Design, synthesis and antistaphylococcal activity of marine pyrrole alkaloid derivatives

Article abstract of DOI:10.3109/14756366.2013.793183

A novel set of 16 hybrids of bromopyrrole alkaloids with aroyl hydrazone were designed, synthesized and evaluated for antibacterial and antibiofilm activities against methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43866), methicillin-susceptible

Full text of DOI:10.3109/14756366.2013.793183

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(3): 401–407
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.793183
!
SHORT COMMUNICATION
Design, synthesis and antistaphylococcal activity of marine pyrrole
alkaloid derivatives
Rajesh A. Rane, Niteshkumar U. Sahu, Chetan P. Shah, and Nishant K. Shah
S. P. P. School of Pharmacy and Technology Management, NMIMS university, Mumbai, India
Abstract
Keywords
A novel set of 16 hybrids of bromopyrrole alkaloids with aroyl hydrazone were designed,
synthesized and evaluated for antibacterial and antibiofilm activities against methicillin-
resistant Staphylococcus aureus (MRSA; ATCC 43866), methicillin-susceptible Staphylococcus
aureus (MSSA; ATCC 35556) and Staphylococcus epidermidis (SE, S. epidermidis ATCC 35984).
Of the 16 tested hybrids, 14 exhibited equal or superior antibiofilm activity against MSSA
and MRSA relative to standard vancomycin. Compound 4m showed highest potency with
antibiofilm activity of 0.39 mg/mL and 0.78 mg/mL against MSSA and MRSA, respectively.
Thus, this compound could act as a potential lead for further development of new
antistaphylococcal drugs.
Antistaphylococcal activity, bromopyrrole
alkaloids, hydrazide derivatives
History
Received 6 November 2012
Revised 9 March 2013
Accepted 3 April 2013
Published online 10 May 2013
Introduction
published14,15. Synthesized 1,3,4-oxadiazole derivatives (E1–E8)
showed antimicrobial activity against representative Gram-
negative and Gram-positive bacteria. Herein, eight of twenty
tested derivatives showed equal antibacterial activity compared
to standard ciprofloxacin against S. aureus (MIC – 1.56 mg/mL).
We further derivatized 1-methyl-4,5-dibromopyrrole core with
4-thiazolidinone feature and tested for its antibiofilm potential
against few Gram-positive bacteria. Antibiofilm activity of
4-thiazolidinones, F1 and F2 (MIC ¼ 0.78 mg/mL) was 3-fold
superior than standard vancomycin (MIC ¼ 3.125 mg/mL),
while activity of compounds F3, F4, F5 and F6 was 2-fold
(MIC ¼ 1.56 mg/mL) against S. aureus biofilm. Compounds F1–F7
showed equal antibiofilm activity against S. epidermidis com-
pared to standard Vancomycin (MIC ¼ 3.125 mg/mL) (Figure 1a).
Aroyl hydrazones constitute an important class of privileged
scaffold for new drug development. Aroyl hydrazone derivatives
have been known to possess diverse array of biological effects like
anticonvulsant, analgesic, anti-inflammatory, antimalarial and
antitumor activity. Also, aroyl hydrazone moiety is common
among many of antimicrobial and antimycobacterial agents16.
High-throughput screening performed on a library of small
molecules indicated aroyl hydrazone as biofilm development
inhibitor17 (Figure 1a).
As extension of our previous work and with the aim to obtain
more effective compounds, we thought to be of interest to design
and synthesize a series of molecular hybrids of 1-methyl-4,5-
dibrompyrrole core and aroyl hydrazone as anti-staphylococcal
agents (Figure 1b). The phenyl ring of these hybrids is substituted
with different groups or atoms in order to obtain a good
discrimination of antistaphylococcal activity among the com-
pounds. All the new compounds were tested for antibacterial and
antibiofilm activity against bacterial strains of S. aureus,
including MRSA and SE.
Staphylococcus aureus is a major cause of metal-biomaterial
infections, whereas Staphylococcus epidermidis (SE) is found
mostly in polymer-biomaterial-associated infections1. Biofilm
growth of methicillin-resistant S. aureus (MRSA) is common
during hemodialysis2 and among patients bearing indwelling
medical devices3. In the USA, 12–25% of MRSA catheter-related
blood stream infections result in mortality, with annual patient
care costs projected to be as high as $460 million4. When
enclosed within a biofilm, MRSA exhibits increased resistance to
multiple classes of antibiotics5. Current strategies for targeting
staphylococcal pathogens includes interference with cell–cell
signaling by modulating quorum sensing6, targeting slime
formation by inhibiting bacterial cell wall synthesis7, impairing
adhesion using biosurfactants8 and immunotherapy9. The chal-
lenge of staphylococcal strains resistant to most or all conven-
tional antibiotics could be faced by developing new antibacterial
agents with different chemical characteristics and mechanism of
action with respect to current antibiotics.
Bromopyrrole alkaloids represent an important family of
marine alkaloids produced by marine sponges with diverse
biological activities10–13. Many of these molecules are identified
by the presence of 4,5-dibromopyrrole ring with oroidin as their
prototype alkaloid known for its antibiofilm activity10. Recently, a
few reports on synthesis of marine bromopyrrole alkaloid
derivatives containing 1,3,4-oxadiazole and thiazolidinone fea-
tures for their antimicrobial and antibiofilm properties have been
Address for correspondence: Rajesh A. Rane, S. P. P. School of Pharmacy
and Technology Management, NMIMS university, Vile Parle, Mumbai-
400056, India. Tel: +91 92242 94690. E-mail: rajeshrane_uict@
yahoo.co.in

402 R. A. Rane et al.
J Enzyme Inhib Med Chem, 2014; 29(3): 401–407
(a)
(b)
Figure 1. (a) Reported bromopyrrole alkaloids and aroyl hydrazones with antibacterial and antibiofilm activities. (b) Design of novel N0-arylidene-4,5-
dibromo-1-methyl-1H-pyrrole-2-carbohydrazides using molecular hybridization approach.
