Computational Biology and Chemistry 78 (2019) 330–337

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Computational Biology and Chemistry

journal homepage: www.elsevier.com/locate/cbac

Design, synthesis, antimicrobial activity and computational studies of novel T

azo linked substituted benzimidazole, benzoxazole and benzothiazole
derivatives
Virendra R. Mishraa, Chaitannya W. Ghanavatkara, Suraj N. Malib, Shahnawaz I. Qureshia,b,
⁎ ⁎
Hemchandra K. Chaudharib, , Nagaiyan Sekara,
a
Department of Dyestuff Technology, Institute of Chemical Technology, Matunga (E), Mumbai, Maharashtra, 400 019, India
b
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga (E), Mumbai, Maharashtra, 400 019, India

A R T I C LE I N FO A B S T R A C T

Keywords: Novel azo linked substituted benzimidazole, benzoxazole, and benzothiazole were synthesized by diazo coupling
Heterocyclic azo dyes and characterized by 1H NMR, elemental analysis, FTIR and UV–vis spectroscopy. The newly synthesized
REMA assay compounds were evaluated for invitro antibacterial activity against Staphylococcus aureus and Escherichia Coli
Molecular docking strains by Resazurin microtiter assay method (REMA). The minimum inhibitory concentration (MIC in μg/mL)
Density functional theory
were used to express the antibacterial activities. The azo linked compounds exhibited good to moderate or high
antibacterial activities in vitro. Computational studies were performed to correlate HOMO-LUMO gap with
antibacterial activity. The comparative molecular docking studies revealed better insights into binding me-
chanisms.

1. Introduction (Coburn et al., 1987).

The problem of antimicrobial drug resistance to antibiotics like β-
lactam antibiotics, quinolones, and macrolides is widely known (Raja
and Prabakaran, 2010). There is still an increase in mortality, mor-
bidity, and antibiotics treatment failure due to a variety of infectious
conditions caused by resistant microbials (Özkay et al., 2010). There-
fore, there is a need to develop effective antimicrobial agents to over-
come these issues (Mali and Chaudhari, 2018). Heterocyclic compounds
containing benzimidazole, benzoxazole, and benzothiazole moieties are
attracting researchers due to their structural and photophysical prop-
erties (Tao et al., 2012; Wang et al., 2015). These scaffolds are also
utilized in a wide range of pharmacological activities (Demmer and
Bunch, 2015; Keri et al., 2015; Liu et al., 2018). Benzimidazole con-
taining structures are “privileged substructures” (Mohanty et al., 2018).
UK-1 [bis (benzoxazole)] is a natural product consisting of the 2-(2'-
hydroxyphenyl) benzoxazole core and it has anticancer activity (Fig. 1).
The studies relating to the metal binding of UK-1 revealed that it has
capability to bind biologically important metal ions (Kumar et al.,
2002). A derivative of UK-1, 1 is also found with metal mediated DNA-
binding capability (McKee and Kerwin, 2008). The analogues of 2-ar-
ylbenzazoles 2 and 3 show the cytotoxic activities (Fig. 1) (Aiello et al.,
2008). Compound 4 is a promising agent for periodontal diseases
On the other hand, azo compounds are utilized in cosmetics as well
as pharmaceutical research and are involved in a number of biological
reactions, such as inhibition of DNA, RNA and protein synthesis, ni-
trogen fixation and carcinogenesis (Hassan et al., 2017). Sulfonamide
azo dyes were the first chemotherapeutic agent used for the cure of
bacterial infection in human. A series of azo dye were synthesized as a
potential antimicrobial agent (Al-Rubaie and Mhessn, 2012; Karci et al.,
2009; Saad et al., 2017; Yazdanbakhsh et al., 2012). Most of these dyes
show only in vivo activity (Al-Rubaie and Mhessn, 2012). To get invitro
activity in azo dyes, they should either form a metal complex
(Mahmoud et al., 2017, 2016) or contain heterocyclic ring (Afifi et al.,
2017; Shridhar et al., 2011).
In the present work, we report six novel heterocyclic azo dyes linked
to benzimidazole, benzoxazole and benzothiazole scaffolds. Dyes are
evaluated for in vitro antibacterial activity against both gram-positive
and gram-negative organisms Staphylococcus aureus and Escherichia Coli
strains respectively by REMA assay method. To understand the binding
activity, molecular docking studies were performed against protein
targets as reported in the literature for benzimidazole, benzoxazole and
benzothiazole scaffolds (Bassyouni et al., 2012; Bax et al., 2010; Liu
et al., 2018; Malashkevich et al., 1999). Computational studies are
performed to understand the relation between the HOMO-LUMO gap

