A new chiral boron-dipyrromethene (BODIPY)-based fluorescent probe: molecular docking, DFT, antibacterial and antioxidant approaches

Article abstract of DOI:10.1080/07391102.2019.1701555

A new chiral BODIPY-based fluorescent compound, 5-bromo-4,4-difluoro-3(S)-1-phenylethyl)amino) BODIPY, 4 was synthesized for biomedical applications. Optical, antimicrobial, antioxidant properties of the compound 4 are investigated. The partition coefficient suggested that the compound 4 has the potential to be developed as an active antibacterial and antioxidant candidate. In this context, antibacterial assay was carried out for compound 4 against various bacterial strains, revealing maximum inhibition zone (24 ± 2.19 mm) in Escherichia coli. Moreover, results of antioxidant activity of compound 4 revealed IC₅₀ values to be greater than ascorbic acid. Molecular docking has given brief insight about the binding of the compound 4, suggesting that it has a strong potential to inhibit bacterial target enzymes viz., DNA gyrase, enzymes in the type II fatty acid synthesis and Ddl (d-alanine: d-alanine ligase) in peptidoglycan synthesis. The molecular geometry and electrostatic potential of compound 4, was established by DFT (Density Functional Theory) calculations. AbbreviationsBBB blood?brain barrierBDE bond dissociation energyBODIPY boron–dipyrromethaneDdl D-alanine:D-alanine ligaseDDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinoneDFT density functional theoryDNA deoxyribonucleic acidDPPH 1,1?diphenyl?2?picrylhydrazylNBSN-bromo succinimideROS reactive oxygen speciesUV–vis ultraviolet–visibleFMO frontier molecular orbitalsHOMO highest occupied molecular orbitalLUMO lowest unoccupied molecular orbital Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2019.1701555

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
A New Chiral Boron–dipyrromethene
(BODIPY)–based Fluorescent Probe: Molecular
docking, DFT, Antibacterial and Antioxidant
approaches
Rua B. Alnoman, Shazia Parveen, Mohamad Hagar, Hoda A. Ahmed & Julian
G. Knight
To cite this article: Rua B. Alnoman, Shazia Parveen, Mohamad Hagar, Hoda A. Ahmed & Julian
G. Knight (2019): A New Chiral Boron–dipyrromethene (BODIPY)–based Fluorescent Probe:
Molecular docking, DFT, Antibacterial and Antioxidant approaches, Journal of Biomolecular
Structure and Dynamics, DOI: 10.1080/07391102.2019.1701555
To link to this article: https://doi.org/10.1080/07391102.2019.1701555
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A New Chiral Boron–dipyrromethene (BODIPY)–based Fluorescent Probe: Molecular
docking, DFT, Antibacterial and Antioxidant approaches
Rua B. Alnomana*, Shazia Parveena*, Mohamad Hagara,b, Hoda A. Ahmeda,c, Julian G. Knightd
aFaculty of Science, Chemistry Department, Taibah University, Yanbu Branch, 46423, Yanbu, Saudi
Arabia.
bFaculty of Science, Chemistry Department, Alexandria University, Alexandria, Egypt.
cFaculty of Science, Department of Chemistry, Cairo University, Cairo, Egypt.
dSchool of Chemistry, Bedson Building, Newcastle University, Newcastle upon Tyne, NE1 7RU,
UK.
*Corresponding authors: email: rua‒b‒n@live.co.uk (R.B. Alnoman), shazia021@gmail.com (S.
Parveen).
Abstract
A new chiral BODIPY–based fluorescent compound, 5‒bromo‒4,4‒difluoro‒3(S)‒1‒
phenylethyl)amino)BODIPY, 4 was synthesized for biomedical applications. Optical, antimicrobial,
antioxidant properties of the compound 4 are investigated. The partition coefficient suggested that
the compound 4 has the potential to be developed as an active antibacterial and antioxidant
candidate. In this context, antibacterial assay was carried out for compound 4 against various
bacterial strains, revealing maximum inhibition zone (24±2.19 mm) in Escherichia coli. Moreover,
results of antioxidant activity of compound 4 revealed IC50 values to be greater than ascorbic acid.
Molecular docking has given brief insight about the binding of the compound 4, suggesting that it
has a strong potential to inhibit bacterial target enzymes viz., DNA gyrase, enzymes in the type II
fatty acid synthesis and Ddl (D‒alanine:D‒alanine ligase) in peptidoglycan synthesis. The molecular
geometry and electrostatic potential of compound 4, was established by DFT (Density Functional
Theory) calculations.
Keywords: BODIPY, antibacterial, antioxidant, molecular docking, DFT
Abbreviations:
BBB
blood‒brain barrier
BDE
bond dissociation energy
Boron–dipyrromethane
BODIPY
Ddl
D‒alanine:D‒alanine ligase
2,3–dichloro–5,6–dicyano–1,4–benzoquinone
Density functional theory
Deoxyribonucleic acid
DDQ
DFT
DNA

