Synthesis, X-ray crystal structure elucidation and Hirshfeld surface analysis ofN-((4-(1H-benzo[d]imidazole-2-yl)phenyl)carbamothioyl)benzamide: Investigations for elastase inhibition, antioxidant and DNA binding potentials for biological applications

Article abstract of DOI:10.1039/d0ra02501a

The interest in the present study pertains to the development of a new compound based upon a benzimidazole thiourea moiety that has unique properties related to elastase inhibition, free radical scavenging activity and its DNA binding ability. The title compound,N-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-3-benzoyl thiourea (C₂₁H₁₈N₄O₂SH₂O:TUBC), was synthesized by reacting an acid chloride of benzoic acid with potassium thiocyanate (KSCN) along with the subsequent addition of 4-(1H-benzo[d]imidazol-2-yl)benzenamineviaa one-pot three-step procedure. The structure of the resulting benzimidazole based thiourea was confirmed by spectroscopic techniques including FTIR,¹H-NMR,¹³C-NMR and single crystal X-ray diffraction and further examined by Hirshfeld surface analysis. TUBC was also investigated by using bothin silicomethodology including molecular docking for elastase inhibition along with quantum chemical studies andin vitroexperimental methodology utilizing elastase inhibition and free radical scavenging assay along with DNA binding experiments. Docking results confirmed that TUBC binding was within the active region of elastase. In comparison to the reference drug oleanolic acid, the low IC₅₀value of TUBC also indicated its high tendency towards elastase inhibition. TUBC scavenged 80% of DPPH˙ radicals which pointed towards its promising antioxidant activity. TUBC-DNA binding by DFT, docking, UV-visible spectroscopy and viscosity measurements revealed TUBC to be a potential drug candidate that binds spontaneously and reversibly with DNAviaa mixed binding mode. All theoretical and experimental findings pointed to TUBC as a potential candidate for a variety of biological applications.

Full text of DOI:10.1039/d0ra02501a

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PAPER
Synthesis, X-ray crystal structure elucidation and
Hirshfeld surface analysis of N-((4-(1H-benzo[d]
imidazole-2-yl)phenyl)carbamothioyl)benzamide:
investigations for elastase inhibition, antioxidant
and DNA binding potentials for biological
applications†
Cite this: RSC Adv., 2020, 10, 20837
a
d
Mamoona Rafiq,b Rabail Ujan,c Aamer Saeed,
*
*
Nasima Arshad,
Shahid I. Farooqi,a Fouzia Perveen,e Pervaiz Ali Channar,
Saba Ashraf,
d
f
Qamar Abbas,g Ashfaq Ahmed,b Tuncer Hokelek,h Manpreet Kauri
and Jerry P. Jasinskii
The interest in the present study pertains to the development of a new compound based upon
a benzimidazole thiourea moiety that has unique properties related to elastase inhibition, free radical
scavenging activity and its DNA binding ability. The title compound, N-(4-(1H-benzo[d]imidazol-2-yl)
phenyl)-3-benzoyl thiourea (C21H18N4O2SH2O:TUBC), was synthesized by reacting an acid chloride of
benzoic acid with potassium thiocyanate (KSCN) along with the subsequent addition of 4-(1H-benzo[d]
imidazol-2-yl)benzenamine via
a one-pot three-step procedure. The structure of the resulting
benzimidazole based thiourea was confirmed by spectroscopic techniques including FTIR, 1H-NMR, 13C-
NMR and single crystal X-ray diffraction and further examined by Hirshfeld surface analysis. TUBC was also
investigated by using both in silico methodology including molecular docking for elastase inhibition along
with quantum chemical studies and in vitro experimental methodology utilizing elastase inhibition and free
radical scavenging assay along with DNA binding experiments. Docking results confirmed that TUBC
binding was within the active region of elastase. In comparison to the reference drug oleanolic acid, the
low IC50 value of TUBC also indicated its high tendency towards elastase inhibition. TUBC scavenged 80%
of DPPHc radicals which pointed towards its promising antioxidant activity. TUBC–DNA binding by DFT,
docking, UV-visible spectroscopy and viscosity measurements revealed TUBC to be a potential drug
candidate that binds spontaneously and reversibly with DNA via a mixed binding mode. All theoretical and
experimental findings pointed to TUBC as a potential candidate for a variety of biological applications.
Received 18th March 2020
Accepted 27th May 2020
DOI: 10.1039/d0ra02501a
rsc.li/rsc-advances
organic chemistry.1 Thioureas have been evaluated over the last
four decades and a variety of derivatives having a wide range of
1 Introduction
biological, agricultural and pharmaceutical applications have
been reported.2 Among all of these, thioureas exhibit antiviral,3
The thiourea nucleus is quite distinctive in its reactivity and
a very predominant functional group in the eld of synthetic
aDepartment of Chemistry, Allama Iqbal Open University, Islamabad-44000,
Pakistan. E-mail: nasimaa2006@yahoo.com; nasima.arshad@aiou.edu.pk
bDepartment of Chemistry, Women University of Azad Jammu and Kashmir, Bagh,
Pakistan
cDr. M. A. Kazi Institute of Chemistry, University of Sindh, Jamshoro, Pakistan
dDepartment of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan.
E-mail: aamersaeed@yahoo.com
gDepartment of Physiology, University of Sindh, Jamshoro, 76080, Pakistan
hDepartment of Physics, Faculty of Engineering, Hacettepe University, Beytepe-
Ankara, 06800, Turkey
iDepartment of Chemistry, Keene State College, 229 Main Street, Keene, NH, 03435,
USA
† Electronic supplementary information (ESI) available: NMR spectra & CIF
report. CCDC 1986282. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/d0ra02501a
eResearch Center for Modeling and Simulations, National University of Sciences and
Technology (NUST), Islamabad, Pakistan
fSulaiman Bin Abdullah Aba Al-Khail-Centre for Interdisciplinary Research in Basic
Science (SA-CIRBS), International Islamic University, Sector H-10, Islamabad,
Pakistan
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Fig. 1 Structures of benzimidazole and thiourea conjugated fluorescent hybrid receptor (A), anti-bacterial agent (B) and carbonic anhydrase
inhibitors (C).
antibacterial,4 antitubercular,5 antiproliferative,6 antioxidant,7 was prepared in 10% aqueous DMSO, while a stock solution of
anticancer,8 neuroprotection,9 antidepressant,10 appetite sh salmon-DNA (FS-ds-DNA) (Sigma-Aldrich) was prepared in
suppressant,11 antiproliferative,12 cytotoxic,13 antimalarial,14 double deionized water. DNA purity was conrmed with the
anti-HIV,15 anti-inammatory,16 and antiallergic17 activities.