⁎
Corresponding authors.
E-mail address: n.sekar@ictmumbai.edu.in (N. Sekar).

https://doi.org/10.1016/j.compbiolchem.2019.01.003
Received 2 December 2018; Received in revised form 4 January 2019; Accepted 7 January 2019
Available online 07 January 2019
1476-9271/ © 2019 Elsevier Ltd. All rights reserved.
V.R. Mishra et al. Computational Biology and Chemistry 78 (2019) 330–337

Fig. 1. Potential therapeutic/diagnostic agents for UK-1 analogues, 2-Arylbenzothiazoles and 2-arylbenzazole from literature.

Fig. 2. UV–vis Absorption spectra of 7 (a–c) and 8 (a–c) in DCM solvents at

room temperature.

Table 2
Invitro antibacterial activities of finally synthesized compounds 7(a–c) and
8(a–c).
Molecule ID. Bacteria MIC (μg/mL)

Scheme 1. Synthesis scheme of novel Heterocyclic Azo Dyes. Gram-positive (Staphylococcus Gram-negative (Escherichia
aureus) coli)

7a 625 625
Table 1
7b 625 625
Electronic spectral data for 7(a–c) and
7c 625 625
8(a–c). 8a 78.125 78.125
Dyes Wavelength 8b 1250 625
λabs. (nm) 8c 625 312.5
Ciprofloxacin 50 25
7a 488
7b 486
7c 487 purification. Thin layered chromatography (TLC 0.25 mm E-Merck si-
8a 466
lica gel 60 F254 pre-coated plates) was used to monitor the reactions,
8b 465
8c 466 which were visualized with UV light. Melting points were measured on
standard melting point apparatus from Sunder industrial product,
Mumbai and are uncorrected. 1H NMR spectra were recorded on a
and the activity of the molecule. 500 MHz, 400 MHz, and 300 MHz, 13C NMR spectra were recorded on
the 126 MHz instrument of Agilent Technology. The absorption spectra
of the compounds were recorded on a Perkin Elmer Lambda-25 spec-
2. Materials and methods trophotometer.

2.1. Materials and equipment 2.2. Computational method

All the required chemicals and solvents were acquired from com- Density Functional Theory (DFT) was used to optimize the ground
mercial sources (SD Fine Chemical Ltd.) and used without any further state of compounds using the Gaussian 09 package (Frisch et al., 2009)

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V.R. Mishra et al.
Fig. 3. Graphical representation of the antibacterial activity of synthesized compounds 7(a–c) and 8(a–c).
Fig. 4. Crystal structure of target receptor 1D7U along with standard drug Ciprofloxacin.
and the popular hybrid functional B3LYP. The B3LYP functional is a
combination of Becke’s three parameter exchange functional (B3)
(Becke, 1993) with the nonlocal correlation functional of Lee, Yang,
and Parr (LYP) (Lee et al., 1988). The basis sets used for all atoms were
the popular Pople’s split-valence basis sets. The B3LYP functional is
used with the double zeta basis set 6-31 g (d) for optimization of mo-
lecules in the gas phase and in DMSO.
2.3. Molecular modelling
The molecular docking studies were carried out using the Glide
module incorporated into Schrodinger’s molecular modeling software
(Glide, 2017). All the molecular properties were calculated using the
Qikprop module (Qikprop, 2017). The analysis of binding free energies
corresponding with the docked complexes was carried out using the
Prime MMGBSA module (Prime, 2017) installed on Linux based com-
puter having 16 GB RAM.
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Computational Biology and Chemistry 78 (2019) 330–337
2.4. Antimicrobial screening using Resazurin microtiter assay method
(REMA)
The nutrient broth (3.25 g) was dissolved in 250 mL of water. The
media was sterilized by moist heat sterilization method using an au-
toclave. The freshly prepared and sterilized media was used for eva-
luation. S. aureus and E. coli culture were inoculated in nutrient broth
and kept in an incubator for 24 h at a temperature of 37 °C, OD580
adjusted to 0.1 (approx. 107 cells/mL). Stock solution (5 mg/mL) of the
standard drug Ciprofloxacin was prepared in 2 mL of DMSO. Stock so-
lution (5 mg/mL) of the test compounds (dyes) was prepared by dis-
solving the test compound in 100 μL of DMSO and volume was made up
to 1 mL with nutrient broth. 100 μL of a stock solution of the standard
compound was taken into an Eppendorf tube and volume was made up
to 1 mL with nutrient broth. From this 100 μL of solution was serially
diluted into each well of 96 microtiter plate which previously contained
100 μL of nutrient broth. Dilution of a stock solution of test compound
V.R. Mishra et al. Computational Biology and Chemistry 78 (2019) 330–337