Materials and methods
were recorded on JEOL AL-300 FT-NMR spectrometer (Tokyo,
Japan) with dimethyl sulfoxide (DMSO)-d6 as solvent using
tetramethyl silane as internal reference. Mass spectra were
obtained on Thermo Finnigan LCQ advantage max (LCMS)
(Washington). Compounds 1, 2 and 3 were prepared according to
reported methods9,10. Evaporations of final product solutions
were done under vacuum with a rotatory evaporator.
Chemistry
Instrumentation
All reactions were carried out under an inert nitrogen atmosphere
˚
under anhydrous conditions and by using molecular sieves (4 A
1/16 inches pellets), ethanol and acetonitrile were freshly distilled
from CaCl2. All chromatographic solvents were distilled before
use. Silica gel of 60–120 and 200–400 mesh was used for column
and flash chromatography. Some of the starting materials were
Synthesis of 4,5-dibromo-N0-arylidene-1-methyl-1H-pyrrole-2-
carbohydrazide analogs 4
obtained from S. D. Fine Pvt. Ltd. (Mumbai, India), SRL Appropriate aldehydes (27.5 mmol) and compound
3 (6,
(Mumbai, India), Spectrochem (Mumbai, India)/Aldrich (India) 27.5 mmol), were stirred in 30 mL absolute ethanol in a round-
and some of them were prepared in the laboratory and were used bottomed flask. A quantity of 1–2 mL of triethylamine was added
without further purification. All melting points were recorded on to the reaction mixture and was refluxed for 4–7 h. Most of the
Thermomik Campbell electronics (India), having oil-heating 4,5-dibromo-1-methyl-1H-pyrrole-2-carbohydrazide was con-
system and were uncorrected. Analytical thinlayer chromatog- sumed at this time (monitored by TLC), the reaction mixture
raphy (TLC) was carried out on precoated SiO2 plates (silica gel was concentrated under reduced pressure and the obtained solid
60, F 254, Merck, India). Fourier transform infrared spectroscopy residue was partitioned between 50 mL water and 50 mL chloro-
spectra were recorded on Perkin Elmer RX I spectrometer using form to compound 4 in organic layer. Column purification using
KBr pellets. All the nuclear magnetic resonance (NMR) spectra flash chromatography (SiO2, 10% MeOH/CHCl3) of the crude

DOI: 10.3109/14756366.2013.793183
Design, synthesis and antistaphylococcal activity of marine pyrrole alkaloid derivatives 403
product yielded pure 4,5-dibromo-N0-arylidene-1-methyl- (m, 5H ArH), 8.43 (s, 1H azometine H), 11.62 (s, 1H CONH); 13
1Hpyrrole-2-carbohydrazide. The obtained solid was recrystal- NMR (75 MHz, DMSO-d6): ꢀ 157.3, 137.2, 135.6, 134.1, 128.6,
C
lized from MeOH.
128.4, 127.9, 127.4, 126.4, 126, 114.5, 94.6, 37.9; MS m/z:
409.9427 [M]þ, 411.9430 [M þ 2]þ, 413.9432 [M þ 4]þ.
4,5-dibromo-N0-(furan-2-ylmethylene)-1-methyl-1H-pyrrole-2-
carbohydrazide (4a). Yield: 78%; m.p.: 188–190 ꢀC; IR (KBr):
Vmax/cm 1620 (C¼C), 1643 (CH¼N), 1715 (C¼O), 3206 (NH);
1HNMR (300 MHz, DMSO-d6): ꢀ 3.59 (s, 3H pyrrole N-CH3), 6.43
(s, 1H pyrrole 3H), 6.7–7.4 (m, 3H ArH), 8.40 (s, 1H azometine H),
11.52 (s, 1H CONH); 13C NMR (75 MHz, DMSO-d6): ꢀ 157.4,
149.3, 144.1, 134.3, 127.8, 126.3, 118.6, 114.5, 112.6, 94.6, 37.8;
MS m/z: 373.9152 [M]þ, 375.9059 [M þ 2]þ, 377.9063 [M þ 4]þ.
4,5-dibromo-N0-(2,6-dichlorobenzylidene)-1-methyl-1H-pyrrole-
2-carbohydrazide (4b). Yield: 78%; m.p.: 256–258 ꢀC; IR
(KBr): Vmax/cm 1628 (C¼C), 1644 (CH¼N), 1719 (C¼O),
3209 (NH); 1HNMR (300 MHz, DMSO-d6): ꢀ 3.56 (s, 3H pyrrole
N-CH3), 6.46 (s, 1H pyrrole 3H), 6.9–7.67 (m, 3H ArH), 8.42 (s,
1H azometine H), 11.57 (s, 1H CONH); 13C NMR (75 MHz,
DMSO-d6): ꢀ 157.4, 138.1, 133.5, 130.5, 129.4, 128.3, 127.6,
126.2, 114.5, 94.5, 37.7; MS m/z: 451.8491 [M]þ, 453.8492
[M þ 2]þ, 455.8494 [M þ 4]þ.
