Synthesis, structure, photophysical, electrochemical properties and antibacterial activity of brominated BODIPYs as an inhibitor of DNA gyrase B of S. aureus

Article abstract of DOI:10.1142/S1088424619500433

BODIPYs with 3-thienyl and 4-acetamido phenyl groups substituted at the meso-position are subjected to regioselective bromination using three equivalents of N-bromosuccinimide (NBS) to yield their 2-mono and 2,6-di bromoderivatives. Their photophysical, e

Full text of DOI:10.1142/S1088424619500433

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2019; 23: 1–10
DOI: 10.1142/S1088424619500433
Published at http://www.worldscinet.com/jpp/
Synthesis, structure, photophysical, electrochemical
properties and antibacterial activity of brominated
BODIPYs as an inhibitor of DNA gyrase B of S. aureus
a
b†
a
Dijo Prasannan , Chennakkandathil Sareena , Chellaiah Arunkumar
b
and Suchithra Tharamel Vasu *
a
Bioinorganic Materials Research Laboratory, Department of Chemistry, National Institute of Technology Calicut,
NIT Campus, P.O., Calicut, India-673 601
School of Biotechnology, National Institute of Technology Calicut, NIT Campus, P.O., Calicut, India-673 601
b
Received 8 December 2018
Accepted 28 February 2019
ABSTRACT: BODIPYs with 3-thienyl and 4-acetamido phenyl groups substituted at the meso-
position are subjected to regioselective bromination using three equivalents of N-bromosuccinimide
(
NBS) to yield their 2-mono and 2,6-di bromoderivatives. Their photophysical, electrochemical
and antimicrobial properties are investigated. This paper presents a mechanistic investigation of the
antibacterial effect of brominated BODIPYs, particularly against Staphylococcus aureus. Fluorescence
microscopic images reveal that the compounds are internalized effectively within the bacterial cells,
making it an ideal antibacterial drug. Morphological analysis of the bacterial cells after the treatment
with the test compounds showed that the compounds did not affect the cell membrane or cell wall and
the antibacterial effect of these compounds is achieved via a different mechanism. The most effective
compound was selected to explore the target of action. Molecular docking studies were performed on
2
2 selected proteins in S. aureus and the in silico results were validated by in vitro experiments. It was
observed that the supercoiling activity of DNA gyrase was completely inhibited by the 2,6-dibromo-
,3,5,7-tetramethyl-8-(4-acetamido)-4-bora-3a,4a-diaza-s-indacene, 3c by forming H-bonds with the
1
ASP 81 residue of the enzyme.
KEYWORDS: BODIPYs, DNA gyrase B, Staphylococcus aureus, antibacterial studies, docking,
fluorescence imaging.
flexible functionalization nature have a distinctive
INTRODUCTION
position [1]. BODIPYs have become one of the
cornerstones in biotechnology, due to their prospective
applications in biomolecular labeling [2], medical
diagnosis, drug delivery [3], cellular pH [4] andviscosity
sensing [5]. Their high tissue penetration with minimal
autofluorescence from biological samples makes them
good candidates for use in biology. The biological
properties of BODIPYs, particularly their antimicrobial
and anticancer properties, have been well reported in
literature [6–8]. BODIPYs featuring iodine atoms on the
2,6-positions used in in vitro photodynamic antimicrobial
chemotherapy (PACT) have been proven to be effective
against S. xylosus and E. coli under mild conditions
[9]; antimicrobial activity of 2,6-diiodo-substituted
Out of the numerous available flurophores such as
rhodamine, fluorescein, phthalocyanine and squaraine,
4
,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPYs)
dyes with their superior photophysical properties and
*
Correspondence to: Suchithra Tharamel Vasu, School of
Biotechnology, National Institute of Technology Calicut, NIT
Campus P.O., Calicut, India-673 601, tel.: +91 (495) 2285318
(
lab), +91 9744244461 (mobile), fax: +91 (495) 2287250,
email: drtvsuchithra@nitc.ac.in
†
b
Current address: School of Biotechnology, National Institute
of Technology Calicut, NIT Campus P.O., Calicut, India-673
6
01, tel.: +91 (495) 2285318 (lab), +91 9744244461 (mobile),
fax: + 91 (495) 2287250.
Copyright © 2019 World Scientific Publishing Company

2
D. PRASANNAN ET AL.
Scheme 1. Synthesis of brominated BODIPYs
BODIPY has been reported against P. aeruginosa [10].
Halogenated BODIPY dyes due, to their heavy atom
effect, display efficient intersystem crossing (ISC) and
act as photosensitizers to exhibit photocytotoxicity [11,
activity of cationic BODIPY derivatives is well des-
cribed [9, 20, 21]; hence the need for investigating the
mechanism of bacterial cell disruption in uncharged
BODIPY species is also necessary. Here, we explore
the mechanism of action via in silico analysis, and this
has been validated by in vitro studies. To the best of
our knowledge, this paper reports for the very first time
2,6-dibromo BODIPY as a novel inhibitor of DNA gyrase
of S. aureus, and we anticipate that the compound 3c is a
promising lead structure for various medical applications.