DPPH
NBS
1,1‒diphenyl‒2‒picrylhydrazyl
N–bromo succinimide
ROS
Reactive oxygen species
UV–vis
FMO
HOMO
LUMO
1.
Ultraviolet–visible
frontier molecular orbitals
Highest occupied molecular orbital
Lowest unoccupied molecular orbital
Introduction
Infectious diseases still remain the leading worldwide cause of mortality and morbidity,
despite the advent of antibiotics and vaccines (Vicente et al., 2006). The major reason hampering the
efforts to control microbial infections is the emergence and proliferation of drug resistant pathogens,
demanding the quest for complementary approaches to the current drug‒based treatments. Besides, a
paucity of effective therapies for many globally important microbial infections drives the need for
development of novel approaches for either treatment or prevention of infection.
Fluorescent probes have recently attracted considerable attention owing to their simple and
rapid implementation, as well as their high sensitivity and high spatial resolution (Lakowicz 2006).
They are markers of genesis of living organisms (Yang et al., 2014), medical diagnostics (Kobayashi
et al., 2010), cancer cells (Luo et al., 2011), and different ions (Sunahara et al., 2007). Boron–
dipyrromethene (BODIPY) possesses a lipophilic cation and a very prevalent fluorescent group
characterized by strong absorption in the visible and near–infrared region (Kamkaew et al., 2013),
high photo–stability, low sensitivity to environmental variation, and an easily modified basic core
with tunable optical properties (Loudet et al., 2007). Due to its high fluorescence quantum yield,
wide wavelength assimilation and ease of modification, BODIPY are used as a fluorescent tag in
tumor imaging and malignancy–targeted therapy (Gao et al., 2016; Gupta et al., 2017). Notably, one
of the most prospective trends is the application of BODIPY dyes as photosensitizers in a
photodynamic therapy (Kamkaew et al., 2013). Scheme 1 shows the central heterocyclic core of
BODIPY. It is an established fact that the incorporation of fluorine atoms into a drug allows
simultaneous modulation of the pharmacodynamic properties of drugs viz., electronic, lipophilic and
steric parameters (Pucelik et al., 2016). The lipophilicity of fluorine can enhance the cell membrane
permeability thereby improving the bioavailability of drug candidates (Isanbor et al., 2006). The
pharmacokinetic properties of drugs can be improved by (i) strongly electronegative fluorine that
possibly increases the binding ability between the ligand and target and (ii) the stable C–F bond
contributes to the metabolic stability of the drugs in body (Lazzeri et al., 2004). However,
incorporating fluorine into the BODIPY core with a view to improving their therapeutic outcome has
been seldom reported (Watley et al., 2015).
As far as biological applications are concerned, fluorophores absorbing at longer wavelength
are of much importance because at higher wavelength absorption by cells and water, light scattering
and auto–fluorescence are reduced significantly, allowing for deeper light penetration, resulting in
efficient therapy and diagnosis (Gai et al., 2016; Loudet et al., 2007). BODIPY’s high tissue
penetration with minimal auto–fluorescence 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 the literature (Gayathri et al., 2014; Tapia et al.,
2016). A variety of BODIPY derivatives have been reported to exhibit good cancer mitochondrial

localization selectivity due to the higher mitochondrial membrane potential of cancer cells (Gao et
al., 2016). It is known that cancer cells exhibited an increased antioxidant capacity response to the
oxidative stress induced by drug treatment in some drug–resistant cases (Weniger et al., 2011).
Therefore, simultaneously increasing mitochondrial oxidative stress and decreasing the antioxidant
response capacity has emerged as an effective strategy in normal cancer therapy and some drug
resistance cases (Akhdar et al., 2012). Moreover, one of our interest is in studying the theoretical
calculations for predicting the molecular geometry which controls the properties of prepared
materials in relating the calculated data with the experimental findings (Ahmed, et al., 2019a; Hagar
et al., 2019 a; Hagar et al., 2020).
Herein this paper, we report the synthesis, characterization, molecular docking studies on
various bacterial enzymatic targets, DFT calculations, and biological activities, viz., antibacterial and
antioxidant assays of a new fluorescent BODIPY based compound.
2.
Experimental
2.1
Materials and methods
All chemicals were purchased from the Aldrich and Alfa Aesar chemical companies and were
used as supplied except where indicated. All reactions were carried out under an atmosphere of
nitrogen in pre–dried glassware. Where necessary, solvents were dried prior to use. Dichloromethane
was distilled from calcium hydride under nitrogen immediately prior to use. Tetrahydrofuran was
distilled from sodium wire and benzophenone ketyl immediately prior to use. Acetonitrile was
distilled from K2CO3, under an atmosphere of nitrogen.
2.2
Instrumentation
IR spectra were recorded on a Varian 800 FT–IR Scimitar Series infrared spectrometer. 1H, 13C
NMR spectra were recorded on a JEOL ECS 400 or a Bruker Avance 300 MHz instrument. All 1H
NMR were referenced relative to CDCl3 (δH = 7.26, δC = 77.16). Data for 1H NMR spectra are
reported as follows: chemical shifts measured in ppm from internal tetramethylsilane on the δ scale,
integration, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, p = pentet and m
= multiplet). 13C NMR chemical shifts are reported in ppm from tetramethylsilane on the δ scale,
using solvent resonance as the internal standard [deuterated chloroform (CDCl3) at 77.16 ppm]. Mass
spectrometry analysis was performed using a Micromass LCT premier Mass Spectrometer in
Electrospray (ES) mode. Thin layer chromatography was performed on EM reagent 0.25 mm silica
gel 60F plates. Visualization was accomplished with UV light and aqueous potassium permanganate
(VII) solution. UV–vis and fluorescence spectra were recorded at 20°C on a Shimadzu UV–1800.
Relative fluorescence quantum yields (Φ) were determined using dilute solutions with an absorbance
below 0.1 at the excitation wavelength and using Rhodamine 6G (Φf = 0.95) or Rhodamine B (Φf =
0.65) in ethanol as standard.
2.3
Syntheses
2.3.1 Synthesis of 2,2’–((4–Methylphenyl)methylene)bis(1H–pyrrole) (1).
4–Methylbenzaldehyde (1.60 ml, 1.73 g, 13.6 mmol) was added to pyrrole (24.0 mL, 346
mmol) and the mixture was stirred at room temperature for 25 min. BF3.Et2O (0.14 g, 1.40 mmol)
was added to the mixture and stirred for 30 min. The solvent was removed under reduced pressure
and the resulting material was partitioned between CH2Cl2 (50 mL) and NaOH (200 mL). The
organic extract was dried (MgSO4) and the solvent was removed under reduced pressure. The brown
oil crude product was purified by column chromatography (petroleum ether:ethyl acetate:Et3N
9:1:0.1) to give the title compound 1 as a tan solid (2.2 g, 69%) (Scheme 2).