4,5-dibromo-N0-(4-chlorobenzylidene)-1-methyl-1H-pyrrole-2-
carbohydrazide (4h). Yield: 87%; m.p.: 216–218 ꢀC; IR (KBr):
Vmax/cm 1612 (C¼C), 1644 (CH¼N), 1712 (C¼O), 3209 (NH);
1HNMR (300 MHz, DMSO-d6): ꢀ 3.58 (s, 3H pyrrole N-CH3),
6.46 (s, 1H pyrrole 3H), 6.87–7.4 (m, 4H ArH), 8.42 (s, 1H
azometine H), 11.59 (s, 1H CONH); 13C NMR (75 MHz, DMSO-
d6): ꢀ 157.2, 146.9, 136.3, 131.2, 130.2, 128.4, 127.4, 126.4,
114.5, 94.5, 37.8; MS m/z: 417.8879 [M]þ, 419.8881 [M þ 2]þ,
421.8883 [M þ 4]þ.
4,5-dibromo-N0-(2-hydroxy-4-methoxybenzylidene)-1-methyl-1H-
pyrrole-2-carbohydrazide (4i). Yield: 76%; m.p.: 194–196 ꢀC;
IR (KBr): Vmax/cm 1620 (C¼C), 1641 (CH¼N), 1719 (C¼O),
3212 (NH), 3440 (OH); 1HNMR (300 MHz, DMSO-d6): ꢀ 3.28 (s,
3H OCH3), 3.57 (s, 3H pyrrole N-CH3), 6.47 (s, 1H pyrrole 3H),
5.38 (s, 1H OH), 6.9–7.42 (m, 3H ArH), 8.38 (s, 1H azometine
H), 11.54 (s, 1H CONH); 13C NMR (75 MHz, DMSO-d6): ꢀ
164.4, 162.2, 157.3, 146.2, 133.2, 127.9, 126.2, 114.2,
110.8,107.2,103.2, 94.2, 55.8, 37.7; MS m/z: 429.9325 [M]þ,
431.9328 [M þ 2]þ, 433.9331 [M þ 4]þ.
4,5-dibromo-N0-(4-(dimethylamino)benzylidene)-1-methyl-1H-
pyrrole-2-carbohydrazide (4c). Yield: 80%; m.p.: 216–218 ꢀC;
IR (KBr): Vmax/cm 1623 (C¼C), 1646 (CH¼N), 1718 (C¼O),
1
3212 (NH); HNMR (300 MHz, DMSO-d6): ꢀ 3.12 (s, 6H CH3),
4,5-dibromo-1-methyl-N0-(thiophen-2-ylmethylene)-1H-pyrrole-2-
carbohydrazide (4j). Yield: 78%; m.p.: 230–232 ꢀC; IR (KBr):
Vmax/cm 1614 (C¼C), 1637 (CH¼N), 1716 (C¼O), 3208 (NH);
1HNMR (300 MHz, DMSO-d6): ꢀ 3.58 (s, 3H pyrrole N-CH3),
6.47 (s, 1H pyrrole 3H), 6.67–7.2 (m, 3H ArH), 8.41 (s, 1H
azometine H), 11.60 (s, 1H CONH); 13C NMR (75 MHz, DMSO-
d6): ꢀ 157.4, 144.8, 130.6, 128, 127.4, 126.9, 125, 124.9, 114.5,
94.6, 37.8; MS m/z: 389.8836 [M]þ, 391.8839 [M þ 2]þ,
393.8841 [M þ 4]þ.
4,5-dibromo-N0-(4-methoxybenzylidene)-1-methyl-1H-pyrrole-2-
carbohydrazide (4k). Yield: 80%; m.p.: 115–117 ꢀC; IR (KBr):
Vmax/cm 1619 (C¼C), 1646 (CH¼N), 1710 (C¼O), 3204 (NH);
1HNMR (300 MHz, DMSO-d6): ꢀ 3.12 (s, 3H OCH3), 3.54 (s, 3H
pyrrole N-CH3), 6.43 (s, 1H pyrrole 3H), 6.74–7.1 (m, 4H ArH),
8.49 (s, 1H azometine H), 11.58 (s, 1H CONH); 13C NMR
(75 MHz, DMSO-d6): ꢀ 162.8, 157.7, 146.7, 130.1, 127.3, 126.0,
114.5, 114.3, 94.3, 55.7, 37.8; MS m/z: 413.9376 [M]þ, 415.9379
3.58 (s, 3H pyrrole N-CH3), 6.42 (s, 1H pyrrole 3H), 6.7–7.4 (m,
4H ArH), 8.43 (s, 1H azometine H), 11.67 (s, 1H CONH); 13C
NMR (75 MHz, DMSO-d6): ꢀ 157.8, 153.5, 146.9, 128.1, 127.5,
126.1, 123.4, 114.6, 112.1, 94.7, 42, 37.6; MS m/z: 426.9690
[M]þ, 428.9693 [M þ 2]þ, 430.9697 [M þ 4]þ.
4,5-dibromo-N0-(4-fluorobenzylidene)-1-methyl-1H-pyrrole-2-
carbohydrazide (4d). Yield: 74%; m.p.: 204–206 ꢀC; IR (KBr):
Vmax/cm 1618 (C¼C), 1641 (CH¼N), 1715 (C¼O), 3206 (NH);
1HNMR (300 MHz, DMSO-d6): ꢀ 3.59 (s, 3H pyrrole N-CH3),
6.43 (s, 1H pyrrole 3H), 6.9–7.8 (m, 4H ArH), 8.40 (s, 1H
azometine H), 11.64 (s, 1H CONH); 13C NMR (75 MHz, DMSO-
d6): ꢀ 165.3,157.5,146.5, 130.3, 129.4, 127.4, 126.3, 115, 114.2,
94.6, 37.7; MS m/z: 401.9178 [M]þ, 403.9180 [M þ 2]þ,
405.9182 [M þ 4]þ.