help of a UV-visible spectrophotometer. The absorbance ratio
Moreover, the presence of imidazole and benzamide moie- A260/A280 was evaluated as 1.813 which assured the DNA purity.24
ties play essential roles for the synthesis of various metal The DNA concentration was found to be 1.59 ꢀ 10ꢁ4 M by using
complexes.18,19 In the thiourea structure, nitrogen atoms of the values of absorbance at 260 nm, and a molar extention
imidazole, whereas the nitrogen and carbonyl oxygen atoms of coefficient (3) 6600 cmꢁ1 Mꢁ1 in Beer's equation, while keeping
the benzamide moiety provide a platform by acting as chelating the path length constant at 1 cm.
ligands to form complexes of transition metal ions. Biological
activities of the benzimidazole functionalized thioureas have
been studied extensively.20 Benzimidazole and thiourea conju-
gated uorescent hybrid receptors as shown in Fig. 1 have been
reported for the selective recognition of a phosphate ion even in
the presence of other anions.21 Chiral benzimidazole function-
alized thioureas were synthesized and tested for antibacterial
and anticancer activities,22 Fig. 1. Carbonic anhydrase inhibi-
tion was also carried out by the use of benzimidazole based
thioureas23 as revealed in Fig. 1.
Keeping in view the importance of benzimidazole function-
alized thioureas, in the current scenario, we have synthesized
a novel benzimidazole based acyl thiourea. We believe that the
extensive studies on this novel molecule will catch the attention
of the scientic community especially since it will be of great
interest for those who have an interest in heterocyclic organic
synthesis with extensive biological applications and particularly
for supramolecular systems. X-ray single crystal, Hirshfeld
surface analysis, elastase inhibition, free radical scavenging
activity, and DNA binding studies have been carried out here to
highlight the incredible characteristics of the target compound,
TUBC.
2.2 Instrumentations
A Gallenkamp melting point apparatus (MP-D) was used to
determine the melting point through an open capillary method.
FTIR analysis was performed using a Bruker FT-IR Bio-Rad-
1
Excalibur Series Mode No. FTS 300 MX spectrometer. The H
NMR and 13C NMR spectra were recorded on a Bruker 300 MHz
NMR spectrometer in deuterated DMSO and CDCl3 solutions
with tetramethylsilane (TMS) as an internal reference. HPLC-MS
analysis was achieved by an LC Agilent system 1200 series
instrument and the elemental analyses were conducted using
an LECO-183 CHNS analyzer. A UV-visible spectrophotometer
(Shimadzu-1800; TCC-240, Japan), with temperature controller
to maintain a required temperature of the sample (in quartz
cuvette) and an automated Schott Gerate digital viscometer
(Model; AVS 310) were used for DNA binding experiments.
2.3 Synthesis procedure for N-(4-(1H-benzo[d]imidazol-2-yl)
phenyl)-3-benzoylthiourea – TUBC
The new thiourea derivative (TUBC) was synthesized using
different chain length acid chlorides that produced their
respective isothiocyanates. The acid chloride (5.0 mmol) in
anhydrous acetone (50 mL) was added dropwise to the
suspension of potassium thiocyanate (10.0 mmol) in acetone
(25 mL) at room temperature. The reaction mixture was cooled
2 Experimental
2.1 Materials and methods
All chemical and reagents are of analytical grade and purchased to room temperature. A solution of 4-(1H-benzo[d]imidazol-2-yl)
from Sigma-Aldrich. Synthesis of the title compound was benzenamine (5.0 mmol) in anhydrous acetone was added
monitored on silica gel plates (aluminum pre-coated Kiesel 60 dropwise and the reaction mixture was further reuxed for 2–3
F254) and an Rf value was calculated. In DNA binding experi- hours (Scheme 1). The progress of the reaction was monitored
ments, a stock solution of the synthesized compound (TUBC) through TLC. On completion, the mixture was poured into cold
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Scheme 1 Synthesis of TUBC, C21H18N4O2S$H2O.
water and the solid precipitates of title molecule were ltered
2.7 In silico methodology
and recrystallized from ethanol.
2.7.1 Molecular docking for elastase inhibition. The crystal
structure of porcine pancreatic elastase was accessed from the
Protein Data Bank (PDB) (www.rcsb.org) with PDBID 7EST. The
retrieved target protein structure was minimized by using UCSF
Chimera 1.6rctool.28 The stereochemical properties of elastase
and a Ramachandran plot and values were also accessed from
the PDB. A hydrophobicity graph of the target protein was
generated by Discovery Studio 4.1 Client.29 The synthesized
chemical structure of TUBC was sketched in ACD/ChemSketch
and minimized by UCSF Chimera 1.6rctool. The PyRx docking
tool was employed to perform the molecular docking experi-
ment.30 The grid box center values of (center_x ¼ 4.5741, cen-
ter_y ¼ 56.7946 and center_z ¼ ꢁ5.5085) and size values for all
three coordinates were adjusted as (size_x ¼ 53.2441, size_y ¼
43.0418 and size_z ¼ 49.3944). The default exhaustiveness value
¼ 8 was used to maximize the binding conformational analysis
of ligands within the active region of target protein. Docking
analysis was done on the basis of lowest binding energy (kcal
molꢁ1) values and structure activity relationship (SAR) analyses.
The three dimensional (3D) graphical depiction was accom-
plished by Discovery Studio (2.1.0).
2.7.2 Quantum chemical studies. Quantum chemical
calculations were carried out on the ground state for the
molecule using crystal structure data as the starting geometry
and using Gaussian 09 soware with WebMo Pro employed for
graphics visualization.31,32 The TUBC compound was optimized
for the hybrid B3LYP functional with the 6-31+G(d) basis set. In
the electronic structure DFT method, the B3LYP functional has
attained popularity and shows tremendous reliability for
structural properties of synthetic and natural products due to its
efficient balance between computational cost and accuracy.33
2.7.3 Molecular docking for DNA binding. The Molecular
Operating Environment (MOE 2017: Chemical computing Inc.)
tool was employed to sketch and optimize the structure of TUBC
compound at the MOPAC 7.0 level of theory. Structures were
sketched using the molecular builder of the MOE suite and
fetched into the database aer geometry relaxation. The X-ray
crystallographic structure of DNA possessing PDB ID: 1QTI
2.4 Characterization data
Yellow solid; yield: 72%; Rf: 0.63; mp: 235 ꢂC; FTIR (cmꢁ1): 3050
(Sp2C–H), 1680 (C]O), 1620 (C]N), 1241 (C]S), 3350 (N–H),
Ar C]C(1550); 1H NMR: (DMSO-d6, 300 MHz); d ¼ 12.79 (s, 1H,
NH, D2O exchangeable), 11.66 (s, 1H, NH, D2O exchangeable),
10.98 (s, 1H, NH, D2O exchangeable), 8.23–7.70 (m, J ¼ 8.4 5H,
Ar–H), 7.92 (d, J ¼ 8.4 Hz, 2H, Ar–H), 7.68 (d, J ¼ 7.1 Hz, 2H, Ar–
H), 7.24 (d, J ¼ 7.2 Hz, 2H, Ar–H), 7.10 (d, J ¼ 8.1 Hz, 2H, Ar–H);
13C NMR: (DMSO-d6, 75 MHz); 179.34, 168.75, 151.15, 139.76,
133.67, 132.58, 129.20, 128.94, 128.23, 127.26, 124.75, 122.65.
