Experimental and theoretical studies of 2-Mercaptobenzothiazole with 2-Bromomethylmesitylene and 1,4-Bis(bromomethyl)durene

Article abstract of DOI:10.1016/j.molstruc.2020.128894

Biologically important 2-(2,4,6-trimethylbenzylthio)benzo[d]thiazole[BTM1] and 2-(4-((benzo[d]thiazol-2-ylthio)methyl)-2,3,5,6-tetramethylbenzylthio)benzo[d]thiazole [BTD2] were synthesized and characterised using FT-IR, NMR and single-crystal XRD (SCXRD). SCXRD revealed that both compounds were crystallized as a triclinic system and were associated through weak intermolecular interactions like H-bondings (CH^…N, sp³-, and sp²-CH^…π), π-π stackings and Vander Waals interactions. These weak intermolecular interactions in BTM1 and BTD2 were studied using Crystal Explorer and Gaussian. The interaction of title compounds with epidermal growth factor receptor (EGFR)tyrosine kinase protein was performed using AutoDock Vina. Molecular docking study revealed an efficient interaction of compounds with the protein resulting in good anti-cancer activity. DNA binding studies were explored against calf thymus (CT) DNA using a spectrophotometric titration method and was found that benzothiazole compounds showed hyperchromism and good binding efficiency. In-vitro anti-microbial activities against some bacterial and fungal strains were investigated. BTM1exhibited a better inhibition against bacterial (Bacillus subtilis and Staphylococcus aureus) and fungal (Aspergillus niger) strains than BTD2.

Full text of DOI:10.1016/j.molstruc.2020.128894

Journal Pre-proof
Experimental and Theoretical Studies of 2-Mercaptobenzothiazole
with 2-Bromomethylmesitylene and 1,4-Bis(bromomethyl)durene
Caroline Daisy , R.Nandini Asha , G.S. Suresh Kumar ,
E. Vadivel , N. Bhuvanesh , B.Ravindran Durai Nayagam
PII:
S0022-2860(20)31219-9
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128894
MOLSTR 128894
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Journal of Molecular Structure
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22 January 2020
9 July 2020
13 July 2020
Please cite this article as: Caroline Daisy , R.Nandini Asha , G.S. Suresh Kumar , E. Vadivel ,
N. Bhuvanesh , B.Ravindran Durai Nayagam , Experimental and Theoretical Studies of 2-
Mercaptobenzothiazole with 2-Bromomethylmesitylene and 1,4-Bis(bromomethyl)durene, Journal of
Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128894
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Experimental and Theoretical Studies of 2-Mercaptobenzothiazole with 2-
Bromomethylmesitylene and 1,4-Bis(bromomethyl)durene
Caroline Daisya, R.Nandini Ashaa, G.S. Suresh Kumarb, E.Vadivelc, N.Bhuvaneshd and
B.Ravindran Durai Nayagama*
aDepartment of Chemistry and Research centre, Popes’ College (Autonomous), Sawyerpuram-628251,
Affiliated to Manonmaniam Sundaranar University, Tirunelveli-627012, Tamil Nadu, India.
bPhotosciences and Photonics Section, CSIR-National Institute for Interdisciplinary Science and
Technology (NIIST), Thiruvananthapuram -695 019, Kerala, India
cPost-Graduate Department of Chemistry, Dnyanaprasarak Mandal's College and Research Centre,
Assagao, Mapusa-403507, Goa, India.
dDepartment of Chemistry, Texas A&M University, College Station, TX 77842, USA
Corresponding author: e-mail:bravidurai@gmail.com, Phone no: +919443290387

Graphical Abstract
Abstract
Biologically important2-(2,4,6-trimethylbenzylthio)benzo[d]thiazole[BTM1] and
2-(4-((benzo[d]thiazol-2-ylthio)methyl)-2,3,5,6-tetramethylbenzylthio)benzo[d]thiazole
[BTD2]were synthesized and characterised using FT-IR, NMR and single-crystal XRD
(SCXRD). SCXRD revealed that both compounds were crystallized as a triclinic system and
were associated through weak intermolecular interactions like H-bondings (CH…N, sp3-, and
sp2-CH…π), π-π stackings and Vander Waals interactions. These weak intermolecular
interactions in BTM1 and BTD2 were studied using Crystal Explorer and Gaussian. The
interaction of title compounds with epidermal growth factor receptor (EGFR)tyrosine kinase
protein was performed using AutoDock Vina. Molecular docking study revealed an efficient
interaction of compounds with the protein resulting in good anti-cancer activity. DNA
binding studies were explored against calf thymus (CT) DNA using a spectrophotometric
titration method and was found that benzothiazole compounds showed hyperchromism and
good binding efficiency. In-vitro anti-microbial activities against some bacterial and fungal
strains were investigated. BTM1exhibited a better inhibition against bacterial (Bacillus
subtilis and Staphylococcus aureus) and fungal (Aspergillus niger) strains than BTD2.