4,5-dibromo-1-methyl-N0-(4-nitrobenzylidene)-1H-pyrrole-2-car-
bohydrazide (4e). Yield: 80%; m.p.: 242–246 ꢀC; IR (KBr): Vmax
/
cm 1620 (C¼C), 1644 (CH¼N), 1714 (C¼O), 3204 (NH); 1HNMR [M þ 2]þ, 417.9382 [M þ 4]þ.
(300 MHz, DMSO-d6): ꢀ 3.53 (s, 3H pyrrole N-CH3), 6.41 (s, 1H
pyrrole 3H), 6.8–7.8 (m, 4H ArH), 8.41 (s, 1H azometine H), 11.58 4,5-dibromo-N0-(3,4-dimethoxybenzylidene)-1-methyl-1H-pyr-
(s, 1H CONH); 13C NMR (75 MHz, DMSO-d6): ꢀ 157.6, 150.4, role-2-carbohydrazide (4l). Yield: 85%; m.p.: 220–222 ꢀC; IR
146.5, 139.7, 127.4, 126.4, 124, 123.8, 114.5, 94.3, 37.8; MS m/z: (KBr): Vmax/cm 1621 (C¼C), 1644 (CH¼N), 1713 (C¼O), 3214
428.9124 [M]þ, 430.9127 [M þ 2]þ, 432.9129 [M þ 4]þ.
(NH); 1HNMR (300 MHz, DMSO-d6): ꢀ 3.14 (s, 3H OCH3), 3.23
(s, 3H OCH3), 3.59 (s, 3H pyrrole N-CH3), 6.46 (s, 1H pyrrole
3H), 6.67–7.14 (m, 3H ArH), 8.48 (s, 1H azometine e H), 11.62
(s, 1H CONH); 13C NMR (75 MHz, DMSO-d6): ꢀ 157.8, 152.2,
149.8, 146.7, 130.7, 127.5, 126.2,122.6, 114.6, 111.8, 109.3, 94.6,
56.2, 37.8; MS m/z: 443.9482 [M]þ, 445.9484 [M þ 2]þ,
447.9487 [M þ 4]þ.
4,5-dibromo-N0-(4-(2,6-dichlorophenoxy)benzylidene)-1-methyl-
1H-pyrrole-2-carbohydrazide (4m). Yield: 88%; m.p.: 212–
214 ꢀC; IR (KBr): Vmax/cm 1620 (C¼C), 1642 (CH¼N), 1717
(C¼O), 3207 (NH); 1HNMR (300 MHz, DMSO-d6): ꢀ 3.55 (s, 3H
pyrrole N-CH3), 6.47 (s, 1H pyrrole 3H), 7.2–7.9 (m, 7H ArH),
8.47 (s, 1H azometine H), 11.64 (s, 1H CONH); 13C NMR
(75 MHz, DMSO-d6): ꢀ 159.4, 157.7, 146.8, 146.6, 129.8, 129.1,
128.9, 127.5, 127.2, 126.8, 126.0, 117.7, 114.5, 94.6, 37.9; MS
m/z: 543.8755 [M]þ, 545.8757 [M þ 2]þ, 547.8760 [M þ 4]þ.
4,5-dibromo-N0-(4-hydroxybenzylidene)-1-methyl-1H-pyrrole-2-
carbohydrazide (4f). Yield: 88%; m.p.: 234–236 ꢀC; IR (KBr):
Vmax/cm 1624 (C¼C), 1643 (CH¼N), 1721 (C¼O), 3204 (NH),
3410 (OH); 1HNMR (300 MHz, DMSO-d6): ꢀ 3.58 (s, 3H pyrrole
N-CH3), 6.49 (s, 1H pyrrole 3H), 5.34 (s,1H OH), 6.64–7.63
(m, 4H ArH), 8.48 (s, 1H azometine H), 11.66 (s, 1H CONH); 13C
NMR (75 MHz, DMSO-d6): ꢀ 160.3, 157.8, 146.5, 130.4, 127.5,
126.1, 125.9, 116.5, 114, 94.3, 37.8; MS m/z: 399.9221 [M]þ,
401.9223 [M þ 2]þ, 403.9225 [M þ 4]þ.
4,5-dibromo-1-methyl-N0-(3-phenylallylidene)-1H-pyrrole-2-car-
bohydrazide (4g). Yield: 78%; m.p.: 218–220 ꢀC; IR (KBr):
Vmax/cm 1610 (C¼C), 1649 (CH¼N), 1723 (C¼O), 3213 (NH);
1HNMR (300 MHz, DMSO-d6): ꢀ 3.55 (s, 3H pyrrole N-CH3),
6.44 (s, 1H pyrrole 3H), 6.2-7.12 (dd, 2H CH¼CH), 7.4–7.8

404 R. A. Rane et al.
J Enzyme Inhib Med Chem, 2014; 29(3): 401–407
4,5-dibromo-N0-(2,5-dihydroxybenzylidene)-1-methyl-1H-pyrrole- visible bacterial growth) tube from the MIC assay, onto
2-carbohydrazide (4n). Yeild: 80%; m.p.: 230–232 ꢀC; IR (KBr): substance-free Mueller–Hinton agar plates. The plates were
Vmax/cm 1622 (C¼C), 1645 (CH¼N), 1709 (C¼O), 3209 (NH), incubated at 37 ꢀC for 24 h, and the MBC was defined as the
3440 (OH); 1HNMR (300 MHz, DMSO-d6): ꢀ 3.54 (s, 3H pyrrole lowest concentration of compounds that produced subcultures
N-CH3), 5.28 (s, 1H OH), 5.36 (s, 1H OH), 6.43 (s, 1H pyrrole 3H), growing no more than five colonies on each plate.