Fig. 5. Crystal structure of target receptor 2XCS along with standard drug Ciprofloxacin.

Fig. 6. Protein interface of target 1D7U with a surface binding site for most docked 8b.

Fig. 7. Protein inerface of target 2XCS with the surface binding site for most docked 8c.

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V.R. Mishra et al. Computational Biology and Chemistry 78 (2019) 330–337

Table 3
The Docking scores and interactions of 7(a–c) and 8(a–c) to the proposed target receptor S. aureus Gyrase complex with DNA (PDB ID: 2XCS) and dialkylglycine
decarboxylase (PDB ID: 1D7U).
Comp.ID PDB ID:2XCS PDB ID:1D7U

Docking Residues involved glide energy Docking Residues involved glide energy MMGBSA dG
score score XP Bind
XP (Kcal/ (Kcal/mol) energy (Kcal/
mol) mol)

−1.01
7a −4.253 DG10:DG9(PI-PI STACKING), −49.83 – −50.352 −45.273
DC11(H-BOND)
7b −4.792 DC11:DG10(PI-PI STACKING), −49.826 −3.605 TRP138(PI-PI STACKING), −59.645 −51.083
DC11(H-BOND) LYS 272:GLY111
(H-BOND)
7c −7.139 DC11:DG10(PI-PI STACKING), −57.825 −3.989 TRP138(PI-PI STACKING)(PI-CATION); −52.158 −42.462
DG10(SALT BRIDGE,H-BOND) SER215(H-BOND)
8a −3.962 DC11:DG10(PI-PI STACKING), −41.72 −3.909 TYR20:GLN52(H −38.384 −58.419
(least DC11:DG10(H-BOND), -BOND),ARG406:LYS272(SALT BRIDGE),
dock) TRP138(PI-PI STACKING)
8b −4.77 DC11:DG10(PI-PI STACKING), −42.245 −4.187 ALA112(H-BOND) −38.255 −74.635
DC11:DG9(H-BOND) (most dock) GLY111(H-BOND)
8c −7.648 DC11:DG10(PI-PI STACKING), −48.406 −0.021 GLY211(H-BOND) −43.94 −52.485
(most DC11:DG10(H-BOND),DG9(PI- (least dock)
dock) CATION)
Ciprofloxacin −3.523 DG9:DG8(H-BOND,SALT BRIDGE) −41.993 −3.066 ALA112(H-BOND) −38.12 −35.625
TRP138(PI-CATION)

Table 4
ADME predictions for compounds by QikProp.
COMP.ID MW volume QPlogPo/w QPPCaco #metab %Human PSA Rule #stars
Oral Absorption Of
Five

7a 541.728 1624.989 6.671 1910.984 4 100 71.357 2 3

7b 525.667 1622.934 6.221 1319.418 4 93.304 81.228 2 2
7c 524.682 1643.017 6.31 1155.924 3 92.795 85.095 2 2
8a 418.512 1340.094 4.763 697.677 5 100 79.82 0 1
8b 402.452 1303.894 4.187 652.056 5 100 88.814 0 0
8c 401.467 1314.426 4.209 547.276 4 100 90.187 0 0

#stars = Number of property or descriptor values that fall outside the 95% range of similar values for known drugs, QPlogPo/w = Predicted octanol/water partition
coefficient, QPPCaco = Predicted apparent Caco-2 cell permeability in nm/sec. Percent Human Oral Absorption = Predicted human oral absorption on 0–100%
scale., Rule of Five = Number of violations of Lipinski’s rule of five, volume = Total solvent-accessible volume in cubic angstroms using a probe with a 1.4 Å radius,
PSA = Van der Waals surface area of polar nitrogen and oxygen atoms, #metab = Number of likely metabolic reactions.