12]. Heavy atoms like bromine could induce triplet-state
formation, and the generation of reactive singlet oxygen
species has been the key cytotoxic agent in the therapeutic
process, fostering cell death by an apoptotic and/or
necrotic mechanisms [13]. Apart from this key cytotoxic
mechanism, no other well-established therapeutic
mechanism has been reported to foster cell death in
presence of halogenated BODIPYs. Hence, it is very
interesting to report here the bioactivity of brominated
BODIPYs where the photosensitizing property is not
responsible for their antibacterial properties.
The emergence of multidrug-resistant strains of
pathogens demands strategies for developing antibacterial
agents with novel mechanisms of action. Cellular
microenvironments are complex, thus identifying the
exact mode and target leading to intracellular toxicity
is a topic of investigation. Among the different targets
identified, DNA gyrase is a well-known pharmacological
target for antibacterial drugs because it has not been found
in eukaryotes. This prokaryotic type II topoisomerase
consists of the subunits GyrA and GyrB, with different
functional domains. The GyrA subunit is principally
involved in DNA breakage and recombination of
the supercoiling reaction, whereas the GyrB subunit
is responsible for the ATP hydrolysis reaction. The
derivatives of dibromopyrrolamides, including coumarin,
thiophene, pyrazole and quinolone, are potent against
gram-positive strains and are well established [14–17].
The rise of methicillin-resistant Staphylococcus aureus
explains the current relevance of new classes of DNA
gyrase inhibitors [18].
RESULTS AND DISCUSSION
The precursors, dipyrromethane and BODIPYs, were
obtained in fairly good yield, 52–68% [13]. Bromination
reactions were done according to procedures from the
literature to afford 2,6-dibromo derivatives as the major
product [13]. The overall synthetic route is outlined in
Scheme 1. The structures were confirmed by IR, NMR
spectroscopy and mass spectrometry, as presented in the
Supplementary information, Figs S1 to S20. The B–F,
B–N, and –C=N, of the BODIPY core were spotted as IR
-1
vibrations near 1532, 1400, and 1734 cm respectively.
The β–H of pyrrole ring [13], observed near 775 and
-1
771 cm in 1a and 1c, was absent in corresponding
dibrominated product 1b. The broad peak at 7.86 ppm
and one proton singlet at 5.51 ppm respective of –NH
and meso-proton in dipyrromethane 1 were absent in
BODIPY 1a. The absence of the two proton intensities
respective of the β-pyrrole proton signals indicates
successful dibromination at 2,6-pyrrolic position [13].
Photophysical properties
The photophysical properties of the synthesized
BODIPY derivatives are summarized in Table 1. With
increasing numbers of bromine atoms, a systematic batho-
chromic shift is observed in both absorption and emission
profiles. The S –S transition at 505 nm for compound
The present work focuses on the antibacterial activity
of 2,6-dibromo BODIPYs with a representative member
of gram-positive bacteria, S. aureus. As the electronic
structure of a molecule has a key role in inducing toxicity,
BODIPYs containing a thiophene ring and an amide
group were selected for the study [19]. The antimicrobial
0
1
1a in dichloromethane is red shifted by 13 nm with the
incorporation of one bromine atom at β-positions; 518 nm
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SYNTHESIS, STRUCTURE, PHOTOPHYSICAL, ELECTROCHEMICAL PROPERTIES AND ANTIBACTERIAL ACTIVITY
3
Table 1. Absorption and emission properties of compounds 1–2 (a–c), 3a and 3c in DCM, at λex = 480 nm and slit width, 9. The
molar extinction coefficient (e) and fluorescence quantum yields (F ) of compounds were calculated in toluene. Fluorescein in
f
aqueous NaOH (0.1 N, F = 0.90) at λ = 480 nm was used as the reference
f
ex
Compound
λabs (nm)
λem (nm)
Ff
log e
Maximum brightness
Singlet oxygen
quantum yield (FD)
Phototoxic power (PP)
3
-1
.