M.p. 108–110°C. Rf = 0.23 (petroleum ether:ethyl acetate:Et3N 9:1:0.1). 1H NMR (300 MHz,
CDCl3) δ 7.94 (bs, 2H, NH), 7.17–7.11 (m, 4H), 6.67–6.74 (m, 2H), 6.18 (q, J=2.8 Hz, 2H), 5.92–
5,97 (m, 2H), 5.47 (s, 1H), 2.35 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 139.15, 136.68,
132.80, 129.43, 128.37, 117.21, 108.48, 107.18, 43.66, 21.15. IR (neat): υmax/cm−1: 3655, 3342,
2980, 2981, 1682, 1553, 1513, 1111, 717. HRMS–ES Calcd for C16H16N2 + H+: 237.1392, found:
237.1397.
2.3.2 Synthesis of 1,9–Dibromo–5–(4–methylphenyl)dipyrrin (2).
2,2'–((4–Methylphenyl)methylene)bis(1H–pyrrole), 1 (1.00 g, 4.24 mmol) was dissolved in
dry THF (100 mL) and cooled to –78 ᵒC. N–Bromosuccinimide (1.52 g, 8.54 mmol) was added to
the mixture in two portions over 1 h. DDQ (0.98 g, 4.3 mmol) in THF (10 mL) was added to the
mixture dropwise over 10 min. The reaction mixture was warmed to room temperature and the
solvent was removed under reduced pressure. The orange crude product was purified by column
chromatography (CH2Cl2) to give the title compound 2 as a light brown solid (1.33 g, 80%).
M.p. 112–114°C. Rf = 0.92 (CH2Cl2). 1H NMR (300 MHz, CDCl3) δ 12.47 (s, 1H), 7.26 (d, J = 8.5
Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 6.44 (d, J = 4.2 Hz, 2H), 6.26 (d, J = 4.2 Hz, 2H), 2.37 (s, 3H,
CH3). IR (neat): υmax/cm−1: 3130, 2980, 1717, 1573, 1595, 1414, 1308, 1246, 1111, 1041, 778.
HRMS–ES Calcd for C16H12N2Br2 + H+: 390.9445, found: 390.9454.
2.3.3 Synthesis of 3,5‒dibromo‒4,4‒difluoro‒8‒(4‒methylphenyl)‒4‒bora‒3a‒4a‒diaza‒
s‒indacene (3).
The compound 3 is synthesized according to the procedure reported earlier (Alnoman et al.,
2016). N,N–Diisopropylethylamine (2.14 g, 16.6 mmol) and BF3.Et2O (3.14 g, 22.1 mmol) were
added to a solution of 1,9–dibromo–5–(4–methylphenyl)dipyrrin, 2 (1.08 g, 2.76 mmol) in dry
CH2Cl2 (20 mL) and the mixture was stirred at room temperature for 2 h. The mixture was then
washed with 0.1 M NaOH (50 mL) and water (100 mL). The aqueous layer was extracted with
dichloromethane (2 × 150 mL). The combined organic layers were dried (MgSO4) and the solvent
was removed under reduced pressure. The orange crude product was purified by column
chromatography (CH2Cl2:petroleum ether 1:1) to give the title compound 3 as an orange solid (1.05
g, 86%).
M.p. 208–210°C. Rf = 0.30 (petroleum ether:CH2Cl2 1:1). 1H NMR (300 MHz, CDCl3) δ 7.37 (d,
J = 7.9 Hz, 2 H), 7.31 (d, J = 7.8 Hz, 2H), 6.80 (d, J = 4.3 Hz, 2H), 6.52 (d, J = 4.3 Hz, 2H), 2.45 (s,
3H).13C NMR (101 MHz, CDCl3) δ 143.68, 141.67, 135.57, 132.32, 131.78, 130.57, 129.67, 129.41,
122.66, 21.59. 11B NMR (128 MHz, CDCl3) δ –0.36 (t, JB–F = 28.5 Hz). 19F NMR (282 MHz,
CDCl3) δ –146.80 (q, JF–B =28.5 Hz). IR (neat): υmax/cm−1: 3139, 2446, 1530, 1446, 1381, 1312,
11
1093, 1063, 736. HRMS–ES Calcd for C16H11N2 B79Br81Br19F2 + Na+: 462.9227, found: 462.9222.
2.3.4 Synthesis of 3‒((S)‒1‒phenylethyl)amino‒5‒bromo‒4,4‒difluoro‒8‒(4‒
methylphenyl)‒4‒para‒3a‒4a‒diaza‒s‒indacene (4).
3,5‒dibromo‒4,4‒difluoro‒8‒(4‒methylphenyl)‒4‒bora‒3a‒4a‒diaza‒s‒indacene, 3 (0.10 g,
0.23 mmol) was dissolved in dry acetonitrile (13 mL) followed by the addition of (S)‒α‒
methylbenzylamine (0.06 g, 0.45 mmol). The mixture was stirred at room temperature for 24 h. The
solvent was removed under reduced pressure to give the crude product as red oil. The crude product
was purified using column chromatography (petroleum ether: ethyl acetate 8:2) to give the 3‒amino
BODIPY (4) as an orange solid.
Yield: (0.10 g, 91%), m.p. 128‒130
CDCl3) δ 7.38 – 7.27 (m, 5H, Ph), 7.27 (d, J = 7.3 Hz, 2H, Ar‒H), 7.27 (d, J = 7.3 Hz, 2H, Ar‒H),
̊
C, Rf = 0.55 (petrol/ethyl acetate, 4:1). 1H NMR (300 MHz,

