Novel stilbene scaffolds efficiently targetMycobacterium tuberculosisnucleoid-associated protein, HU

Article abstract of DOI:10.1039/d0nj05947a

Novel scaffolds of stilbene were identified as inhibitors ofMycobacterium tuberculosisby targeting the nucleoid-associated protein, HU, using molecular docking. Based on the proposed combinatorial libraries I to VI, structures I and III had significantly greater docking binding energy that was comparable to that of the reference ligand, protein HU, fromMycobacterium tuberculosis.Using these docking results, 18 compounds were synthesized, characterized and evaluated forin vitroantitubercular (anti-TB) efficacy in theMycobacterium tuberculosisstrain, H37Rv. Thein vitroscreening results indicated a significant positive correlation between the docking binding efficacy (r²> 0.5) and clogp. Compounds3f,3dand4fwere ranked as top scoring ligands that interacted with amino acids ARG 53, ARG 55, PRO 81, PHE 79, and LYS 13, where the -NO₂or -Cl substitution at theparaposition of the 3-phenyl ring was essential for interacting of the HU protein. The hydrogen bonding with ARG 55 and LYS 13 of these compounds was similar to that with the reference ligand that inhibits the HUM_tbprotein. Compounds3d,3f, and4fwere evaluated as active leads, with MIC90 values of 21.3, 23.2 and 44.1 μM, respectively. The above mentioned compounds were also evaluated for antibacterial and antifungal efficacy in a panel of selected bacteria and fungi. Compound3dhad efficacy (MIC90: 6.82 μM) inS. aureusandE. coli. Compound3fwas also efficacious inE. coliandA. Niger, with an MIC90 value of 7.42 μM for both microorganisms. The fluoro-phenyl derivatives,3iand4i, were efficacious inC. albicans(MIC90 values of 8.2 and 7.8 μM, respectively) andA. niger(MIC90 values of 4.1 and 3.1 μM, respectively). Our results suggest that substitutions at theparaposition of 3-phenyl acryl derivatives with -NO₂and -Cl significantly affected the binding interactions with the HUMtb protein in the docking studies. Furthermore these compounds had antitubercular and antimicrobial efficacy. The substituted phenyl acrylic acid and hydrazides could be inhibitors of the HUMtb protein ofMycobacterium tuberculosis.

Full text of DOI:10.1039/d0nj05947a

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Novel stilbene scaffolds efficiently target
Mycobacterium tuberculosis nucleoid-associated
protein, HU†
Cite this: New J. Chem., 2021,
45, 10683
Ramalingam Peraman,*a Geethavani Meka,a Naresh Babu Chilamakuru,a
b
Vinay Kumar Kutagulla,a Saloni Malla,b Charles R. Ashby Jr.,c Amit K. Tiwari
and Padmanabha Reddy Yiragamreddya
*
Novel scaffolds of stilbene were identified as inhibitors of Mycobacterium tuberculosis by targeting the
nucleoid-associated protein, HU, using molecular docking. Based on the proposed combinatorial libraries I to
VI, structures I and III had significantly greater docking binding energy that was comparable to that of the
reference ligand, protein HU, from Mycobacterium tuberculosis. Using these docking results, 18 compounds
were synthesized, characterized and evaluated for in vitro antitubercular (anti-TB) efficacy in the
Mycobacterium tuberculosis strain, H37Rv. The in vitro screening results indicated a significant positive
correlation between the docking binding efficacy (r2 4 0.5) and clogp. Compounds 3f, 3d and 4f were
ranked as top scoring ligands that interacted with amino acids ARG 53, ARG 55, PRO 81, PHE 79, and LYS 13,
where the –NO2 or –Cl substitution at the para position of the 3-phenyl ring was essential for interacting of
the HU protein. The hydrogen bonding with ARG 55 and LYS 13 of these compounds was similar to that with
the reference ligand that inhibits the HUMtb protein. Compounds 3d, 3f, and 4f were evaluated as active
leads, with MIC90 values of 21.3, 23.2 and 44.1 mM, respectively. The above mentioned compounds were also
evaluated for antibacterial and antifungal efficacy in a panel of selected bacteria and fungi. Compound 3d had
efficacy (MIC90: 6.82 mM) in S. aureus and E. coli. Compound 3f was also efficacious in E. coli and A. Niger,
with an MIC90 value of 7.42 mM for both microorganisms. The fluoro-phenyl derivatives, 3i and 4i, were
efficacious in C. albicans (MIC90 values of 8.2 and 7.8 mM, respectively) and A. niger (MIC90 values of 4.1 and
3.1 mM, respectively). Our results suggest that substitutions at the para position of 3-phenyl acryl derivatives
with –NO2 and –Cl significantly affected the binding interactions with the HUMtb protein in the docking
studies. Furthermore these compounds had antitubercular and antimicrobial efficacy. The substituted
phenyl acrylic acid and hydrazides could be inhibitors of the HUMtb protein of Mycobacterium tuberculosis.
Received 5th December 2020,
Accepted 6th May 2021
DOI: 10.1039/d0nj05947a
rsc.li/njc
drug interactions associated with these regimens.1–3 Drug
resistant strains of TB are one of the most life-threatening
1. Introduction
Currently, the treatment of multidrug resistant (MDR) and airborne infectious diseases in the world and the development
extensively resistant strains of Mycobacterium tuberculosis is of drugs for this disease is considered a high priority.4,5 It has
problematic due to resistance, the use of multidrug, long-term been postulated that drugs developed for the treatment of DR-TB
regimens that decrease the likelihood of compliance and thus should (a) be bactericidal; (b) be efficacious; (c) have novel
therapeutic efficacy and the adverse effects and potential drug- mechanisms of action; (d) low or minimal toxicity; (e) eradicate
the infection in a short period of time (f) possess synergism with
theexisting drugs; (g) preferably interact with numerous targets
a Medicinal chemistry Division, Raghavendra Institute of Pharmaceutical Education
and (h) be non-carcinogenic.6,7
and Research (RIPER)-Autonomous, Anantapur (AP), India.