6.97–7.84 (m, 3H ArH), 8.41 (s, 1H azometine H), 11.66 (s, 1H
CONH); 13C NMR (75 MHz, DMSO-d6): ꢀ 157.8, 153.8, 151.3, Antibiofilm study
146.0, 127.5, 126.0, 120.5, 119.9, 119.5, 116.4, 114.6, 94.6, 37.8;
Detection of biofilm formation by tissue culture plate
MS m/z: 415.9168 [M]þ, 417.9169 [M þ 2]þ, 419.9171 [M þ 4]þ.
method. Tissue culture plate (TCP) assay described by
Christensen et al.19 is most widely used and was considered as
standard test for detection of biofilm formation. In present study,
we screened MRSA (ATCC 43866), MSSA (ATCC 35556) and
SE (ATCC 35984) bacterium for their ability to form biofilm by
TCP method as described by Christensen et al. with a modifi-
cation in duration of incubation, which was extended to 24 h.
Isolates from fresh agar plates were inoculated in respective
4,5-dibromo-N0-benzylidene-1-methyl-1H-pyrrole-2-carbohydra-
zide (4o). Yield: 86%; m.p.: 212–214 ꢀC; IR (KBr): Vmax/cm
1
1625 (C¼C), 1641 (CH¼N), 1711 (C¼O), 3214 (NH); HNMR
(300 MHz, DMSO-d6): ꢀ 3.56 (s, 3H pyrrole N-CH3), 6.42 (s, 1H
pyrrole 3H), 6.67–7.36 (m, 5H ArH), 8.42 (s, 1H azometine H),
11.62 (s, 1H CONH); 13C NMR (75 MHz, DMSO-d6): ꢀ 157.5,
146.7, 133.6, 131.1, 129.1, 128.7, 127.5, 126.1, 114.6, 94.6, 37.9;
media and incubated for 18 h at 37 ꢀC in stationary condition and
diluted 1 in 100 with tryptic soy broth (TSB). Individual wells of
MS m/z: 383.9270 [M]þ, 385.9273 [M þ 2]þ, 387.9276 [M þ 4]þ.
sterile, polystyrene, 96-well flat-bottom tissue culture plates were
filled with 0.2 mL aliquots of the diluted cultures and only broth
4,5-dibromo-N0-(4-hydroxy-3-methoxybenzylidene)-1-methyl-1H-
pyrrole-2-carbohydrazide (4p). Yield: 80%; m.p.: 248–250 ꢀC;
served as control to check sterility and nonspecific binding of
media.
The tissue culture plates were incubated for 18 h and 24 h at
IR (KBr): Vmax/cm 1627 (C¼C), 1649 (CH¼N), 1715 (C¼O),
3211 (NH), 3436 (OH); 1HNMR (300 MHz, DMSO-d6): ꢀ 3.16 (s,
3H OCH3), 3.58 (s, 3H pyrrole N-CH3), 5.34 (s, 1H OH) 6.47 (s,
37 ꢀC. After incubation, content of each well was gently removed
1H pyrrole 3H), 6.7–7.4 (m, 3H ArH), 8.49 (s, 1H azometine H),
11.60 (s, 1H CONH); 13C NMR (75 MHz, DMSO-d6): ꢀ 157.7,
151.2, 149.4, 146.9, 131.0, 127.5, 126.1, 122.9, 117.1, 114.6,
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
112.2, 94.6, 56.2, 37.6; MS m/z: 429.9326 [M]þ, 431.9328
‘‘sessile’’ organisms in the plate were fixed with sodium acetate
(2%) and stained with crystal violet (0.1% w/v). Excess stain was
[M þ 2]þ, 433.9330 [M þ 4]þ.
rinsed off by thorough washing with de-ionized water and plates
were kept for drying. Adherent staphylococcal cells usually
Antibacterial study
formed biofilm on all side wells and were uniformly stained with
crystal violet. Optical densities (ODs) of stained adherent bacteria
were determined with a micro ELISA auto reader (California) at
wavelength 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 were then averaged and standard deviation
was calculated. To compensate for background absorbance, OD
readings from sterile medium, fixative and dye were averaged and
subtracted from all test values. The mean OD value obtained from
The newly prepared compounds were screened for their minimal
inhibitory concentration (MIC), minimal bactericidal concentra-
tion (MBC) and antibiofilm concentration assay against MRSA
(ATCC 43866), methicillin-susceptible S. aureus (MSSA, ATCC
35556) and SE (ATCC 35984) bacterium using a standard tube-
dilution assay (Table 1). MIC assays for the antibacterial activities
of the compounds were performed according to the broth
microdilution (in tubes) method of the Clinical and Laboratory
Standards Institute (CLSI) of America18. MBC of the compounds
was obtained by subculturing 100 mL from each negative (no
Table 1. Antibacterial and antibiofilm test results*.