Anal. calcd for C21H16N4OS: C, 67.72; H, 4.33; N, 15.04; S, 8.61
found: C, 67.50; H, 4.53; N, 15.00; S, 8.63.
2.5 X-ray data collection and structure renement
Crystallographic data were recorded on a Rigaku Oxford
Diffraction Eos CCD diffractometer using Mo Ka radiation (l ¼
˚
0.71073 A) at T ¼ 173(2) K. Multi-scan [empirical absorption
correction using spherical harmonics, implemented in SCALE3
ABSPACK scaling algorithm CrysAlisPro 1.171.38.46] was
applied.25 The structure was solved by direct methods and
rened by full-matrix least squares against F2 using all data
(SHELXT and SHELXL).26 All non-H atoms were rened aniso-
tropically. The O-, N- and C-bound H atoms were positioned
˚
˚
geometrically, with O–H ¼ 0.87 A (for water OH), N–H ¼ 0.88 A
˚
(for NH) and C–H ¼ 0.95 A (for aromatic CH), and constrained
to ride on their parent atoms with Uiso(H) ¼ k ꢀ Ueq(O, N, C),
where k ¼ 1.5 for OH hydrogen atoms and k ¼ 1.2 for all other H
atoms. Full crystallographic data for compound TUBC have
been deposited with the Cambridge Crystallographic Data
Centre (CCDC # 1986282).†
2.6 Hirshfeld surface calculations
The Hirshfeld surface (HS) analysis was carried out by
employing Crystal Explorer program 17.5.27 The structural input
le was obtained in the CIF format. The Hirshfeld surface
distance from the nearest nucleus inside and outside the
surface was measured and represented by di and de, respec-
tively, while a normalized contact distance was represented as
dnorm. White, red and blue colors have been selected for the
˚
and resolution of 2.7 A was downloaded from the Protein Data
Bank.34 1QTI was protonated and optimized expending
a protonate-3D menu. Water molecules attached with the base
pairs of 1QTI were removed. For the purpose of docking anal-
ysis, coordinates of 1QTI were relaxed using the AMBER
Forceeld and semiempirical PM3 approaches. Relaxed coor-
dinates had the minimum energy and a stable conformation for
visualization of dnorm
.
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best scoring function calculations. The TUBC optimized struc- assay solution consisted of 100 mL of DPPH (150 mM), 20 mL of
ture was subjected to methodical molecular docking taking increasing concentration of test compounds and the volume
DNA as a receptor at default parameters with an RMS gradient was adjusted to 200 mL in each well with methanol. The reaction
of 0.01 kcal molꢁ1 using Site Finder to nd out the active sites of mixture was then incubated for 30 minutes at room tempera-
1QTI. A number of docking runs were completed to attain the ture. Ascorbic acid (Vitamin C) was used as a reference inhib-
nal docking poses as perfect as possible. At each step of the itor. The assay measurements were carried out by using
simulation, the energy of the interaction of TUBC with 1QTI was a microplate reader (OPTIMax, Tunable) at 517 nm. The reaction
determined. The remaining parameters were kept as a default.35 rates were compared, and the percent inhibition caused by the
presence of tested inhibitors was calculated. Each concentra-
tion was analyzed in three independent experiments run in
triplicate.
2.8 In vitro experimental methodology
2.8.1 Elastase inhibition assay. The elastase (elastase from
porcine pancreas – Sigma Aldrich) inhibition activity was per-
formed following an already reported method with some
modications.36–38 In order to perform the inhibition of elastase
activity, the amount of released p-nitroaniline, which was
hydrolyzed from the substrate (N-succinyl-Ala-Ala-Ala-p-nitro-
anilide) by elastase, was determined by measuring the absor-
bance at 410 nm. In detail, 0.8 mM solution of N-succinyl-Ala-
Ala-Ala-p-nitroanilide was prepared in a 0.2 M Tris–HCl buffer
(pH 8.0) and this buffer (130 mL) was added to the test sample
(10 mL) in a 96 well microplate.39 The microplate was pre-
2.8.3 DNA binding experiments. UV-visible spectroscopic
titrations for TUBC were performed by adding increasing
concentrations of DNA (10–80 mM) in an optimized concentra-
tion (2.34 ꢀ 10ꢁ4 M). The sample solution was allowed to stay
within the cuvette for at least ve minutes so that an equilib-
rium between the compound and DNA could be maintained at
the desired temperature of 37 ꢂC. Viscosity measurements were
carried out to observe the structral changes in the DNA duplex
in the presence of the titled compound. The viscosity of 10 mM
DNA (ho) was measured on a digital viscometer and then small
changes in DNA viscosity (h) were noted upon addition of TUBC
from 10 to 110 mM. Experimental conditions were set similar as
for the spectroscopic experiments.
ꢂ
incubated for 10 min at 25 C before an elastase (0.0375 unit
per mL) stock solution (10 mL) was added. Aer enzyme addi-
tion, the microplate was kept at 25 ꢂC for 30 min, and the
absorbance was measured at 410 nm using a microplate reader.
All experiments were carried out in triplicate.