-1
F × e/10 M cm
1
a
502
518
543
518
543
567
0.23
0.09
0.04
4.80
4.62
4.32
14.48
3.74
0.75
0.10
0.63
0.26
6310
26263
5432
1
b
1
c
2
a
502
516
0.05
4.58
1.82
0.12
4562
2
b
518
540
502
529
548
568
510
542
0.07
0.03
0.42
0.13
4.78
4.73
4.76
4.96
1.63
4.36
0.64
0.88
0.10
0.97
38564
47259
5754
2
c
3
a
24.58
12.48
3
c
88465
1
.2 x 10-5
for 2-mono-(1c) and 543 nm for 2,6-di-bromo (1b) deri-
1
a
b
vative. The geometries of the ground state (S ) and the first
1.0 x 10-5
0
1
excited state (S ) are similar; hence the emission spectrum
1
0
0
0
0
.8 x 10-5
1c
of the BODIPY is the mirror image of the S to S of the
0
1
.6 x 10-5
dye. No notable solvatochromic effect was observed
for absorption or emission with solvents of varying
refractive indices, Table S1. Halogenated BODIPYs are
well known for singlet oxygen production by heavy atom
.4 x 10-5
.2 x 10-5
0.0
effects; the singlet oxygen quantum yield (F ) computed
D
are comparable to that of recent reports in the literature
0.2 x 10-5
-
[
22]. The singlet oxygen production in brominated
-
0.4 x 10-5
BODIPYs is proven by diphenylisobenzofuran (DPBF)
titration [13]. When DPBF is irradiated in the presence of
BODIPYs, the singlet oxygen ( O ) species generated in
-0.6 x 10-5
0.0
-0.5
-1.0
-1.5
-2.0
1
2
Potential (V)
the presence of BODIPYs deplete DPBF molecules and
can be followed by measuring the decrease in absorbance
of DPBF at 410 at regular irradiation intervals. The plot
of absorbance vs. irradiation time reflects the singlet
oxygen yield of the compounds as shown in Fig. S21. The
phototoxic power (PP) of the BODIPY molecules are also
shown in Table 1 [13, 23, 24]. As there are many factors
contributing to the complete antimicrobial efficacy,
a higher PP value alone does not guarantee effective
antibacterial efficacy. Photo images and representative
normalized optical and fluorescence (dash) profiles of
BODIPYs, 1a, 1b and 1c in toluene at room temperature
are shown in the Supplementary information, Figs S22
and S23 respectively.
Fig. 1. Cyclic voltammogram of BODIPYs 1a–1c in dichloro-
methane containing 0.1 M tetrabutylammonium hexafluoro
phosphate as the supporting electrolyte at 100 mV/s
with the increase of the number of bromine atoms on the
β-position [13] as shown in Table 2.
Single crystal X-ray diffraction studies
Compounds, 1b, 2a, 3a and 3c, crystallized in acetone/
cyclohexane, chloroform/hexane and chloroform/cyclo
hexane respectively, are characterized by X-ray crystal
structure analyses and the data are presented in Table 3.
.
Compound 3a was crystallized in orthorhombic (3a ꢀ
Electrochemical studies
.
.
H O) and triclinic (3a ꢀDMF ꢀCH COOH) crystal sys-
2
3
Electrochemical studies were performed to analyze
the effect of bromine groups on the BODIPY core,
and the cyclic voltammograms of all BODIPYs show
one reversible reduction and one irreversible reduction.
A representative voltammogram of 1a–1c is shown in
Fig. 1. A successive anodic shift in the reduction wave
potential is observed in all the series 1–2 (a–c), 3a and
tems with different solvent molecules in the crystal
lattices. The molecular structures of compounds, 1b,
2a, 3a and 3c are shown in Fig. 2. The mono-bromo
(1b) and di-bromo (3c) derivatives are well stabilized by
two types of interactions, namely, (thienyl)C∙∙∙Br (3.448 Å),
(thienyl)H∙∙∙F (2.479 Å) and (phenyl)C∙∙∙Br (3.451 Å) and
(phenyl)H∙∙∙F (2.560 Å) respectively as shown in Figs 2a
and 2e. Moreover, the existence of strong hydrogen
3
c, indicating that the BODIPY becomes easier to reduce
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4
D. PRASANNAN ET AL.
Table 2. Electrochemical data of dyes, 1a–3c
Compound Reduction potential (V)
largest contribution to the Hirshfeld surfaces in each
crystal, as presented in Fig. 3.
I
II
Antibacterial properties
1
a
-0.84
-0.70
-0.55
-0.85
-0.75
-0.61
-1.25
-1.02
-1.80
-1.68
-1.48
-1.83
-1.67
-1.54
-1.72
-1.84
All the compounds (1 mg/mL) were tested for
antimicrobial activity against S. aureus under light and
dark conditions, Table 4 [13]. The percentage zone
of inhibition observed in agar well diffusion for 1c,
1
b
1
c
2
a
2
b, 2c and 3c is promising and is comparable to that
2
b
of the commercially available antibiotic vancomycin.
Interestingly, the antibacterial activities were comparable
under light and dark conditions, which clearly indicate
that it is not the photosensitizing activity of brominated
BODIPYs which is responsible for the antibacterial
activity. Hence, further evaluation of antibacterial activity
was done under ambient light conditions. The minimum
inhibitory concentration (MIC) was determined using
the broth dilution method in 96-well microtitre plates
as well as a resazurin reduction assay [25, 26]. Both
the assays showed same values of MIC, and the order
is, 1b ≈ 1c > 2b ≈ 2c > 3c; the highest being observed
for unbrominated derivatives (1a, 2a and 3a), 100 mg/mL
and the lowest, 20 mg/mL for 3c as shown in Table 4.
The presence of two bromine atoms and an amide
group in the molecule may impart a synergistic effect to
2
c
3
a
3
c
bonding in two of the crystal structures of 3a is one of
the key factors for stabilizing the molecules (2.574 Å
in 3a ꢀH O and 2.202 Å in 3a ꢀDMF ꢀCH COOH). The
interactions present in the crystals of 1b, 2a, 3a and 3c
were quantified using Crystal Explorer 3.1. Hirshfeld
surface analysis provides a 3D picture of close contacts
present in the crystals which are summarized in a 2D
fingerprint plot, Fig. S24. The H–H (23–51%), C–H
.