MSSA
S. epidermidis
antibiofilm
concentration
MIC (mg/mL)
MRSA
antibiofilm
concentration
MIC (mg/mL)
MSSA
MBC (MIC)
(mg/mL)
S. epidermidis
MBC (MIC)
MRSA
MBC (MIC)
(mg/mL)
Antibiofilm
concentration
MIC (mg/mL)
S. No.
R
(mg/mL)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
furan-2-yl-
1.56 (0.39)
3.125 (1.56)
3.125 (0.39)
3.125 (0.39)
3.125 (0.39)
3.125 (0.39)
12.5 (3.125)
3.125 (0.39)
3.125 (0.39)
3.125 (0.39)
3.125 (0.39)
3.125 (1.56)
1.56 (0.195)
3.125 (1.56)
3.125 (1.56)
3.125 (1.56)
3.125 (1.56)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
25 (12.5)
3.125 (1.56)
1.56 (0.78)
3.125 (1.56)
3.125 (1.56)
3.125 (1.56)
3.125 (1.56)
3.125 (1.56)
6.25 (3.125)
1.56 (0.78)
3.125 (1.56)
6.25 (3.125)
1.56 (0.78)
3.125 (1.56)
1.56 (0.78)
3.125 (1.56)
6.25 (3.125)
3.125 (1.56)
6.25 (3.125)
0.78
1.56
0.78
0.78
0.78
0.78
6.25
0.78
1.56
1.56
1.56
1.56
0.39
1.56
12
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
3.125
1.56
1.56
1.56
3.125
3.125
1.56
2,6-dichlorophenyl-
4-dimethyaminophenyl-
4-fluorophenyl-
4-nitrophenyl-
4-hydroxyphenyl-
cinnamyl-
4-chlorophenyl-
2-hydroxy-4-methoxyphenyl-
thiophen-2-yl-
4-methoxyphenyl-
3,4-dimethoxyphenyl-
4-(2,6-dichlorophenoxy)phenyl-
2,3-dihydroxyphenyl-
phenyl-
25
3.125
3.125
1.56
3.125
3.125
0.78
3.125
12.5
3.125
6.25
4o
4p
Standardy
4-hydroxy-3-methoxyphenyl
3.125
3.125
*The initiating dilution was 100 mM.
yFor all assays, standard used was vancomycin.

DOI: 10.3109/14756366.2013.793183
Design, synthesis and antistaphylococcal activity of marine pyrrole alkaloid derivatives 405
Table 2. Screening of various staphylococcal strains for biofilm formation by TCP method in tryticase soy broth with 1% glucose
at 18 and 24 h of incubation.
No. of isolates
MRSA (ATCC 43866)
MSSA (ATCC 35556)
Incubation time (hours)
SE (ATCC 35984)
Biofilm formation (OD570 nm
)
18
24
18
24
18
24
High (40.240 ꢁ 0.020)
1
6
54
4
8
62
12
21
92
17
28
92
5
11
64
7
18
70
Moderate (0.120–0.240 ꢁ 0.024)
Weak/non (50.12 ꢁ 0.014)
media control well was deducted from all the test OD values
(Table 2).
(a) 10
8
6
4
2
0
Antibiofilm assay
In the antibiofilm concentration experiment, overnight culture of
bacterium (MRSA (ATCC 43866), MSSA (ATCC 35556) and SE
(ATCC 35984) was diluted 1:10 in TSB (OD 600 ¼ 0.6–0.8), then
was diluted 1:200 in Mueller–Hinton. The bacterial suspension
was inoculated into the wells of sterile 96-well polystyrene
microtiter plates (Falcon) incubated at 37 ꢀC for 6 h. The plates
with young biofilm were washed gently four times with sterile
PBS before adding fresh TSB containing the various concentra-
tions of compounds and incubated at 37 ꢀC for 16 h. The initiate
dilution was 200 mM. MIC values were defined as the lowest
concentration at which no visible growth is observed. For all
assays, reference standard used was vancomycin20.
Concentration in µg/mL
(b) 10
In order to study effectiveness of most active compound 4m on
older biofilm (24 h) of MSSA, MRSA and SE, log reduction in the
viable count was determined according to Filoche et al.21 with
some modifications. In brief, 100 mL of nutrient medium and
10 mL of microorganism cultures were placed in 96-well plates
(CellstarÕ) and incubated for 24 h at 37 ꢀC in order to establish
biofilm formation. Following growth (24 h), the supernatant was
removed and each well was rinsed with sterile saline three times.
Subsequently, the first experimental well was filled with double-
strength nutrient medium, while the other wells were filled with
single-strength nutrient medium (150 mL). The ethanol extract or
volatile oil (150 mL) was added to the first well. Positive and
negative controls were used. Double-fold serial dilution was then
carried out across the plate and the plates were incubated for 24 h
at 37 ꢀC. After incubation, cultures were removed from the wells
showing no turbidity and wells were rinsed three times with
sterile saline to remove unattached bacteria. Attached cells were
removed and quantified by viable cell counts. In order to do this,
attached cells were removed from wells’ surface using a sterile
needle. This step was validated by staining the recovered wells
with crystal violet (1%) and visual examination after rinsing three
times with saline. Viable counts were performed using 10-fold
serial dilution. Attached microbial populations were expressed as
colony forming unit per well (CFU/well). The method for each
strain was carried out in triplicate and the standard mean of error
was calculated. The graphical result for log reduction of CFU/mL
versus concentration is presented in Figure 2(a, b and c).
8
6
4
2
0
Concentration in µg/mL
(c) 10
8
6
4
2
0
Concentration in µg/mL
Results and discussion
Figure 2. (a) Evaluation of antibiofilm activity of compound 4m by
Log reduction in viable counts of MSSA (ATCC 35556). (b) Evaluation
of antibiofilm activity of compound 4m by Log reduction in
viable counts of MRSA (ATCC 43866). (c) Evaluation of antibiofilm
activity of compound 4m by Log reduction in viable counts of SE
(ATCC 35984).
