Novel synthetic organic compounds inspired from antifeedant marine alkaloids as potent bacterial biofilm inhibitors

Article abstract of DOI:10.1016/j.bioorg.2015.06.001

In this paper, we have reported seventeen novel synthetic organic compounds derived from marine bromopyrrole alkaloids, exhibiting potential inhibition of biofilm produced by Gram-positive bacteria. Compound 5f with minimum biofilm inhibitory concentratio

Full text of DOI:10.1016/j.bioorg.2015.06.001

Bioorganic Chemistry 61 (2015) 66–73
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel synthetic organic compounds inspired from antifeedant marine
alkaloids as potent bacterial biofilm inhibitors
Rajesh A. Rane a,1, Rajshekhar Karpoormath a, ,1, Shital S. Naphade b,1, Pavankumar Bangalore c,1
,
⇑
Mahamadhanif Shaikh a, Girish Hampannavar a
a Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
b Government College of Amravati, Maharashtra 444601, India
c SPP School of Pharmacy and Technology Management, Vile Parle, Mumbai 400056, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 January 2015
Available online 12 June 2015
In this paper, we have reported seventeen novel synthetic organic compounds derived from marine bro-
mopyrrole alkaloids, exhibiting potential inhibition of biofilm produced by Gram-positive bacteria. Com-
pound 5f with minimum biofilm inhibitory concentration (MBIC) of 0.39, 0.78 and 3.125 lg/mL against
MSSA, MRSA and SE respectively, emerged as promising anti-biofilm lead compounds. In addition, com-
pounds 5b, 5c, 5d, 5e, 5f, 5h, 5i and 5j revealed equal potency as that of the standard drug Vancomycin
(MBIC = 3.125 lg/mL) against Streptococcus epidermidis. Notably, most of the synthesized compounds dis-
played better potency than Vancomycin indicating their potential as inhibitors of bacterial biofilm. The
cell viability assay for the most active hybrid confirms its anti-virulence properties which need to be fur-
ther researched.
Keywords:
Marine alkaloids
Bromopyrrole
1,3,4-Oxadiazoles
Biofilm inhibitors
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
planktonic bacteria arising from adhered populations, but are inef-
fective towards those bacteria hidden in biofilms [10].
A global trend of indiscriminate and continuous use of antibi-
otics for the treatment of bacterial infections has led to the devel-
opment of bacterial resistance for most of the existing
antimicrobials [1]. This resistance has led researchers worldwide
to search for new molecules active against bacterial pathogens. A
promising approach for the development of a new generation of
antimicrobial drugs has started from the studies of host–pathogen
interactions, prompting a shift of drug targets from bacterial sur-
vival to virulence control. For example, receptors involved in bac-
terial adherence to surfaces as well as signal systems controlling
bacterial group behaviour of population, organised in biofilms
(such as quorum sensing (QS) molecules) stands as potential
non-conventional targets for microbial control [2]. Antimicrobial
drugs directed against such non-conventional targets do not risk
bacterial survival avoiding the development of resistance [3]. Var-
ious organic compounds interfering with bacterial QS were identi-
fied as potent QS inhibitors [4–9]. On the other hand, conventional
antibiotics eradicates the symptoms of an infection by eliminating
Bromopyrrole alkaloids are a family of marine alkaloids repre-
sents a fascinating example of large variety of secondary metabo-
lites formed by marine sponges as a defense towards bacterial
biofouling leading to their protection against aquatic feedant.
These compounds are known to be involved in defense mechanism
of sponges against fishes and other aquatic feedants. They do so by
their inhabitant antibiotic as well as antibiofilm action. For
instance, Oroidin has been reported to inhibit the growth of
P. aeruginosa at 190
ited Gram-positive S. aureus growth at 0.005
action. Dihydronsventrin and Sventin were reported as biofilm
inhibitors at 51 and 74 M against P. aeruginosa. In addition,
lM. Petabromopseudilin derivative (II) inhib-
lg/mL by antibiotic
l
Ageliferin, Dibromophakelline, Mauritamide B and Agelanesin C
all are reported as QS inhibitors at 11.29, 17.99, 36.76 and
36.30 lM respectively (Fig. 1) [11–12]. These metabolites are
identified by their signature 4,5-dibromopyrrole motif with
Oroidin as there structural prototype reported as antibiofilm lead
compound. Structure–activity relationship on synthetic library
inspired form these class of alkaloids indicated that N-methylation
of the pyrrole core led to an increased antibiofilm activity towards
medically relevant Gram-negative bacterium P. aeruginosa as well
as Gram-positive bacterium S. aureus [13,14].
⇑
Corresponding author.
E-mail address: karpoormath@ukzn.ac.za (R. Karpoormath).
1
These authors contributed equally for this paper.
http://dx.doi.org/10.1016/j.bioorg.2015.06.001
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73
67
Fig. 1. Some reported bromopyrrole alkaloids and 1,3,4-oxadiazole with biofilm inhibiting, antibacterial, quorum sensing (QS) inhibiting activity.