Key Words: Benzothiazole; Weak bonding interactions; Hirshfeld surface analysis; DFT;
Molecular docking; DNA binding.
Highlights
 BTM1 and BTD2 were crystallized as a triclinic system and well associated.
 DFT calculations revealed that BTD2 had higher stability than BTM1.
 Molecular docking exposed BTM1 and BTD2 as efficient anti-cancer drugs.
 DNA binding studies displayed that both benzothiazole compounds had
hyperchromism and had good binding efficiency with CT-DNA.
 When compared with BTD2, BTM1 exhibited good activity against the microbes that
were taken for study
.

1. Introduction
Benzothiazole (BT) derivatives were studied and found to have a broad-spectrum of
biological applications like analgesic, anticancer, antioxidant, anti-inflammatory, anti-
diabetic, anti-convulsant, anti-tubercular, anti-malarial, anti-leishmanial, anti-histaminic,
antibacterial, anti-viral, antifungal, etc., [1-3]. 2-Mercaptobenzothiazole(2-MBT) is a
significant skeleton familiar for its association with numerous biological activities and its
derivatives that are manufactured worldwide for an extensive variety of medical applications
[4,5]. Incorporation of benzyl moiety into 2-MBT is noteworthy for anticancer and
antibacterial activities. Modifications on the benzyl moiety lead to a large number of
compounds showing diverse pharmacological activities [6-7].
Hydrogen bondings, π-π stacking interactions, and other weak forces are vital for
structural stability, the function of DNA and RNA, and self-assembly or molecular
recognition processes when extended structures are formed from building blocks with
aromatic moieties [8-11].“Hirshfeld surfaces” afford a remarkable approach of exploring
packing modes and intermolecular (building block) interactions in molecular crystals
[12].“2D plots” are a visual representation of all building block connections and are exclusive
for a given crystal structure and polymorph [13].
Density functional theory (DFT) is a quantum mechanical computational modelling
technique utilized in chemical, physical, and material sciences to inspect the electronic/
nuclear structure in the ground state of many-body systems, specifically atoms, molecules,
and condensed phases. Among the ever-increasing number of DFT methods, the hybrid

functional „B3LYP‟ has emerged as a good compromise in computational cost, coverage, and
accuracy in results. Thus, it has become a standard method for investigating the molecular
properties of organic molecules in the gas phase [14-18].
Docking biologically active compounds into the binding site of a receptor and
exploring the binding affinity of the compound-receptor complexes are the strategy structure-
based drug designs. It helps to develop better drug candidates with improved activity [19].
Epidermal growth factor receptor (EGFR, ErbB-1and HER1 in humans) is a transmembrane
protein that is a receptor for members of the EGF family of extracellular protein ligands.
EGFR kinase is suitable for therapeutic intrusion because of its significant character of
abnormal signalling in cancer and its involvement in regulating growth, migration and
apoptosis of tumour cells [20-22]. Some benzothiazole derivatives were designed and
proposed as potential EGFR tyrosine kinase inhibitors in cancer treatment [23].
Looking into the biological importance of 2-mercaptobenzothiazole, the present work
concentrates on the syntheses of novel 2-(benzylthio)benzothiazole compounds and
investigates on the weak bonding interactions using SCXRD results, CrystalExplorer,
Gaussian and in silico studies. Newly prepared benzothiazole derivatives were explored for
DNA binding and anti-microbial activities.
2. Experimental
2.1. General Methods
All chemicals and solvents were acquired from commercial sources with high purity
and analar grade and used as received unless otherwise noted. The melting point of title
compounds was measured in an open capillary tube. Infrared spectra were recorded on a
JASCO 4600 (Japan) FT-IR spectrometer (in KBr pellet) with a scan range from 400 to 4000
cm-1.1H- and 13C-NMR spectra were recorded with a Bruker Avance III, 400 MHz, 9.4 Tesla
superconducting magnet using CDCl3 as a solvent. Chemical shifts were reported as δ (ppm)

