Fungal biofilm inhibition by piperazine-sulphonamide linked Schiff bases: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1002/ardp.201700354

We report the synthesis of some new piperazine-sulphonamide linked Schiff bases as fungal biofilm inhibitors with antibacterial and antifungal potential. The biofilm inhibition result of Candida albicans proposed that the compounds 6b (IC₅₀ = 32.1 μM) and 6j (IC₅₀ = 31.4 μM) showed higher inhibitory activity than the standard fluconazole (IC₅₀ = 40 μM). Compound 6d (MIC = 26.1 μg/mL) with a chloro group at the para position was found to be the most active antibacterial agent of the series against Bacillus subtilis when compared with the standard ciprofloxacin (MIC = 50 μg/mL). Compound 6j (MIC = 39.6 μg/mL) with an OH? group at the ortho position showed more potent antifungal activity as compared to that of the standard fluconazole (IC₅₀ = 50 μM) against C. albicans. Thus, the synthesized compounds 6a–k were found to be potent biofilm inhibitors as well as active antibacterial and antifungal agents. The molecular docking study of the synthesized compounds against the secreted aspartyl protease (SAP5) enzyme of C. albicans exhibited good binding properties. The in silico ADME properties of the synthesized compounds were also analyzed and showed their potential to be developed as potential oral drug candidates.

Full text of DOI:10.1002/ardp.201700354

Received: 9 November 2017
Revised: 26 February 2018
Accepted: 26 February 2018
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DOI: 10.1002/ardp.201700354
FULL PAPER
Fungal biofilm inhibition by piperazine-sulphonamide linked
Schiff bases: Design, synthesis, and biological evaluation
1
2
3
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Rajendra H. Patil
Firoz A. Kalam Khan
Kaivalya Jadhav
4
5
5
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Manoj Damale
Siddique Akber Ansari
Hamad M. Alkahtani
5
6
7
3
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Azmat Ali Khan
Shantanu D. Shinde
Rajesh Patil
Jaiprakash N. Sangshetti
1
Department of Biotechnology, Savitribai
Phule Pune University, Pune, India
Abstract
2
Department of Pharmaceutical Chemistry,
We report the synthesis of some new piperazine-sulphonamide linked Schiff bases as
fungal biofilm inhibitors with antibacterial and antifungal potential. The biofilm
inhibition result of Candida albicans proposed that the compounds 6b (IC50 = 32.1 μM)
and 6j (IC50 = 31.4 μM) showed higher inhibitory activity than the standard fluconazole
Oriental College of Pharmacy, Navi Mumbai,
India
3
Y. B. Chavan College of Pharmacy, Dr. Rafiq
Zakaria Campus, Aurangabad, India
4
Shreeyash Institute of Pharmaceutical
(
IC50 = 40 μM). Compound 6d (MIC = 26.1 μg/mL) with a chloro group at the para
Education and Research, Aurangabad, India
5
position was found to be the most active antibacterial agent of the series against
Bacillus subtilis when compared with the standard ciprofloxacin (MIC = 50 μg/mL).
Compound 6j (MIC = 39.6 μg/mL) with an OH─ group at the ortho position showed
more potent antifungal activity as compared to that of the standard fluconazole
Department of Pharmaceutical Chemistry,
College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia
6
Department of Pharmacy, BITS, Pilani, India
7
Sinhgad Technical Education Society's, Smt.
Kashibai Navale College of Pharmacy,
Kondhwa (Bk), Pune, India
(
IC50 = 50 μM) against C. albicans. Thus, the synthesized compounds 6a–k were found
to be potent biofilm inhibitors as well as active antibacterial and antifungal agents. The
molecular docking study of the synthesized compounds against the secreted aspartyl
protease (SAP5) enzyme of C. albicans exhibited good binding properties. The in silico
ADME properties of the synthesized compounds were also analyzed and showed their
potential to be developed as potential oral drug candidates.
Correspondence
Dr. Jaiprakash N. Sangshetti, Y. B. Chavan
College of Pharmacy, Dr. Rafiq Zakaria
Campus, Aurangabad 431 001 (M.S.), India.
