vitro biofilm inhibition and antibacterial activities and of linezolid-like Schiff bases 5(a-j)
Entry
Inhibition of P.
Minimum inhibitory concentration (MIC) (µg/mL)
B. subtilis E. coli P. aeruginosa
(NCIM-2063) (NCIM-2256) (NCIM-2036)
aeruginosa biofilm
(
IC50 µM)
5
5
a
39.39± 0.98
31.09± 1.24
27.46± 0.84
31.61± 1.16
27.87± 1.34
29.06± 0.94
35.87± 0.75
12.97± 0.33
15.63± 0.20
15.93± 0.18
30± 1.65
20± 1.25
20± 1.32
25± 1.65
20± 1.40
35± 1.80
40± 2.36
5± 0.25
35± 1.10
30± 1.45
25± 0.67
30± 1.85
20± 1.14
40± 1.75
35± 1.15
10± 0.50
10± 0.55
2± 0.15
20± 0.85
20± 1.15
30± 1.25
20± 1.36
20± 0.75
25± 1.50
30± 1.55
2.5± 0.15
3.5± 0.18
2.5± 0.15
25± 1.20
b
5
c
5
5
5
5
5
5
d
e
f
g
h
i
10± 0.60
3± 0.18
Linezolid
Ciprofloxacin ND
50± 1.75
25± 0.95
Experiments were performed in triplicates and compared to DMSO-treated controls; Standard
errors were all within 10% of the mean; ND: not done.
15

Table 3
In silico physicochemical pharmacokinetic parameters important for good oral bioavailability of
synthesized compounds 5(a-i)
Entry
%
TPSA n-
MV
MW
miLog P n-ON
n-OHNH Lipinski’s
2
ABS (A )
ROTB
acceptors donors
violations
Rule
-
-
-
-
< 500 ≤ 5
< 10
3
< 5
0
≤ 1
0
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
96.98 34.83
98.56 30.25
97.90 32.15
86.47 65.29
94.06 43.30
94.98 40.62
94.98 40.62
95.98 37.72
95.98 37.72
3
3
3
3
5
3
3
3
3
261.77 284.33 3.51
266.70 302.32 3.67
288.84 353.22 4.77
277.81 316.33 2.54
312.86 344.38 3.15
290.75 323.37 3.66
290.75 274.11 3.66
315.14 369.82 4.46
331.71 383.85 4.86
3
0
0
3
0
0
5
2
0
5
0
0
4
1
0
g
h
i
4
1
0
4
0
0
4
0
0
%
ABS: percentage absorption, TPSA: topological polar surface area, n-ROTB: number of
rotatable bonds, MV: molecular volume, MW: molecular weight, miLog P: logarithm of
partition coefficient of compound between n-octanol and water, n-ON acceptors: number of
hydrogen bond acceptors, n-OHNH donors: number of hydrogen bonds donors.
16

Figure 1. Biosynthesis of signal molecules HHQ and PQS.
17

F
OH
O
O
N
N
N
H
N
FabH inhibitor
O
HO
Good for antibacterial activity
O
YKAs3003
Linezolid
O
O
F
F
Ar
OH
O
N
N
N
N
Linezolid-like Schiff bases 5(a-i)
HN
Where Ar= Phenyl/substituted phenyl,
indolyl, furanyl and substituted quinolinyl
Ciprofloxacin
Figure 2. Design of linezolid-like Schiff base derivatives 5(a-i).
18

Figure 3. Inhibition of P. aeruginosa biofilm (FESEM images). FESEM analysis of P.
aeruginosa biofilm (control) shows bunches of cells surrounded by biofilm. However, in
presence of Schiff bases 5h and 5i individual cells were observed, indicating an inhibition of
biofilm formation.
19

Figure 4. Structural features of linezolid-like Schiff bases 5(a-i) for antibacterial activity.
20

5
a
5h
21

5i
Linezolid
Figure 5. Molecular docking study of compounds 5a, 5h, 5i and linezolid with PqsD enzyme of P. aeruginosa
(
PDB ID: 3H76). Ligands are shown in red color. Hydrogen bonds are shown as gree
22

F
F
a
O
NH
+
F
No2
O
N
NO2
3
1
2
b
F
F
Ar
c
O
N
NH2
O
N
N
4
5(a-i)
Where Ar =
Cl
Cl
OCH3
OH
OCH3
F
OH
5
a
5d
5e
5b
5c
N
N
Cl
O
Cl
N
H
5f
5g
CH3
5
h
5i
Scheme 1. Synthesis of linezolid-like Schiff derivatives 5(a-i): (a) i-Pr
2
EtN, AcOEt; (b)
HCO NH , 10% Pd/C, THF/MeOH; (c) Ar-CHO, EtOH, AcOH.
2
4
23

Graphical abstract
Biofilm inhibition of linezolid-like Schiff bases: Synthesis, biological activity,
molecular docking and in silico ADME prediction
a,
a
b
b
Jaiprakash N. Sangshetti *, Firoz A. Kalam Khan , Rajendra H. Patil , Sayali D. Marathe ,
b
c
Wasudev N. Gade , Devanand B. Shinde
a
Dr. Rafiq Zakaria Campus, Y.B. Chavan College of Pharmacy, Aurangabad 431001 (M.S.),
India
b
Department of Biotechnology, University of Pune, Pune 411007 (M.S.), India
c
Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad 431004 (M.S.), India
*
Corresponding author. Tel.: +91 240-2381129.
E-mail address: jnsangshetti@rediffmail.com (J. N. Sangshetti).
24