6.67 (d, J = 4.9 Hz, 1H, pyrrole‒H), 6.57 (br d, J = 6.3 Hz, 1H, NH), 6.35 (d, J = 6.3 Hz, 1H,
pyrrole‒H), 6.33 (d, J = 6.3 Hz, 1H, pyrrole‒H), 5.98 (d, J = 6.0 Hz, 1H, pyrrole‒H), 4.76 (pent, J =
6.9 Hz, 1H, NCH), 2.42 (s, 3H, ArCH3), 1.66 (d, J = 6.8 Hz, 3H, CH3CH).13C NMR (400
MHz,CDCl3) δ 161.37, 142.97, 139.53, 135.91, 133.90, 133.23, 132.25, 130.88, 130.30, 129.16,
129.08, 128.01, 125.64, 120.99, 116.84, 115.32, 111.86, 54.77, 24.31, 21.46. HRMS‒ES Calcd for
11
C24H22N3 B79BrF2 [M+H]+: 480.1063. Found: 480.1058. IR (neat): υmax cm‒1: 3396, 2360, 1599,
1520.
Several conditions were examined for the synthesis of 4,4‒difluoro‒3,5‒bis(((S)‒1‒
phenylethyl)amino)BODIPY 5. Initially, 2 equivalents of (S)‒α‒methylbenzylamine I were added to
3,5‒dipromoBODIPY 3 in dry acetonitrile at room temperature for 22 h. The 1H NMR spectrum for
the crude product showed a large amount of (S)‒α‒methylbenzylamine I but this was easily removed
by an acid workup. Purification using column chromatography gave the mono substitution product,
3‒bromo‒difluoro‒5‒(((S)‒1‒phenylethyl)amino)BODIPY 4 in excellent yield (95%).
2.4
Partition Coefficient (log P).
The values of the partition coefficient and other molecular properties of compounds 3 and 4
were calculated by molinspiration software (https://www.molinspiration.com).
2.5
Antibacterial activity
Compound 4 was screened for in vitro antibacterial activity against two Gram‒negative
[Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853)] and two Gram‒positive
[Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (MTCC 121)] bacterial strains. The
agar well diffusion method of Perez et al., was adopted for measuring the antibacterial assays (Perez
et al., 1990). All cultures were routinely maintained on NA (nutrient agar) and incubated at 27°C for
overnight in NB (Nutrient broth). After incubation, 0.1 ml aliquot of bacterial culture was spread
uniformly with the help of spreader on NA plates. Wells of 8 mm diameter were punched into the
agar medium, sealed with soft agar and loaded with different concentrations of the metal complexes
(0.1 ml). The plates were then incubated for 24 h at 37°C and the resulting zones of inhibition (mm)
were measured. DMSO solvent was used as a positive control.
2.6
Antioxidant activity
The antioxidant assay of compound 4 was also studied by using DPPH free radical and
hydrogen peroxide as previously reported (Miliauskas et al., 2004; Ruch et al., 1989).
2.6.1 DPPH free radical scavenging activity
The DPPH free radical assay is a very significant and appropriate method which provided an
easy and rapid way for the estimation of the antioxidant activity of the test compounds. Method is
based on the spectrophotometric measurement of the color loss of the DPPH due to its consumption
by the antioxidant species present in the tested sample. An aliquot from the test solutions (0.1 mL)
were mixed with 2.9 mL DPPH solution in methanol (~9 x 10‒5 mol/L). The DPPH radical inhibition
in the presence of the antioxidant species was followed by monitoring the decrease of absorbance at
515 nm, for 2 hours. It is a stable free radical that can accept hydrogen ion or electron on reaction
with antioxidant compound and it became a stable diamagnetic molecule (Patel et al., 2012). The
change in color after the reaction of 100 min at 30°C revealed that antioxidant moiety was present in
compound.
2.6.2 Hydrogen peroxide scavenging activity

As is well known, H2O2 is very reactive among all the oxygen species, hydrogen peroxide
assay was also carried out to find the antioxidant potential of the compound 4. The scavenging
capabilities of target compound 4 against hydrogen peroxide were evaluated using UV‒Vis.
Spectrophotometer (Nayab et al., 2016).
2.7
Molecular Docking
The rigid molecular docking studies were performed by using HEX 8.0 software (Macindoe
et al., 2010; Mustard et al., 2005), which is an interactive molecular graphics program for calculating
and displaying feasible docking modes of interacting drug molecule with biological target of interest.
Antibacterial targets were selected, based on previously reported studies on the compounds identified
in the active fraction. The crystal structures of target enzymes viz., E. coli DNA gyrase (PDB ID:
1KZN), FabG (PDB ID: 1I01), FabZ (PDB ID: 1U1Z), FabI (PDB ID: 5CG1) and Ddl (PDB ID:
2I80) were procured from the Protein Data Bank (http://www.rcsb.org/pdb) in PDB format.
Visualization of the docked pose has been done by using CHIMERA (www.cgl.ucsf.edu/chimera).
2.8
DFT calculations
Gaussian 09 software was used for DFT calculations for the studied compounds (Frisch et al.,
2009). DFT/B3LYP methods using 6‒31G (d,p) basis set was selected for the calculations. The
geometries were optimized by minimizing the energies with respect to all geometrical parameters
without imposing any molecular symmetry constraints. The structures of the optimized geometries
had been drawn with Gauss View (Dennington et al., 2009). Also, calculations frequencies were
carried out by the same level of theory. The frequency calculations showed that all structures were
stationary points in the geometry optimization method with none imaginary frequencies.
3.
Results and Discussion
3.1
Synthesis and characterization
The 3,5–dibromo BODIPY 3 was prepared in good yield using a standard synthetic sequence
(Alnoman et al., 2016) from pyrrole and p–tolualdehyde via boron trifluoride–etherate catalyzed
condensation to the dipyrromethane, 1 (Scheme 2).
Regioselective bromination of the dipyrromethane with NBS followed by DDQ oxidation led
to the corresponding 1,9–dibromo dipyrromethene, 2 which was reacted with BF3 to give the
dibromo–BODIPY, 3. Subsequent nucleophilic substitution of the dibromo BODIPY, 3 with (S)–α–
methylbenzylamine, I gave 3–bromo–4,4–difluoro–5–(((S)–1–phenylethyl)amino)BODIPY, 4 in
excellent yield (95%) (Scheme 3). Under the mild conditions used, there was no sign of the 3,5‒
diamino BODIPY, 5 which would result from double nucleophilic substitution. It is expected that the
strongly electron donating amino substituent in the mono‒substitution product 4 deactivates the
BODIPY towards further nucleophilic attack.
The structure of the BODIPY 4 was confirmed with spectroscopic methods (1H & 13C NMR, IR
and LC–MS). In the 1H NMR spectrum the presence of methylbenzylamine in the product is
confirmed by three characteristic proton signals observed at δ=6.57, 4.76 and 1.66 ppm which
corresponding to the NH, CH and CH3 respectively. Moreover, the 13C NMR spectrum of the
BODIPY 4 showed the appearance of two signals corresponding to the CH3 and CH group of
methylbenzylamine at δ 24.31, 54.77 ppm, respectively. The NMR spectra of compounds 1‒4 are
reported in Figures S1‒S4 (Supplementary Info).