Email: jnsangshetti@rediffmail.com
Funding information
King Saud University, Grant number: RG-
K E Y W O R D S
1439-010
antibacterial, fungal biofilm inhibition, piperazine, Schiff bases, sulphonamide
1
|
INTRODUCTION
emergence of antimicrobial resistant microbial strains in the last
decades constitutes a substantial need for the development of new
[
1–4]
Discovery of potent and effective antimicrobial drugs indicates most
important developments in therapeutics, not only in the management
of serious infections, but also in the control and treatment of some
infectious complications of other therapeutic modalities such as cancer
chemotherapy and surgery. In last decade, microbial infection becomes
an important complication and a main cause of morbidity and mortality
in immuno-compromised patients such as those suffering from cancer,
AIDS, and tuberculosis and in organ transplantation cases. In spite of
the availability of large antimicrobial drugs for the treatment, the
classes of antimicrobial agents.
The therapeutic resistance phenomena, which are very often
associated with the biofilm formation is one of the major problem
[5]
related to the treatment of Candida albicans infections. Biofilms are
defined as complex microbial communities that are encapsulated in a
matrix of extracellular polymeric substances. They develop when such
community of microorganisms irreversibly adheres to an inert or living
surface. Contactwith a solid surface triggersthe expression of a panel of
enzymes, which catalyze the formation of sticky polysaccharides that
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Arch Pharm Chem Life Sci. 2018;e1700354.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201700354

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PATIL ET AL.
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promotethe surface colonizationandthe microbialcells protection. This
adherent community is considered an important virulence factor
because it is difficult to eradicate and often responsible for treatment
2
|
RESULTS AND DISCUSSION
Chemistry
2.1
|
[6]
failures. Indeed, the biofilm represents a physical barrier that prevents
drugs from entering and expressing their activity.
2.1.1
|
Synthesis of the title compounds
Schiff bases are considered as an important antimicrobial scaffold.
Our group reported the synthesis and screening of linezolid-like Schiff
The Schiff bases 6a–k were synthesized by refluxing a mixture of 2-
ethoxy-5-(4-methylpiperazin-1-ylsulfonyl)benzaldehyde 4 (1.0 mmol)
and of various substituted aromatic amines 5a–k (1.0 mmol) in 15 mL
absolute ethanol using glacial acetic acid (3.0 mmol) as catalyst
[7]
bases as inhibitors of biofilm formation and antibacterial agents.
[8]
Yuan et al. investigated the in vitro activity of taurine-5-bromosa-
licylaldehyde Schiff base (TBSSB) against methicillin-resistant
Staphylococcus aureus (MRSA); as the therapy for MRSA infections is
becoming more difficult because of multidrug resistance and strong
biofilm forming properties and Schiff bases have attracted attention as
promising antibacterial agents. Also piperazine and sulphonamide
containing compounds have been reported as important scaffolds for
(
Scheme 1). After completion of reaction as indicated by TLC, the
reaction mixture was poured in petri plate and allowed to stand
overnight. The solid substance obtained was collected and recrystal-
lized from ethanol. All the derivatives were obtained by similar method
by treating with corresponding amines. The physical data of
synthesized compounds are presented in Table 1. The synthesized
compounds were obtained in good yield (82–90%) within 10–12 h.
Purity of the synthesized compounds was checked by thin layer
chromatography (TLC) and melting points were determined in open
[9]
antibacterial and antifungal agents. Hatnapure et al. prepared and
screened a series of novel piperazine derivatives of biological interest
for their antibacterial and antifungal activity and many compounds
were shown to have potent antibacterial and antifungal activities when
compared with standard ciprofloxacin and miconazole. Zoumpoulakis
1
capillaries on melting point apparatus and were uncorrected. H NMR,
1
3
C NMR, and mass spectral analysis confirmed the formation of the
[10]
et al.
designed the synthesis of a series of novel sulfonamide
synthesized compounds and the data also suggested the proposed
structures (Supporting Information).
compounds and tested in vitro for antibacterial and antifungal activity
and some analogues exhibited very promising results especially as
antifungal agents. Better antifungal activity than commercial ketoko-
nazole and bifonazole were observed. Thus, we decided to synthesize a
series of some new Schiff bases with piperazine and sulphonamide-
coupled scaffolds as antimicrobial agents.