3 Results and discussion
3.1 Chemistry
2.8.2 Free radical scavenging assay. Radical scavenging
activity was determined by modifying an already reported FT-IR analysis show characteristic absorption bands for N–H at
method by 2,2-diphenyl-1 picrylhydrazyl (DPPH) assay.40,41 The 3215–3350 cmꢁ1, C–H aromatic at 3033–3080 cmꢁ1, C]O of
Table 1 Experimental details for the title compound
Empirical formula
Formula weight
Temperature
Wavelenght
Crystal system
Space group
C21H16N4OS$H2O
390.45
173(2) K
˚
0.71073 A
Monoclinic
P21/n
˚
Unit cell dimensions
a ¼ 6.9104 (3) A
˚
b ¼ 19.6432 (7) A
˚
c ¼ 14.0505 (5) A
b (ꢂ)
Volume
100.235 (4)
1876.90 (13) A
3
˚
Z
4
Density (calculated)
Absorbtion coefficient
F (000)
Crystal size
Ɵ Range for data collection
Index ranges
Reections collected
Independent reections
Renement method
Data/restraints/parameters
Goodness-of-t on F2
Final R indices
[I > 2s(I)]
1.382 Mg mꢁ3
0.20 mmꢁ1
816
0.54 ꢀ 0.14 ꢀ 0.12 mm3
2.5–32.7ꢂ
ꢁ6 # h # 10, ꢁ24 # k # 29, ꢁ18 # l # 21
12 574
6185 [R(int) ¼ 0.0244]
Full-matrix least-squares on F2
4364/0/256
1.027
R1 ¼ 0.0570
wR2 ¼ 0.1497
R indices (all data)
Largest diff. peak and hole (e A
R1 ¼ 0.0856 wR2 ¼ 0.1744
0.379/ꢁ0.408
ꢁ3
˚
)
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Table 2 Hydrogen-bond geometry (A˚, ꢂ) for the title compounda
D–H/A
D–H
H/A
D/A
D–H/A
N1–H1/O1Wi
N2–H2/O1
0.88
0.88
0.88
0.95
0.95
0.95
0.87
0.87
2.33
1.85
1.96
2.81
2.55
2.61
2.49
1.94
3.0761 (19)
2.6067 (17)
2.8103 (16)
3.449 (2)
3.2104 (17)
3.461 (2)
142
142
162
125
127
149
153
162
N4–H4/O1Wv
C4–H4A/O1Wi
C10–H10/S1
C20–H20/O1vi
O1W–H1WA/S1i
O1W–H1WB/N3
3.2911 (13)
2.7832 (17)
a
Symmetry codes: (i) ꢁx + 1, ꢁy + 1, ꢁz + 1; (v) x ꢁ 1/2, ꢁy + 1/2, z ꢁ 1/2;
(vi) ꢁx + 1/2, y ꢁ 1/2, ꢁz + 3/2.
Fig. 3 A partial view of the crystal structure of C21H18N4O2S H2O,
viewed along the a-axis. The N–H/O, O–H/N, O–H/S, hydrogen
bonds and weak C–H/S and C–H/O intermolecular interactions are
shown as dashed lines (see Table 2). Only the H atoms involved in
hydrogen bonding have been included for clarity.
carbonyl at 1680–1748 cmꢁ1, C]N of imine at 1542–1694 cmꢁ1
1
and C]C (Ar) 1532–1575 cmꢁ1. The H NMR spectrum of the
title compound involved three characteristic broad singlets for
a N–H proton which appeared at d 12.79, 11.66 ppm, 10.98 ppm,
and characteristic 2H doublets of 1,4-disubstitued phenyl ring
protons appeared at d 7.92 and 7.10 ppm respectively, whilst
doublets for two phenyl ring protons of benzimidazole
appeared at d 7.68 and 7.24 ppm. The aromatic protons of
benzoyl ring gave the most deshielded multiplet at d 8.23–
7.70 ppm. All of these signals suggest the formation of the target
compound. In the 13C NMR spectrum signals for carbon (C]S)
appeared at d 179.34 ppm, a signal for carbonyl carbon was
shown at d 168.75 ppm, and signal for imine carbon appeared at
d 151.15 ppm. All these signals propose the formation of the
desired compound. 1H NMR and 13C NMR spectra are provided
as Fig. S1 and S2 (in the ESI†).
respectively. The asymmetric unit contains only one crystallo-
graphically independent molecule and one uncoordinated
water molecule in the unit cell as depicted along with the atom-
numbering scheme in Fig. 2, where the uncoordinated water
molecule is linked to a benzimidazole nitrogen via a hydrogen
bond (O–HW/NBnzimd; where W ¼ water, Bnzimd ¼ benz-
imidazole), (Table 2).
The mean planes of the benzene rings [A (C3–C8) and B (C9–
C14)] are oriented at a dihedral angle of A/B ¼ 10.43(6)ꢂ. Atom
C2 is co-planar with the benzene ring A (C3–C8) with
˚
a maximum deviation of 0.015(2) A, while atoms N2 and C15 are
co-planar with the benzene ring B (C9–C14) with maximum
˚
˚
deviations of 0.019(2) A (for atom N2) and ꢁ0.019(2) A (for atom
C15). The mean planes of rings C (N3/N4/C15/C16/C21) and D
(C16–C21) are oriented at a dihedral angle of C/D ¼ 0.77(7)ꢂ. So,
the benzimidazole moiety, (N3/N4/C15–C21), is essentially
3.2 Description of crystal structure
The single crystal X-ray structural determination of the titled
compound conrms the assignment of its structure from
spectroscopic data. The experimental details including the
crystal data, data collection and renement are summarized in
Table 1. The hydrogen-bond geometry, the selected interatomic
distances and the selected bond lengths, bond angles together
with the torsion angles are given in Tables 2, 3 and 4,
˚
planar to within 0.010(2) A (r.m.s. deviation ¼ 0.0067) with atom
C19 deviating the most from the mean plane, and atom C12 is
co-planar with the benzimidazole moiety with a maximum
˚
deviation of 0.045(2) A. The mean planes of the benzene rings [A
(C3–C8) and B (C9–C14)] are oriented with respect to the
benzimidazole moiety at dihedral angles of 3.57(5) and 7.23(6)ꢂ,
Fig. 2 The molecular structure of the title compound, C21H18N4O2S H2O, with the atom-numbering scheme for the asymmetric unit.
Displacement ellipsoids are drawn at the 50% probability level.
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respectively. There are intramolecular N–H/O hydrogen bonds any p-stacking interactions.42,43 In the HS plotted over dnorm
and weak C–H/S intermolecular interactions present in the Fig. 4, (Top), the white surface indicates contacts with distances
molecule (Table 2). equal to the sum of van der Waals radii, and the red and blue
,
In the crystal structure, the uncoordinated water molecules colours indicate distances shorter (in close contact) or longer
are linked via intermolecular N–H/OW and O–HW/S hydrogen (distinct contact) than the van der Waals radii, respectively.44
bonds (Table 2). The molecules are further linked via additional The bright-red spots appearing near S1, O1W and hydrogen
weak O–H/N intermolecular interactions (Table 2) as well as by atoms H1, H4, H1WA indicate their roles as the respective
signicant weak p/p stacking interactions [Cg1/Cg2 donors and acceptors in the dominant N–H$$$O, C–H$$$O and
2
1
3.8046(2)1 A and 3.5836(2) A; Cg2/Cg4 3.6867(2) A and O–H$$$S intermolecular interactions. The shape-index of the
˚
˚
˚
2
2
1
˚
˚
˚
3.6795(2) A; Cg2/Cg5 3.4822(2) A and 3.5953(2) A; where Cg1 HS is a tool to visualize the p$$$p stacking by the presence of
¼ N3/C15/N4/C16/C21, Cg2 ¼ C3/C8, Cg4 ¼ C16/C21; C5 ¼ adjacent red and blue triangles; if there are no adjacent red and/
N3/C15/N4/C16/C20; symmetry 1 ¼ 1 ꢁ x, ꢁy, ꢁz and 2 ¼ 2 ꢁ x, or blue triangles, then there are no p$$$p interactions. Fig. 4,
ꢁy, ꢁz] resulting in a three-dimensional supramolecular (Bottom) clearly suggests that there are no p$$$p interactions in
network (Fig. 3). Hydrogen bonding, weak intermolecular the title compound.