with tetramethylsilane as an internal standard. DNA binding experiments were done using
Jasco V-650 UV spectrophotometer.
2.2. Synthesis of 2-bromomethylmesitylene (M1),1,4-bis(bromomethyl)durene(D2),and 2-
benzylthiobenzothiazole derivatives (BTM1 and BTD2):
2-Bromomethylmesitylene (M1) and 1,4-bis(bromomethyl)durene (D2) were
synthesized using the reported procedure [24].
2.2.1. Synthesis of 2-bromomethylmesitylene (M1):
Mesitylene (12.0 g, 0.10 mol), paraformaldehyde (3.08 g, 0.10 mol), 50 mL of glacial
acetic acid and 20 mL of 31 wt% HBr/acetic acid solutions were stirred at 80oC. After 2 h,
the reaction mixture was poured into 100mL of water. The product 2-bromomethylmesitylene
was filtered and vacuum-dried at room temperature.
2.2.2. Synthesis of 1,4-bis(bromomethyl)durene (D2):
Durene (13.42 g, 0.10mol) and paraformaldehyde (6.15 g, 0.20 mol) were dissolved in
50 mL of glacial acetic acid and 40 mL of a 31wt% HBr/acetic acid solution. The reaction
mixture was kept for 8 h at 120oC and poured into 100 mL of water. The solid was filtered
and dried under vacuum at room temperature.
2.2.3. Synthesis of 2-benzylthiobenzothiazole derivatives (BTM1 and BTD2):
BTM1 was synthesized from sodium salt of 2-mercaptobenzothiazole (2-MBT, 0.189
g, 1 mmol) with 2-bromomethylmesitylene (M1, 0.213 g, 1 mmol) in ethanol (10 mL) and
was heated at 80°C and stirred continuously for 1 h. The solid was filtered and vacuum-dried.
The dried solid was recrystallized using chloroform. Pure BTM1 was filtered and dried.
Likewise, BTD2 was prepared from sodium salt of 2-mercaptobenzothiazole (2-MBT, 0.378
g, 2 mmol) and1,4-bis(bromomethyl)durene (D2,0.226 g,1 mmol) (Scheme 1).
2.2.3.1. 2-(2,4,6-trimethylbenzylthio)benzo[d]thiazole [BTM1]

Yellow crystalline solid; melting point: 92-94 °C; IR (KBr) vmax: 3058, 2942, 2905,
1605 cm-1; 1H-NMR (400 MHz, CDCl3, δppm): 7.93–6.88 (m, 6H, Ar-H), 4.67 (s, 2H, CH2),
13
2.46–2.37 (m, 6H, 2‟,6‟ CH3), 2.27 (m, 3H, 4‟ CH3); C-NMR (400 MHz, CDCl3, δppm):
167.2, 153.2, 137.6, 135.1, 129.1, 128.1, 126.0, 124.1, 121.4, 120.9, 77.4, 77.0, 76.5, 32.9,
20.9, 19.6.
Scheme 1. Synthesis of BTM1 and BTD2
2.2.3.2. 2-(4-((Benzo[d]thiazol-2-ylthio)methyl)-2,3,5,6-tetramethylbenzylthio)benzo[d]thi-
azole [BTD2]
Pale yellow crystalline solid; melting point: 165-167 °C; IR (KBr) vmax: 3100–2750,
1
1774, 1672, 1596 cm-1; H NMR (400 MHz, CDCl3, δppm): 7.98–7.33 (m, 8H, Ar-H), 4.78
13
(s, 4H, 2CH2), 2.423 (s, 12H, 2‟,3‟,5‟,6‟ CH3); C NMR (400 MHz, CDCl3, δppm): 167.0,
153.2, 135.1, 134.3, 131.3, 126.1, 124.3, 121.4, 121.0, 34.4, 16.5.
2.3. X-ray structure determination
Single crystals obtained were characterised using a BRUKER Quest X-ray (fixed-Chi
geometry) diffractometer. The goniometer was controlled using the APEX3 software suite
[25]. The absorption correction program SADABS [26] was employed to correct the data for
absorption effects. Systematic reflection conditions and statistical tests of the data suggested

the space group P-1. A solution was obtained readily using XT/XS in APEX3 [27-30]. The
absence of additional symmetry and voids was confirmed using PLATON (ADDSYM). The
structure was refined (weighted least-squares refinement on F2) to convergence [27-30].
Olex2 was employed for the final data presentation and structure plots [31]. Mercury 3.0 was
used for generating structures and molecular interaction drawings. Crystallographic data for
the structures reported in this paper have been deposited in the Cambridge crystallographic
data centre. BTM1(CCDC no.1448446) and BTD2(CCDC no.1495365).
2.4. Hirshfeld surface analysis
Intermolecular interactions as well as surface properties of the pro-molecule of BTM1
and BTD2 were defined by Hirshfeld surface [32] and 2D fingerprint plots [33] were
successfully acquired from CrystalExplorer-17.5 [34] using the cif files of SCXRD data.
2.5. Computational methodology
The single-crystal structure files (cif file) of the title compounds were converted to
mol2 files using Mercury 3.0. These files were used as input files in the Gaussian 09W
software package [35] for energy and other calculations. Theoretical calculation of BTM1
and BTD2 in the gas phase was performed using the analytical gradient method of DFT with
Becke‟s three parameters (B3) exchange functional together with the Lee-Yang-Parr (LYP)
non-local correlation functional, symbolized B3LYP [15] using 6-311G basis set
implemented in Gaussian 09W software package [35]. Parameters such as ionization potential
(I), electron affinity (A), electronegativity (χ), electrochemical potential (µ), hardness (η),
softness (ζ), and electrophilicity index (ω) were calculated using previous reports [36-38].
HOMO, LUMO and molecular electrostatic potential (MEP)surfaces were generated using
GaussView 5.0.
2.6. Molecular Docking Studies