Further analysis of the purified compound showed the presence of a signal at 3396 cm−1 in
the IR spectrum, which confirmed that the molecule contains an NH group. High resolution mass
spectrometry of BODIPY 4 gave a signal at m/z 480.1063 which corresponds to a molecular formula
11
of C24H22N3 B79BrF2.
3.2
Optical Properties
One of the features of BODIPY compounds is that structural modifications may be easily
made, which affords additional opportunities to vary their optical properties. The photophysical
properties of selected BODIPY compounds 3 and 4, are summarized in Table 1. The 3‒and 5‒
substituents significantly influence both the absorption and emission spectra (Figure 1). As shown in
Table 1 the dibromo BODIPY, 3 shows an absorbance at 522 nm, typical for a simple BODIPY. In
comparison to the parent BODIPY 3, the presence of the amino group at 3 position of the BODIPY
core displays a slightly blue‒shifted absorption maximum (510 nm) and reduced quantum yield (0.07
vs. 0.44 for the dibromide) which can be attributed in part to the free rotation of the aryl group in the
mezo–position and partly due to the influence of the amine.
3.3 Partition Coefficients (log P)
Lipophilicity of a drug candidate provides insight into inter‒ and intramolecular forces
affecting the drug delivery across the bilipid membrane as well as its interaction with the target
biomolecule. Thus, lipophilicity is an important factor in determining the pharmacokinetics and
pharmacodynamics of a drug entity. The cytotoxic activity of the drug molecule is correlated with its
lipophilicity and aqueous solubility‒a higher lipophilicity facilitates better cellular uptake and
enhanced activity against the tested cancer cell lines (Mahendiran et al., 2018). Table 2 depicts the
biomolecular properties of compounds 3 and 4. Topological polar surface area (TPSA) i.e. surface
belonging to polar atoms and molecular weight are the descriptors which correlate with passive
molecular transport through membranes that allows prediction of route of transport of drugs through
the barrier membranes the intestine and blood‒brain barrier (BBB). The Lipinski's Rule of Five
indicates the physicochemical properties that are prerequisite for drug development; so that the
absorption or permeation of a molecule is more likely when the molecular weight is under 500
gmol−1, the value of log P is lower than 5, and the molecule has at most 5 H–donor and 10 H–
acceptor atoms (Lipinski et al., 1997). The log P value for the compound 4 was calculated and was
found to be 3.37 while it was –1.13 for compound 3. This suggested that the compound 4 has the
potential to be developed as an orally active drug like candidates and may be potentially active
antibacterial and antioxidant drug candidate.
3.4 Antibacterial activity
To overcome the resistance due to bacterial infections, compound 4 was evaluated for
efficient bioactivity against various bacterial strains. The antibacterial activity of 4 was evaluated by
measuring the diameter of the zone of inhibition (mm). Table 3 and Figure 2, displays the
antibacterial activity of compound 4. The results revealed that the compound 4 exhibited a
significant antibacterial activity (inhibition zone 24±2.19 mm) against Gram negative bacterial strain,
E. coli.
3.5
Antioxidant activity
In a normal cell, prooxidant and antioxidant tend to remain in equilibrium, which can be
changed towards the prooxidants if production of ROS (reactive oxygen species) is increased or if
the antioxidants are decreased. This stage is referred to as oxidative stress (Gulcin 2012; Koksal et
al., 2008). As our understanding of the role of free radicals in human diseases have increased,
antioxidants have attracted broader interest because of their role in inhibiting ROS‒relayed reactions