interactions and van der Waals contacts along with weak p/p
The overall two-dimensional ngerprint plot, Fig. 5a, and
stacking interactions are the dominant interactions in the those delineated into H/H, H/C/C/H, C/C, H/S/S/H,
crystal packing.
H/O/O/H, C/N/N/C, H/N/N/H and C/O/O/C
contacts45 are illustrated in Fig. 5b–i, respectively, together with
their relative contributions to the Hirshfeld surface. The most
important interaction is H/H contributing 45.4% to the overall
crystal packing due to the large amount of the short interatomic
H/H contacts (Table 3), which is reected in Fig. 5b as widely
3.3 Hirshfeld surface analysis
The analysis of calculated Hirshfeld surfaces is invaluable since
it provides additional insight into the weak intermolecular
interactions in the crystal structure. Thus, a Hirshfeld surface
(HS) analysis was carried out by to investigate the locations of
atom–atom short contacts with potential to form hydrogen
bonds and the quantitative ratios of these interactions besides
˚
scattered points of high density with the tip at de ¼ di 1.00 A. In
the absence of C–H/p interactions in the crystal, the pair of
characteristic wings resulting in the ngerprint plot delineated
into H/C/C/H contacts with 12.9% contribution to the HS,
Fig. 4 (Top) View of the three-dimensional Hirshfeld surface of the title compound plotted over dnorm in the range ꢁ0.5953 to 1.5115 a.u.
(Bottom) Hirshfeld surface of the title compound plotted over shape-index.
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Fig. 5 The full two-dimensional fingerprint plots for the title compound, showing (a) all interactions, and delineated into (b) H/H, (c) H/C/C/
H, (d) C/C, (e) H/S/S/H, (f) H/O/O/H, (g) C/N/N/C, (h) H/N/N/H and (i) C/O/O/C interactions. The di and de values are the closest
internal and external distances (in A˚) from given points on the Hirshfeld surface contacts.
˚
Fig. 5c, and the pair of thin edges at de + di ꢃ 2.95 A result from 1.8% contribution to the HS have a symmetric distribution of
interatomic H/C/C/H contacts (Table 3). The C/C contacts points, with the scattered points of low density, and is viewed as
˚
assigned to short interatomic C/C contacts (Table 3) with pair of spikes with the tips at de + di ꢃ3.40 A.
11.9% contribution to the HS appear as a symmetric distribu-
The Hirshfeld surface representations with the function
˚
tion of points in Fig. 5d, with the tips at de + di ¼ 3.31 A. The H/
d
norm plotted onto the surface are shown for H/H, H/C/C/H,
S/S/H interactions, Fig. 5e, has an 10.1% contribution to the C/C, H/S/S/H, H/O/O/H and C/N/N/C interactions in
total HS area of the molecule with a symmetric distribution of Fig. 6a–f, conrming that the HS surface analysis provides a full
points, and is viewed as pair of spikes with the tips at de + di ¼ understanding of the intermolecular interactions in a facile and
˚
2.35 A result from short interatomic H/S/S/H contacts (Table immediate way.
3). The H/O/O/H contacts (Table 3) in the structure with 9.8%
The Hirshfeld surface analysis conrms the importance of H-
contribution to the HS has a symmetric distribution of points, atom contacts in establishing the packing. The large number of
˚
Fig. 5f, with the tips at de + di ¼ 2.81 A. The symmetrical N–H$$$O, O–H$$$S and C–H$$$O interactions suggest that van
distribution of points in the ngerprint plot delineated into C/ der Waals interactions, hydrogen bonding and weak intermo-
N/N/C contacts (Table 3), Fig. 5g, the 5.3% contribution to the lecular interactions play the major roles in the crystal packing.46
overall crystal packing is viewed as pair of tiny spikes with the
˚
tips at de + di ¼ 3.36 A. In the ngerprint plot delineated into
3.4 Elastase inhibition docking studies
H/N/N/H contacts Fig. 5h, the 2.7% contribution to the HS
arises from the interatomic H/N/N/H contacts listed in Table
TUBC was docked against elastase. Docking energy analyses was
carried out to predict the best tted conformational position of
synthesized compound TUBC within the active region of a tar-
geted protein. The predicted docked complex was evaluated on
˚
3, and is viewed as pair of spikes with the tips at de + di ꢃ 3.33 A.