Table 5
In-silico distribution and toxicity profile of the compounds as obtained from the admetSAR server.
Entry p-gp substrate/inhibitor CYP-2C9 substrate/ CYP-2D6 substrate/ CYP-3A4 substrate/ CYP-1A2 CYP-2C19 AMES Test Carcinogenicity
probability inhibitor inhibitor inhibitor inhibitor inhibitor

7a Substrate/Non inhibitor Non-substrate Non-substrate Substrate/inhibitor Inhibitor Inhibitor toxic Non-carcinogen

7b Substrate/Non inhibitor Non-substrate Non-substrate Substrate/inhibitor Non-Inhibitor Inhibitor toxic Non-carcinogen
7c Substrate/Non inhibitor Non-substrate Non-substrate Non Substrate/inhibitor Inhibitor Inhibitor Non-toxic Non-carcinogen
8a Substrate/Non inhibitor Non-substrate Non-substrate Non Substrate/inhibitor Inhibitor Inhibitor Non-toxic Non-carcinogen
8b Substrate/Non inhibitor Non-substrate Non-substrate Substrate/inhibitor Inhibitor Inhibitor toxic Non-carcinogen
8c Substrate/Non inhibitor Non-substrate Non-substrate Non Substrate/inhibitor Non-Inhibitor Non-Inhibitor Non-toxic Non-carcinogen

Table 6 (dyes) is not required because of the high concentration of test com-
HOMO-LUMO energy gap of 7(a–c) and 8(a–c). pound used in the assay. 20 mg of Resazurin solution was dissolved in
Dyes HOMO Energy LUMO Energy ELUMO-EHOMO
100 mL of sterile water.

Hartee eV Hartee eV eV
2.4.1. Assay protocol
7a −0.1926 −5.24 −0.0884 −2.41 2.83 100 μL of nutrient broth was added to each well of the microtiter.
7b −0.1915 −5.21 −0.0875 −2.38 2.83
100 μL test compound was added to the first well of a particular con-
7c −0.1929 −5.24 −0.0879 −2.38 2.84
8a −0.1899 −5.17 −0.0782 −2.13 3.04 centration (1250 μg/mL) and serially diluted (two-fold dilution) till
8b −0.1903 −5.18 −0.0778 −2.12 3.06 fifth well i.e. four dilutions. 100 μL of homogenized bacterial cell cul-
8c −0.189 −5.14 −0.0767 −2.09 3.05 ture suspension (105 cells per well) was added to all the wells (well with
or without drug). After this, the plate was labeled and it was incubated
at 37 °C for 20–24 h. After 24 h, 30 μL of 0.02 % Resazurin solution was