The two-dimensional (2D) chemical structures of the title compounds were sketched
using ChemDrawUltra and converted to pdb files using Chem3D Ultra.
The X-ray crystal structure of epidermal growth factor receptor (EGFR) kinase bound
with the small molecule 6-acrylamido-4-anilinoquinazoline, an irreversible kinase inhibitor
(PDB ID: 1M15)[23] was retrieved from the RCSB protein data bank
(http://www.rcsb.org/pdb) for molecular docking study. Protein and grid preparation were
made using the AutoDock Tool 4.2 [39]. Polar hydrogen atoms were added to the protein
kinase receptor structure and then the Gasteiger charge was computed and added,
accordingly. Molecular docking study of benzothiazole derivatives was performed with the
AutoDockVina [40]. Chain A of EGFR is constituted by 327 amino acids. This chain was
chosen for docking. Iressa was used as a reference anticancer drug. The binding site required
for the action of the EGFR kinase domain is Leu-718-Thr-854.
The protein was loaded into AutoDockTool 4.2, creating a PDBQT file that contains a
protein structure with hydrogens in all polar residues. The docking site on the protein target
was defined by establishing a grid box with the dimensions X: 74 Y: 60 Z: 62 Å, with a grid
spacing of 0.372 Å, centered on X: 52.45 Y:0.316 Z: 21.46 Å. The best conformation was
chosen with the lowest docked energy after the docking search was completed. Ten runs were
performed in all cases per each ligand structure, and for each run the best pose was saved.
The interactions of complex EGFR protein-ligand conformations, including hydrogen bonds
and hydrophobic interactions, were analysed using Discovery studio 4.0 client[41] and
PyMol[42].
2.7. DNA Binding Studies
DNA binding studies were performed using a spectrophotometric titration method
[43]. Experiments involving the interaction of compounds with CT-DNA were performed
using double distilled water with tris(hydroxymethyl)aminomethane (Tris, 5 mM) and

sodium chloride (50 mM). The pH was adjusted to 7.2 using hydrochloric acid. A solution of
CT-DNA in the Tris-NaCl buffer gave a ratio of about 1.8 to 1.9 of absorbance at 260 and
280 nm, indicating that the DNA was sufficiently free of protein. The absorption titration
experiments were performed by maintaining 50 μg/mL of the concentration of the title
compound as constant but with variable CT-DNA concentration from 10 to 50 μM. While
measuring the absorption spectra, equal amounts of DNA were added to both complex and
reference solutions in order to eliminate the absorbance of DNA itself. The data were then fit
into the following equation and the intrinsic binding constant Kb was calculated [44] in each
case.
[DNA] / (εa-εf) = [DNA] / (εb-εf) + 1/[Kb(εb-εf)]
where [DNA] is the concentration of DNA in base pairs, εa the apparent absorption
coefficient, εf the extinction coefficient of the free compound and εb the extinction coefficient
of the compound when fully bound to DNA respectively. In the plots of [DNA]/ (εa-εf) vs
[DNA], the ratio of a slope to the intercept gives the Kb value.
2.8. Antimicrobial studies
Antimicrobial studies have also been carried out. The Agar Well diffusion method
was used in antibacterial assay against the gram-positive bacteria Bacillus subtilis and gram-
negative bacteria Staphylococcus aureus. For antifungal screening, the disc diffusion method
[45] was used against fungi Candida albicans and Aspergillus niger. Gentamicin was used as
a standard drug for antibacterial screening and amphotericin B for antifungal screening.
3. Results and Discussion
3.1. Crystal Structure Analysis Using SCXRD
Single crystals of the title compounds were collected which were then subjected to
SCXRD analysis. Crystallographic data and structure refinement details of BTM1 and BTD2
are shown in Table 1. Fig. 1 shows molecular geometries and thermal ellipsoids. Cell content