.
.
2
3
(
6.5–18%) and H–F (11.1–21%) interactions make the
Table 3. Crystal data and structure refinement parameters for compounds, 1b, 2a, 3a and 3c
.
.
.
Parameters
1b
2a
3a ꢀH O
3a ꢀDMF ꢀCH COOH
3c
2
3
Molecular formula
Formula weight
CCDC number
Temperature/K
Crystal system
Space group
λ/Å
C H BBrF N S
C H BF N O
C H BF N O
C H B F N O
C H BBr F N O
13
8
2
2
17 14
2
3
21 24
2
3
2
47 55
2
4
7
5
21 20
2
2
3
352.99
1577779
293 (2)
325.12
1022866
296 (2)
Triclinic
P-1
399.24
1033432
296 (2)
895.60
1058023
293 (2)
539.03
1577778
293 (2)
Monoclinic
Orthorhombic
P2 2 2
Monoclinic
Monoclinic
P2 /n
1
P2 /c
1
P2 /n
1
1
1
1
0.71073
10.5117 (8)
7.2016 (6)
0.71073
0.71073
9.3409 (7)
9.5069 (6)
23.1828 (17)
90
0.71073
10.7156 (14)
45.965 (5)
9.4560 (12)
90
0.71073
17.2407 (7)
7.8770 (4)
17.4257 (9)
90
a/Å
9.6236 (4)
13.0176 (5)
13.3553 (6)
109.777 (2)
93.596 (2)
95.930 (2)
1557.44 (11)
4
b/Å
c/Å
17.4185 (15)
90
a (°)
β (°)
95.658 (3)
90
90
93.827 (3)
90
113.9710 (10)
90
g (°)
90
3
Volume/Å
1312.17 (19)
4
2058.7 (3)
4
4647.1 (10)
4
2162.39 (18)
4
Z
-
3
Densitycalcd /mg m
F (000)
1.787
1.387
1.288
1.280
1.656
696
672
840
1888
1072
q range (°)
2.350 to 25.000
2314/181
1.182
2.551 to 28.315
7442/436
0.990
1.76 to 25.99
4046/280
1.069
2.101 to 25.000
7902/676
2.157 to 24.996
3807/275
1.024
Data/parameters
2
GOF on F
1.064
a
1
b
2
R , wR [I > 2sꢀ(I)]
0.0421, 0.0995
0.0512, 0.1167
0.0330, 0.0810
0.0785, 0.1745
0.0331, 0.0794
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SYNTHESIS, STRUCTURE, PHOTOPHYSICAL, ELECTROCHEMICAL PROPERTIES AND ANTIBACTERIAL ACTIVITY
5
.
.
.
Fig. 2. Molecular structures (40% probability level) of (a) 1b, (b) 2a, (c) 3a ꢀH O, (d) 3a ꢀD MF ꢀC H COOH and (e) 3c
2
3
improve the antibacterial efficacy of the compound. This
is reflected in the MIC value. The minimum bactericidal
concentration (MBC) [27] values computed by time-kill
assay also showed lower MBC values, 60–80 mg/mL
for brominated derivatives. The brominated BODIPY
derivative 3c which shows fairly good MIC and MBC
was chosen for further study.
superoxides and hydroxyls were examined. The presence
of superoxide radicals and hydroxyl radicals (HO )
·
was assayed by the reduction of p-nitrobluetetrazolium
chloride (NBT) and terephthalic acid, respectively [30].
However, under these experimental conditions, the
presence of such radicals was not detected. Nevertheless,
·
OH has an exceedingly short lifetime; as soon as they
The mode of action of BODIPY as antimicrobial
photodynamic therapeutic agent is well reported [28].
However, in the present study, the antibacterial effect was
observed in dark; hence raising a need to investigate the
role of other reactive radicals in cell depletion. The free-
radicals generated during the excitation of fluorescent
BODIPY molecules react with surrounding cellular
components and induce toxicity in living cells [29]. Such
reactions being oxygen-dependent, radical species such as
are formed, they may react with DMSO in water to form
·
·
CH and CH S(OH)(O ). This possibility cannot be ruled
3
3
out; hence more detailed studies are required and are is
underway.
In silico modeling and docking studies were performed
using the Schrodinger 10.4 MacroModel [31]. The protein
target for compound 3c in S. aureus was retrieved from
the Kyoto Encyclopedia of Genes and Genomes (KEGG)
database resource and previous reports in the literature, in
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D. PRASANNAN ET AL.
H-F
C-C
H-H
H-O
C-H
H-S
C-N
H-N
Others
3
c
3
a⋅H O
2
3
a⋅DMF⋅CH COOH
3
2
a
1
b
0
20
40
60
80
100
%
Interaction
.
.
.
Fig. 3. Percentage contribution of various interactions of compounds, 1b, 2a, 3a ꢀH O, 3a ꢀD MF ꢀC H COOH and 3c
2
3
Table 4. Percentage zone of inhibition, MIC and MBC values
of BODIPYs against S. aureus
[32]. Certain antibacterials inhibit gyrase-catalyzed
supercoiling of DNA by inhibiting the gyrase ATPase
reaction by competing with ATP for binding to GyrB.