Hence continuing with our quest focused on development of
novel antibiofilm molecules, we have described in this short com-
munication, the identification of seventeen novel compounds cap-
able of inhibiting biofilm formation for a panel of Gram-positive
and Gram-negative pathogenic bacterium. These compounds, con-
sisting of N-methyl-4,5-dibromopyrrole motif integrated with
1,3,4-oxadiazole were chosen for the current study based on their
resemblance to known marine antibiofilm agents, QS inhibitors as
well as reported antibiotics (Figs. 1 and 2) [13–17]. Based on the
potent activity of synthesized adducts towards bacterial biofilm
formation, a structure–activity analysis was developed, that can
direct future studies involved in the search of novel compounds
for biofilm control. Further cell viability data against human VERO
cells much above the bacterial MBIC concentrations warrants their
suitability for the development of new antibiofilm drugs for thera-
peutic uses.
Br
N
N
N
CH3
Br
Het/Ar
O
R
Br
Br
N
2. Materials and methods
N
N
CH3
O
2.1. Synthesis and purification of the compounds
R
In the present work, proposed compounds were synthesized
utilizing the reaction sequence as shown in Scheme 1.
Trichloroacetylation of 1-methylpyrrole using equimolar quantity
of trichloroacetyl chloride gave an excellent yield of 2-trichloroace
R = SH or S-alkyl or aryl or heteroaromatic ring
Fig. 2. Design of novel 2-(1-methyl-4,5-dibromopyrrol-2-yl)-5-substituted-1,3,4
oxadiazoles.

68
R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73
Br
CCl3COCl
COCCl3
COCCl3
anhydrous ether
Br2/Chloroform
N
CH3
N
CH3
Br
N
CH3
K2CO3
3
1
2
NH2NH2 . H2O
CONHNH2
RT, 1 h
Br
Br
Br
R-COOH,
O
Br
N
R
N
POCl3, reflux
H3C
H3C
N
5
N
4
5a - R = 2-hydroxyphenyl-
5b - R = benzyl-
CS2/KOH
Ethanol,reflux 12 hr
5c - R = 4-amino phenyl-
5d - R = 4-chloro phenyl-
Br
Br
5e - R = 4-methoxy phenyl-
5f - R = 2,4- dichlorophenyl-
5g - R = styryl-
O
N
SH
H3C
N
N
5h - R = 2-chloro-5-fluorophenyl-
5i - R = 4-nitrophenyl-
6
5j - R = 4,5-dibromo-1H-pyrrol-2-yl-
5k - R = 2-methoxy-4-vinylphenol
5l - R = pyridin-4-yl-
Alkyl/Aryl halide,NaOH
Ethanol,stir for 10 hr
5m - R = phenyl
Br
Br
O
N
SR
H3C
N
N
7
7a - R = methyl-
7b - R = ethyl-
7c - R = phenyl-
Scheme 1. Synthesis of 2-(1-methyl-4,5-dibromopyrrol-2-yl)-5-substituted-1,3,4-oxadiazoles.
tyl-1-methylpyrrole 2 [18]. Bromination of 2 using two equivalent
of bromine in chloroform gave 4,5-dibromo-2-trichloroacetyl-1-
methylpyrrole 3 in excellent yield [18]. 4,5-Dibromo-2-trichloro-
acetyl-1-methylpyrrole 3 on stirring with excess of hydrazine
hydrate at room temperature gave its acid hydrazide derivative 4
the ATR technique. The 1H NMR and 13C NMR spectra were recorded
on a Bruker AVANCE 400 (Bruker, Rheinstetten/Karlsruhe, Germany)
spectrometer using CDCl3 and/or DMSO-d6. High-resolution mass
spectra (HRMS) were recorded on an Autospec mass spectrometer
with electrospray ionization (ESI) operating at 70 eV using
post-acceleration photomultiplier detector.
in good yield. Compound
4 on condensation with equimolar
quantities different aromatic and hetero-aromatic acids in presence
of excess of phosphorus oxychloride at reflux condition gave
2-(4,5-dibromo-1-methylpyrrol-2-yl)-5-aryl-1,3,4-oxadiazoles 5.
5-(4,5-Dibromo-1-methyl-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6
was synthesized by reaction of 4 with carbon disulfide and alco-
holic potassium hydroxide at reflux condition. Compound 6 was
S-alkylated by reaction with different aryl/alkyl halides in alcoholic
sodium hydroxide to give S-alkylated derivatives 7 (Scheme 1).
2.2.1. General procedure for synthesis of 2,2,2-trichloro-1-(1-methyl-
1H-pyrrol-2-yl) ethanone 2
In a three-necked round-bottomed flask equipped with a sealed
mechanical stirrer, a dropping funnel, and an efficient reflux con-
denser with a calcium chloride drying tube was charged with
225 g (1.23 mol) of trichloroacetyl chloride and 200 ml of anhy-
drous diethyl ether. The solution was stirred while 77 g
(1.20 mol) of freshly distilled N-methylpyrrole in 640 ml of anhy-
drous ether was added over 3 h; the heat of reaction causes the
mixture to reflux. Following the addition, the mixture was stirred
for 1 h before 100 g (0.72 mol) of potassium carbonate in 300 ml
of water was slowly added through the dropping funnel. The layers
were separated, and the organic phase was dried with magnesium
sulphate and filtered. The solvent was removed by distillation on a
steam bath, and the residue was dissolved in 225 ml of hexane. The
dark solution was cooled on ice to induce crystallization. The tan
solid was collected and washed with 100 ml of cold hexane, giving
189–196 g (77–80%) of 1. 2-Pyrrolyltrichloromethyl ketone: m.p.
64–66 °C [18].