in both compounds is displayed in Fig. 2a (BTM1) and Fig. 3a (BTD2).
In BTM1, the torsion angles of C(4)-C(5)- C(6)-S(1) and C(16)-C(5)-C(6)-S(1) are -
85.4(2) and 92.2(2) respectively. In it the benzothiazole ring was perpendicular to the
mesitylene ring. In BTD2, the torsion angles of S(2)-C(8)-C(9)-C(10) and S(2)-C(8)-C(9)-
C(14) are -86.36(15)° and 91.65(15)° for one benzothiazole ring with the durene ring. The
torsion angles C(11)-C(12)-C(19)-S(3) and C(13)-C(12)-C(19)-S(3) of the second
benzothiazole ring with the durene ring are 90.95(15)° and 89.15(15)°. It indicated that the
two benzothiazole rings are perpendicular to the durene ring. Also, the two benzothiazole
rings adopted a stepped cis conformation with respect to the durene ring. In BTM1, the
benzothiazole ring was present above the mesitylene ring plane where terminal H-atom (six-
membered ring, H17) of benzothiazole ring existed on same plane. However, in BTD2, most
parts of the two benzothiazole rings were above the durene ring plane and few parts of the
rings (C3C4 and C24C25) were below that plane. This proved the cis-conformation of
benzothiazole rings in BTD2.
The interplanar angle (14.37°) between benzothiazole ring-I and ring-II of BTD2
disclosed that the two rings did not lie perfectly on the same plane. Each benzothiazole ring
was in a different direction to the vertical plane of durene moiety (vertical plane through
C8C9C12C19). Tilt angles (ring-I, 4.66° and ring-II, 11.09°) of the vertical plane of durene
moiety and benzothiazole ring plane indicated that ring-I was placed below and ring-II
[above (N2C26C25C24C23) and below (C20S4C21C22)] passed through this plane. This
exposed that BTD2 is crystallized ina symmetrical structure with a slight distortion.
The orientation of benzothiazole ring from mesitylene moiety was explored from the
distances between the hetero atoms (S/N) in benzothiazole ring and mesitylene ring plane
(horizontal). The S-atom within the benzothiazole ring was close to this plane (distance
between S2 and mesitylene ring plane is 0.263 Å) in BTM1. But in BTD2, N-atoms (N1 and

N2) in benzothiazole rings (I and II) were nearer to the horizontal durene ring plane (ring-I:
0.343 Å and ring-II: 0.250 Å). This showed that the orientation of benzothiazole ring in
BTM1 was opposite to those in BTD2.
Torsion angles were used to identify the orientation of substituents, the positive (+)
angle for clockwise direction and the negative (−) angle for counter-clockwise direction.
Torsion angles of BTM1[C5-C6-S1-C7, 163.4(2)°; C6-S1-C7-S2, 1.5(2)°; C6-S1-C7-N1, and
-178.0(2)°] andBTD2[ring-I: C9-C8-S2-C7,-177.6(1)°; C8-S2-C7-S1,-176.0(1)°; C8-S2-C7-
N1,and 4.4(2)° and ring-II: C12-C19-S3-C20, -179.8(1)°; C19-S3-C20-S4,-174.7(1)°; C19-
S3-C20-N2,and 6.5(2)°] also confirmed that their benzothiazole rings were organized in the
opposite direction. The benzothiazole ring of BTM1 was in the clockwise direction (positive
torsion angle) while each benzothiazole ring of BTD2 was in the counter-clockwise direction
(negative torsion angle) concerning mesitylene and durene moieties respectively.
3.1.1. Non-covalent Interactions and Supramolecular Self-assembly
Crystal structural analysis of compounds revealed the presence of abundant non-
covalent (weak bonding) interactions such as intermolecular H-bonds (CH…N, sp3-, and sp2-
CH…π), π-π stacking, and Van der Waals forces. The hydrogen-bonding parameters [d(H…A)
>> d(D-H)] exposed that these bonds were weak H-bonds (Table 2) [46].
Weak intermolecular H-bonding was observed in BTM1 [H16…N1(A), 2.660 Å] with
the hydrogen atom (H16) in the benzene ring of one benzothiazole moiety as a donor and the
nitrogen atom (N1) in the thiazole ring of another neighbouring benzothiazole moiety as an
acceptor. This created a centrosymmetric eight-membered ring motif through another unit of
BTM1(A) with Etter‟s notation (8) like acid dimers, amide dimers etc.,(Fig. 2b) [47]. The
interplanar distance and the angle between benzothiazole and benzothiazole(A) rings [1.034
Å and angle 0°] made a z-type arrangement. On the other hand, this sort of H-bond was not
found in BTD2.