Their binding site lies in the 24-kDa amino terminal sub-
domain of GyrB. The DNA gyrase inhibitory activity of
Compound
Zone of inhibition (%)
MIC
MBC
(mg/mL) (mg/mL)
Light
Dark
3
c was assessed in an in vitro supercoiling assay by gel
1
a
14.44
17.78
19.44
13.89
21.67
22.22
14.44
18.89
21.00
12.00
16.11
20.00
12.00
15.56
17.78
14.44
15.55
21.00
60
40
40
80
40
40
80
20
10
100
80
electrophoresis [33]. It was observed that the supercoiling
activity was inhibited completely by the compound
even at MIC concentration, 20 mg/mL, showing that 3c
is a potent inhibitor of the enzyme in vitro as shown in
Fig. 4c.
The BODIPY, being neutral, easily penetrates cell
membranes and is likely to accumulate into subcellular
membranes due to its lipophilicity. The fluorescence
microscopic images shown in Fig. 5c show complete
internalization of the compound inside the cells and con-
firm that the antimicrobial activity of the test compound
1
b
1
c
80
2
a
100
60
2
b
2
c
60
3
a
100
60
3
c
Vancomycin
20
3
c against S. aureus is due to their efficient uptake by the
bacterial cells. In addition, the morphological changes
induced by 3c on S. aureus was evaluated by scanning
electronic microscopy (SEM) [34], as shown in Figs 5d
and 5e. The images clearly showed regular well defined
clustered and smooth surfaced S. aureus cells and no
difference in morphology was observed between treated
and untreated cells. Hence, it could be inferred that due to
exposure of the compound 3c, the membrane morphology
of the bacterial cells is unaffected.
ADMET properties (absorption, distribution, meta-
bolism, excretion and toxicity) evaluate the drug likeness
of a molecule based on a set of in silico guidelines [35].
Here, ADMET predicts optimum PhysChem and oral
absorption properties for the molecule 3c. The results
are well in accordance with RO5, Veber and Egan rules
as shown in Figs 6a and 6b and Table 5. However,
according to the Pfizer rule, 3c shows cytotoxicity in in
order to understand the mode of interaction between 3c
and target proteins in S. aureus as shown in Table S2. The
molecule 3c exhibited a maximum glide score of -4.89
with DNA gyrase B (Table S2). The H bond between the
–NH group of 3c and the ASP 81 residue of the enzyme
was the major stabilizing interaction between the ligand
and the enzyme in its active site, Figs 4a and 4b. Positi-
vely charged and polar interactions also play a role in the
binding of ligand 3c into the active site of the protein.
Hydrophobic interaction from residues surrounding the
ligand is yet another major main stabilizing factor for the
complex.
The in silico results were further affirmed by in vitro
experiments with 3c and the purified protein. The type
II topoisomerase DNA gyrase can introduce negative
supercoils into DNA at the expense of ATP hydrolysis
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SYNTHESIS, STRUCTURE, PHOTOPHYSICAL, ELECTROCHEMICAL PROPERTIES AND ANTIBACTERIAL ACTIVITY
7
Fig. 4. (a) 2D, (b) 3D images of docking 3c with DNA gyrase B subunit of S. aureus and (c) Gel Electrophoresis showing the
inhibition of supercoiling activity of DNA gyrase of S. aureus at different concentrations of the test compound; lane C-1-negative
control (relaxed DNA alone), lane C-2-positive control (relaxed DNA + enzyme), lane a–c (relaxed DNA + enzyme + different
concentrations of the compound, 3c (a) 20 (b) 40 and (c) 60 mg/mL
Fig. 5. Fluorescence microscopic images of bacterial cells at 100x showing the uptake of 3c by S. aureus cells (a) untreated cells
(
(
b) bright field image and (c) cells treated with 3c. Scanning electron microscopy (SEM) images of S. aureus (d) untreated and
e) treated with 40 mM of 3c
Fig. 6. (a) PhysChem filter positioning and (b) oral absorption estimation of 3c
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8
D. PRASANNAN ET AL.
Table 5. The ADMET properties of BODIPY 3c
was rotary evaporated. The resulting crude product was
purified by silica gel column chromatography using
chloroform/ hexane and afforded the pure compound as
an orange solid in good yield. The BODIPY (100 mg)
was then subjected to NBS bromination (3 equiv.) in
dichloromethane at room temperature overnight. The
reaction mixture was washed with water and extracted in
CH Cl . The extract was dried over anhydrous Na SO ,
and the solvent was rotary evaporated. This was then
purified by column chromatography on silica/neutral
alumina gel using chloroform/hexane or chloroform/
acetone.
The BODIPYs 1a and 2a were dark orange powders,
3a was green, and all appeared orange-green in organic
solvents. The 2,6-di-bromo derivatives 1b and 2b were
bright pink, both in the solid form and in solution whereas
3c and all other 2-monobromo derivatives were pinkish
orange as, shown in Fig. S22.