2.2. Chemistry
All reactions were carried out in anhydrous conditions (Molecu-
lar sieves, 4 Å 1/1600 pellets) under inert nitrogen atmosphere.
Freshly dried and distilled solvents (Ethanol and CAN) over CaCl2
were used. Silica gel of 60–120 mesh and 200–400 mesh was
obtained from for column and flash chromatography. Some of the
starting materials were obtained from commercially available sup-
pliers and were used without further purification. Analytical Thin
layer Chromatography (TLC) was carried out on precoated plates
of SiO2 (silica gel 60, F 254, Merck). The IR spectra were recorded
on a Bruker Alpha FT-IR spectrometer (Billerica, MA, USA) using

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73
69
2.2.2. General procedure for synthesis of 2,2,2-trichloro-1-(4,5-
dibromo-1-methyl-1H-pyrrol-2-yl) ethanone 3
144.9, 135.6, 127.3, 116.8, 115.9, 107.6, 105.3, 97.5, 27.5; MS
m/z: 395.9075 (M+), 397.9095 (M2+), 399.9105 (M4+).
In a three-necked round-bottom flask equipped with a sealed
mechanical stirrer, a dropping funnel, and an efficient reflux con-
denser with a calcium chloride drying tube was charged with
3.0 g (1.0 mol) of 2,2,2-trichloro-1-(1-methyl-1H-pyrrol-2-yl)-
ethanone 2 in 70 ml of chloroform. The solution was stirred in
an ice-bath (0–5 °C) with the help of mechanical stirrer until
the solution becomes homogeneous. After that 1.8 ml (2.0 mol)
of Bromine (Br2) was added to above solution over a period of
2 h dropwise with the help of dropping funnel. When addition
of bromine is completed, the mixture was stirred at room tem-
perature until the HBr gas formed is completely evolved. The
reaction mixture was partitioned with 50 ml of water. The
organic phase was dried over sodium sulphate and solvent was
evaporated in vaccuo. Crystals of brominated pyrrole were col-
lected and further recrystallized from n-hexane, giving 4.5–5.0 g
of 2,2,2-trichloro-1-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl) etha-
none 3, m.p. 142–144 °C [18].
2-(4-Chlorophenyl)-5-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-
1,3,4-oxadiazole (5d) Yield: 70%; m.p.: 172–174 °C; IR (KBr) Vmax
cmꢀ1: 1239 (CAOAC of 1,3,4-oxadiazole), 1603 (C@N), 3074 (CAH
aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.51 (s, 3H pyrrole
NACH3), 6.54 (s, 1H pyrrole 3H), 7.5–8.0 (m, 4H ArH); 13C NMR
(75 MHz, DMSO-d6): d 164.7, 132.8, 130.9, 129.3, 128.7, 125.2,
106.6, 103.4, 97.9, 27.3; MS m/z: 414.8655 (M+), 416.8658 (M2+),
418.8661 (M4+).
2-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-5-(4-methoxyphenyl)-
1,3,4-oxadiazole (5e) Yield: 60%; m.p.: 203–205 °C; IR (KBr) Vmax
cmꢀ1: 1238 (CAOAC of 1,3,4-oxadiazole), 1615 (C@N), 2909
(CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.47 (s, 3H
pyrrole NACH3), 4.19 (s, 3H OCH3), 6.36 (s, 1H pyrrole 3H),
7.1–8.0 (m, 4H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.8,
163.5, 134.2, 128.4, 115.9, 115.6, 108, 105.1, 96.2, 55.4, 27.9; MS
m/z: 410.9208 (M+), 412.9022 (M2+), 414.9028 (M4+).
2-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-5-(2,4-dichlorophenyl)-
1,3,4-oxadiazole (5f) Yield: 60%; m.p.: 197–199 °C; IR (KBr) Vmax
cmꢀ1: 1236 (CAOAC of 1,3,4-oxadiazole), 1605 (C@N), 3083
(CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.58 (s, 3H
pyrrole NACH3), 6.74 (s, 1H pyrrole 3H), 7.1–8.1(m, 3H ArH); 13C
NMR (75 MHz, DMSO-d6): d 164.6, 135.7, 135.3, 134.5, 133.4,
131.4, 129.8, 127.6, 108.3, 104.2, 97.6, 27.2; MS m/z: 448.8105
(M+), 450.8115 (M2+), 452.8121 (M4+).
2-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-5-styryl-1,3,4-oxadia
zole (5g) Yield: 80%; m.p.: 200–202 °C; IR (KBr) Vmax cmꢀ1: 1229
(CAOAC of 1,3,4-oxadiazole), 1620 (C@N), 2999 (CAH aromatic);
1H NMR (300 MHz, DMSO-d6): d 3.49 (s, 3H pyrrole NACH3), 6.33
(s, 1H pyrrole 3H), 6.67 (d, 1H CH@CH), 6.72 (d, 1H CH@CH),
7.0–7.7 (m, 5H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.6,
163.6, 137.2, 133.7, 133.1, 128.9, 128.6, 127.7, 124.5, 108.3,
100.5, 96.3, 27.5; MS m/z: 406.9100 (M+), 408.9102 (M2+),
410.9108 (M4+).