and their help in protecting the human body against damage by ROS (Kancheva et al., 2013).
Antioxidants could prevent or impede the oxidation of substrates even in substantially lower
concentration than the oxidized substrates providing protection against free radicals and effects of
ROS (Gulcin et al., 2007; Gulcin et al., 2008). In biological systems, uncontrolled accumulations of
hydrogen peroxide leads to the formation of oxygen free radicals which cause immense damage to
cells membrane. The antioxidant compounds donate the electrons to hydrogen peroxide and
neutralize it into water molecule (Sakthikumar et al., 2018). Compound 4 exhibited promising DPPH
(2,2’‒diphenyl‒1‒picrylhydrazyl) and H2O2 radical scavenged antioxidant activity (Table 4). Figure
3 shows the graphical representation of variation of antioxidant activity for the compound 4 (%
DPPH and % H2O2 inhibition) as a function of concentration and comparison with ascorbic acid as
the standard. The results revealed that the IC50 values of compound 4, were found to be 27.2 and 29.9
respectively for DPPH and H2O2 inhibition. These IC50 values were greater than IC50 value of
ascorbic acid used as a standard (~26.68). Many factors such as, planarity, bond angle, steric effects,
intramolecular hydrogen bonding, bond dissociation energy (BDE), dipole moment etc. are held
responsible for higher antioxidant activities (Sakthikumar et al., 2018).
3.6
Molecular Docking
Molecular docking studies were carried out for compound 4 to further ascertain the binding
modes to the target enzymes. Since, major antibacterial constituents present in the active fraction
belong to the group of flavonoids, three targets have been identified to act on by flavonoids (George
et al., 2019). They are, (i) DNA gyrase involved in DNA synthesis (Islam et al., 2019; Plaper et al.,
2003), (ii) Enzymes in the type II fatty acid synthesis‒ β‒ketoacyl ACP reductase (FabG), 3R‒
hydroxyacyl ACP dehydratase (FabZ) and trans 2‒enoyl ACP reductase (FabI) (Tasdemir et al.,
2006; Zhang et al., 2008) and (iii) D‒alanine:D‒alanine ligase (Ddl) in peptidoglycan synthesis
(Fakhar et al., 2016; Wu et al., 2008).
DNA gyrase is one of the members of topoisomerase II enzymes that controls the topological
state of DNA in cells. It plays a vital role in replication of DNA by coupling with the ATP
(Adenosine triphosphate) and helping in the supercoiling of the DNA. The binding of drug and
inhibition of this enzyme is responsible for the bactericidal effects of the respective drugs. The
compounds 4 was docked into the active sites of E. coli DNA gyrase subunit b. E. coli DNA Gyrase
contains 378 amino acid residues. Docked poses of compound 4 in DNA gyrase, with lowest binding
energy are shown in Figure 4. The binding affinity was evaluated by the binding free energy in
Kcal/mol. The molecular docking results with DNA gyrase (1KZN) revealed that the new BODIPY
compound 4 fits in the binding socket and the binding sites recognized constituted of amino acid–
Lys162, Gly75, Asp74, Gln72, Gln135, Val71, Val–133, Thr165, Glu50 and Ala47. The binding
energy was calculated to be –10.23 Kcal/mol.
Flavonoids inhibit the enzymes involved in fatty acid synthesis. Unlike humans, bacteria, and
other prokaryotes, have Type II Fatty Acid Synthase system (FAS); consisting of many enzymes and
each responsible for a specific step in fatty acid synthesis. Hence, multiple enzyme containing Type
II FAS pathway would be a potential target for the development of many antibacterial compounds
(Heath et al., 2004) and also improve the possibility for identifying broad spectrum antibiotics
(Zhang et al., 2006). The three pertinent targets involved in the fatty acid biosynthesis are FabZ that
catalyzes short chain and long chain of saturated and unsaturated β‒hydroxyacyl‒ACPs, FabI that is
responsible to reduce enoylthioester to an acyl moiety and FabG catalyzing the first reductase step in
fatty acid biosynthesis. Figures 5 (a‒c) display the best docked poses for compound 4 with enzymes
in Type II FAS pathway.
Moreover, among many different targets responsible for inhibition of cell wall synthesis in
bacteria, D‒alanine:D‒alanine ligase (Ddl) is one of the key enzyme involved in peptidoglycan
synthesis for catalyzing the ligation of D‒alanine‒D‒alanine (Bruning et al., 2011). Quercetin was

reported for its ability to inhibit D‒alanine:D‒alanine ligase enzyme (Plaper et al., 2003). The results
of molecular docking with Ddl is shown in Figure 6.
The binding energies along with the binding sites of compound 4 with different enzymatic
targets have been summarized in Table 5. According to the minimum energy principle, the lowest
energy leads to highest stability of complex formed by ligand and receptor (Bharti et al., 2018). This
was further confirmed by the DFT calculations performed.
3.7
DFT calculations
3.7.1 Molecular Geometry
The theoretical DFT calculations were performed in gas phase by DFT/B3LYP method at 6‒
31G basis set. Since the absence of the imaginary frequency of all optimized structures, they were
approved to be stable. The results of the theoretical calculations for all compounds 1‒4 revealed the
non‒planar geometry for all compounds (Figure 7). The tolyl ring A is not coplanar with the other
rings B, C and D for all compounds. The twist angle (ϴ) for compounds 1 between the tolyl A and
the pyrrole ring B and C was 89.20 and 87.05, respectively. However, 59.97° was the ϴ angle
between the planes passing through the pyrrole rings B and C. Recently, we have reported that the
molecular geometry could affect the characteristics that are important in many applications
(Alnoman et al., 2019; Nafee et al., 2019 a; Nafee et al., 2019 b).
The oxidation reaction of 1 with bromine affords dibromo compound 2 with more planar
geometry of the bis pyrrole moiety, 5.17° between rings B and C while that between A and B, A and
C was 63.06° and 63.32°, respectively. This result could be explained in the term of better
conjugation of the π‒cloud of the pyrrole rings with the conjugated C=C group. Consequently, the
high conjugation leads to more enforcement of the conjugated system to be more planar. The
presence of a H‒bond (1.94507 Å) between the N‒atoms of the pyrrole rings is also explained
coplanarity of the rings B and C. The complexation reaction of 2 efficiently gave diflouroborane 3
without any significant effect on all twist angles, only ≈ 3° lower than the free ligand 2.
The nucleophilic substitution of 3 with I to afford 4 has a significant impact on the planarity.
The twist angle between tolyl ring A and ring B decreases to 54.15° instead of 60.16° of compound 3
however, the angle between the pyrrole rings increases to 6.05° instead of 2.30°, this could be
explained in the term of the steric hindrance of the attached chiral compound, (S)‒α‒
methylbenzylamine I.
The calculated DFT thermal parameters, dipole moment and the polarizability of the
investigated compounds 1‒4 are tabulated in Table 6. As shown in the table, as expected the
oxidation reaction of 1 with bromine will affords a more polar compound 2. The polarity as well as
the polarizability of the dibromo compound (2) is higher than that of 1. The increment of the
polarizability could be explained in terms of the extra‒conjugation of 2 under the oxidation reaction
by the presence C=C group between the pyrrole rings. However, the complexation reaction of 2 to
afford dibromo BODIPY 3 does not significantly affect the polarizability, 264.30 for compound 2
and 277.4 for 3. While, the attachment of the boron atom increases the polarity of dibromo BODIPY
3 to 8.92 Debye instead of 4.35 Debye for reduced reactant 2. Finally, the nucleophilic substitution
reaction of a chiral (S)‒α‒methylbenzylamine I does not impact the dipole moment of 3 but it highly
increases the polarizability, 358.2 instead of 277.4 for 3. The higher polarizability of 4 could be
explained due to the mesomeric effect of the primary amino group attached (Scheme 4).