2
.2
|
Biological assays
In vitro biofilm inhibition assay
2
.2.1
|
Taking into account all of the aforementioned, and as a part of our
[
11,12]
ongoing work toward identifying biologically active molecules
we
The synthesized compounds 6a–k were evaluated for anti-biofilm
activity to explore a possible role of piperazine-sulphonamide
linked Schiff bases in inhibiting/impeding the formation of biofilm
in C. albicans using MTT assay method. This method is based on the
fact that the higher the biofilm formation, the greater is the extent
of absorption of crystal violet and, thus, the less is the
effectiveness of the compounds. Fluconazole was used as standard
for the comparison of biofilm inhibition activity. The IC50 value
(concentration that decreased biofilms by 50%) of synthesized
compounds is presented in Table 2.
report the synthesis of a series of novel piperazine-sulphonamide
linked Schiff bases, and the study of their effects on inhibition of
Candida albicans biofilm. Also antifungal activity was evaluated against
Candida albicans. Antibacterial activity was screened for two Gram-
negative bacteria namely, Escherichia coli and Pseudomonas aeruginosa,
and two Gram-positive bacteria, namely Staphylococcus aureus and
Bacillus subtilis. We have also evaluated the synthesized compounds
for in silico ADMET prediction and results showed that compounds
could be exploited as oral drug candidates.
SCHEME 1 Synthesis of the title compounds 6a–k

PATIL ET AL.
| 3 of 8
TABLE 1 Physical data for the piperazine-sulphonamide linked Schiff bases 6a–k
Entry
R
Mol. formula
Yield (%)
87
Rf value
0.71
0.68
0.65
0.60
0.70
0.76
0.65
0.68
0.72
0.70
0.62
Mp (°C)
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
p-OCH
3
C
21
C
20
C
21
C
20
C
20
C
20
C
20
C
21
C
21
C
20
C
20
H
H
H
H
H
H
H
H
H
H
H
27
25
25
N
N
N
3
3
3
O
O
O
4
3
5
S
S
S
110
H
82
90–92
p-COOH
p-Cl
90
220
24ClN
3
O
3
S
85
110–112
80
o-NO
m-NO
p-NO
o-CF
m-CF
2
24
24
24
24
24
25
25
N
N
N
4
O
4
O
4
O
5
5
5
S
83
2
S
S
84
140–144
138–140
110–114
130–134
140–142
98–100
g
h
i
2
84
3
F
3
N
3
3
O
3
S
S
84
3
F
3
N
O
3
84
j
o-OH
p-OH
N
N
3
O
4
S
S
85
k
3
O
4
86
The synthesized compounds 6a–k had shown good biofilm
2
─NO at meta and para position in compounds 6f and 6g showed great
inhibition activity (IC50 range = 31.4–169.1 µM) against C. albicans
strain. Compounds 6b (IC50 = 32.1µM), 6i (IC50 = 37.2 µM), 6j
decrease in activity. Among all the synthesized compounds, compound
6j was found to be the most active biofilm inhibitory compound. As
observed from activity data, compounds 6a, 6j, and 6k with electron
(
IC50 = 31.4 µM), and 6k (IC50 = 39.5 µM) were found to show potent
biofilm inhibition activity against C. albicans when compared with
standard fluconazole (IC50 = 40.0 µM). The compounds 6a
donating groups like ─OH and ─OCH
compounds 6c–h with electron withdrawing groups (except 6i) like
─Cl, ─NO , ─COOH, and ─CF on the phenyl group.
3
were more active than
(
IC50 = 46.2 µM) and 6d (IC50 = 47.5 µM) had shown significant biofilm
2
3
inhibition activity when compared with standard fluconazole. The
compound 6j having ─OH at the ortho position was found to be more
potent than unsubstituted phenyl analogue 6b, while compounds 6i
2
.2.2 Field emission scanning electron microscopy
|
analysis
and 6k having ─CF
respectively, were found to be less potent compared to unsubstituted
phenyl analogue 6b. Compounds 6a, 6c, and 6d with ─OCH , ─COOH,
and ─Cl at the para position, respectively, and compound 6h with ─CF
at the ortho position showed somewhat decreased in activity, while
3
at the meta position and ─OH at para position,
After identifying the lead with potent anti-biofilm activity and to better
understand the biofilm inhibition by compounds, we carried out the
field emission scanning electron microscopy (FESEM) analysis of most
active anti-biofilm compound 6j (IC50 = 31.4 μM). Result revealed that
3
3
TABLE 2 In vitro biofilm inhibition, antibacterial and antifungal activities of piperazine-sulphonamide linked Schiff bases 6a–k
Antibiofilm activity, IC50 (μM)
Antibacterial activity, MIC (μg/mL)
Antifungal activity, MIC (μg/mL)
P. aeruginosa
E. coli
B. subtilis
S. aureus
C. albicans
Entry
C. albicans (NCIM-3471)
(NCIM-2036)
(NCIM-2256)
(NCIM-2063)
(NCIM-2901)
(NCIM-3471)
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
46.2
32.1
53.6
47.5
76.2
169.1
121.1
66.3
37.2
31.4
39.5
–
39.0
40.0
115.0
175.0
122.0
119.6
188.4
49.2
181.6
74.5
68.5
50.0
–
158.2
228.1
225.1
197.0
111.2
92.6
36.6
34.7
47.2
26.1
40.9
62.3
94.6
33.6
29.2
37.66
41.1
50.0
–
195.0
125.5
112.2
147.3
98.5
82.2
51.1
56.0
75.0
84.1
247.8
244.3
185.2
45.0
39.6
47.2
–
118.6
174.8
35.7
g
h
i
95.5
190.1
84.0
85.4
j
131.4
99.16
50.0
129.2
111.9
25.0
k
CP
FA
40.0
–
–
50.0
CP, ciprofloxacin; FA, fluconazole. Experiments were performed in triplicates and compared to DMSO-treated control. Standard errors were all within 10% of
the mean.