2.2.3. General procedure for synthesis of 4,5-dibromo-1-methyl-1H-
pyrrole-2-carbohydrazide 4
To a round bottom flask equipped with a sealed mechanical stir-
rer, solution of 2,2,2-trichloro-1-(1-methyl-1H-pyrrol-2-yl)etha
none and 30 ml of ethanol was added while stirring until the solu-
tion becomes homogeneous. To the above mixture 2.5 equivalents
(3–4 ml) hydrazine hydrate (99% v/v) was added and stirred at
room temperature till precipitate starts to appear. The whole reac-
tion mixture was poured over crushed ice. The solid precipitated
was filtered under vacuum and dried to obtain 4,5-dibromo-1-me
thyl-1H-pyrrole-2-carbohydrazide 4; m.p. 250–252 °C [18].
2.2.4. General procedure for synthesis of 2-(4,5-dibromo-1-methyl-
1H-pyrrol-2-yl)-5-aryl-1,3,4-oxadiazole analogues 5
Appropriate aromatic/heteroaromatic acid (1 mmol) and
4
(1 mmol) were stirred in 20 ml phosphorous oxychloride in a
round bottom flask. It was then refluxed for 20 h until the 4,5-dibro
mo-1-methyl-1H-pyrrole-2-carbohydrazide was consumed (moni-
tored by TLC). The reaction mixture was poured on crushed ice to
extract 5 in organic layer. Column purification using flash chro-
matography (SiO2, 10% MeOH/CHCl3) of the crude product yielded
pure 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-aryl-1,3,4-oxa
diazole analogues, which was further recrystallized from MeOH.
2-(5-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole-
2-yl) phenol (5a) Yield: 60%; m.p.: 135–137 °C; IR (KBr) Vmax
cmꢀ1: 1233(CAOAC of 1,3,4-oxadiazole),1611 (C@N), 3038 (CAH
aromatic), 3440 (OH phenol); 1H NMR (300 MHz, DMSO-d6): d
3.59 (s, 3H pyrrole NACH3), 5.3 (s, 1H phenol OH), 6.42 (s, 1H
pyrrole 3H), 6.7–7.8 (m, 4H ArH); 13C NMR (75 MHz, DMSO-d6):
d 167.5, 164.3, 157.8, 130.1, 128.9, 126.6, 121.5, 117.3, 108.6,
107.4, 100.8, 97.4, 27.3; MS m/z: 396.8901 (M+), 398.8910 (M2+),
400.8916 (M4+).
2-Benzyl-5-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadia
zole (5b) Yield: 70%; m.p.: 158–160 °C; IR (KBr) Vmax cmꢀ1: 1232
(CAOAC of 1,3,4-oxadiazole), 1608 (C@N), 2995 (CAH aromatic);
1H NMR (300 MHz, DMSO-d6): d 3.41 (s, 3H pyrrole NACH3), 4.32
(s, 2H), 6.34 (s, 1H pyrrole 3H), 6.9–7.4 (m, 5H ArH); 13C NMR
(75 MHz, DMSO-d6): d 165.2, 164.5, 135.4, 133.3, 128.6, 128.1,
124.7, 108.1, 100.3, 96.8, 27.6; MS m/z: 394.9121 (M+), 396.9125
(M2+), 398.9127 (M4+).
2-(2-chloro-5-fluorophenyl)-5-(4,5-dibromo-1-methyl-1H-pyrrol-
2-yl)-1,3,4-oxadiazole (5h) Yield: 70%; m.p.: 178–180 °C; IR (KBr):
Vmax cmꢀ1: 1237 (CAOAC of 1,3,4-oxadiazole), 1623 (C@N),
3015 (CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.56 (s,
3H pyrrole NACH3), 6.22 (s, 1H pyrrole 3H), 7.1–8.0 (m, 3H ArH);
13C NMR (75 MHz, DMSO-d6): d 164.4, 160.5, 138.8, 134.2, 127.4,
127.1, 116.3, 115.8, 105.3, 101.6, 98.1, 27.7; MS m/z: 432.8266
(M+), 434.8269 (M2+), 436.8272 (M4+).
2-(4,5-Dibromo-1-methyl-1H-pyrrol-2yl)-5-(4-nitrophenyl)-
1,3,4-oxadiazole (5i) Yield: 60%; m.p.: 287–289 °C; IR (KBr) Vmax
cmꢀ1: 1240 (CAOAC of 1,3,4-oxadiazole), 1619 (C@N), 2935
(CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.61 (s, 3H
pyrrole NACH3), 6.34 (s, 1H pyrrole 3H), 7.1–8.4 (m, 4H ArH);
13C NMR (75 MHz, DMSO-d6): d 164.3,147.9, 135.3, 132.1, 131.6,
128.4,103.4, 100.5, 95.2, 27.6; MS m/z: 425.9799 (M+), 427.9800
(M2+), 429.9801 (M4+).
2,5-bis(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole
(5j) Yield: 50%; m.p.: 210–212 °C; IR (KBr) Vmax cmꢀ1: 1245
(CAOAC of 1,3,4-oxadiazole), 1628 (C@N), 2940 (CAH aromatic);
1H NMR (300 MHz, DMSO-d6): d 3.52 (s, 6H pyrrole NACH3), 6.46
(s, 2H pyrrole 3H); 13C NMR (75 MHz, DMSO-d6): d 164.5, 136.2,
106.5, 103.6, 96.8, 27.6; MS m/z: 525.8100 (M+), 527.7080 (M2+),
529.7085 (M4+).