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V.R. Mishra et al.
Fig. 8. HOMO-LUMO plot of 8 (a–c).
added, the plate was observed after 30 min.
2.5. Synthesis
2.5.1. Intermediates 3(a–c) were synthesized as reported (Holler et al.,
2002)
2.5.1.1. General method for preparation of 4(a–c). Substituted aromatic
amine 3(a–c) (2 mmol.) was dissolved in 30% or 70% H2SO4. It is either
a clear solution or dispersion depending on the amine used. The
reaction mixture was cooled to 0–5 °C. Sodium nitrite (2.2 mmol) was
dissolved in water and added dropwise to the reaction mixture for
about 15 min. under mechanical stirring. The reaction mixture was
stirred for 1.5 h at the same temperature. Completion of diazotization
was monitored using starch-iodide paper as an external indicator.
0.01 g of urea was added to destroy the excess nitrous acid present
after completion of diazotization.
2.5.1.2. General method for preparation of 7(a–c) and 8(a–c). N, N-
Dibutyl-4-phenylthiazol-2amine, and 3-(diethylamino) phenol were
dissolved in propionic acid and acetic acid mixture (1:5). Clear
reddish to orange solution was obtained. The reaction mixture was
cooled to 0–5 °C, diazotized amine 4(a–c) was added slowly with
constant stirring maintaining the temperature at 0–5 °C and pH between
5–6 using sodium acetate. After additional stirring for 2.5–3 h the
precipitate was filtered and dried after thorough washing with water.
Dyes 7(a–c) and 8(a–c) were recrystallized by ethanol.
2.6. Experimental data
2.6.1. 2-(Benzothiazol-2-yl)-4-[(2-(dibutylamino)-4-phenylthiazol-5-yl)
diazenyl] phenol (7a)
Reddish orange solid, Yield: 85%, m.p. = 174–176 °C. IR (KBr)
cm−1 3795 (−OH), 3056 (Ar−CH), 1542 (N = N), 1458(C = N),
1322(C–N). 1H NMR (500 MHz, DMSO-d6) δ 12.00 (s, 1 H), 8.56 (d,
J = 2.3 Hz, 1 H), 8.30 (d, J = 7.8 Hz, 2 H), 8.17 (d, J = 7.8 Hz, 1 H),
8.09 (d, J = 8.1 Hz, 1 H), 7.76 (dd, J = 8.8, 2.3 Hz, 1 H), 7.57 (dd,
J = 12.9, 7.4 Hz, 3 H), 7.52 – 7.45 (m, 2 H), 7.20 (d, J = 8.8 Hz, 1 H),
1.68 (q, J = 15.0, 7.6 Hz, 4 H), 1.38 (q, J = 14.6, 7.3 Hz, 4 H), 0.95 (t,
J = 7.4 Hz, 6 H). 13C NMR (126 MHz, DMSO-d6) δ 168.55, 164.29,
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Computational Biology and Chemistry 78 (2019) 330–337
160.02, 157.35, 154.3, 151.88, 146.04, 140.06, 135.18, 134.29,
130.37, 130.03, 128.92, 127.01, 125.65, 125.42, 125.41, 123.07,
122.78, 122.54, 119.64, 118.41, 46.96, 28.0, 20.03, 14.20. Elemental
analysis: (C30H31N5OS2) Calculated: C, 66.51; H, 5.77; N, 12.93;
Found: C, 66.56; H, 5.77; N, 12.94
2.6.2. 2-(Benzoxazol-2-yl)-4-[(2-(dibutylamino)-4-phenylthiazol-5-yl)
diazenyl] phenol (7b)
Reddish orange solid, Yield: 82% m.p. = 178–180 °C. IR (KBr)
cm−1 3056 (Ar−CH), 1530 (N = N), 1472(C = N), 1329 (C–N). 1H
NMR (500 MHz, DMSO-d6) δ 11.43 (s, 1 H), 8.28 (d, J = 6.5 Hz, 3 H),
7.88 (d, J = 7.8 Hz, 2 H), 7.83 (d, J = 8.3 Hz, 1 H), 7.55 (d, J = 7.7 Hz,
2 H), 7.50 (d, J = 7.5 Hz, 3 H), 7.25 (d, J = 8.9 Hz, 1 H), 1.68 (br s,
4 H), 1.37 (q, J = 14.5, 7.0 Hz, 4 H), 0.95 (t, J = 7.2 Hz, 6 H).
Elemental analysis: (C30H31N5O2S) Calculated C, 68.55; H, 5.94; N,
13.32. Found: C, 68.59; H, 5.94; N, 13.33
2.6.3. 2-(1H-Benzoimidazol-2-yl)-4-[(2-(dibutylamino)-4-phenylthiazol-
5-yl) diazenyl] phenol (7c)
Reddish orange solid, Yield: 80% m.p. = 234–236 °C. IR (KBr)
cm−1 3789 (−OH), 3301(N-H), 3065 (Ar-CH), 1530 (N = N),
1444(C = N), 1300(C–N). 1H NMR (400 MHz, DMSO-d6) δ 13.41 (s,