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PATIL ET AL.
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in absence of compound, cells of C. albicans were seen as enmeshed-
covered structures, and cells were enclosed, a typical of biofilm
structures. When the cells of C. albicans were subjected to inhibitory
concentration of compound 6j, 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
activity. Unsubstituted phenyl analogue 6b showed significant activity
against P. aeruginosa and B. subtilis than activity against E. coli and
3
S. aureus. Introduction of ─OCH at the para position of phenyl 6a led
to increase in the antibacterial activity against P. aeruginosa, while
other substitutions showed decreased activity for the same organism.
The substituted phenyl analogue had shown increased activity against
E. coli. Compound 6d with ─Cl at the para position, compound 6h with
[13]
C. albicans is quorum sensing (QS) mediated (Figure 1).
3 3
─CF at the ortho position and compound 6i with ─CF at the meta
position gave potent compounds against B. subtilis and showed the
increased antibacterial activity, at the same time other substitutions
decreased the activity for the same organism. Compound 6c having
2
.2.3 In vitro antifungal activity
|
Minimum inhibitory concentration (MIC) values for antifungal activity
against C. albicans were determined using standard agar method using
fluconazole as standard. Dimethyl sulfoxide was used as solvent
control. The results of in vitro antifungal activity (Table 2) showed that
synthesized compounds 6a–k have moderate to good activity. Also our
results demonstrated that most potent biofilm inhibitors 6a, 6i–k
showed a significantly potent antifungal activity against C. albicans
when compared with standard fluconazole. Comparison of antifungal
activity of compounds with that of antifungal drug fluconazole
─COOH at the para position, compounds 6e and 6f having ─NO
the ortho and meta position, respectively, compounds 6h and 6i having
─CF at the ortho and meta position, respectively, and ─OH at the para
2
at
3
position in compound 6k showed increased activity against S. aureus
while other substitutions showed decrease in activity for the same
organism. Among all the synthesized analogues, compound 6d was
found to be the most active compound against B. subtilis. Compound
6h had shown broad spectrum of antibacterial activities. As observed
from activity data (Table 2), compounds 6a, 6j, and 6k with electron
(
MIC = 50.0 µg/mL) showed that compound 6b (MIC = 51.1 µg/mL)
had same antifungal profile against C. albicans. Substituted phenyl
analogue ─CF at the meta position in compound 6i (MIC =
5.0 µg/mL) and ─OH at the ortho and meta position in compound
j (MIC = 39.6 µg/mL) and 6k (MIC = 47.2 µg/mL) showed increased
donating groups like ─OH and ─OCH
compounds 6c–h with electron withdrawing groups (except 6h and 6i)
like ─Cl, ─NO , and ─COOH on the phenyl group.
3
were more active than
3
2
4
6
2.3
|
Molecular docking analysis
activity when compared with standard drug fluconazole. All the other
synthesized compounds like 6a, 6c–h were found less active than
fluconazole. Among all synthesized, compound 6j (MIC = 39.6 µg/mL)
was found to be the most active compound against C. albicans.