4-(2-(5-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazol-
2-yl)vinyl)-2-methoxyphenol (5k) Yield: 75%; m.p.: 160–162 °C; IR
4-(5-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole-
(KBr) Vmax cmꢀ1
: 1231 (CAOAC of 1,3,4-oxadiazole), 1627
2-yl) aniline (5c) Yield: 65%; m.p.: 183–185 °C; IR (KBr) Vmax cmꢀ1
:
(C@N), 2950 (CAH Aromatic), 3441 (OH phenol); 1H NMR
(300 MHz, DMSO-d6): d 3.58 (s, 3H pyrrole NACH3), 3.89 (s, 3H
OCH3), 5.2 (s, 1H OH), 6.54 (s, 1H pyrrole 3H), 6.70 (d, 1H CH@CH),
6.73(d, 1H CH@CH), 7.0–7.38 (m, 3H ArH); 13C NMR (75 MHz,
DMSO-d6): d 164.5, 160.6, 149.4, 147.2, 133.8, 130.8, 128.9,
1235 (CAOAC of 1,3,4-oxadiazole), 1610 (C@N), 2989 (CAH
aromatic), 3320 (NH NH2); 1H NMR (300 MHz, DMSO-d6): d 3.54
(s, 3H pyrrole NACH3), 6.44 (s, 1H pyrrole 3H), 6.2 (s, 2H NH2),
6.9–7.6 (m, 4H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.6,

70
R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73
124.1, 122.8, 116.2, 109.5, 107.3, 100.3, 96.4, 55.9, 27.1; MS m/z:
452.9320 (M+), 454.9325 (M2+), 456.9328 (M4+).
1222 (CAOAC of 1,3,4-oxadiazole), 1616 (C@N), 2699 (SH group);
1H NMR (300 MHz, DMSO-d6): d 3.58 (s, 3H pyrrole NACH3), 6.43
(s, 1H pyrrole 3H), 7.14–7.47 (m, 5H ArH); 13C NMR (75 MHz,
DMSO-d6): d 166.4, 160.8, 136.2, 128.4, 127.4, 126.7, 123.3,
105.5, 100.9, 97.8, 28.1; MS m/z: 412.8632 (M+), 414.8654 (M2+),
416.8658 (M4+).
2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(pyridin-4-yl)-
1,3,4-oxadiazole (5l) Yield: 70%; m.p.: 240–242 °C; IR (KBr) Vmax
cmꢀ1: 1245 (CAOAC of 1,3,4-oxadiazole), 1613 (C@N), 2899
(CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.7 (s, 3H pyrrole
NACH3), 6.41 (s, 1H pyrrole 3H), 7–8.27 (m, 4H pyridine); 13C NMR
(75 MHz, DMSO-d6): d 164.2, 149.9, 143.9, 128.4, 121.8, 107.1,
100.5, 97.2, 27.3; MS m/z: 381.9001 (M+), 383.9006 (M2+),
385.9009 (M4+).
2.3. Antibiofilm study
2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-phenyl-1,3,4-oxadiazole
(5m) Yield: 80%; m.p.: 270–272 °C; IR (KBr) Vmax cmꢀ1: 1239
(CAOAC of 1,3,4-oxadiazole), 1614 (C@N), 1710 (C@O), 2931
(CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.55 (s, 3H
pyrrole NACH3), 6.38 (s, 1H pyrrole 3H), 7.44–7.62 (m, 5H ArH);
13C NMR (75 MHz, DMSO-d6): d 177.9, 164.9, 160.3, 157.8, 149.3,
135.2, 132.7, 128.7, 128.4, 127.2, 123.8, 123.2, 117.8, 115.3,
107.2, 100.9, 97.6, 27.6; MS m/z: 474.9022 (M+), 476.9026 (M2+),
478.9033 (M4+).
2.3.1. Detection of biofilm formation by tissue culture plate (TCP) assay
Detection of biofilm formation was confirmed by Tissue culture
plate (TCP) assay as described in literature [13]. Tissue culture plate
(TCP) assay as described in literature is most widely used and was
considered as standard test for detection of biofilm formation
[11]. In our present study, we have screened Methicillin-sensitive
Staphylococcus aureus (MSSA; ATCC 35556), Methicillin-resistant
Staphylococcus aureus (MRSA; ATCC 43866), Streptococcus epider-
midis (SE; ATCC 35984) and Pseudomonas aeruginosa (PA) bacterium
for their ability to form biofilm by TCP method. Isolates from fresh
agar plates were inoculated in respective media and incubated for
18 h at 37 °C in stationary condition and diluted 1 in 100 with tryp-
tic soy broth (TSB). Individual wells of sterile, polystyrene, 96-well
flat-bottom tissue culture plates were filled with 0.2 mL aliquots of
the diluted cultures and only broth served as control to check steril-
ity and nonspecific binding of media.
The tissue culture plates were incubated for 18 h and 24 h at
37 °C. After incubation, content of each well was gently removed
by tapping the plates. The wells were washed four times with
0.2 mL of phosphate buffer saline (PBS, pH 7.2) to remove free
floating ‘‘planktonic’’ bacteria. Biofilms formed by adherent ‘‘ses
sile’’ organisms in the plate were fixed with sodium acetate (2%)
and stained with crystal violet (0.1% w/v). Excess stain was rinsed
off by thorough washing with de-ionized water and plates were
kept for drying. Adherent staphylococcal cells usually formed bio-
film on all side wells and were uniformly stained with crystal vio-
let. Optical densities (ODs) of stained adherent bacteria were
determined with a micro ELISA auto reader (California) at wave-
length of 570 nm (OD 570 nm). These OD values were considered
as an index of bacteria adhering to surface and forming biofilms.