The choice of a drug target is a critical step in developing an
E-mail: drramalingamp@gmail.com
b Department of Pharmacology & Experimental Therapeutics, College of Pharmacy & efficacious drug. Therefore, based on the literature related to
Pharmaceutical Sciences, The University of Toledo, Toledo, OH 43614, USA.
suitable drug targets, we found that small proteins (polypeptides
E-mail: Amit.Tiwari@utoledo.edu
c Department of Pharmaceutical Sciences, St. John’s University, Queens, NY, 11439,
containing r100 amino acids), especially nucleoid-associated
proteins (NAPs), may be novel targets for drug discovery as they
are involved in information storage, cell signalling, metabolism
and cell growth in microorganisms.8 Examples of such bacterial
USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj05947a
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small proteins (SPs) are Pseudomonas type III repressor A (PtrA)
and Pseudomonas type III repressor B (PtrB), which are involved
in the suppression of the type III secretion system that is
activated due to DNA damage in Pseudomonas aeruginosa.9
Furthermore, they are crucial proteins in the protection of spore
DNA from damage in the dormant spores of Bacillus and
Clostridium spp.10 Zinc-metalloprotease 1 (Zmp1) is an extra-
cellular enzyme that is essential for Mycobacterium tuberculosis
intracellular survival and pathogenesis.11
Nucleoid-associated proteins (NAPs), such as HU, DNA-binding
protein from starved cells (Dps), and nucleoid associated protein,
which are present in Mycobacterium spp, broadly regulate gene
expression and affect mycobacterial resistance to anti-tuberculosis
drugs.12–14 In Mycobacterium, HUMtb (HU-coding gene; gene id:
15610123) is involved in chromosome organization, as well as
determining cellular architecture.13 Interestingly, the compound
stilbene alters the HU-DNA interface in the N-terminal domain of
Mycobacterial HU, resulting in the inhibition of HU–DNA binding,
disrupting the nucleoid architecture of M. tuberculosis.15 This
shows that HU is a potential target for the development of
tuberculosis therapies.
Fig. 1 Designed structures (classes I–VI) as inhibitors of the Mycobacter-
ium tuberculosis nucleoid-associated protein HU.
as the reported toxicity of stilbene results from its high lipo-
Numerous studies indicate that stilbene is one of the most philicity and tissue penetration and (2) improve absorption,
promising pharmacophores due to its natural abundance in a distribution, metabolism and excretion.35
variety of medicinal plants,16,17 with more than 400 isolated
The chemical libraries were obtained (I to VI) based on four
natural stilbenes, including resveratrol (3,40,5-trihydroxy stilbene), substitutions: R1, R2, R3 and R4 on the phenyl rings of the 2,3-
pterostilbene (3,5-dimethoxy-40-hydroxystilbene), piceatannol (trans- diphenyl acryl derivatives. These acryl derivatives had acceptable
3,5,30,40-tetrahydroxystilbene) and pinosylvin (3,5-dihydroxy-trans- lipophilicity (logP o 5), as well as good synthetic feasibility.
stilbene).18 Stilbene-based compounds have been reported to have Thus, the designed libraries (I to VI) were considered for
anti-cancer,19 anti-bacterial,20 anti-fungal,21 anti-viral,22 anti- molecular docking and the structures were 3/2 substituted
tubercular,23 anti-leprotic,24 anti-inflammatory,25 anti-platelet,26 phenyl acrylic acid (Classes I and II), 3/2 phenyl substituted acrylic
anti-diabetic,27 anti-convulsant,28 PPAR agonistic,29 cardio- acid hydrazide (Class III and Class IV) and 3/2 substituted acryl
protective,30 neuro-protective31 and anti-melanogenic efficacies.32 amides (Classes V and VI). Thus, classes I, III and V were 3-phenyl
Notably, stilbenoids are anti-microbial compounds that are substituted, whereas Classes II, IV and VI were 2-phenyl substi-
synthesized de novo to protect plants from fungal and other micro- tuted. The compound library is shown in Table 1.
bial infections.33 Furthermore, stilbene significantly decreases the
2.2 Molecular docking
formation of bio-films by certain microorganisms, which decreases
the likelihood of drug resistance.34 Currently, only a limited number The HU protein structure of Mycobacterium tuberculosis H37Rv
of studies have been conducted to identify molecules that target (code: 4DKY) consists of two identical chains, Chain A and Chain
MtbHU and disrupt its biological activity. This is possibly due to the B. They are represented by 1 unique sequence entity and each chain
common assumption that large and adaptive interfaces, such as contains 106 amino acid residues.36 Each structure from the
HU’s DNA binding surface, cannot be targeted.15 The aim of this designed library was docked with HUMtb using Auto Dock tools
study was to establish strategies to target NAPs with aryl substituted (The Scripps Research Institute, La Jolla, CA, USA) and Discovery
stilbenes and their divergent mechanisms for interacting with the studio visualization software (Biovia, San Diego, CA, USA). The
HUMtb protein.23 Therefore, we evaluated the in vitro efficacy of 3-phenyl substituted derivatives, i.e., Classes I, III and V (binding
novel stilbene derivatives as inhibitors of HUMtb in Mycobacterium energies ꢀ4 to ꢀ6.3 kcal molꢀ1), had binding energies comparable
tuberculosis. We also evaluated in vitro antibacterial and antifungal to that of the reference ligand, 4,40-[(E)-ethene-1,2-diylbis(7-
efficacy of our stilbenes derivatives.
sulfonylimino)]dibenzoic acid (binding energy ꢀ7.0 kcal molꢀ1).