Structure–activity relationship (SAR) revealed that compounds 6a, 6j,
The synthesized compounds 6a–k showed very good binding
interactions with the active site of SAP5 enzyme. The docking
interactions have been studied into substrate binding site pocket,
namely, S4, S3, S2, S1, S1′, S2′, and S3′ of SAP5. The docking
interactions indicated that compounds were held deep into these
pockets by combination of various hydrophobic, van der Waal's
interactions and charge interactions. The most active (fungal biofilm
inhibitors) compounds 6b, 6i–k were held into substrate binding
pockets by forming a most number of interactions namely van der
Waal's interactions with active site amino acid residues such as Ile12,
Trp51, Asp86, Gly220, Thr221, Asn249, Phe281, Thr283, Glu295,
Arg297, and Ile305. 4-Methylpiperazine ring buried deep into active
site and mostly had formed the hydrophobic interactions with active
site residues such as Lys50, Trp51, Arg52, Gly85, Asp86, Thr221,
and 6k with electron donating groups like ─OH and ─OCH
active than compounds 6c–h with electron withdrawing groups
except 6i) like ─Cl, ─NO , ─COOH, and ─CF on the phenyl group.
3
were more
(
2
3
2
.2.4 In vitro antibacterial activity
|
MIC values for antibacterial activity were determined using standard
agar method using ciprofloxacin as standard. Dimethyl sulfoxide was
used as solvent control. From activity data (Table 2), the synthesized
compounds 6a–k had exhibited moderate to good antibacterial
FIGURE 1 Inhibition of C. albicans biofilm (FESEM images). FESEM analysis of C. albicans biofilm (control) shows bunches of cells
surrounded by biofilm. However, in presence of compound 6j individual cells were observed, indicating an inhibition of biofilm formation

PATIL ET AL.
| 5 of 8
Thr222, Ile223, Asn249, Thr283, Arg229, Ser301, and Ile305. The
nitrogen atom of imine group (─CN─) had formed strong charge
interactions with amino acid residues such as Trp51, Arg52, Gly85,
Asp86, Thr221, Thr222, Tyr225, Phe281, Thr283, Glu295, Arg297,
and Ile305. The most active compound 6j (Figure 2) had shown very
good binding affinity, that is −52.81 kcal/mol. The compound 6j had
shown very strong non-covalent interactions with amino acids such as
Ile12, Trp51, Arg52, Asp86, Thr222, Ile223, Tyr225, Asn249, Phe281,
Thr283, Glu295, Arg297, and Ile305.
Native inhibitor of SAP5 that is PepA was also docked into binding
pockets of SAP5 and had shown number of non-covalent interactions
such as van der Waal's and hydrophobic interactions but no charge
interactions (Figure 2). The binding affinity of PepA was least, that is
−
25.78 kcal/mol when compared with all synthetic compounds. The
amino acid residues such as Glu10, Lys83, Asp86, Gly131, Phe281,
Glu300, and Asp303 had shown van der Waals interactions with two
─
CO functional groups and aliphatic nitrogen atoms. The amino
acids like Ala11, Ile12, Trp51, Lys193, and Leu216 had formed
hydrophobic interactions with terminal ─CH groups of PepA. The
3
mode of interactions of active synthetic compounds when compared
with co-crystallized complex (PepA) of SAP5 found that important
amino acid residues such as Ile12, Lys83, Gly85, Asp86, Gly220,
Thr221, Thr222, Thr222, Ile223, Tyr225, and Ile305 had interacted
with active compounds and PepA by forming van der Waal's,
hydrophobic and charge interactions.