Experiment was performed in triplicate and repeated three times,
the data was then averaged and standard deviation was calculated.
To compensate for background absorbance, OD readings from ster-
ile medium, fixative and dye were averaged and subtracted from
all test values. The mean OD value obtained from media control
well was deducted from all the test OD values as given in Table 2.
2.2.5. General procedure for synthesis of 5-(4, 5-dibromo-1-methyl-
1H-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6
In a two necked round bottom flask equipped with sealed
mechanical stirrer and an efficient reflux condenser, 4,5-dibro
mo-1-methyl-1H-pyrrole-2-carbohydrazide 4 (0.003 mol) in etha-
nol (20 ml) was added. To this solution potassium hydroxide
(0.006 mol) and carbon disulfide (4 ml) was added with stirring.
The reaction mixture was refluxed for 12 h. The solvent was
removed under reduced pressure; the residue was treated with
water and then filtered. The filtrate was cooled and neutralized
to pH 6 using dilute hydrochloric acid. The precipitated product
was filtered, washed with water and dried to obtain 5-(4,5-dibro
mo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6.
5-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole-
2-thiol (6) Yield: 80%; m.p.: 190–192 °C; IR (KBr) Vmax cmꢀ1: 1242
(CAOAC of 1,3,4-oxadiazole), 1625 (C@N), 2751 (SH group).
1H NMR (300 MHz, DMSO-d6): d 3.41 (s, 3H pyrrole NACH3), 3.
2(s, 1H SH), 6.61(s, 1H pyrrole 3H); 13C NMR (75 MHz, DMSO-d6): d
164.4, 160.5, 127.9, 107.5, 100.2, 96.8, 27.5; MS m/z: 336.8369
(M+), 338.8376 (M2+), 340.8379 (M4+).
2.2.6. General procedure for synthesis of 2-(4,5-dibromo-1-methyl-
1H-pyrrol-2-yl)-5-(alkylnarylthio)-1,3,4-oxadiazole 7
In a two necked round bottom flask 5-(4,5-dibromo-1-me
thyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6 (1 mmol), alkyl/aryl
halide (1 mmol) and sodium hydroxide (5 mmol) in ethanol
were stirred for 10 h at room temperature. The solvent was
removed under reduced pressure. The residue obtained was
dried to give 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(alkyln
arylthio)-1,3,4-oxadiazole 7.
2.3.2. Antibiofilm assay
For antibiofilm concentration experiment, overnight cultures of
bacterium Methicillin-sensitive Staphylococcus aureus (MSSA; ATCC
35556), Methicillin-resistant Staphylococcus aureus (MRSA; ATCC
43866), Streptococcus epidermidis (SE; ATCC 35984) and Pseu-
domonas aeruginosa (PA; ATCC 27853) were diluted (1:10) in TSB
(tryptic soy broth) (OD 600 = 0.6–0.8) and were then diluted to
1:200 in Mueller–Hinton. The bacterial suspension was inoculated
into sterile 96-well polystyrene microtiter plates (Falcon) incu-
bated at 37 °C for 6 h. The plates with young biofilm were washed
gently four times with sterile PBS before adding fresh TSB contain-
ing various concentrations of compounds and incubated at 37 °C for
2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(methylthio)-1,3,4-ox
adiazole (7a)Yield: 80%; m.p.: 186–188 °C; IR (KBr) Vmax cmꢀ1
:
1247 (CAOAC of 1,3,4-oxadiazole), 1629 (C@N), 2749 (SH); 1H
NMR (300 MHz, DMSO-d6):
d 3.41 (s, 3H pyrrole NACH3),
6.57(s, 1H pyrrole 3H), 2.36(s, 3H); 13C NMR (75 MHz, DMSO-d6):
d 164.8, 160.2, 126.2, 107.8, 100.4, 96.9, 27.3, 16.9; MS m/z:
350.8567 (M+), 352.8571 (M2+), 354.8575 (M4+).
2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(ethylthio)-1,3,4-oxa
diazole (7b) Yield: 55%; m.p.: 150–152 °C; IR (KBr) Vmax cmꢀ1
:
1229 (CAOAC of 1,3,4-oxadiazole), 1632 (C@N), 2751 (SH); 1H
NMR (300 MHz, DMSO-d6): d 1.3 (t, 3H), 3.1(m, 2H), 3.62 (s, 3H
pyrrole NACH3), 6.62(s, 1H pyrrole 3H); 13C NMR (75 MHz,
DMSO-d6): d 165.3, 160.1, 127.3, 106.5, 100.3, 97.1, 27.9, 27.3,
16.8; MS m/z: 364.8676 (M+), 366.8682 (M2+), 368.8688 (M4+).