In contrast, the molecules with 2-phenyl substitutions, i.e.,
Classes II, IV and VI, had very low binding energies (ꢀ1.0 to
ꢀ3.0 kcal molꢀ1) and thus the 2-phenyl substituted derivatives
were not synthesized. The binding energy (kcal molꢀ1) and
inhibition constants (mM) for the active libraries (Table 1) were
2. Results and discussion
2.1 Compound library
Given the importance of the stilbene core in the inhibition of determined for HUMtb
.
HUMtb,15 six classes of chemical structures (class I to VI) were
Initially the reference ligand, the trans-stilbene compound
designed as shown in Fig. 1. The stilbene core was modified to 4,40-[(E)-ethene-1,2-diylbis({5[(phenylcarbonyl)amino]benzene-
produce 2,3-diphenyl acryl derivatives to (1) reduce the toxicity, 2,1-diyl}sulfonylimino)]dibenzoic acid,15 was docked using 10
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Table 1 A compound library and the molecular docking data for HUMtb
Comp.
code
Docking energy Inhibition
X
R1 R2
R3
H
R4
(kcal molꢀ1
)
constant nM
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
6g
6h
6i
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
ꢀ5.65
ꢀ4.66
ꢀ5.58
ꢀ5.71
71.92
381.75
80.96
65.07
516.44
31.13
157.03
169.7
125.97
665.06
1630
203.02
465.62
408.54
278.35
398.98
1040
213.39
630.89
1920
435.24
610.36
565.06
258.50
1850
1280
620.54
1600
1850
742.52
1480
1290
1140
1750
1980
OCH3 OH
H
H
Cl
Cl
OCH3 OCH3 OCH3 ꢀ4.48
NO2
H
H
F
H
OCH3 OH
H
H
H
H
H
H
H
H
H
H
H
ꢀ6.15
ꢀ5.19
ꢀ5.14
ꢀ5.32
ꢀ4.33
ꢀ3.80
ꢀ5.04
ꢀ4.55
CN
OH
H
OH
NHNH2
NHNH2
NHNH2
H
Fig. 2 (A) Surface electrostatic potential of HU protein – red colour
indicates the H-bond acceptor region, blue colour indicates the H-bond
donor region and yellow colour indicates the hydrophobic region of the
binding pocket; (B), (C) and (D) binding poses of the standard trans-
stilbene derivative, 4,40-[(E)-ethene-1,2-diylbis({5[(phenyl carbonyl)
amino]benzene-2,1-diyl}sulfonylimino)]dibenzoic acid. (B) Conformation
1 with HUMtb (Chain B; PDB gene code: 4dky). The dotted violet lines
indicate hydrophobic interactions with amino acid residues ILE 32, PHE 50,
PHE 79, PRO 81, PHE 85, LYS 86, VAL 89 and SER 90; (C) conformation 2
with HUMtb (Chain B; PDB gene code: 4dky). The dotted green line
indicates H-bonding interactions with amino acid residue ARG 53 and
the dotted violet lines indicate hydrophobic interactions with amino acid
residues VAL 29, PHE 79, PRO 81, LYS 86 and VAL 89; (D) conformation 3
with HUMtb (Chain B; PDB gene code: 4dky). The dotted green line
indicates the H-bonding interaction with amino acid residue LYS 13 and
the dotted violet lines indicate hydrophobic interactions with amino acid
residues MET 1, LEU 6, LEU 14, ALA 24, VAL 28, PHE 49 and PHE 79.
Cl
Cl
NHNH2 Cl
NHNH2
NHNH2
NHNH2
NHNH2
NHNH2
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
GluA
GluA
GluA
GluA
GluA
GluA
GluA
GluA
GluA
—
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
—
—
OCH3 OCH3 OCH3 ꢀ4.62
NO2
H
H
F
H
OCH3 OH
H
H
H
H
H
H
H
H
H
H
H
ꢀ4.85
ꢀ4.64
ꢀ4.07
ꢀ5.01
ꢀ4.04
ꢀ3.89
ꢀ4.15
ꢀ4.31
CN
OH
H
H
Cl
Cl
OCH3 OCH3 OCH3 ꢀ4.09
NO2
H
H
F
H
OCH3 OH
H
H
H
H
H
H
H
H
H
H
H
ꢀ5.15
ꢀ3.82
ꢀ3.65
ꢀ4.20
ꢀ3.60
ꢀ3.28
ꢀ4.25
ꢀ4.11
CN
OH
H
H
Cl
Cl
OCH3 OCH3 OCH3 ꢀ3.80
NO2
H
H
F
—
—
H
H
H
H
H
—
—
ꢀ5.05
ꢀ3.45
ꢀ2.61
ꢀ3.85
ꢀ7.25
ꢀ6.98
carbon-1, decreased the favourable binding pose to HUMtb
.
CN
OH
H
—
—
Indeed, the binding energy was less than ꢀ4.0 kcal molꢀ1 for
most of the compounds, with an amide linkage bearing amino
acids (5a–i and 6a–i), except for those compounds with –NO2
and –Cl substitutions at the para position of the 3-phenyl ring.