2 H), 8.45 (s, 1 H), 8.27 (d, J = 6.6 Hz, 2 H), 7.69 (br s, 3 H), 7.51 (br s,
2 H), 7.45 (d, J = 6.4 Hz, 1 H), 7.28 (s, 2 H), 7.13 (d, J = 8.8 Hz, 1 H),
1.66 (br s, 4 H), 1.34 (br s, J = 6.6 Hz, 4 H), 0.93 (t, J = 6.9 Hz, 6 H).
Elemental analysis: (C30H32N6OS) Calculated: C, 68.67; H, 6.15; N,
16.02. Found: C, 68.69; H, 6.15; N, 16.03
2.6.4. 2-(Benzothiazol-2-yl)-4-[(4-(diethyl amino)-2-hydroxyphenyl)
diazenyl] phenol (8a)
Reddish brown solid, Yield: 80% m.p. = 134–136 °C. IR (KBr)
cm−1 3794 (−OH), 3044 (Ar-CH), 1592 (N = N), 1492(C = N),
1320(C–N). 1H NMR (500 MHz, DMSO-d6) δ 13.25 (s, 1 H), 11.95 (s,
1 H), 8.60 (d, J = 2.0 Hz, 1 H), 8.15 (d, J = 7.8 Hz, 1 H), 8.11 (d,
J = 8.1 Hz, 1 H), 7.86 (d, J = 6.8 Hz, 1 H), 7.62 (d, J = 9.1 Hz, 1 H),
7.59 – 7.52 (m, 1 H), 7.46 (t, J = 7.6 Hz, 1 H), 7.20 (d, J = 8.8 Hz, 1 H),
6.53 (d, J = 8.9 Hz, 1 H), 6.14 (s, 1 H), 3.46 (q, J = 7.0 Hz, 4 H), 1.15
(t, J = 6.9 Hz, 6 H).13C NMR (126 MHz, DMSO-d6) δ 164.15, 157.11,
156.69, 151.83, 135.30, 129.95, 128.95, 126.95, 125.59, 124.94,
V.R. Mishra et al.
122.74, 122.55, 122.49, 122.46, 120.85, 120.21, 119.77, 118.30,
117.39, 44.94, 13.12. Elemental analysis: (C23H22N4O2S) Calculated:
C, 66.01; H, 5.30; N, 13.39. Found: C, 68.06; H, 5.31; N, 13.38
2.6.5 2-(Benzoxazol-2-yl)-4-[(4-(diethyl amino)-2-hydro-
xyphenyl) diazenyl] phenol (8b)
Brownish yellow solid, Yield: 78% m.p. = 198–200 °C. IR (KBr)
cm−1 3796 (−OH),2964 (Ar-CH), 1616 (N = N),
1465(C = N),1322(C–N) 1H NMR (300 MHz, DMSO-d6) δ 13.18 (s,
1 H), 11.48 (s, 1 H), 8.41 (s, 1 H), 7.97 (d, J = 8.5 Hz, 1 H), 7.90 (br s,
2 H), 7.61 (d, J = 8.8 Hz, 1 H), 7.50 (br s, 2 H), 7.26 (d, J = 9.0 Hz,
1 H), 6.49 (d, J = 8.8 Hz, 1 H), 6.12 (s, 1 H), 3.46(br s,4 H), 1.15 (t,
J = 6.8 Hz, 6 H). Elemental analysis: (C23H22N4O3) Calculated: C,
68.64; H, 5.51; N, 13.92. Found: C, 68.66; H, 5.51; N, 13.93
2.6.6 2-(1H-Benzoimidazol-2-yl)-4-[(4-(diethyl amino)-2-hy-
droxyphenyl) diazenyl] phenol (8c)
Reddish brown solid, Yield: 78% m.p. = 150–152 °C. IR (KBr)
cm−1 3789 (−OH), 3057 (Ar-CH), 1601 (N = N), 1465(C = N), 1336
(C–N). 1H NMR (400 MHz, DMSO-d6) δ 13.38 (s, 2 H), 8.47 (s, 1 H),
7.81 (d, J = 8.3 Hz, 1 H), 7.69 (br s,1 H), 7.56 (d, J = 8.8 Hz, 1 H), 7.28
(br s, 3 H), 7.13 (d, J = 8.7 Hz, 1 H), 6.46 (d, J = 8.4 Hz, 1 H), 6.09 (br
s, 1 H), 5.71 (s, 1 H), 3.42(br s,4 H),1.12 (t, 6 H). Elemental analysis:
(C23H23N5O2) Calculated: C, 68.81; H, 5.77; N, 17.44. Found: C, 68.86;
H, 5.77; N, 17.45
3. Result and discussion
3.1. Synthesis and spectral characterization
The synthetic pathways adopted for the preparation of novel azo
dyes containing benzothiazole, benzoxazole and benzimidazole are
outlined in Scheme 1. 5-Aminosalicylic acid was reacted with 2-ami-
nothiophenol, 2-aminophenol, and o-phenylenediamine via cyclisation
to produce intermediates 3(a–c) in good yield. The intermediates
3(a–c) were diazotized using conc. H2SO4 and sodium nitrite at 0–5 °C
and coupled with compound 5 or 6 in acidic condition at 0–5 °C. The
desired azo dyes 7(a–c) and 8(a–c) were obtained in good yields.
IR spectra of 7(a–c) and 8(a–c) showed a peak (−OH) stretching
between 3700 and 3800 cm−1, 1520 and 1620 cm−1 for azo group,
–C = N stretching vibrations between 1450–1500cm−1. The aromatic
CeH stretching vibrations were observed among 3060–3170 cm−1 in
the form of a medium band.
1
H NMR spectra, of 7(a–c) and 8(a–c) are taken in DMSO (d6). They