The C–H…π interactions act as weak hydrogen bonds and aromatic cloud/carbon acts
as H-acceptor. The aliphatic (sp3) C–H…πand aromatic(sp2) C–H…π interactions can deeply
affect the characteristics of the systems. The intermolecular interaction energy depends on the
character of the molecular fragments, C-H as well as π-groups. The stronger the proton
donating ability of the CH group, the larger the stabilizing effect [48]. For typical cases
involving sp3(aliphatic) and sp2(aromatic) CH groups as the hydrogen donor, the energy of a
one-unit C-H…π hydrogen bond is ca. 1.5–2.5 kcal mol-1. Interactions involving aromatic
CHs are stronger than the aliphatic ones [48,49].
The sp3 C-H of benzyl moiety and benzothiazole ring of BTM1 associated to make
two sp3C–H...π and four sp3C–H…C [sp3C–H...π: 2.968 Å for H9…centroid of benzothiazole
ring (B) and H9(B)…centroid of benzothiazole ring; and sp3CH…C: 2.865 Å for H9…C12(B)
and H9(B)…C12 and,2.771 Å for H9…C13(B) and H9(B)…C13] interactions with the adjacent
molecule, BTM1(B). The aromaticsp2C-H..π of BTM1 was formed with BTM1(C) and
BTM1(D) through benzyl moiety and benzothiazole ring [two sp2C–H…π:3.327 Å for
H17…centroid of benzyl ring(C) and H17(D)…centroid of benzyl ring, and two sp2 C–H…C:
2.831 Å for H17…C4(C) and H17(D)…C4]. These sp3 C–H…π and sp2 C–H…π interactions
developed a typical edge-to-face, specifically T-shaped arrangements through benzyl moiety
and benzothiazole ring with four neighbouring BTM1 molecules.
But in BTD2, two sp3C–H…π [H16c…centroid of benzyl ring(A) and
H16c(A)…centroid of benzyl ring; 2.874 Å] and two sp3C–H…C [H16c…C13(A) and
H16c(A)…C13; 2.724 Å] created between sp3 C–H of benzyl moiety of BTD2 and benzyl
moiety of BTD2(A) developed parallel-displaced arrangements [28] (Fig.3b). Two aromatic
sp2 C–H…π were formed between the terminal benzothiazole rings [sp2 C–H…π: 3.694 Å for
C5–H5…centroid of benzothiazole ring(B) and C5(C)–H5(C)…centroid of benzothiazole ring,
and sp2 C–H…C: 2.868Å for H5…C23(B) and H5(C)…C23] of BTD2 with other BTD2 (B and

C). The terminal H-atom of benzothiazole ring and the centroid of neighbouring
benzothiazole ring formed 1-D chain networks (3.694 Å) with parallel-displaced or slip
arrangements [50].
BTM1 and BTD2 exhibited π–π stacking supramolecular interactions in the crystal
due to their aromatic moieties with short contact distance < 3.7 Å (Table 3). Three π–π
stacking interactions [Oneπ–π: 3.967 Å, centroid of benzyl…centroid of benzyl (E);3.674 Å
for C4…C5(E) and C5…C4(E); 3.606 Å for C4…C16(E) and C16…C4(E) and, Twoπ–π: 3.925
Å for the centroid of benzo5…centroid of benzo6(F) and the centroid of benzo6…centroid of
benzo5(F); 3.552 Å for C7…C12(F) and C12…C7(F); and 3.626 Å for C13…C13(F)] were
observed between benzyl moiety and benzothiazole ring of BTM1 with BTM1(E) and
BTM1(F) (Fig. 2c). These π–π stacking interactions created the perpendicular arrangement of
benzyl moiety and benzothiazole ring in BTM1 and these intermolecular interactions
generated a parallel-displaced or slip stacking arrangement with neighbouring molecules,
BTM1(E) and BTM1(F) [50].
Analysis of stacking interaction indicated eight types of π…π stacking modes in
BTD2, i.e one benzyl…benzyl [centroid…centroid (A), 4.519 Å; C11…C11(A), 3.682 Å;
C11…C12(A), 3.642 Å; and C12…C11(A), 3.642 Å]; three benzo6…benzo6 [5.972 Å for
centroid…centroid(A);3.627
Å
for C3…C24(A) and C24…C3(A); 4.884
Å
for
centroid…centroid(B/C); 3.395 Å for C5…C23(B/C)]; and four benzo5…benzo6 [3.658 Å for
centroid…centroid(D/E),C7…C23(D/E), 3.670 Å; C2…C26(D/E), 3.500 Å; C2…C25(D/E),
3.513 Å; C3…C25(D/E), 3.415 Å; C3…C26(D/E), 3.437 Å; and C5…C20(D/E), 3.544].This
displayed the existence of π–π stacking supramolecular interactions in the crystal due to
aromatic benzyl-benzyl moieties of BTD2 and BTD2(A) (Fig.3b) and
benzothiazole…benzothiazole rings of BTD2 with BTD2(B-E). The stacking pairs are not
completely parallel to each other. So these π–πstacking interactions created face-to-face 3-D

parallel-displaced or slip stacking supramolecular arrangements throughout the crystal [50].
One BTM1 molecule is connected to six other molecules [BTM1(A-F)] through
weak C–H...N, C–H…π, and π–π interactions. These weak perpendicular stacking interactions
propagated the herringbone mode of packing in BTM1(Fig. 2c and 2d)[50]. However, one
BTD2 molecule is in contact with nine other molecules [BTD2(A-I)] through weak C–H…π
hydrogen bonds, π–π stacking and Van der Waals interactions. The weak intermolecular
interactions and parallel-displaced stacking lead to the tub-like packing arrangements in
BTD2 (Fig. 3c and 3d) [50].
3.2. Hirshfeld Surface and 2D Fingerprint Plot Analysis
Hirshfeld surfaces (HS) reflected intermolecular connections visually offering a
hitherto unseen picture of molecular shape in a crystalline environment. The surfaces
encoded evidence about all intermolecular interactions simultaneously, but sophisticated
interactive illustrations are required to extract the information most efficiently. The mapping
took advantage of the triangulation of the “Hirshfeld surfaces” and plotted the fraction of
points on the surface as a function of the closest distances from the point to nuclei inside and
outside the surface. In this manner, all interaction types (H-bonding, C–H…π interactions, π–π
stacking, close and distant Van der Waals contacts) are readily identifiable, and it becomes a
standard method to analyse crystal packing and intermolecular interactions in crystals
[12,13].
“The dnorm values were mapped onto the HS exploiting, a red–blue-white colour
pattern: the red areas indicated closer contacts with negative dnorm value while the blue zones
indicated longer contacts with positive dnorm value, and the white regions displayed the Van
der Waals separation with a dnorm value of zero” [12]. Fig.4f displays surfaces that have been
mapped over the dnorm range of -0.084 to 1.385 Å in BTM1. The red spots on the dnorm
surface indicated the existence of hydrogen bonding interactions, i.e., C–H…N. Other colour