Chemistry
In this study, 1-methyl-4,5-dibromo-1H-pyrrole-2-carbohydrazide
derivatives were synthesized utilizing the reaction sequence, as
shown in Scheme 1. Trichloroacetylation of 1-methylpyrrole gave

406 R. A. Rane et al.
J Enzyme Inhib Med Chem, 2014; 29(3): 401–407
Scheme 1. Synthesis of N0-arylidene-4,5-dibromo-1-methyl-1H-pyrrole-2-carbohydrazides.
an excellent yield of compound 122. Compound 1 was brominated substituted phenyl ring with oxygen linker that adds drug-like
using bromine in chloroform to give compound 223. Compound 2 features to it. Next, the molecules with substitution at ortho- or
was further converted into corresponding hydrazide compound 3 meta-position of aromatic rings did not exhibit increased potency
by stirring with hydrazine hydrate at room temperature. relative to para-substituted molecules. Further, heteroaromatic ring
Compound 3 on condensation with respective aldehydes gave system was incorporated as in compounds 4a and 4j instead of
4,5-dibromo-1-methyl-1H-pyrrole-2-carbohydrazides, i.e. com- phenyl ring. Both these derivatives showed better activity
1
pound 4. The spectral data (infra red (IR), H NMR and mass indicating that a suitable substituted heteroaromatic ring system
spectra (MS)) of all synthesized compounds were in agreement can increase the potency of the compounds. Increasing the length of
with the proposed structures. The MS of all the compounds aroyl hydrazone linker using cinnamyl substituent decreases the
exhibited the [Mþ] as molecular ion peak, confirming the antibiofilm activity relative to compound 4o.
molecular weight. Presence of dibromo was confirmed with the
Antibiofilm activity of compounds 4a, 4c, 4d, 4e, 4f and 4h
presence of [M þ 2]þ, [M þ 4]þ peaks in mass spectra. The H- was 3-fold superior (MIC ¼ 0.78 mg/mL) to that of standard
NMR spectra of all compounds 4a–4p showed single at ꢀ 3.5 vancomycin (MIC ¼ 3.125 mg/mL) against MSSA, while that of
corresponding to N-CH3 attached to pyrrole core. Proton at third compounds 4b, 4i, 4j, 4k, 4l and 4n (MIC ¼ 1.56 mg/mL) was
position of pyrrole core resonates at ꢀ6.4. N-H proton of 2-fold superior than MSSA. For antibiofilm activity against
CONHN¼CH group resonated in between ꢀ11.7 and 11.5. The MRSA; compounds 4a, 4b, 4c, 4f and 4j exhibited 3-fold potency
azometine proton to which aromatic ring B is attached showed a (MIC ¼ 1.56 mg/mL) compared to standard vancomycin
singlet at around ꢀ 8.5–8.3. The aryl protons of ring B resonated (MIC ¼ 6.25 mg/mL), whereas activity of compounds 4d, 4e, 4h,
1
in the range of ꢀ 7.9–6.6.
4i, 4k, 4l, 4n and 4p was found to be 2-fold superior to that of
standard. Hybrid compound (4m) displayed four times the
potency relative to standard vancomycin against biofilms of
MSSA and MRSA with MIC of 0.39 mg/mL and 0.78 mg/mL,
respectively. Further, the antistaphylococcal potential of most
active hybrid compound (4m) was validated by determining
antibacterial and antibiofilm MICs of its intermediates. The
antibacterial and antibiofilm MICs obtained for 1-methyl-1H-
pyrrole-2-carbohydrazide compound 3 and 4-(2,6-dichlorphe-
noxy)-benzaldehyde against all tested bacterium were ꢂ100 mg/
mL. All the tested compounds showed less antibiofilm activity
against SE (MIC ¼ 25 mg/mL) compared to standard vancomycin.
All the compounds exhibited similar antibacterial profile on
planktonic and biofilm stages, which can be beneficial as it
increases their spectrum of activities.
Biological results
The synthesized series of compounds showed some promising
antibiofilm activity especially against MSSA and MRSA. As
indicated in Table 1, most of synthesized compounds showed
potent to moderate antibacterial and antibiofilm activity against all
tested Gram-positive bacterium. These derivatives are more active
against biofilm produced by MRSA and MSSA compared toSE. All
tested compounds exhibited equal or more antibiofilm potency
against MSSA and MRSA compared to standard vancomycin,
except compounds 4g and 4o. In general, substitution of benzene
ring at 4-position is favorable for antibacterial and antibiofilm
activity. Substituents likes fluoro, chloro, nitro, hydroxyl, methoxy
and 4-(2,6-dichlorophenoxy)phenyl placed at 4-postion of benzene
ring, resulted in favorable effect on antibiofilm activity relative to
the unsubstituted compound 4o. Herein, 4-(2,6-dichlorophenox-
Conclusion
y)phenyl-substituted compound 4m was more active compared to In conclusion, 16 novel hybrids of aroyl hydrazone and marine
other compounds (MIC: 0.39 mg/mL MSSA, 0.79 mg/mL MRSA). bromopyrrole alkaloids were designed, synthesized and evaluated
Activity of compound 4m may be attributed to the presence of para- for their antistaphylococcal activity. The synthesized hybrids

DOI: 10.3109/14756366.2013.793183
Design, synthesis and antistaphylococcal activity of marine pyrrole alkaloid derivatives 407
prostheses to silicone rubber by rhamnolipid biosurfactants. J Appl
Microbiol 2006;100:470–80.
9. Bryers JD, Ratner BD. Bioinspired implant materials befuddle
bacteria. ASM News 2004;70:232–7.