FIGURE 2 Molecular docking study of compound 6j and PepA
with SAP5 enzyme of C. albicans (PDB ID: 2QZX). Ligands are
shown in red color ball and sticks
2.4
|
ADMET prediction
Due to unfavorable absorption, distribution, metabolism, elimination,
and toxicity (ADMET) properties, many potential therapeutic agents
may fail to reach the clinical stage. Therefore, in silico study was carried
out for assessment of ADMET parameters and result obtained is
presented in Table 3. The data obtained for all the synthesized
compounds 6a–k were within the range of accepted values. None of
the synthesized compounds had violated the Lipinski's rule of five for
its variants. The value of polar surface area (PSA), logP, and H/C ratio
for synthesized compounds 6a–k were within the accepted range thus
indicating for good oral bioavailability. The parameters like number of
rotatable bonds and number of rigid bonds are linked with intestinal
absorption result, and the result showed that all synthesized
TABLE 3 Physicochemical pharmacokinetic parameters important for agents to have good oral bioavailability of synthesized compounds 6a–k
Entry
MW
LogP
3.73
3.72
3.42
4.38
4.05
4.05
4.05
4.34
4.74
3.43
3.43
PSA
79.8
70.5
107
70.5
115
115
115
61.3
70.5
90.8
90.8
n-Rot bond
n-Rig bond
HBD
0
HBA
5
Rings
Ratio H/C
0.38
0.35
0.428
0.4
Toxicity
NT
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
417.18
387.49
431.50
421.94
433.50
433.50
433.50
411.44
455.49
403.49
403.49
7
6
7
6
7
7
7
5
7
6
6
21
21
22
21
22
22
22
21
21
21
21
3
3
3
3
3
3
3
3
3
3
3
0
4
NT
1
6
NT
0
4
NT
1
6
0.5
NT
1
6
0.5
NT
g
h
i
1
6
0.5
NT
0
3
0.473
0.476
0.4
NT
0
4
NT
j
1
5
NT
k
1
5
0.4
NT
MW, molecular weight; LogP, logarithm of partition coefficient of compound between n-octanol and water; PSA, polar surface area; n-Rot bond, number of
rotatable bonds; n-Rig bond, number of rigid bonds; HBA, hydrogen bond acceptors; HBD, hydrogen bond donor; NT, non-toxic.

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PATIL ET AL.
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compounds 6a–k had good absorption. Also, all the synthesized
compounds 6a–k were found to be non-toxic.
chlorosulfonylbenzaldehyde 3. The compound 3 on reaction with 1-
methylpiperazine in presence of triethylamine as a base in methylene
dichloride as solvent reacted to give 2-ethoxy-5-(4-methylpiperazin-1-
ylsulfonyl)benzaldehyde 4 in good yield (90%). The Schiff bases 6a–k
were synthesized by refluxing a mixture of compound 4 (1.0 mmol) and
of various substituted aromatic amines 5a–k (1.0 mmol) in 15 mL
3
|
CONCLUSION
In conclusion, we have synthesized a series of new piperazine-
sulphonamide linked Schiff bases 6a–k in good yield. All the synthesized
compounds were tested for fungal biofilm inhibition activity, antibacte-
rial and antifungal activities. Based on the activity data, SAR for the
series has been developed. Interestingly, compounds 6b, 6i–k had
shown good results for fungal biofilm inhibition activity. Also,
compounds 6i–k had shown very good potential for the development
of novel antifungal agents. Compounds 6a, 6b, 6d, 6h, and 6i were
shown to have potent antibacterial activity and can serve as important
pharmacophores for the design and development of new leads as
antibacterial agent. The mechanism for fungal biofilm inhibition is
demonstrated by molecular docking study and result had shown good
binding interactions with SAP5. In silico ADME study of synthesized
compounds indicated that compounds had potential to develop as good
oral drug-like candidate. Thus, suggesting that the compounds from the
present series can serve as important gateway for the design and
development of new good oral drug-like antimicrobial agents.
[
14]
absolute ethanol using glacial acetic acid (3.0 mmol) as catalyst.
After completion of reaction as indicated by TLC, the reaction mixture
was poured in petri plate and allowed to stand overnight. The solid
substance obtained was collected and recrystallized from ethanol. All
the derivatives were obtained by similar method by treating with
corresponding amines.
4.2
|
Biological assays
In vitro biofilm inhibition assay
4.2.1
|
The piperazine-sulphonamide linked Schiff bases 6a–k were evaluated
for anti-biofilm via evaluating the metabolic activity of cells by
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In
5
this assay, 10 cells/well of C. albicans (yeast form) were inoculated in
RPMI in presence of various concentrations (0–100 μM) of compounds
6
a–k and standard drug fluconazole, and incubated for 24 h at 30°C.
After incubations, the media was removed, and biofilm was washed
with phosphate buffer. A 100 μL of MTT (1 μg/μL) was added to each
well, and incubated in dark for 3 h. After 3 h, the clear solution (without
violet granules) was removed and 100 μL of dimethylsulfoxide was
added to each well, and optical density recorded at 575 nm. The
concentration that decreased biofilms by 50% (IC50) was computed
4
4
4
|
EXPERIMENTAL
Chemistry
.1
|
.1.1 General
|
[15,16]
from growth inhibition curve.