spots have also been observed due to the existence of some close contacts, such as C…H,
C…C, H…H, S…H, C…S, and C…N in BTM1 and BTD2 molecules. The red spots on the di
and de surface indicated the existence of close contacts available inside and outside the
surface. Weak intermolecular interactions were observed on N1, C4, C12, C13, H9, H17, and
H19 atoms in BTM1andC5, C13, C23, H4, H5, H8A, H15B, H16A, H16C, H17A, H19B,
and H25 atoms in BTD2. One BTM1 molecule was surrounded by six BTM1 molecules and
one BTD2 was encircled by nine BTD2 molecules. Surface area and volume of BTD2
(565.25 Å3 and 503.56 Å2) were higher than BTM1 (379.77Å3and 344.22 Å2).
Fig. 5 illustrates 2D plots for BTM1 and BTD2. They typically cover much greater
ranges in both de and di (from 1.0 to almost 2.8Å) which indicated the presence of aromatic
compounds and this is attributed to the different types of interactions, as well as the dramatic
anisotropic shape of these molecules. The shortest contact in BTM1 and BTD 2was quite
close to1.2 Å, the generally acknowledged Van der Waals radius for hydrogen [12,13].
Fig.4 exhibits surfaces that have been mapped over the dnorm range of BTM1 (-0.084
to 1.385 Å) and BTD2 (-0.112 to 1.329 Å). The d spots for two weak dimeric C–H…Naromatic
on the Hirshfeld surface mapped with dnorm confirmed the presence of short intermolecular
interactions between molecular pairs in BTM1 (Fig.4f). The red spots on the di near N-atom
(Fig. 4d) and de near aromatic H-atom[C9–H16] (Fig. 4e) surface of BTM1 directed the
existence of close contacts available inside and outside the surface. The long spikes present in
the 2D fingerprinting plot (Fig. 5b) which are characteristic of hydrogen bonds (H…N 7.7%)
in BTM1 were found to have similar contributions over the Hirshfeld surfaces [51]. Hirshfeld
surfaces (Fig. 4hand 4i, absence of red spots near N-atoms) and 2D fingerprinting plots (Fig.
5g, absence of long spikes) of BTD2 confirmed the absence of C–H…Naromatic hydrogen
bonding. This further validated the directionality of N-atom in benzothiazole rings.

The dominant intermolecular connection was the C-H…π (C-H…C) contact for
molecular packing in a herringbone arrangement. Compounds were stabilized through C–
H…π interactions which were visible on the de surface as well as in fingerprint plots. The
„bright-orange‟ spots and a broad depression on the de surface above the π-electron cloud of
aryl rings were indicators of C–H…π interactions (Fig. 4e and 4k) [52]. Moreover, these
interactions appeared as a red concave region on the surface around the acceptor atom and a
corresponding blue region around the C–H donor. These C–H…π interactions were also
visible as a pair of „wings‟ in the top-left and bottom-right region of the 2D fingerprint plots
(Fig. 5c and 5h) [53,54]. The top-left region (di< de) corresponds to the points on the surface
around the C–H donor and the bottom-right region (de< di) for π-acceptor. So, the shape of
the wings and the sum of the di and de pointed out the importance of these connections. The
decomposition of the fingerprint plots corresponding to C–H…π interactions comprised 20.1%
and 17.7% of the total Hirshfeld surface area (HSA) ofBTM1 and BTD2 respectively.
The „shape index‟ which is the measurement of shape can be sensitive to very subtle
changes in the surface shape. The red triangles on the shape index signified concave regions
and represented atoms of the π-πstacked molecule above them, while the blue triangles
denoted convex regions indicating the ring atoms of the molecule inside these surfaces. The
large flat region delineated by a blue outline on the curvedness surfaces indicated π–π
stacking of molecules [55,56]. From this, it is clear that the crystal structure of BTM1 and
BTD2exhibited stacking interactions (Fig. 4b and 4h). The “curvedness” mapped on the HS
disclosed that the largest regions of flat curvedness appear for BTM1 andBTD2 (Fig. 4c and
4i). From „shape index‟ and „curvedness‟, we can get the proportion of π–π stacking
interactions observed on benzyl moiety and benzothiazole ring in compounds. These results
were in agreement with the 2D fingerprint plots (Fig. 5d and 5i). The C…C contacts in BTM1
(3.5%) and BTD2 (4.8%) revealed π–π stacking interactions.