show multiplet peaks in the region of 7.10–8.60 ppm due to the pre-
sence of aromatic protons. In (7a-c) one singlet peak and in 8(a–c) two
singlet peaks in the region of 11–14 ppm are due to hydroxyl proton.
The aliphatic protons are in the range 0.9–1.7 ppm.
13
C NMR of 7a and 8a are taken in DMSO (d6). It shows benz-
thiazole C (N=C-S) around 164–169 ppm, (C-O-) around 153–157 ppm,
aromatic carbon is the range of 117–155 ppm and aliphatic carbon is
the range of 10–50 ppm.
3.2. UV-Visible study
UV–visible studies of 7(a–c) and 8(a–c) were performed in DCM in
order to explore their λmax. values at a concentration of 10 μM. From,
Table 1 it is observed that 7(a–c) showed a relatively more red shift in
absorbance than 8(a–c). All the azo compounds displayed λmax in the
range of 465–488 nm (Fig. 2).
3.3. Biological evaluation (Antimicrobial Activity)
All the newly synthesized compounds 7(a–c) and 8(a–c) were
subjected to antimicrobial testing. The MIC was determined by micro-
broth dilution method i.e. Resazurin microtiter assay method (REMA), a
micro-broth dilution technique against gram-positive (Staphylococcus
aureus) and gram-negative (Escherichia coli) bacteria. Ciprofloxacin was
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Computational Biology and Chemistry 78 (2019) 330–337
used as a standard (50 μg/mL on S.aureus) (25 μg/mL on E.coli).
MIC was determined by visual inspection of color change from blue/
blackish (inhibition) to pink (growth). Blackish color forms instead of
blue because the test compound is a dye which itself has red, yellow or
brown in color. The MIC values of all the synthesized dyes are shown in
Table 2. Molecule 8a is most active dyes in the series against both gram-
positive and gram-negative organisms (MIC of 78.125 μg/mL). While 8c
showed good activity against gram-negative organism (MIC of
312.5 μg/mL) (Fig. 3).
3.4. Molecular docking and ADMET predictions
To rationalize the results from invitro antibacterial studies we car-
ried out flexible ligand docking using the Glide module (Glide, 2017).
The crystal structures of necessary target proteins were obtained from
the protein data bank (PDB ids-2XCS and 1D7U) for evaluation of an-
timicrobial mechanisms (Fig. 4–7). One of the highest bacterial targets
is dialkylglycine decarboxylase (PDB id-1D7U). Another crystal struc-
ture used was S. aureus Gyrase complex with GSK299423 and DNA. All
the synthesized novel azo linked heterocycles were docked into the
corresponding binding pockets of these proteins (Table 3). The com-
pound 8c was obtained as the most docked compound (docking score of
−7.648 kcal/mol) for target S. aureus Gyrase complex with DNA (PDB
id-2XCS) (Fig. 7). It interacted with DC11, DG10 (PI-PI STACKING),
DC11, DG10 (H–BOND), DG9 (PI-CATION) residues. However, the
docking score for standard drug ciprofloxacin was obtained at
−3.523 kcal/mol having interaction with DG9, DG8 (H-BOND, SALT
BRIDGE) residues (Fig.). The compound 8b was obtained as most
docked (docking score: -4.187 kcal/mol) one for target dialkylglycine
decarboxylase (PDB id-1D7U) with ALA112 (H-BOND), GLY111 (H-
BOND) interacting residues (Fig. 6). The docking score for standard
compound ciprofloxacin for target dialkylglycine decarboxylase was
found to be -3.066 kcal/mol (Fig. 4). We have also calculated the
MMGBSA dG Bind energy using Prime module of Schrödinger, LLC, and
NY programme (Prime., 2017) (Table 3). It was revealed that dg bind