All the starting material used in synthesis of title compounds and
solventswerepurchasedfromSigmaorAvrasynthesisandusedwithout
further purification. The purity of the synthesized compounds was
checked by ascending thin layer chromatography (TLC) on silica gel-G
4
(
.2.2
|
Field emission scanning electron microscopy
FESEM) analysis
(
Merck) coated aluminum plates, visualized by iodine vapor. The melting
To further confirm the impedance of biofilm, surface topography of the
biofilm was observed in presence of title compounds by FESEM (SEI
points of synthesized compounds were measured in open capillary
1
3
1
5
tubes. C NMR and H NMR spectra were recorded on 400 MHz
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 for mass spectra.
NOVA, NANO-SEM, 450, USA). For this purpose, 10 cells/well of
C. albicans were inoculated in RPMI media in presence of IC50 value of
6j compound, and incubated for 24 h at 30°C. After incubations, the
media was removed, and biofilm was washed with phosphate buffer
followed by series of ethanol concentrations (10–100%, each for
15 min), dried, mounted on aluminum stubs conductive carbon cement,
and finally coated with a gold film. The biofilm with planktonic cells
were observed under FESEM at 1000× magnifications.
The InChI codesof the investigated compoundstogether with some
biological activity data are also provided as Supporting Information.
4
.1.2 Synthesis of the title compounds
|
4
.2.3 In vitro antifungal activity
|
The synthetic approach applied is outlined in Scheme 1. Initially to a
suspension of salicylaldehyde and aqueous sodium hydroxide solution,
diethyl sulfate was slowly added dropwise to obtain 2-ethoxybenzal-
dehyde 1. Ethyl orthoformate and ammonium chloride were added to
Antifungal activity was determined by standard agar dilution method
[
17]
as per CLSI (formerly, NCCLS) guidelines.
The synthesized
compounds and standard fluconazole were dissolved in DMSO
solvent. The medium yeast nitrogen base was dissolved in phosphate
buffer pH 7 and it was autoclaved at 110°C for 10 min. With each set
a growth control without the antifungal agent and solvent control
2
-ethoxybenzaldehyde 1 in presence of ethanol to produce
diethylacetal of 2-ethoxybenzoic aldehyde 2 which on chlorosulpho-
nation in presence of chlorosulphonic acid at 0°C yielded 2-ethoxy-5-

PATIL ET AL.
| 7 of 8
DMSO were included. The fungal strains were freshly subcultured on
to Sabouraud dextrose agar (SDA) and incubated at 25°C for 72 h.
The fungal cells were suspended in sterile distilled water and diluted
further developed for drug design programs. We have also assessed
parameters like number of rotatable bonds (>10) and the number of
rigid bonds which signify that the compound may have good oral
5
[24]
to get 10 cells/mL. Ten microliters of standardized suspension was
bioavailability and good intestinal absorption.
inoculated onto the control plates and the media incorporated with
the antifungal agents. The inoculated plates were incubated at
ACKNOWLEDGMENTS
2
5°C for 48 h. The readings were taken at the end of 48 and 72 h.
The MIC was the lowest concentration of drug preventing growth of
macroscopically visible colonies on drug containing plates when
there was visible growth on the drug free control plates.
The authors S.A.A. and H.M.A. 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-
1
439-010. The author R.P. would like to acknowledge UPE-II, and
4.2.4
|
In vitro antibacterial activity
Departmental Research and Development Programme.
All the synthesized compounds were screened for in vitro antibacterial
activity. Minimum inhibitory concentration (MIC) values were deter-
mined using method recommended by National Committee for Clinical
Laboratory Standards (NCCLS). In vitro antibacterial activities of the
synthesized compounds 6a–k were tested in nutrient broth (NB) for
bacteria by the twofold serial dilution method. Seeded broth (broth
containing microbial spores) was prepared in NB from 24 h old
bacterial cultures on nutrient agar (Hi-media) at 37 ± 1°C. The bacterial
suspension was adjusted with sterile saline to a concentration of
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
ORCID
Jaiprakash N. Sangshetti
http://orcid.org/0000-0002-9064-4111
−
4
−5
1
× 10 –10 colony forming units (CFU). The synthesized com-
pounds and standard drug ciprofloxacin were prepared by twofold
serial dilutions to obtain the required concentrations of 400, 200, 100,
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4.4
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How to cite this article: Patil RH, Kalam Khan FA, Jadhav K,
et al. Fungal biofilm inhibition by piperazine-sulphonamide
linked Schiff bases: Design, synthesis, and biological
evaluation. Arch Pharm Chem Life Sci. 2018;1–8.
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