Nevertheless, 5a–i and 6a–i lacked H-bonding interactions and
790.12
4.85
7.68
Std-I
Std -II
—
conformations for HUMtb, and among them, conformations 1, 2 their binding poses were dissimilar from the reference ligand.
and 3 (Fig. 2) were analysed due to their favourable binding Among compounds 3a–i, 3f had higher binding energy
energies (ꢀ8.1, ꢀ7.25 and ꢀ6.98 kcal molꢀ1). Conformation 1 (ꢀ6.15 kcal molꢀ1) and formed H-bonding and hydrophobic
had hydrophobic interactions, whereas conformations 2 and 3 interactions with the HUMtb protein (Fig. 3). Furthermore, 3f
had both hydrophobic and hydrogen bonding (H-bonding) inter- had interactions similar to the reference ligand conformation 2,
actions with amino acid residues ARG 53 and LYS 13 of HUMtb where it interacted with ARG 53, PRO 81, ARG 55 (H-bonding) and
(Fig. 2). The hydrophobic interactions in both conformations 1 ALA 78. Therefore, compound 3f was considered a promising
and 2 were similar, i.e., with the amino acid residues PHE 79, compound from the 3a–i series, which was shown to have in vitro
PRO 81, LYS 86 and VAL 89. However, the hydrophobic interaction antitubercular efficacy (MIC90 = 23.2 mM).
of the phenyl ring of the ligand with PHE 79 and PRO 81 was
In contrast, the active compound 3d had hydrophobic
noted as a unique characteristic for all ten conformations. This interactions with the HUMtb protein (Fig. 3), where it interacted
interaction is due to the aromatic phenyl ring of phenylalanine with ILE 32, VAL 33, VAL 36, HIS 37 and PRO 77. The
and the pyrrolidine ring of proline amino acids.
interaction with ILE 32 was similar to that of the reference
Docking analysis of the chemical library (Table 1) indicated ligand conformation 1. In addition, 3d interacted with HIS 37
that compounds 3a–i and 4a–i had higher binding energies due to the presence of an ortho –Cl group. The compounds from
than the remaining classes of compounds (5a–i and 6a–i) with the 4a–i series had no hydrogen bonding interactions, except
the HU protein. Bulky groups, such as an amino acid linkage at for compound 4f (binding energy of ꢀ4.85 kcal molꢀ1) and it
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Fig. 3 Compound 3f: A binding pose of compound 3f (brown colored)
with HUMtb (Chain A; PDB gene code: 4dky). The dotted green line
indicates the H-bonding interaction of the nitro group of the ligand with
amino acid residue ARG 55, and the dotted violet lines indicate hydrophobic
interactions with ARG 53, ALA 78, ARG 80 and PRO 81. Compound 3d:
A binding pose of compound 3d (brown colored) with HUMtb (Chain A; PDB
gene code: 4dky). The dotted violet lines indicate hydrophobic interactions
with amino acid residues ILE 32, VAL 33, VAL 36, HIS 37 (with ortho Chlorine)
and PRO 77. Compound 4f: A binding pose of compound 4f (brown
colored) with HUMtb (Chain B; PDB gene code: 4dky). The dotted green
line indicates the H-bonding interaction of the nitro group of ligands with
amino acid residue LYS 13. The dotted violet lines indicate hydrophobic
interactions with amino acid residues LEU 10, VAL 25 and VAL 28.
Scheme 1 Synthetic route for the synthesis of compounds 3a–i and 4a–i.
2.4 Characterization
The synthesized compounds 3a–i and 4a–i were characterized
by physical and spectral techniques. The compounds were
yellow to orange coloured crystals and were stable under light
and at room temperature. Except for the nitro compounds
(3f and 4f), the remaining compounds had melting points
4160 1C. Compounds 3b, 3e, 4b and 4e had very high melting
points (4300 1C). Among the series, the acid hydrazide derivatives
(4a–i) had a relatively higher melting point compared to the acid
derivatives (3a–i) as a result of the increased inter-molecular
hydrogen bonding. The calculated log P values (c log p) of
the compounds were in good agreement (r2 4 0.8) with the
retardation factor (Rf value from the TLC technique). The
clog p values of the compounds were in the range of 1–5, thereby
satisfying the ‘‘Lipinski rule’’ in terms of hydrogen bond donors
(HBDs), hydrogen bond acceptors (HBAs), partition coefficient
(log P) and molecular weight (MW). The physical data for the
compounds are shown in Table 2.
formed a hydrogen bond with amino acid residue LYS 13
(Fig. 3), which is similar to reference ligand conformation 3.
Therefore, 4f is considered to be an active compound (MIC90 =
44.1 mM) from the 4a–i series.
2.3 Chemical synthesis
From the proposed classes (I to VI) of chemical structures, we
synthesized classes I and III due to their favorable binding
energy in molecular docking poses with HUMtb
.
The stilbene derivatives class I (3a–i) and class III (4a–i) were
synthesized (Scheme 1) as 3-(substituted phenyl)-2-phenylacrylic
acid (3a–i) and 3-(substituted phenyl)-2-phenylacrylic acid
hydrazide (4a–i). Compounds 3a–i were synthesized using a
previously published synthesis scheme,36 where different
aldehydes (a–i) were allowed to react with phenyl acetic acid
under anhydrous conditions using acetic anhydride as a solvent
and triethyl amine as a catalyst.