Finally, the C/O/O/C contacts, Fig. 5i, in the structure with
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Table 3 Selected interatomic distances (A˚) for the title compounda
Table 4 Selected bond lengths (A˚), bond and torsion angles (ꢂ) for the
title compound
S1/C10
3.2106 (17)
3.2917 (13)
2.56
C3/C15i
C3/C16i
C4/C21i
C4/C16iv
C5/C18iv
C5/C20i
C5/C17iv
C6/C17i
C6/C18i
C7/C17i
C7/C16i
C9/C9i
3.544 (2)
S1/O1Wi
S1/H10
3.833 (2) Parameters
3.586 (3)
X-ray analysis
S1/H1WAi
O1/N2
2.49
3.413 (3)
3.575 (3)
3.596 (3)
3.580 (3)
3.492 (3)
3.562 (3)
3.476 (3)
3.517 (3)
3.637 (2)
3.718(2)
2.91
˚
The bond lengths (A)
S1–C1
O1–C2
N1–C1
N1–C2
N2–C1
N2–C9
N3–C15
N3–C21
N4–C15
N4–C16
1.6632 (17)
1.2217 (19)
1.3919 (19)
1.376 (2)
1.376 (2), 1.4085 (18), 1.3229 (17)
1.388 (2), 1.3601 (18)
1.3710 (18)
2.6062 (17)
2.8103 (16)
3.0764 (18)
2.7848 (18)
3.2917 (13)
2.49
2.61
1.85
2.33
1.96
O1W/N4ii
O1W/N1i
O1W/N3
O1W/S1i
O1/H8
O1/H20iii
O1/H2
O1W/H1i
O1W/H4ii
C9/C14i
C1/H10
O1W/H4Ai 2.82
O1W/H11ii 2.81
C2/H2
C4/H1
C11/H4
2.36
2.47
2.74
The bond angles (ꢂ)
O1–C2–N1
N4/C8i
N4/C3iv
N1/H4A
3.442 (2)
3.379 (2)
2.59
122.12 (14)
121.74 (15)
116.14 (14)
118.26 (12)
127.94 (12)
113.79 (14)
129.09 (14)
104.97 (12)
107.46 (12)
126.12 (13)
114.97 (13)
112.49 (12)
124.71 (13)
122.79 (12)
O1–C2–C3
N1–C2–C3
N1–C1–S1
N2–C1–S1
N1–C1–N2
C2–N1–C1
C15–N3–C21
C15–N4–C16
N2–C9–C10
N2–C9–C14
N3–C15–N4
C12–C15–N3
C12–C15–N4
C15/H1WB
C21/H1WB
H1/H1WAi
H1/H4A
H1/H1WBi
H1WA/H4ii
H1WB$$$H4ii
H2/H14
2.98
2.81
2.22
1.96
2.55
2.37
2.57
2.20
N1/H1WAi 2.84
N3/H1WB
N3/H13
N4/H11
C1/C14i
C1/C13i
C2/C15i
C2/C12i
1.94
2.62
2.62
3.496 (2)
3.383 (3)
3.610 (2) 3.550 (2) H4/H11/H14/H19iii 2.17
2.24
a
Symmetry codes: (i) ꢁx + 1, ꢁy + 1, ꢁz + 1; (ii) x + 1/2, ꢁy + 1/2, z + 1/2;
(iii) ꢁx + 1/2, y + 1/2, ꢁz + 3/2; (iv) ꢁx, ꢁy + 1, ꢁz + 1.
The torsion angles (ꢂ)
the basis of minimum energy values (kcal molꢁ1) and interac- C4–C3–C2–N1
17.0 (2)
C4–C3–C2–O1
C8–C3–C2–N1
C8–C3–C2–O1
C2–N1–C1–N2
S1–C1–N1–C2
ꢁ163.18 (17)
ꢁ163.93 (16)
15.9 (2)
tion patterns like hydrogen and hydrophobic interactions.
Binding pocket and ligand conformational analysis is shown in
Fig. 7. Docking results showed that TUBC exhibits good binding
energy values (kcal molꢁ1) against the receptor with little uc-
3.9 (2)
ꢁ175.18 (14)
tuations. Prior research showed that the standard error mean of S1–C1–N2–C9
0.4 (3)
docking energy for Autodock is 2.5 kcal molꢁ1
.
C1–N2–C9–C10
C1–N2–C9–C14
C11–C12–C15–N4
C13–C12–C15–N3
C13–C12–C15–N4
ꢁ11.8 (3)
170.60 (17)
ꢁ7.0 (2)
ꢁ7.8 (2)
3.5 In vitro elastase inhibition studies
173.16 (15)
The inhibitory potential of TUBC against elastase enzyme was
evaluated according to the following formula:
populated piperidines.47–51 It can be attributed that the presence
of NH functional group capable of exhibiting basic, nucleo-
philic and H-bonding interactions thereby making it more
hydrophilic whilst the amidic carbonyl can interact as a H-bond
donor system. The elastase inhibition activity of TUBC is
pointing towards its candidacy to be used in skin care and
cosmetic industry.
Elastase inhibition activity (%) ¼ (ODcontrol ꢁ ODsample ꢀ 100)/
ODcontrol
where, ODcontrol and ODsample represent the optical densities in
the absence and presence of a TUBC, respectively. Oleanolic
acid was used as the standard inhibitor for elastase. The ob-
tained results in terms of IC50 (mM) values are presented in
Table 5. The compound TUBC showed very potent inhibitory
activity against this enzyme, as it was evident from its lower IC50
value, relative to oleanolic acid (standard).
3.6 Free radical scavenging
TUBC showed signicant elastase inhibition as compared to
reference oleanolic acid. This is in line with the fact that acyl The radical scavenging activity of TUBC was evaluated at a value
thioureas are well known for exhibiting a wide range of bioac- compared with the standard (Vitamin C) in Table 5. TUBC has
tivities. The elastase inhibition of TUBC is comparable to those shown signicant scavenging activity against DPPHc even at
of others acyl thioureas including 1-butanoyl, 1-pentanoyl and concentration (100 mg mLꢁ1), which showed it to be a potential
1-heptanoyl as well as the 3-phenylcoumarins and densely antioxidant.
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Fig. 6 The Hirshfeld surface representations with the function dnorm plotted onto the surface for (a) H/H, (b) H/C/C/H, (c) C/C, (d) H/S/S/
H, (e) H/O/O/H and (f) C/N/N/C interactions.
Fig. 7 Elastase inhibition; binding pocket and ligand (TUBC) conformational analysis.
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electronic transitions.35 The calculated molecular orbital ener-
gies (eV) for the surfaces of the frontier molecular orbitals are
shown in Fig. 9 (Table 6).
Table 5 Elastase and free radical scavenging inhibitory activity of
TUBCa
Free radical %
Compound
TUBC
Elastase IC50 + SEM (mM) scavenging (100 mg mLꢁ1
)
In HOMO the electronic clouds are distributed primrily on
the p-disubstituted benzene and benzo[d]imidazol rings. In
LUMO, they are located on the phenyl ring and thiourea group.
In LUMO+1 they are located primarily on the phenyl and p-
disubstituted benzene rings while in LUMO+2 they are delo-
calized primarily on the phenyl ring. In HOMOꢁ1 the electron
clouds are located on the thiourea group. Surface plots for the
highest occupied molecular orbitals (HOMOꢁ1 and HOMO) act
as electron donors and the three lowest unoccupied molecular
orbitals (LUMO, LUMO+1 and LUMO+2) act as electron
acceptors.
10.532 ꢄ 1.083
80.915 ꢄ 0.519
—
Oleanolic acid 13.451 ꢄ 0.014
Vitamin C
—
94.823 ꢄ 0.046
a
SEM ¼ standard error of the mean; values are expressed in mean ꢄ
SEM.