3.7.2 Frontier molecular orbitals and molecular electrostatic potential
The frontier molecular orbitals (FMOs) energy difference is an important parameter for many
applications (Braga et al., 2019; Chi et al., 2019; Coleone et al., 2019; Jung et al., 2019; Ortega et
al., 2019; Wang et al., 2020). Moreover, the energy level of HOMO (highest occupied molecular
orbital) and LUMO (lowest unoccupied molecular orbital) is highly affected by the electronic nature
of the attached groups. Furthermore, estimation of the energy gap between FMOs could be used in
prediction of many important parameters such as polarizability (α), global softness (S) and chemical
hardness (η) (Ahmed et al., 2019 b; Hagar et al., 2019 b, Hagar et al., 2019 c). Figure 8 shows the
predicted ground state isodensity surface plots for the FMOs and their energy differences of the
prepared compounds 1‒4.
As shown from Table 7, the electron densities of the HOMO orbitals are mainly localized on
the pyrrole rings with little sharing of the tolyl ting for compounds 1, 2. On the other hand, the
electron densities of the LUMO orbitals are similarly localized on the pyrrole rings for all
compounds except for compound 1 which has a high contribution of the tolyl ring in LUMO. The
energy level of FMO as well as their energy gap and the global softness (S) were significantly
affected by conjugation of the π‒ system and the electronic nature of the attached groups. The
oxidation reaction of compound 1 significantly decreases the energy gap between the FMO, and this
could be explained in terms of the conjugation. Moreover, attachment of highly electron withdrawing
Br atoms of 2 moves the frontier molecular orbitals to lower energy with respect to the reduced
compound 1. The complexation reaction of 2 by boron has a small lowering effect on the energy
levels of the frontier molecular orbital and their gap. However, the introducing of the electron
releasing alkylamino group of 4 raises the levels of the FMO to higher energy with a decrement in
the energy gap due to the mesomeric effect of the primary N‒atom. Consequently, it can be
emphasized that structure modification of the molecules in affects their electronic nature may be
enable controller the characteristics and optimization for the desired applications.
The charge distribution map for the compounds 1‒4 was calculated under the same basis sets
according to molecular electrostatic potential (MEP) (Figure 9). The red region (negatively charged
atomic sites) was distributed on the pyrrole rings for compounds 1, 2, however the introduction of
the bromine atoms increases their electrophilicity. While, the tolyl rings showed the least negatively
charged atomic sites (blue regions). As shown from figure 8, there is a significant effect of the
attached groups on the charge distribution. The complexation reaction strongly shifted the
electrophilic center to the boron instead of the pyrrole ring. On the other hand, there is no impact of
the (S)‒α‒methylbenzylamino on the charge distribution of 3‒bromo‒4,4‒difluoro‒5‒(((S)‒1‒
phenylethyl)amino)BODIPY 4.
4.
Conclusions
A new chiral fluorescent BODIPY compound 4, has been synthesized and characterized. The
partition coefficient value and other molecular properties were calculated and the results revealed
that compound 4 has a log P value of 3.37, which was ˂5, in accordance with Lipinski’s rule of five.
Further, the antibacterial assays performed for BODIPY compound 4 against various bacterial
strains, the results revealed that maximum zone of inhibition of 24±2.19 mm was found in the case
of Gram negative bacterial strain, E. coli. Moreover, the DPPH and H2O2 radical scavenged
antioxidant activity illustrated that the IC50 values of compound 4, were found to be greater than
ascorbic acid. In order to ascertain the binding values, molecular docking studies on various bacterial
enzymatic targets have been performed. Moreover, DFT calculations were carried out to understand
the molecular geometry and the molecular electrostatic potential of the investigated compound.

Acknowledgements
Authors gratefully acknowledge the Deanship of Scientific Research, Taibah University for support
of our research group (60229).
Conflicts of Interest
The authors have no conflicts to declare.
Supplementary Information
Supplementary data associated with this article can be found, in the online version.
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Scheme 1. Structure of BODIPY, 4,4–Difluoro–4–bora–3a,4a–diaza–s–indacene.
Scheme 2: Synthesis of 3,5‒dibromo BODIPY compound 3.
Scheme 3: Synthesis of 5‒bromo‒4,4‒difluoro‒(((S)‒1‒phenylethyl)amino)BODIPY compound, 4.

Scheme 4: Resonance structures of compound 4.

Figure 1. Absorption spectrum in terms of the molar absorption coefficient (ε) and normalised
fluorescence spectrum for 3 (heavy lines), and 4 (faint lines) in CHCl3.

Figure 2. Zones of inhibition of compound 4 against bacterial strains.