The chemical structure of the compounds was established
by UV, IR, 1H-NMR, 13C-NMR and mass (LC-ESI-MS) spectral
data (Table 2). The characteristic maxima (lmax) in the
UV-visible spectra of all compounds indicated the presence of
the stilbene chromophore. A bathochromic shift was observed
during the transformation of the acid derivatives (3a–i) to acid
hydrazides (4a–i). The infra-red (FT-IR) spectra of the com-
pounds confirmed the presence of functional groups, including
O–H stretching (E3500 cmꢀ1
)
and N–H stretching
The transformation of compounds 3a–i to 4a–i was conducted
as previously reported,37 using a microwave (200 MW), where
acethydrazide reacted with compounds 3a–i in the presence of
acetic acid. The % yield of 4a–i (57–65%) was greater than that
for 3a–i (52–58%). The chemical test (decolorization of pink
color of KMnO4) for unsaturation (CQC) was positive. The
product formation during the progress of the reaction was
monitored using thin layer chromatography (TLC, using silica
gel GF254 coated plates). A total of 18 compounds (3a–i and 4a–i)
(E3400 cmꢀ1), for 3a–i and 4a–i, respectively. In addition, the
characteristic absorption for a carbonyl group (CQO stretch-
ing) was observed at E1700 and E1680 cmꢀ1 for acid (3a–i)
and amide (4a–i) linkages, respectively. The 1H-NMR spectra
indicated the chemical shift values for a carboxylic proton
(–COOH) and amine proton (–NH2) of 3a–i and 4a–i of
ꢀ
ꢀ
E10 ppm and E8.5 ppm, respectively. The ppm value for
QCH (H3) was E6 ppm. The J value for H3 was observed for
ꢀ
were synthesized and purified using column chromatography. coupling with the aromatic protons H13 and H17. The Hz
The purity of the compounds was confirmed using reversed value for JH3–H13 and JH3–H17 is presented in Table 2. 13C-NMR
phase HPLC with a C18 column.
spectra showed the corresponding chemical shift values for
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CQC (C2 and C3) and CQO (C1) to be E99, E100, and Gram-positive and Gram-negative bacteria (Table 3). Notably,
E170 ppm, respectively. The up-field effect for C1 (E165 ppm) compound 3e, which contains a para-OCH3 substitution, had
was observed for compounds 4a–i due to the mesomeric effect of greater efficacy only in E. coli.
the CO–NH group.
The antifungal efficacy assay indicated that compounds 3f,
The observed 1H-NMR chemical shift for H3 at E6 ppm and 3i and 4i produced a greater inhibition of fungal growth
13C-NMR chemical shift values for C2 and C3 (CQC) of E99 and compared to other compounds (Table 3).
E100, respectively, indicated an E conformation rather than a
Compound 3f was efficacious in E. coli and A. niger, with an
Z
conformation, which was in agreement with the MIC90 value of 7.42 mM but had relatively low efficacy in
literature,37,38 and was due to the 3-phenyl substitutions in P. aeruginosa and P. vulgaris, with MIC90 4 13 mM (Table 3).
the phenyl ring system at the 3rd position. The mass spectra Compound 3d was efficacious (MIC90 = 6.82 mM) in S. aureus
were obtained using the electro-spray ionization technique and E. coli (Table 3). The fluoro-phenyl derivatives, 3i and 4i,
using a positive mode. The obtained m/z value for molecular were efficacious in C. albicans and A. niger with MIC90 values of
ions (M+) or quasi molecular ions (M + H) agreed with the 8.2 and 7.8 mM, respectively (Table 3). The NO2 substitution at
molecular weight of the proposed structure.
the 3-phenyl position produced a 5-fold increase in antifungal
efficacy and a fluorine substitution produced a 9-fold increase
in antifungal efficacy (Table 3). This increases due to the
contribution of –NO2 and –F groups to high membrane perme-
ability of the synthesized molecule and this is in accordance
with the earlier report of Olender et al.39
2.5 In vitro antibacterial and antifungal efficacy assays
The synthesized active derivatives were evaluated for antibacterial
and antifungal efficacy. The antibacterial efficacy was determined
in Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC
25922), Proteus vulgaris (ATCC 13315) and Pseudomonas aeruginosa
(ATCC 27853), using the micro-dilution assay method at concen-
2.6 In vitro antitubercular screening
trations from 0.5–8 mg mlꢀ1. The evaluation of the antifungal The results indicated that compounds 3a–i are significantly
efficacy of the synthesized compounds was done in Aspergillus more potent than 4a–i (MIC90 4 40 mM). Compounds 3d and 3f
niger (ATCC 16404) and Candida albicans (ATCC 10231), using had the greatest efficacy among all of the derivatives, with
concentrations of 0.5–8.0 mg mlꢀ1 (Table 3).