3.7 Density functional theory (DFT) studies
The structural geometry of TUBC has been simulated and
optimized using DFT/B3LYP to determine its geometric and
The HOMO–LUMO gap is a very important parameter for
quantum chemistry. When the gap is small, the molecule is
highly polarizable and has high reactivity. Therefore, these
calculations provide important insight into the reactivity and
site selectivity of the molecular framework. The observed
experimental absorption spectrum (Fig. 11, in Section 3.8)
shows two band envelopes with lmax values located at 340 and
300 nm. Electronic transitions are geneally paired between the
electronic parameters (bond lengths, bond angles, EHOMO
,
ELUMO). The optimized geometries of TUBC have been depicted
in Fig. 8 (Top), wheres the symmetric charge distribution on
each individual atom of TUBC has been indicated. The frontier
molecular orbital (FMO) analysis using the quantum mechan-
ical approach is also a popular way to predict molecular
Fig. 8 (Top) The optimized geometry of TUBC at the B3LYP/6-31G (d,p) level of theory, (Bottom): molecular electrostatic potential surfaces
(MESP) of TUBC.
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Fig. 9 Schematic molecular orbital (MO) diagram of TUBC representing HOMO, LUMO and HOMO–LUMO energy gap (DE).
Table 6 Experimental and calculated energies of molecular orbitals for C21H16ON4S and associated electronic transitions
Experimental lmax (nm eVꢁ1
)
f
MO contribution
Calculated lmax (nm eVꢁ1
)
256/4.84
256/4.84
216/5.74
256/4.84
216/5.74
216/5.74
1.60
1.80
1.80
1.60
1.80
1.80
HOMO / LUMO
346/3.58
281/4.39
255/4.85
304/4.06
254/4.88
232/5.33
HOMO / LUMO+1
HOMO / LUMO+2
HOMOꢁ1 / LUMO
HOMOꢁ1 / LUMO+1
HOMOꢁ1 / LUMO+2
various molecular orbitals of the ground and excited states used to map the molecular electrostatic potential surfaces
corresponding to these two band envelopes as indicated in (MESP) of TUBC and are presented in Fig. 8 (Bottom). It is
Table 4. In this case these represent transitions of electron evident from the Fig. 8 that negative potential is concentrated
density from molecular orbitals in the p-disubstituted and on the oxygen, sulfur and nitrogen atoms which reects elec-
benzo[d]imidazol rings and thiourea group (HOMO and tron transfer from O, S and N. Dispersion of the potential for
HOMOꢁ1) to molecular orbitals in the phenyl ring and thiourea TUBC ranges from ꢁ0.156 to 0.0156 esu indicating electrophilic
group, collectively. Therefore, the rst absorption band enve- and nucleophilic centers in this range.
lope at 340 nm is assigned to overlapping contributions
Quantum chemical DFT studies of TUBC revealed that based
primarily from HOMO / LUMO and HOMOꢁ1 / LUMO. The on EHOMO and ELUMO values, it is considered to be good electron
second absorption band at 300 nm is assigned to overlapping acceptor, and could accept electrons from DNA base pairs.
contributions from HOMO / LUMO+1, HOMO / LUMO+2,
HOMOꢁ1 / LUMO+1, and HOMOꢁ1 / LUMO+2, respec-
3.8 Molecular docking for studies DNA binding
tively. The energy gap between HOMO and LUNO is 3.58 eV
The molecular mechanism for the interactions of TUBC with
suggesting greater reactivity of TUBC for interaction with DNA
DNA was interpreted using molecular docking to simulate the
molecule. [HOMO to LUMO+1 is 4.39 eV; HOMO to LUMO+2 is
modes of interactions between the TUBC and DNA. Pose view
analysis and conformations of TUBC with the lowest free energy
are shown in Fig. 10. Binding free energy (DG) and binding
constant “Kb” values for TUBC were calculated and are given in
Table 7. Fig. 10 (Le) indicate a mixed mode of TUBC interac-
tions with DNA. Ligplot indicate 2D interactions of TUBC with
4.85 eV; HOMOꢁ1 to LUMO is 4.06 eV; HOMOꢁ1 LUMO+1 is
4.88 eV; HOMOꢁ1 to LUMO+2 is 5.33 eV, respectively.
The molecular electrostatic potential (MEP) was also
computed at the B3LYP/6-31+G(d,p) level of theory. The opti-
mized geometry at the B3LYP/6-31G(d,p) level of theory was
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Fig. 10 (Left) Molecular docked complex of TUBC and (Right) its ligplots calculated at the PM3 semi-empirical level of theory.
Table 7 Theoretical data for TUBC–DNA binding obtained from
compounds, the compound with higher partition coefficient
forms a stronger complex.35,52,53 S log P values for TUBC in Table
7 depicted efficient binding of TUBC with the DNA. Another
important steric descriptor is molar refractivity (MR) which is
measure of polarizability of molecule.53 TUBC possesses
a higher MR value, which also indicates signicantly strong
complex formation (Table 7).
molecular docking
Molecular docking
Complex
Kb Mꢁ1
DG kJ molꢁ1
ꢁ23.91
TUBC–DNA
1.60 ꢀ 104
Electronic descriptors
3.9 UV-visible spectroscopic DNA binding studies
EHOMO
ELUMO
(kcal
(kcal
Eele (kcal Evander (kcal EIP (kcal ETotal (kcal
Compound-DNA binding could be visualized through UV-visible
spectroscopy by performing a titration experiment and it is
considered a more reliable way for studying interactional
behavior of a compound towards DNA. Titration of varying
concentrations of DNA on a xed concentration of a compound
results in absorption intensity change as well as in wavelength
shi. These changes in the spectral response of a compound
before and aer DNA additions not only describe interaction via
formation of a compound-DNA adduct, but the extent and nature
of interactions could also be predicted by using absorption data
to determine kinetic and thermodynamic parameters. A decrease
in absorption peak intensity (hypochromism) in the absorption
spectrum of a compound is usually observed when it binds with
DNA through intercalation.54,55 In present DNA binding studies,
a drop in the absorption peak intensity of TUBC compound's
spectra in DNA presence was observed along with the shiing of
a peak towards lower wavelength, Fig. 11 (Le). The extent of
hypochromism was evaluated to be 39.71% and a blue shi of
21 nm aer the addition of 80 mM of DNA (the highly added DNA
concentration). This personage of hypochromism indicated
binding of TUBC with DNA via intercalative interaction, while
a blue shi pointed towards electrostatic interaction in addition
to intercalation.51,52 Using absorption data in the Bensi–Hilde-
brand equation {Ao/A ꢁ Ao ¼ 3G/3H–G ꢁ 3G + 3G/3H–G ꢁ 3G 1/
Kb[DNA]}, the binding constant ‘Kb’ was calculated as the inter-
cept to slope ratio by plotting a graph between Ao/A ꢁ Ao vs. 1/
[DNA],56 Fig. 11 (Right). The Kb value was further substituted in
the classical van't Hoff equation {DG ¼ ꢁRT ln Kb} to determine
free energy change (DG). Kb and DG values which were evaluated
Complex molꢁ1
)
molꢁ1
)
mol)ꢁ1
molꢁ1
)
molꢁ1
)
molꢁ1
)
TUBC– ꢁ12.10113 ꢁ4.68065 ꢁ1179 573 12.9 227 018 12.10113 ꢁ127064.6
DNA
Steric descriptors
Complex
Hf kcal molꢁ1 MR
S log P Vsurf
Dipole
TUBC–DNA 211.372 067
297.2120 5.17019 141.6177 3.181 483
DNA, Fig. 10 (Right). TUBC furnished vander Waal's interac-
tions with the hydrophobic core of the DNA.