Figure 3. Concentration dependent antioxidant activity of compound 4 and ascorbic acid.

Figure 4. Best docked conformation of compound 4 with antibacterial receptor, E. coli DNA gyrase
(PDB ID: 1KZN). The protein and the DNA backbone are displayed in ribbon and coils. Compound
4 is in light blue spheres. The binding pocket of compound 4 and protein 3D interaction on active
site of E. coli DNA gyrase.

Figure 5. Best docked conformation of compound 4 with antibacterial receptor, (a) β‒ketoacyl ACP
reductase (FabG) (PDB ID: 1I01). The protein is displayed in ribbon and coils. Compound 4 is in
pink spheres; (b) 3R‒hydroxyacyl ACP dehydratase (FabZ) (PDB ID: 1U1Z). The protein is
displayed in multicolor ribbon and coils. Compound 4 is in violet spheres and (c) trans 2‒enoyl ACP
reductase (FabI) (PDB ID: 5CG1). The protein is displayed in red and blue ribbon and coils.
Compound 4 is in light green spheres. The binding pocket of compound 4 and protein 3D interaction
on active sites.

Figure 6. Best docked conformation of compound 4 with antibacterial receptor, D‒alanine:D‒
alanine ligase (Ddl) (PDB ID: 2I80). The protein and the DNA backbone are displayed in light blue
ribbon and coils. The binding pocket of compound 4 and protein 3D interaction on active sites.

Figure 7. Optimized geometrical structure of compounds 1‒4.

Figure 8. The calculated ground state isodensity surface plots for frontier molecular orbitals of
compounds 1‒4.

Figure 9. Molecular electrostatic potentials (MEP) of compounds 1‒4.

Table 1 Spectroscopic data for selected BODIPY compounds 3 and 4.
Compound Solvent λabs/nm λem/nm
ε
Φ
CHCl3
CHCl3
522
510
534
515
93649 0.44
32479 0.07
3
4
*Where, ε = Molar extinction coefficient (Mol‒1cm‒1) and Φ
= quantum yield.
Table 2 Calculation of various biomolecular properties of compounds 3 and 4.
Properties
miLogP
TPSA
Compound 3
Compound 4
‒1.13
8.82
3.37
20.20
n atoms
MW
23
439.89
31
481.17
nON
2
3
nOHNH
n violations
Nrotb
0
0
1
1
0
4
Volume
303.5
405.93
*Where, miLogP = theoretically calculated Log P; TPSA = Total Polar
Surface Area; MW = Molecular Weight; nON = number of hydrogen
bond acceptors; nOHNH = number of hydrogen bond donors; n
violations = number of violated drug‒likeness rules; nrotb = number of
rotating bonds, Volume = molecular volume.
Table 3 In vitro antibacterial screening activity of compound 4 (Compound= 1000µg/ml; DMSO=
30µg/disc).
Compound
Diameter of Inhibition zone (mm)
Gram +ve bacteria Gram –ve bacteria
B. subtilis
21±2.06
‒
S. aureus
19±1.01
‒
E. coli
24±2.19
P. aeruginosa
13±0.94
4
DMSO
‒
‒
*Key to interpretation (10‒20 mM): ˂ 10 mm, inactive; 10‒12 mm,
moderately active; ˃ 12 mm, highly active. DMSO (control) shows no clear
inhibition zone.

Table 4 Antioxidant activity of compound 4.
Compound
DPPH Inhibition
Avg.
IC50
(µg/ml
)
H2O2 Inhibition
Avg.
IC50
(µg/ml)
conc.
(µg/ml)
4
20
40
60
80
20
40
60
80
91. 92. 93. 95.
5
93. 94. 95. 96.
5
93.2
94.9
27.2
26.7
87. 90. 91. 94. 91.0
9
93. 94. 95. 96. 94.9
5
29.9
26.7
3
7
4
2
7
2
Ascorbic
acid
2
5
3
2
5
3
*Avg. = Average percentage inhibition for concentrations 20, 40, 60 and 80 µg/mL recorded after 15
min of incubation.
Table 5 Molecular docking analysis of compound 4 with binding sites in the enzymatic targets and
the binding energies (Kcal/mol).
Targets
PDB ID
Binding sites
Binding
energy
(amino acid residues)
Lys162, Gly75, Asp74, Gln72, Gln135,
Val71, Val–133, Thr165, Glu50, Ala47
Gly127, Val125, Ser126, Ser164, Pro394,
Asp123, Arg395, Leu396, Leu119,
Asn163, Asn120, Gln165
E. coli DNA Gyrase
1KZN
1I01
–10.63
–9.21
β‒ketoacyl ACP
reductase (FabG)
3R‒hydroxyacyl ACP
dehydratase (FabZ)
trans 2‒enoyl ACP
reductase (FabI)
1U1Z
5CG1
Arg8, Glu43, Tyr15, Phe46, Ile41, Pro44,
Pro16, Phe45, Gln48
Gly93, Ile92, His90, Ser91, Ser145,
Glu167, Tyr146, Tyr156, Gly148, Gly121,
Met159, Leu100
–8.95
–9.69
D‒alanine:D‒alanine
2I80
Gly182, Gly352, Asp356, Asp329, Leu181,
Met128, Ala179, Ser127, Glu101, Tyr353.
–8.34
ligase (Ddl)
*Where Lys‒ lysine; Gly‒Glycine; Asp‒Aspartic acid; Gln‒Glutamine; Val‒Valine; Thr‒
Threonine; Glu‒Glutamic acid; Ala‒Alanine; Ser‒Serine; Pro‒Proline; Arg‒Arginine; Leu‒
Leucine; Asn‒Asparagine; Tyr‒Tyrosine; Phe‒Phenylalanine; Ile‒Isoleucine; His‒Histidine
and Met‒Methionine.