energy for most docked compound and target dialkylglycine dec-
arboxylase complex was far better (-74.635 kcal/mol) than target
complex with ciprofloxacin (-35.625 kcal/mol). We have also calcu-
lated all the molecular properties and ADME parameters using the
Qikprop utility (Qikprop, 2017) (Table 4). Except for 7a, all the syn-
thesized compounds were found to have values within an acceptable
range (Table 4). Percentage human oral absorption values were
also > 90%. Furthermore, we also carried out the distribution profiling
and toxicity studies of 7(a-c) and 8(a–c) using admetSAR server (Cheng
et al., 2012) (Table 5). Compound 7a, 7b, 8b were found to be AMES
toxic. All the compounds were obtained as non-carcinogenic. The effect
on various Cytochrome enzymes was displayed in Table 5.
3.5. HOMO–LUMO theory (FMO approach)
All the dyes 7(a–c) and 8(a–c) were optimized in DMSO at B3LYP
6–31 g (d) level of theory. The Highest Occupied Molecular Orbital
(HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) are the
most important orbitals in a molecule. Energy gap (ELUMO – EHOMO)
values reflect the reactivity of molecules (Chauhan et al., 2018).
Therefore, we tried to correlate this reactivity with the biological ac-
tivity. A molecule with high chemical reactivity and low kinetic stabi-
lity is generally accomplished with a small energy gap between frontier
orbitals. Ionization potential is directly related to HOMO energy. So,
when the molecule is energized it easily loses an electron. In the case of
LUMO, it is directly accomplished with electron affinity because it ac-
cepts an electron. The HOMO and LUMO theory were applied to the
novel synthesized azo dyes 7(a–c) and 8(a–c).
From Table 6, it is observed that 8a has a HOMO-LUMO gap
(3.04 eV) which is less compared to 8b and 8c Fig. 8. So, it should
exhibit better biological activity and it is confirmed by REMA assay
V.R. Mishra et al.
method. On the other hand, for 7(a–c) we observed a similar activity by
REMA assay method and it has no correlation with a HOMO-LUMO gap.
4. Conclusion
In summary, a series of novel azo linked heterocycles was synthe-
sized. All the compounds were characterized by various spectral tech-
niques like 1H NMR, elemental analysis, FTIR and UV–vis spectroscopy.
We also concluded from our docking studies that these compounds may
evolve as antimicrobial agents in years to come. Comparative docking
studies on common bacterial targets (PDB ids-2XCS and 1D7U) shows
good docking results as compared to Ciprofloxacin. It was observed that
8a has a HOMO-LUMO gap (3.04 eV) which is less compared to 8b and
8c. So, it should exhibit better biological activity and it is confirmed by
REMA assay method. On the other hand, for 7(a–c) we observed a si-
milar activity by REMA assay method and it has no correlation with a
HOMO-LUMO gap. The complex of the most docked 8b with 1D7U
showed good dg bind energy as compared to a complex of 1D7U with
ciprofloxacin. Finally, in conclusion, we can say that still there is a need
to investigate and synthesize the azo linked substituted to benzimida-
zole, benzoxazole and benzothiazole derivatives.
Acknowledgment
Authors (VRM and CWG) are thankful to the office of the Principal
Scientific Adviser to Government of India for financial support by way
of Junior Research Fellowship.
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