MIC90 values of 21.3 and 23.2 mM, respectively (Fig. 4). The –OH
The results indicate that compounds 3a–i were more efficacious and –OCH3 substitutions decreased antitubercular efficacy,
than compounds 4a–i, and among compounds 3a–i, compounds whereas the para-NO2 and ortho-Cl increased the anti-
3d and 3f were primarily efficacious in Gram-positive and Gram- tubercular efficacy 2–3 fold compared to the parent compound,
negative bacteria (Table 3). Overall, the substitution at the 3-phenyl 3a (Fig. 4). Surprisingly, the hydrazide derivatives were less
ring increased the antibacterial and antifungal efficacy by 2-3-fold potent than the acid-based compounds (3a–i), which may be
(Table 3). Among the substitutions in compounds 3a–i, the ortho Cl, due to the non-interacting nature of the acryl chain, as indicated
para-NO2, para-CN, para-OH and meta-F increased their efficacy in by molecular docking (Fig. 4). The compounds that had c log p
Table 3 Antitubercular, antibacterial and antifungal efficacies (MIC90 values) of the synthesized compounds
Anti-TB activity mM/mg mlꢀ1
Antibacterial activity mM/mg mlꢀ1
Antifungal activity mM/mg mlꢀ1
Comp. code
Mtb H37Rv
SA
EC
PA
PV
CA
AN
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
4e
4f
4g
4h
4i
55.7/12.5
92.6/25
48.4/12.5
21.3/6.25
79.5/25
23.2/6.25
50.1/12.5
52.0/12.5
51.5/12.5
104.9/25
87.9/25
45.8/12.5
40.6/12.5
76.1/25
44.1/12.5
94.9/25
49.1/12.5
48.7/12.5
0.73/0.1
6.854
35.6/8
429.5/48
15.5/4
6.82/2
25.4/8
14.8/4
16.0/4
16.0/4
16.5/4
433.5/48
428.1/48
29.3/8
13.0/4
24.3/8
28.2/8
430.3/48
15.7/4
31.2/8
0.27/0.1
2.219
35.6/8
429.5/48
15.5/4
6.82/2
12.7/4
7.42/2
16.0/4
16.0/4
16.5/4
433.5/48
428.1/48
29.3/8
26.0/8
24.3/8
14.1/4
430.3/48
15.7/4
31.2/8
0.27/0.1
2.365
435.6/48
429.5/48
30.9/8
13.6/4
425.4/48
14.8/4
432.0/48
16.0/4
16.5/4
433.5/48
428.1/48
429.3/48
426.0/48
424.3/48
428.2/48
430.3/48
31.4/8
35.6/8
429.5/48
30.9/8
13.6/4
25.4/8
14.8/4
16.0/4
16.0/4
16.5/4
433.5/48
428.1/48
429.3/48
426.0/48
24.3/8
28.2/8
430.3/48
31.4/8
435.6/48
429.5/48
15.5/4
27.3/8
25.4/8
14.8/4
16.0/4
16.0/4
8.2/2
35.6/8
429.5/48
15.5/4
13.6/4
12.7/4
7.42/2
16.0/4
16.0/4
4.1/1
16.7/4
14.0/4
14.6/4
26.0/8
24.3/8
28.2/8
430.3/48
15.7/4
3.9/1
33.5/8
28.1/8
14.6/4
426.0/48
424.3/48
428.2/48
430.3/48
15.7/4
7.8/2
0.0042/2
2.376
431.2/48
0.54/0.2
1.711
431.2/48
0.54/0.2
1.843
Std
SD
CD
0.0021/2
2.470
1.228
3.408
1.103
1.176
0.850
0.916
1.181
SA – Staphylococcus aureus; EC – Escherichia coli; PV – Proteus vulgaris; PA – Pseudomonas aeruginosa; CA – Candida albicans; AN – Aspergillus niger;
SD – standard deviation; CD – critical difference at P o 0.05 (using Student’s t test) based on the calculated mg mlꢀ1 values.
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determined using the open capillary tube method (Table 2).
The functional groups were characterized by IR spectra using a
Fourier transform-infrared spectroscopy technique (Alpha E,
Bruker, ATR; RIPER, Anantapur, India) at a resolution of
4 cmꢀ1. The protons of the synthesized structures were assigned
by the proton nuclear magnetic resonance spectrum (1H NMR),
using a Varian VXR Unity, with TMS as the internal standard and
DMSO-d6/CDCl3 as solvent. Mass spectroscopy (Shimadzu LCMS
8040, Laila Implex, Vijayawada, India) was used to confirm the
molecular weight of each derivative. All of the chemical reagents
used were of analytical or synthetic grade.
Chemical synthesis was performed by conventional heating
and the reactions were monitored by TLC. Purification was
conducted using double recrystallization, followed by preparative
TLC. The purity of the compounds was assessed prior to biological
screening and spectral analysis using HPLC. The characterization
of the compounds was accomplished using UV, IR, 1H NMR,
13C NMR and LC-MS (ESI) techniques.
Fig. 4 (A) Antitubercular efficacy of stilbene analogs for Mycobacterium
tuberculosis H37Rv using the microplate alamar blue assay (MABA) method.
(B) A scatter plot indicating a significant positive correlation between the clog P
values of compounds and the antitubercular efficacy in Mycobacterium
tuberculosis H37Rv. (C) A scatter plot indicating a significant positive correlation
between binding energy and antitubercular efficacy.
3.2 Molecular docking studies
3.2.1 Ligand preparation. The proposed chemical structures
(i.e. ligands) were drawn in Marvin Sketch 19.8 (ChemAxon,
Budapest, Hungary) and subjected to cleaning in 3D and saved
in a PDB format. The PDB format of the structure was opened in
Note Pad and subjected to energy minimization using the
PRODRG server. The text format of ‘‘all hydrogen’’ was converted
into a PDB format.40,41
values from 2–4 had significantly greater efficacy compared to
the other derivatives. Indeed, there was a significant positive
correlation between the c log p values and antitubercular efficacy
(Fig. 4). The electronegative substitution in the 3-phenyl rings
produced greater antitubercular efficacy compared to other
substitutions (Fig. 4). This finding is in agreement with the
docking binding energy of the compounds (Table 1) and the
in vitro antitubercular efficacy data (Fig. 4).