10. Aiello A, Fattorusso E, Menna M, Taglialatela-Scafati O. Modern
alkaloids structure, isolation, synthesis and biology. Weinheim:
Wiley-VCH; 2008:271.
11. Tasdemir D, Topaloglu B, Perozzo R, et al. Marine natural
products from the Turkish sponge Agelas oroides that inhibit the
enoyl reductases from Plasmodium falciparum, Mycobacterium
tuberculosis and Escherichia coli. Bioorg Med Chem 2007;15:
6834–45.
showed promising activity against MSSA and MRSA. Biological
data revealed that substitution of phenyl ring at 4-postion is
favorable for antibiofilm activity against MSSA and MRSA. The
antibiofilm activity at concentrations as low as 0.39 mg/mL and
0.78 mg/mL shown by compound 4m against MSSA and MRSA,
respectively, indicates that this compound can act as novel lead for
development of more potent antistaphylococcal agent. Further
studies on these compounds and optimization of their structures
leading to novel analogs with superior antimicrobial properties
are ongoing in our laboratories.
12. Xiong S, Pang HD, Fan J, et al. In vitro and in vivo antineoplastic
activity of a novel bromopyrrole and its potential mechanism of
action. Br J Pharmacol 2010;159:909–18.
13. Schillaci D, Petruso S, Cascioferro S, Raimondi MV. In vitro anti-
Gram-positive and antistaphylococcal biofilm activity of newly
halogenated pyrroles related to pyrrolomycins. Int J Antimicrob
Agents 2008;31:380–99.
14. Rane RA, Gutte SD, Sahu NU. Synthesis and evaluation of novel
1,3,4-oxadiazole derivatives of marine bromopyrrole alkaloids as
antimicrobial agent. Bioorg Med Chem Lett 2012;22:6429–32.
15. Rane RA, Sahu NU, Shah CP. Synthesis and antibiofilm activity
of marine natural product-based 4-thiazolidinones derivatives.
Bioorg Med Chem Lett 2012;22:7131–4.
Acknowledgements
R.A.R. thanks Professor R. S. Gaud for providing facility to carry out
these experiments.
Declaration of interest
There is no conflict of interest.
References
1. Leclercq R. Epidemiological and resistance issues in multidrug-
resistant staphylococci and enterococci. Clin Microbiol Infect 2009;
15:224–31.
16. Rollas S, Ku¨c¸u¨kgu¨zel S¸G. Biological activities of hydrazone
derivatives. Molecules 2007;12:1910–39.
17. Junker LM, Clardy J. High-throughput screens for small-molecule
inhibitors of Pseudomonas aeruginosa biofilm development.
J Antimicrob Agents and Chemother 2007;51:3582–90.
18. Matthew A, Cockerill FR, Craig WA, et al. Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically;
approved standard, M7-A7. 7th ed. Vol. 26. Wayne, PA, USA:
Clinical and Laboratory Standards Institute; 2006: 1–49.
19. Christensen GD, Simpson WA, Younger JA, et al. Adherence of
cogulase negative Staphylococi to plastic tissue cultures: a quanti-
tative model for the adherence of staphylococci to medical devices.
J Clin Microbiol 1985;22:996–1006.
20. Pan B, Huang R, Han S, et al. Design, synthesis, and antibiofilm
activity of 2-arylimino-3-aryl-thiazolidine-4-ones. Bioorg Med
Chem Lett 2010;20:2461–4.
21. Filoche SK, Soma K, Sissons CH. Antimicrobial effects of essential
oils in combination with chlorhexidine digluconate. Oral Microbiol
Immunol 2005;20:221–5.
2. Reed SD, Friedman JY, Engemann JJ, et al. Costs and outcomes
among hemodialysis-dependent patients with methicillin-resistant or
methicillin-susceptible Staphylococcus aureus bacteremia. J Infect
Control Hosp Epidemiol 2005;26:175–83.
3. Mohan SS, McDermott BP, Cunha BA. Methicillin-resistant
Staphylococcus aureus prosthetic aortic valve endocarditis with
paravalvular abscess treated with daptomycin. Heart Lung 2005;34:
69–71.
4. Mermel LA. Prevention of intravascular catheter-related infections.
Ann Intern Med 2000;132:391–402.
5. Raad I, Hanna H, Jiang Y, et al. Comparative activities of
daptomycin, linezolid, and tigecycline against catheter-related
methicillin-resistant staphylococcus bacteremic isolates embedded
in biofilm. Antimicrob Agents Chemother 2007;51:1656–60.
6. Cirioni O, Giacometti A, Ghiselli R, et al. Prophylactic efficacy of
topical temporin A and RNAIIIinhibiting peptide in a subcutaneous
rat Pouch model of graft infection attributable to staphylococci with
intermediate resistance to glycopeptides. Circulation 2003;108:
767–71.
22. Bailey DM, Johnson RE, Salvador JU. Pyrrole antibacterial agents.
1. Compounds related to pyoluteorin. J Med Chem 1973;16:
1298–300.
7. Burton E, Gawande PV, Yakandawala N, et al. Antibiofilm activity
of GlmU enzyme inhibitors against catheterassociated uropathogens. 23. Linington RG, Williams DE, Tahir A, et al. Latonduines A and B,
Antimicrob Agents Chemother 2006;50:1835–40.
8. Rodrigues LR, Banat IM, van der Mei HC, et al. Interference in
adhesion of bacteria and yeasts isolated from explanted voice
new alkaloids isolated from the marine sponge Stylissa carteri:
structure elucidation, synthesis, and biogenetic implications.
Org Lett 2003;5:2735–8.