A comprehensive understanding of the physico–chemical
interactions of TUBC, along with several electronic and steric
descriptors were calculated and are provided in Table 7. EHOMO
and ELUMO values give an estimate of the electron-donating or
electron-accepting character of a given compound and, conse-
quently, a compound is considered more electron-donating as
the value of its EHOMO increases and more electron accepting as
the value of its ELUMO decreases.52,53 EHOMO values portrayed
TUBC as electron accepting as compared to DNA base pairs.
While interacting with DNA, TUBC withdraws electrons from
electron rich base pairs and behave as good electron acceptors
determining the reason for a higher binding value of TUBC with
DNA. Steric descriptors also exhibited a soundly good correla-
tion with the binding constant, “Kb”. With the partition coeffi-
cient (S log P) which determines extent of lipophilicity of
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Fig. 11 (Left) UV-visible spectra of TUBC in the presence of fs-DNA (10–80 mM) in 10% aqueous DMSO at 37 ꢂC. (Right) Ao/A ꢁ Ao vs. 1/[DNA] plot
for the determination of binding constant of TUBC–DNA complex by using the Benesi–Hildebrand equation. Arrow direction indicates the trend
in absorption in the presence of increasing DNA concentrations.
to be 2.31 ꢀ 104 Mꢁ1, and ꢁ24.89 kJ molꢁ1, respectively. These viscosity to rise up to a certain compound's concentration and
values of binding parameters agree with that reported for spon- then become constant (no change).63
taneous DNA binding via intercalation.57–59 These values are also
As a best and easy verication method for binding mode
comparable with that reported by our group for a new quinolinyl– investigations, in the present work, we ran viscosity experi-
thiourea hybrid as potential DNA binder.60 Further, binding data ments to measure the DNA viscosity at its xed concentration
from spectral analysis was found to agree with the docking and aer the addition for varying TUBC concentrations. Then
analysis.
viscosity was plotted as the cube root of relative viscosity (h/ho)1/
3 vs. [compound/[DNA] ratio, Fig. 12. A linear rise in the viscosity
was observed until 80 mM addition of TUBC was made and then
no signicant change was observed with further added
concentrations of TUBC were made. i.e., the graph gets straight
aer 80 mM addition of TUBC. Viscosity results veried a mixed
binding mode (intercalation + groove binding/or electrostatic
interaction) and further authenticated our computational and
spectroscopic ndings. The aforementioned results indicate
that this molecule (TUBC) has the possibility to be assayed for
other biological activities in face of its versatile characteristics
as analyzed in this paper.
3.10 Viscosity measurements for DNA binding verication
DNA is biopolymer and the lengthening in a DNA duplex due to
a compound's intercalation could be observed as changes in its
viscosity. Insertion of a compound into DNA base pockets bring
a small rise in DNA viscosity. This small rise, while measuring
DNA viscosity before and aer a compound's addition, is
concentration dependent. If viscosity rises linearly with
a compound's concentration, it veries the intercalation mode
involved in compound – DNA binding.61 In opposition, twisting
in the DNA helix arises if binding occurs through a groove or
electrostatic interaction, which reduces the effective length of
DNA; resulting in a decrease in DNA viscosity.62 However, for
mixed binding mode verication, literature has reported
4 Conclusions
Spectroscopic and single crystal X-ray diffraction studies on the
title compound (C21H18N4O2S$H2O$TUBC) conrmed it to be
new benzimidazole thiourea moiety crystallizing with one
independent molecule and one uncoordinated water molecule
in the asymmetric unit. The uncoordinated water molecules are
linked via intermolecular N–H/OW hydrogen bonds and weak
and O–HW/S intermolecular interactions. The molecules are
further linked via additional weak O–H/N intermolecular
interactions and contain signicant weak p/p stacking inter-
actions resulting in
a three-dimensional supramolecular
network. The Hirshfeld surface analysis of the crystal structure
indicates that the most important contributions for the crystal
packing are from H/H (45.4%), H/C/C/H (12.9%), C/C
(11.9%), H/S/S/H (10.1%) and H/O/O/H (9.8%) interac-
tions. Hydrogen-bonding and van der Waals interactions are
the dominant interactions in the crystal packing. TUBC showed
signicant elastase inhibition as compared to reference ole-
anolic acid. Computational docking results indicated their
binding within the active region of a target protein. Signicant
Fig. 12 Plot of (h/ho)1/3 vs. [compound]/[DNA] at physiological pH (7.0)
and temperature (37 ꢂC).
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radical scavenging activity further proved it to be a potential
antioxidant. Quantum chemical DFT studies of TUBC revealed
that based on EHOMO and ELUMO values, it is considered to be
good electron acceptor, accepting electrons from DNA base
pairs as evident from molecular docking studies. The experi-
mental work further showed TUBC to be a potential drug
candidate that binds spontaneously and reversibly with DNA via
a mixed binding mode. Overall theoretical and experimental
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Correspondance, DNA binding analysis & writing and compiling
original dra by Nasima Arshad; synthesis work by Mamoona
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Aamer Saeed, Pervaiz Ali Channar; DNA binding experimental
work by Shahid I. Farooqi; DFT and molecular docking for DNA
binding by Fouzia Perveen; elastase and antioxidant studies by
Qamar Abbas, Ashfaq Ahmed; Hirshfeld surface studies by
Tuncer Hokelek; crystal structure and analysis by Manpreet
Kaur and Jerry P. Jasinski; Jerry P. Jasinski also help corre-
sponding author for review, proof reading for english and
grammar and in DFT interpretation.
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Conflicts of interest
¨
The authors would like to declare that there is no established 20 A. Saeed, R. Qamar, T. A. Fattah, U. Florke and M. F. Erben,
conict of interest.
Res. Chem. Intermed., 2017, 43, 3053–3093.
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The contributions of all authors' organizations for the facilita-
tion of this collaborative research is highly acknowledged. Jerry
P. Jasinski acknowledges the NSF-MRI program (Grant No.
CHE-1039027) for funds to purchase the X-ray diffractometer.
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