2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(phenylthio)-1,3,4-ox
24 h. The initial dilution was 200 lg/mL and minimum biofilm inhi-
bitory concentration (MBIC) values were defined as the lowest con-
centration at which no visible growth was observed. Positive
control was performed for all the evaluated bacteria. A high level
of biofilm growth was observed in case of positive control. For all
assays, Vancomycin was used as reference standard [13].
adiazole (7c) Yield: 65%; m.p.: 170–172 °C; IR (KBr) Vmax cmꢀ1
:

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73
71
2.3.3. Cell viability test
P
Bacterial growth after treatment was measured by quantifying
cell viability after incubation with 5f. MSSA, MRSA and SE cells were
incubated with 5f at various concentrations as shown in Fig. 3 in
isotonic saline solutions at 37 °C under 200 rpm shaking speed
for 4 h. After the treatment, 100
up to 1 mL, and then from 1 mL, 50
viability was evaluated by the colony-counting method. Briefly, a
series of 20-fold cell dilutions (100 L each) were spread onto LB
lL of reaction mixture was made
lL was taken for plating. Loss of
l
plates and left to grow overnight at 37 °C. Colonies were counted
and compared with those on control plates to calculate changes
in cell-growth inhibition. For control, we used isotonic saline solu-
tion without 5f. All treatments were prepared in triplicate and
repeated at least in three independent experiments.
Q
2.4. Cytotoxicity of compounds (selectivity assay)
The synthesized analogs were further studied for their mam-
malian toxicity (IC50) using VERO cell lines. Viability was assessed,
after 72 h, by the conversion of MTT into formazan product using
Promega Cell Titre 96 non-radioactive cell proliferation assay. All
the hybrids were found to be non-toxic and the data is displayed
in Table 1.
3. Results and discussion
R
3.1. Synthesis of the compounds
The Spectral data (IR, 1H NMR, 13C NMR and MS) of all synthe-
sized compounds were in agreement with the proposed structures.
The MS of all the compounds exhibited the [M+.] as molecular ion
peak, confirming the molecular weight. Bromination of pyrrole ring
was confirmed by the presence of [M2+] and [M4+] peaks in mass
spectra. 1H NMR spectra of these hybrids showed singlet peak of
pyrrole-NACH3 proton in the range of d 3.7–3.4. Thiol group of 6
exhibited NMR peak at d 3.2, while proton peaks of aryl substitu-
tions attached to oxadiazole ring resonated between 8.2 and
6.1 ppm. A proton signal at third position of pyrrole ring was
observed to resonate between d 6.7–6.3. 5-(4,5-dibromo-1-me
thyl-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6 displayed distinct IR
peaks at 2751 cmꢀ1 for ASH group, while for the alkylated product
[5-(4,5-dibromo-1-methyl-pyrrol-2-yl)-1,3,4-oxadiazole-2-methyl
thiol] the ASH signal was absent.
Fig. 3. Cell viability of MSSA (P), MRSA (Q) and SE (R) on treatment with 5f at
different concentrations. These experiments were performed in triplicate
(C = Control).
(MBIC = 12.5 lg/mL) with other compounds in the series revealed
that the presence of substituents like chloro-, fluro-, bromo-,
methoxy-, nitro- and amino- on phenyl ring was beneficial than
the un-substituted phenyl ring. In addition, six compounds (5a,
5b, 5g, 5h, 5k and 5l) presented two-fold higher anti-biofilm
3.2. Quantification of biofilm inhibition activities
activity (MBIC = 1.56 lg/mL) than the standard drug Vancomycin
The respective bacteria were evaluated for their ability to form
biofilm by TCP method and the results are presented in Table 2.
Here ‘‘high’’ biofilm forming isolate were selected for future anti-
biofilm study and the selected isolates reproducibly formed biofilm
in the assay performed. Table 1 shows the results of biofilm inhibi-
tion for each assayed compound as well as the concentrations at
which these inhibition levels were observed. All experiments were
performed in triplicate and the mean values were taken as mini-
mum biofilm inhibitory concentration (MBIC). Most of the synthe-
sized compounds disclosed significant biofilm inhibition against
MSSA and MRSA bacterial strains. In the case of MSSA strain, com-
against MSSA. Compounds with substitution at ortho- or meta-
position of aromatic rings displayed lower antibiofilm activity
relative to para- substituted molecules. Also, hetero-aromatic
substitution at R position decreased the anti-biofilm activity for
this class of compounds.
For the MRSA, most active compound was found to be 5f with
four-time superior antibiofilm activity (MBIC = 0.78
standard Vancomycin (MBIC = 6.25 g/mL). Compounds 5c, 5d,
5e, and 5j showed three-time more potency (MBIC = 1.56 g/ml),
whereas 5h, 5i and 5l exhibited two-time potency
(MBIC = 3.125 g/mL) towards biofilm produced by MRSA as com-
lg/mL) than
l
l
l
pound 5f (MBIC = 0.39
lg/mL) having electron withdrawing sub-
pared to the standard drug. Compounds 5a, 5b, 5g, 5m, 6 and 7a
stituents (2,4-dichlo-) on phenyl ring exhibited maximum biofilm
were found to be equipotent to that of standard drug Vancomycin
inhibition with four-fold greater activity than the standard drug
(MBIC = 6.25 lg/mL). Consequently, substituting phenyl ring at
(MBIC = 3.125
5j) exhibited three-fold superior biofilm inhibition (MBIC =
0.78 g/mL) than standard drug. Generally, substituting phenyl
l
g/mL). Further, five compounds (5c, 5d, 5e, 5i and
para-position with halogen-, methoxy-, nitro- and amino- groups
was beneficial for the biofilm inhibition. On the other hand, substi-
tuting 1,3,4-oxadiazole ring with thio-phenyl and thio-ethyl at sec-
ond position decreased the anti-biofilm activity of compounds 7b
l
ring at para-position was beneficial for inhibition of biofilm pro-
duced by MSSA. Comparison of anti-biofilm activity of 5m
and 7c (MBIC = 25 lg/mL). This could be due to the presence of