Molecular weight
539.02 g/mol
5.01
Rigid bonds
Flexibility
22
0.08
1
a
Log P
b
e
Log D
-0.42
HBD
c
f
Log Sw
-6.37
HBA
4
2
2
2
4
d
tPSA
37.04
2
Veber rule
Egan rule
Good
Good
Rotatable bond
count
a
P refers to partition coefficient, logarithm of the partition coefficient
b
betweenn-octanolandwater,characterizelipophilicity. Disdistribution-
c
coefficient, logD represents logP at pH 7.4. logSw represents the
d
logarithm of compound’s water solubility. Topological polar surface
area is the sum of tabulated surface contributions of polar fragments.
e
f
Hydrogen bond donors and acceptors.
5
-(3-thienyl)dipyrromethane (1).Yield 114 mg (56%).
1
H NMR (400 MHz; CDCl ; Me Si): d ppm 5.51 (1H, s,
3
4
H
silico analysis. Though this seems to impose a limitation
on the antimicrobial application of the compound, the
report on antimicrobial photodynamic therapeutic agents
based upon the brominated BODIPYs affirmed the use
of 3c as antibacterial agent [28] and demanding further
research on its cytotoxic effects. MTT assay was also
carried out to determine the cytotoxicity of compound
meso-H py), 5.95 (2H, s, py), 6.15 (2H, m, py), 6.65 (2H,
d, thio), 6.96 (2H, d, thio), 7.28 (1H, m, thio), 7.86 (2H,
s, py-NH).
5
-(4-Acetamido)dipyrromethane (2). The crude pro-
duct was recrystallized in CHCl /hexane to afford a
3
1
white solid 2. Yield 136 mg (80%). H NMR (400 MHz;
CDCl ; Me Si): d ppm 2.14 (3H, s, -CH ), 7.99 (2H, s,
3
4
H
3
3c. The compound exhibited concentration-dependent
py-H), 6.68 (2H, s, py-H), 6.14 (2H, s, py-H), 5.89 (2H,
cytotoxicity towards A549, human epithelial cell lines.
s, py-H), 5.43 (1H, s, meso H), 7.39 (2H, d, Ar-H), 7.13
(
2H, d, Ar-H).
,3,5,7-Tetramethyl-5-(4-acetamido)dipyrromethane
3). The crude product was recrystallized in CHCl3/
1
EXPERIMENTAL
(
hexane to afford a reddish solid as 3 which was subjected
to BODIPY synthesis without any further purification.
8-(3-thienyl)-4-bora-3a,4a-diaza-s-indacene (1a).
The crude product was purified using chloroform/hexane
(50/50) to afford the pure compound as an orange solid.
Synthesis
To a solution of corresponding aldehyde (100 mg) in
pyrrole (25/50 equiv.) a catalytic amount of TFA (0.1
equiv.) was added and the mixture was degassed with a
1
stream of N for 5 min. The mixture was stirred under
Yield 62.mg (52%). H NMR (400 MHz; CDCl ; Me Si):
2
3
4
3
N atmosphere for 30 min at room temperature and upon
d ppm 6.55–6.56 (1H, d, py-H, J (H, H) = 2.8 Hz),
2
H
3
completion of the reaction was quenched with dilute
NaOH (0.1 M, 5 mL). The product was extracted with
ethyl acetate, washed with water, dried over anhydrous
Na SO and evaporated to dryness under vacuum.
7.11–7.10 (2H, d, py-H, J (H, H) = 3.6 Hz), 7.39–7.38
3
(1H, d, J (H, H) = 4.8 Hz), 7.53–7.51 (1H, m) 7.72 (1H,
d, thio-H, J = 2 Hz), 7.92 (2H, s, py-H). IR (KBr,): n,
-1
cm 3110, 2918, 2669, 1626, 1545, 1394, 1377, 1351,
1363 1150, 775.
2
4
The crude product was purified by silica gel column
chromatography using chloroform/ hexane as the eluent
to give light yellow crystalline solid.
The corresponding dipyrromethane (100 mg) was
dissolved in distilled dichloromethane and was oxidized
with 1 equiv. of DDQ at room temperature under stirring
for 1 h. Triethylamine (30 equiv.) was added and the
8-(4-Acetamido)-4-bora-3a,4a-diaza-s-indacene
(2a). The crude product was purified by silica gel column
chromatography chloroform/hexane = 95/5 to yield the
1
pure orange fluorescent dye.Yield 79 mg (68%). H NMR
(400 MHz; CDCl ; Me Si): d ppm 2.17 (3H, s, -CH ),
3
4
H
3
7.3 (1H, s, NH), 7.93 (2H, s, Ar-H), 7.70–7.68 (2H, d,
3
3
.
solution was stirred for additional 15 min. BF ꢀOEt
Ar-H, J (H, H) = 8.4 Hz), 6.55 (2H, d, py-H, J (H, H) =
3
2
3
(41 equiv.) was then added, and stirring continued at
3.6 Hz), 6.95–6.96 (2H, d, py-H, J (H, H) = 4 Hz), 7.57–
7.55 (2H, d, py-H, J (H, H) = 8.4 Hz). IR (KBr,): n, cm
3
-1
room temperature for 1 h. TLC analysis indicated the
formation of the expected compound as an orange-red
spot. The resulting mixture was then washed with water
and extracted with CH Cl . The organic layers were then
3435, 3246, 2924, 2372, 1670, 1590, 1548, 1411, 1386,
1296, 1259, 1181, 1019, 976, 859, 831, 774.