Currently, only a few studies have been published about the
antitubercular efficacy of stilbene-based compounds, trans-
stilbene compound 4,40-[(E)-ethene-1,2-diylbis({5[(phenylcarbonyl)
amino]benzene-2,1-diyl}sulfonylimino)]dibenzoic acid15 and
diarylethenes23 and their IC50 values were 20 mM and 150 mM,
respectively. Accordingly, our compounds, 3d (–Cl substituted)
and 3f (–NO2 substituted) derivatives exhibited IC50 values of
21.3 mM and 23.0 mM, respectively. Our results indicate that nitro
and halogen substitions are important for the design of effica-
cious antitubercular compounds that inhibit the Mtb HU protein.
3.2.2 Receptor preparation. The protein structure was
downloaded from a protein databank (Research Collaboratory
for Structural Bioinformatics) in a PDB format.36 The non-
standard residues and atoms were removed using UCSF
Chimera 1.13.1 (Resource for Biocomputing, Visualization,
and Informatics (RBVI), University of California, San Francisco)
to yield a receptor complex free of any ligand before docking.42
3.2.3 Docking process. The docking of the proposed structures
was done using the Auto Dock 1.5.6 tool (The Scripps Research
Institute, La Jolla, CA, USA). Initially, the PDB protein is loaded and
water molecules are removed, Kollman charges are added and
hydrogens are set as polar. Each molecule was docked separately
and loaded and torsions were set and saved in a PDBQT format.
The grid box, with dimensions of: X = ꢀ5.249, Y = 6.877 and
Z = 4.206 Å, with a default grid spacing of 0.375 Å, was chosen.
The generated gpf and dpf files were converted into log files using a
command prompt tool. The best conformation was chosen based
on the lowest docked binding energy at the end of the docking
analysis. The best conformation was analyzed for binding sites,
interacting amino acids, hydrogen bonds, hydrophobic interactions
and bond lengths using Biovia Discovery studio.43 The results for
each compound are shown in Table 1.
2.7 Statistical analysis
Statistical analysis of compounds synthesized for anti-bacterial
and anti-fungal activity determined in Staphylococcus aureus,
Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa,
Candida albicans, and Aspergillus niger was conducted using
Student’s t test (Table 3).
3.3 Chemical synthesis
3. Experimental
3.1 Materials and methods
3.3.1 The synthesis of (2E)-3-(substituted phenyl)-2-
phenylacrylic acid and the general procedure for compounds
The purity of the compounds was confirmed using precoated (3a–i). The compounds were synthesised as previously
TLC and HPLC (Agilent LC 1200, C18 (250 mm ꢁ 4.6 mm, described.36 In a 500 ml round bottom flask, 40.5 ml
5
micron)) column techniques. The melting point was (42.4 g., 0.40 mole) of freshly purified benzaldehydes (1a–i),
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54.6 g. (0.40 mole) phenyl acetic acid (2), 40 ml of anhydrous 3.5 The determination of the in vitro antibacterial and
triethylamine and 80 ml of acetic anhydride were combined antifungal efficacy using micro-dilution testing
and refluxed gently for 5 hours. Subsequently, the reaction
mixture was subjected to steam-distillation until the distillate
coming over was no longer cloudy and approximately 50 ml of
the distillate was collected. The distillate was discarded and the
aqueous residue was cooled and the solution was separated
from the solid by decantation. The solid was dissolved in
efficacy was determined in a griseofulvin-resistant strain of
1000 ml equal portions of heated 95% ethanol and water. The
mixture was heated to boiling and 2 g of decolorizing carbon was
10231). The obtained MIC90 (90% inhibition, duplicate) mean
added. The hot solution was filtered, and immediately acidified
by Congo red with 6N hydrochloric acid. Finally, the solution was
The antibacterial efficacy was determined in a ciprofloxacin-
resistant strains of Staphylococcus aureus (ATCC 25923), Escherichia
coli (ATCC 25922), Proteus vulgaris (ATCC 13315) and Pseudomonas
aeruginosa (ATCC 27853), using the micro-dilution assay method, at
concentrations of 0.04–9.6 mg mlꢀ1. Similarly, the antifungal
Aspergillus niger (ATCC 16404) and Candida albicans (ATCC
was converted into micromolar concentration (mM) and is reported
in Table 3.
cooled to obtain the targeted compound as crystals. Further-
The above antibacterial and antifungal efficacies were deter-
mined based on a previous publication from our laboratory.44
more, the product was purified by column chromatography. The
homogeneity of the compounds was verified using pre-coated
Broth micro-dilution testing was performed with 96-well,
TLC plates (TLC silica gel 60F254) and HPLC.
round-bottom microliter plates. Each plate included positive
3.3.2 The synthesis of (2E)-3-(substituted phenyl)-2-
phenylacrylic acid hydrazide and the general procedure for
controls (bacteria without antimicrobial compounds), negative
controls (medium only), and serial dilutions of 0.5–8.0 mg mlꢀ1
compounds 4a–i. The compounds were synthesised as pre-
for the test compounds. DMSO was used as a solvent for all of
the compounds. The media used for the bacteria and fungi were
viously reported.37 Compounds 3a–i (20 mmol), acethydrazide
(3.0 g, 40 mmol) and acetic acid (1 ml) were mixed and
Mueller Hinton broth and potato dextrose agar, respectively.
subjected to microwave irradiation at 200 MW (9–13 min) for
an optimal time, depending on the parent compounds (3a–i).
Upon completion of the reaction (monitored by TLC), acetic
The % growth inhibition for the bacteria was determined using
an absorbance value (OD600) obtained at 600 nm. The percentage
inhibition data were obtained for each well using the absorbance
acid was removed in a vacuum and ice water (3 ml) was added
of a blank (media) and a positive control. The absorbance for %
to the residue. The crude product was collected by filtration and
growth inhibition of the test compounds for the fungi was
recrystallized using appropriate solvents. The compounds were
measured at 530 nm using absorbance (OD530) after the addition
purified by column chromatography. The homogeneity of the
of 0.001% resazurin dye and incubation at 35 1C for an
compounds was verified using pre-coated TLC plates (TLC silica
additional 2 h. The growth inhibition percentage of 90% was
gel 60F254) and HPLC.
considered to be the minimum inhibitory concentration (MIC).