72
R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73
Table 1
Antibiofilm results: (MBIC in l
g/mL).a,b
Br
O
Br
N
3C
R
H
N
N
S. no.
R
MSSA
MRSA
S. epidermidis
P. aeruginosa
Cytotoxicity (lM) VERO cells
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
6
7a
7b
2-Hydroxyphenyl-
Benzyl-
1.56
1.56
0.78
0.78
0.78
0.39
1.56
1.56
0.78
0.78
1.56
1.56
12.5
6.25
12.5
12.5
12.5
3.125
6.25
6.25
1.56
1.56
1.56
0.78
6.25
3.125
3.125
1.56
25
3.125
6.25
6.25
6.25
25
6.25
3.125
3.125
3.125
3.125
3.125
25
3.125
3.125
3.125
12.5
12.5
12.5
25
25
12.5
25
NDc
ND
69.0
137.9
248.1
297.0
ND
4-Aminophenyl-
4-Chlorophenyl-
4-Methoxyphenyl-
2,4-Dichlorophenyl-
Styryl-
2-Chloro-5-fluorophenyl-
4-Nitrophenyl-
4,5-Dibromo-1H-pyrrol-2-yl-
2-Methoxy-4-vinylphenol-
Pyridin-4-yl-
25
6.25
12.5
50
6.25
6.25
6.25
50
6.25
50
100
50
ND
78.2
206.5
ND
ND
ND
ND
ND
ND
ND
Phenyl-
Thiol-
Thiomethyl-
Thioethyl-
12.5
25
25
50
50
3.125
7c
Thiophenyl-
25
6.25
Standardb
3.125
ND
Italic values indicates the compounds tested for antibiofilm activity and bold value denotes the most active compounds of the series.
a
The initiate dilution was 200 lg/ml.
For all assay standard used was Vancomycin.
ND – not determined.
b
c
that most the synthesized molecules were antibacterial at more
than three-fold MBIC concentration against respective bacterium.
Further most active compound 5f was subjected to the cell viability
assay at respective MBIC as shown in Fig. 3. It was observed that
there are no effects on the viability of the bacteria at the concentra-
tions at which compound 5f showed antibiofilm activities against
respective bacterium indicating that antibiofilm effects are not
simply due to a decreased number of bacteria but because of the
antivirulence properties of this class of compounds that needs to
be investigated further and is the ongoing work at our laboratory.
Table 2
Screening of various bacterial strains for biofilm formation by TCP method in tryticase
soy broth with 1% glucose at 18 and 24 h of incubation.
Biofilm formation (OD
)
No. of isolates
570nm
MRSA,
ATCC
MSSA,
ATCC
SE,
ATCC
35984
PA
ATCC
27853
43866
35556
Incubation time in hours
18 24 18 24 18 24 18 24
High (>0.240 0.020)
Moderate (0.120–0.240 0.024)
Weak/non (<0.12 0.014)
1
6
4
8
12 17
21 28 11 18 11 20
5
7
5
12
54 62 92 92 64 70 61 79
4. Conclusion
In summary, this study identified compound 5f with MBIC of
0.39, 0.78 and 3.125 lg/mL as promising lead against biofilm pro-
sulphur bridge/linker between 1,3,4-oxadiazole ring and R
(alkyl/aromatic) group.
duced by MSSA, MRSA and SE respectively. Additionally, eight
compounds displayed equal potency with the standard drug Van-
comycin against S. epidermidis. An important observation was that
the compounds which displayed promising biofilm inhibition
activity also exhibited low toxicity towards human VERO cells.
The cell viability assay for the most active hybrid confirms its
anti-virulence properties which need to be further studied. This
could be advantageous for their use in therapeutics and functional-
ization of materials such as biofouling control in closed systems.
Currently, evaluation of such potential along with studies on the
mechanism of biofilm inhibition for these compounds is underway.
Furthermore, we are also working towards consolidation of the
potential use of these compounds on a range of bacterial clinical
isolates chosen in accordance with their foreseen applications.
In case of S. epidermidis (SE) bacterium, compounds with
para-substitution on the benzene ring exhibited equipotent biofilm
inhibition activity (MBIC = 3.125
drug Vancomycin (MBIC = 3.125
l
l
g/mL) compared to the standard
g/ml). Moreover, substitution of
phenyl ring with chloro-, fluro-, bromo-, nitro-, amino- and
methoxy- group (5b, 5c, 5d, 5e, 5f, 5h, 5i and 5j) improved the
anti-biofilm activity against SE. No significant biofilm inhibition
activity was observed for compounds with substitutions at ortho-
and meta- position on the phenyl ring. In addition to this, these
synthesized compounds were also screened against P. aeruginosa.
All compounds displayed less activity (MBIC = 6.25 to 100
towards P. aeruginosa (compared to the standard drug Van-
comycin; MBIC = 3.125 g/mL), indicating that this structural
lg/mL)
l
motif was less potent against biofilm produced by Gram-negative
bacterium as compared to Gram-positive.
Further in order to confirm the antivirulence properties of these
compounds they were evaluated against test microbes for bacteri-
cidal activity (minimum inhibitory concentration). It was observed
Conflict of interest
Authors in this manuscript declare no conflict of interest.

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73
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