1,3,5,7-Tetramethyl-8-(4-acetamido)-4-bora-3a,4a-
diaza-s-indacene (3a). The crude product was purified
2
2
combined, dried over anhydrous Na SO , and the solvent
2
4
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 8–10

SYNTHESIS, STRUCTURE, PHOTOPHYSICAL, ELECTROCHEMICAL PROPERTIES AND ANTIBACTERIAL ACTIVITY
9
-
1
by silica gel column chromatography chloroform/acetone
n, cm 3736, 3442, 2924, 2853, 2372, 2089, 1638, 1597,
9
6
0/10 to yield the pure orange fluorescent dye. Yield
1375, 1351, 1258, 1181, 754. HRMS (ESI-TOF): m/z
483.9463 (calcd. for C H BBr F N O [M–H] 482.91).
2,6-Dibromo-1,3,5,7-tetramethyl-8-(4-Acetamido)-
4-bora-3a,4a-diaza-s-indacene (3c). The compound was
isolated using neutral alumina column using chloroform/
1
+
0 mg (53%). H NMR (400 MHz; CDCl ; Me Si): d
3
4
H
17 12
2
2
3
ppm 1.25 (6H s, py-CH ), 2.54 (6H s, py-CH ), 2.22
3
3
(
3H s, -CH ), 5.97 (2H s, py-H), 7.66–7.68 (2H d, Ar-H,
3
3
3
J (H, H) = 8.4 Hz), 7.26–7.21 (2 H m, Ar-H, J (H, H) =
-
1
1
8
3
9
Hz), 7.40 (1H, s, -NH). IR (KBr): n, cm 3585, 3339,
042, 2922, 2851, 1541, 1468, 1402, 1371, 1083, 1054,
82, 829, 764.
acetone (95/5). Yield 42 mg (30%). H NMR (400 MHz;
3
CDCl ; Me Si): d ppm 7.11–7.13 (2H, d, Ar-H, J
3
4
H
3
(H, H) = 8.4 Hz), 7.63–7.65 (2H, d, Ar-H, J (H, H) =
2
-Bromo-8-(3-thienyl)-4-bora-3a,4a-diaza-s-
8.4 Hz), 2.52 (6H, s, -CH ), 2.16 (3H, s, -CH ), 1.34 (6H,
3
3
1
3
indacene (1b). The brominated derivatives were purified
bycolumnchromatographyonsilicagelusingchloroform/
hexane (50/50) as eluent and afforded the products. Yield
s, -CH3). C NMR (400 MHz; CDCl ; Me Si): d ppm
168.4, 154.0, 141.6, 140.5, 139.3, 130.5, 129.8, 128.6,
120.0, 111.8, 24.7, 13.6–13.9. IR (KBr): n, cm 3851,
3626, 2923, 2852, 1673, 1596, 1537, 1466, 1402, 1348,
1311, 1177, 1000, 763. HRMS (ESI-TOF): m/z 539.9919
3
4
C
-1
1
3
6 mg (30%). H NMR (400 MHz; CDCl ; Me Si): d
3
4
H
3
ppm 6.53–6.54 (1H, d, py-H, J (H, H) = 4 Hz), 6.97
3
+
(
7
(
1H, s, py-H), 7.09–7.10 (1H, d, py-H, J (H, H) = 4 Hz),
(calc. for C H BBr F N O M 539.02).
2
1
20
2
2
3
3
.29–7.31 (1H, d, thio-H, J (H, H) = 4 Hz), 7.46–7.48
1H, m, thio-H), 7.66–7.67 (1H, m, thio-H), 7.70 (1H, s,
13
CONCLUSION
py-H), 7.91 (1H, s, py-H). C NMR (400 MHz; CDCl ;
3
Me Si): d ppm 145.8, 141.9, 142.2, 135.0, 134.0, 133.9,
4
C
In summary, we have described the synthesis and
characterization of series of brominated BODIPYs; their
structural, photophysical and electrochemical properties
have also been explored. The biological properties of the
compounds were investigated against S. aureus and it
was found that the dibromo derivative 3c showed better
antibacterial activity with good MIC and MBC values. M
molecular docking studies were performed and the target
of 2,6-dibromo derivative 3c in S. aureus was identified as
the B subunit of DNA gyrase. In silico target studies were
further validated by in vitro experiments with purified
protein. Through the results obtained from the present
work, it can be concluded that the brominated BODIPY
1
32.5, 130.3, 129.8, 129.6, 127.3, 119.4, 105.8. IR (KBr):
-1
n, cm 3429, 3108, 2962, 2925, 1734, 1642, 1532, 1469,
1
349, 1263, 1211, 845, 812, 771. HRMS (ESI-TOF): m/z
+
341.9475 (calcd. for C H BBrF N S M 351.97).