3.4 Characterization of the synthesized compounds
3.6 The determination of antitubercular efficacy using the
microplate alamar blue assay (MABA)
All compounds were characterized based on physical properties,
including the melting point, retardation factor on silica gel and
c log P values. The structure was established by spectral analysis Compounds were screened in the Mycobacterium tuberculosis
1
using UV, IR, H NMR, 13C NMR and mass (ESI) spectra. The strain, H37Rv (ATCC 27294), using the microplate alamar blue
complete spectral profile of the prototype compound for each assay (MABA) method. A stock solution of 1 mg mlꢀ1 of the test
series is presented below. Furthermore, comparative spectral compounds was prepared in DMSO and was sterilized by
features of all derivatives are appended in Table 2.
passage through 0.22-micron Nylon based membrane filters.
(E)-2,3-Diphenylacrylic acid (3a). lmax: 289 nm, IR (KBr Control samples were pipetted with 50 ml of DMSO, whereas the
pellet, cmꢀ1): 3505 (O–H Str) 3060 (C–H Str Ar-H), 2869 (QC–H positive control samples were pipetted with isoniazid (stock
Str, aliphatic), 1684 (CQO Str), 1604 (CQC str), 833, 802 1 mg mlꢀ1 and suitably diluted to 0.05, 0.1 and 0.2 mg mlꢀ1).
(deformation, out of plane Ar-H) 1410 (C–O str); 1H NMR The inoculum used was a 1 : 100 dilution as this represented
(DMSO-d6, d ppm): 10.12 (s, 1H, –COOH), 6.81–7.82 (m, 9H, 1% of the mycobacterial population (102–103 colony forming
Ar-H); 6.0, 6.1 (dd, 1H, ethylene, JH–H = 5 Hz, 4 Hz), 2.52 (DMSO); units per ml). All compounds were screened for antitubercular
13C NMR (CDCl3, d ppm); 170.02 (CQO) 153.3, 148.3, 137.6, efficacy over the concentration range of 0.8–50 mg mlꢀ1. After
137.0, 128.65, 127.6, 126.4, 121.1, 111.4, 109.8, 99.2; MS (API-ES); 5 days of incubation, 50 mL of a 1 : 1 dilution of Alamar stock
225 (M + 1).
solution was transferred to all wells for a final assay volume of
(E)-2,3-diphenylacrylohydrazide (4a). lmax
:
291 nm; IR: 250 mL per well, yielding a final concentration of 10% v/v of
3398.97(N–H str), 3055.61 (C–H str Ar-H), 2889 (QC–H Str), Alamar blue. The plates were analyzed following exposure to an
1674 (CQO stretching), 1612 (CQC str), 813, 799 (deformation, excitation wavelength of 530 nm and an emission wavelength of
out of plane Ar-H); 1H NMR (DMSO-d6, d ppm): 8.91 (broad, 1H, 590 nm. We also determined whether the test compounds were
NH), 7.68 (s, 2H, NH2), 7.11–7.42 (m, 9H, Ar-H), 5.71, 5.85 (dd, fluorescent at the emission wavelength as this would interfere
1H, ethylene, JH–H = 4 Hz, 2 Hz). 13C NMR (CDCl3, d ppm); with the interpretation of the results. The percent viability was
167.20 (CQO) 154.3, 148.4, 137.2, 136.5, 128.25, 127.1, 126.8, determined based on the fluorescence counts in the presence of
121.7, 110.6, 106.7, 88.8; MS (API-ES): 238.28 (M + 1).
a test compound as a percentage of that in the vehicle control.
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N. B. C., V. K. K, and P. R. Y.; formal analysis, R. P.; investigation,
G. M., N. B. C., V. K. K, and P. R. Y.; resources, R. P.; data curation,
R. P., A. K. T., and S. M.; writing – original draft preparation, R. P.;
writing – review and editing, R. P., S. M., C. R. A. and A. K. T.;
visualization, R. P. and A. K. T; supervision, R. P. and A. K. T.;
project administration, R. P. and A. K. T.; and funding acquisition,
R. P. All authors have read and agreed to the published version of
the manuscript.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This work was supported by the University Grant Commission
(UGC), Government of India, under the UGC Minor Research
Grant Scheme (File No: MRP-7055/16 – SERO/UGC). Spectral
data were provided by Laila implex Pvt. Ltd, Vijayawada (AP),
India. The authors are thankful to DST-FIST Laboratory of
Raghavendra Institute of Pharmaceutical Education and
Research (RIPER), Anantapur (AP), India, funded by Department
of Science and Technology, Govt. of India, for the provided
facility support.
Fig. 5 Structure–activity relationship and structures of potential lead
compounds 3d, 3f and 4f.
A compound was considered to be active only if it produced at
least 90% inhibition of mycobacterial growth (i.e., MIC90).
3.7 Statistical analysis
The antimicrobial screening plates were prepared in duplicate
and Student’s t test was used to analyse the data. Critical
difference data (Table 3) were considered to be statistically
significant if the one-tailed t test value was equal to or greater
than 1.734. The regression analysis was used in finding the r2
value between clogp and antitubercular activity and to establish
a relationship between variables.
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New J. Chem., 2021, 45, 10683–10692
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