13
8
2
2
2
,6-Dibromo-8-(3-thienyl)-4-bora-3a,4a-diaza-s-
1
indacene (1c). Yield 86 mg (55%). H NMR (400 MHz;
CDCl ; Me Si): d ppm 7.05–7.01 (m, 2H, py-H), 7.75
3
4
H
(
s, 2H, py-H), 7.26–7.30 (1H, m, thio-H), 7.48–7.50 (1H,
-1
m, thio-H,), 7.66–7.69 (1H, m, thio-H). IR (KBr): n, cm
3
9
422, 3107, 2922, 2852, 1664, 1544, 1471, 1346, 1252,
97, 981, 966, 913, 848, 717, 782. HRMS (ESI-TOF):
m/z 413.1855 (calcd. for C H BBr F N S 431.89).
1
3
7
2
2
2
2
-Bromo-8-(4-acetamido)-4-bora-3a,4a-diaza-s-
3
c should be further explored as potent antibacterial lead
indacene (2b). The brominated derivatives were separa-
ted by column chromatography on silica gel using
chloroform/acetone (93.75/6.25) as eluent and afforded
structure in various biomedical applications.
Acknowledgments
1
the products. Yield 11 mg (9%). H NMR (400 MHz;
The Department of Science and Technology (DST),
New Delhi, through EMR/2016/002396 project, is
acknowledged for funding. Dr. Babu Varghese, SAIF,
IIT Madras is warmly acknowledged for the single
crystal data collection, structure solution and refinement.
The manuscript was written through contributions
of all authors. All authors have given approval to the
final version of the manuscript. The authors declare
no competing financial interest. It is the author’s
responsibility to obtain written permission to reproduce
previously published material.
CDCl ; Me Si): d ppm 6.50–6.51 (1H, m, py-H), 6.80
3
4
H
3
(
1H, s, py-H), 6.91–6.92 (1H, d, py-H, J (H, H) = 4.4 Hz),
3
7
.40–7.42 (2H, d, Ar-H, J (H, H) = 8.4 Hz), 7.63–7.65
3
(2H, d, Ar-H, J (H, H) = 8.4 Hz), 7.67 (1H, s; py-H), 7.89
(1H, s, py-H), 8.00 (1H, s, NH), 2.15 (3H, s, -CH ). C
13
3
NMR (400 MHz; CDCl ; Me Si): d ppm 210.9, 168.9,
3
4
C
1
1
46.6, 145.7, 141.7, 141.3, 135.1, 134.1, 133.0, 131.6,
30.1, 128.6, 119.5, 105.8, 69.7, 53.8, 31.7, 29.2, 24.5.
-
1
IR (KBr): n, cm 3393, 3115, 2923, 2852, 2372, 2345,
472, 1402, 1299, 1258, 1224, 1183, 758, 745. HRMS
1
(
[
ESI-TOF): m/z 402.0258 (calcd. for C H BBrF N O
1
7
13
2
3
+
M + 1] 403.02).
2
,6-Dibromo-8-(4-acetamido)-4-bora-3a,4a-diaza-s-
Supporting information
1
indacene (2c). Yield 26 mg (18%). H NMR (400 MHz;
CDCl ; Me Si): d ppm 6.89 (1H, s, py-H), 7.43–7.45
Experimental, synthetic procedure, UV-vis, fluore-
3
4
H
1
13
3
scence, H, CNMRspectra,massspectra,electrochemical
data, crystal data, images and tables are given in the
supplementary material. This material is available free
of charge via the Internet at http://www.worldscinet.com/
jpp/jpp.shtml. Crystallographic data have been deposited
(
2H, d, Ar-H, J (H, H) = 8.4 Hz), 7.66–7.67 (2H, d, Ar-H,
3
J (H, H) = 7.6 Hz), 7.74 (1H, s, py-H), 7.83 (1H, s, NH),
1
3
2
.17 (3H, s, -CH ). C NMR (400 MHz; CDCl ; Me Si):
3
3
4
dC ppm 210.8, 168.8, 146.5, 143.7, 141.5, 134.4, 131.6,
28.3, 119.5, 107.1, 69.6, 53.8, 31.7, 29.2, 24.6 IR (KBr):
1
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 9–10

1
0
D. PRASANNAN ET AL.
at the Cambridge Crystallographic Data Centre (CCDC)
16. Hurley KA, Santos TMA, Fensterwald MR, Rajen-
dran M, Moore JT, Balmond EI, Blahnik BJ,
Faulkner KC, Foss MH, Heinrich VA, Lammers
MG, Moore LC, Reynolds GD, Shearn-Nance GP,
Stearns BA, Yao ZW, Shaw JT and Weibel DB.
Med. Chem. Commun. 2017; 8: 942–951.
under numbers CCDC-1577779 (1b), 1022866 (2a),
.
.
.
1
033432 (3a ꢀH O) and 1058023 (3a ꢀD MF ꢀC H COOH)
2
3
and 1577778 (3c). Copies can be obtained on request, free
of charge, via http://www.ccdc.cam.ac.uk/data_request/cif
or from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223-
17. Liu J-J, Sun J, Fang Y-B, Yang Y-A, Jiao R-H and
Zhu H-L. Org. Biomol. Chem. 2014; 12: 998–1008.
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Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 10–10