The percentage contributions (Fig. 6) of these interactions to the relative Hirshfeld
surface areas revealed noticeable involvement of C–H…π (20.1% and 17.7%) and π–π (3.5 %
and 4.8%), whereas a more significant contribution came from H…H contacts (53%and
46.2%), because of the considerable engagement of the hydrophobic groups in the self-
assembly of the molecular framework. This analysis quantified the intermolecular
interactions involved within each crystal structure thus providing a full understanding of the
intermolecular interactions in a facile way [55,56].
There was a distinct splitting of the short H…H fingerprint at de ≈ di ≈ 1.2 Å. This
splitting occurs when there is the shortest contact among three atoms, rather than a direct
two-atom contact [12,13]. Non-uniform distribution (di 2.2-2.5) confirmed the symmetrical
structure with slight distortion in BTD2(Fig. 5f).
3.3. Theoretical Studies
The molecular properties of BTM1 and BTD2 were studied using Gaussian09w. The
energies of compounds were calculated at the B3LYP/6-311G level of theory through the
DFT method in the gas phase that was carried out from the experimental structures [14-18].
Parameters like total energy, EHOMO, ELUMO, ionization potential, electron affinity,
electronegativity, electrochemical potential, hardness, softness, and nucleophilicity are given
in Table 4.
Higher negative (lower) total energy (-1698744.65 Kcal/mol) of BTD2 than BTM1(-
946823.68) revealed that BTD2 is more stable than BTM1. According to the molecular
orbital (MO) theory, HOMO and LUMO are the most important factors affecting the
bioactivity. The interaction between the molecule and the receptor of bacteria is dominated
by π-π stacking and hydrophobic interaction among these frontier molecular orbitals [57,58].
HOMO and LUMO of compounds are demonstrated in Fig. 7. BTM1 showed that the
HOMO and LUMO electron clouds were located only in the benzothiazole ring and not in the

benzyl moiety. This confirmed the formation of intermolecular π-π stacking interactions
between benzothiazole units. But in BTD2, the electron density of HOMO was located in one
benzothiazole (ring-I) and benzyl moiety, and the LUMO in another benzothiazole (ring-II).
It revealed that intermolecular π-π stacking interaction was found between two benzothiazole
rings of neighbouring molecules with benzothiazole ring-I as a donor and ring-II as an
acceptor.
Moreover, the gap between the HOMO and LUMO energy levels (ΔE) of the
molecule was an important parameter that determined the reactivity of the molecule. As ΔE
decreases (especially for the cationic species), the reactivity of the molecule increases leading
to a decrease in the stability of the molecule [57,58]. The HOMO-LUMO energy gaps of both
compounds were nearly similar. Electron affinity and ionization potential were higher for
BTD2 than BTM1.
The nucleophilicity measures the electrophilic power of a molecule. It has been
reported that higher the value of nucleophilicity, higher will be the capacity of the molecule
to donate electrons [59]. The higher nucleophilicity value of BTD2 indicated that it had a
higher capacity to donate electrons than BTM1.
The dipole moment, which is defined as the first derivative of the energy concerning
an applied electric field, is mainly used to study the intermolecular interactions involving the
Van der Waals forces, dipole-dipole forces, etc. BTM1 had a higher dipole moment value and
hence it had stronger intermolecular attraction [60]. The dipole moment of compounds
revealed that the polarity of BTM1 is higher than BTD2.
Molecular electrostatic potential (MEP) maps of compounds were examined using the
same DFT technique (Fig. 7g and 7h). The different values of the electrostatic potential at the
surface were depicted in different colours [61]. Within the MEP of BTM1, the negative
region (red) was found on the nitrogen atom (N1) with a value of -0.05639 a.u and the

maximum positive region (blue) on the hydrogen atom (H16) with a value of 0.05639a.u.
This confirmed the existence of an intermolecular C–H…N interaction in BTM1. But in
BTD2, the MEP revealed the presence of negative region (red) on nitrogen atoms (N1 and
N2) and positive region (yellow) on two benzothiazole rings. This showed face-to-face π–
πintermolecular interactions between the benzothiazole rings of one molecule with another
molecule and therefore the molecule was stabilized by short contacts. SCXRD and Hirshfeld
surface analysis of BTD2 also confirmed the presence of face-to-face π-π intermolecular
interactions.
Mulliken net charges were calculated at the DFT level with the B3LYP/6-311G basis
set in the gas phase using Gaussian09W for molecules (vide supporting information). All
hydrogen atoms in both compounds had a net positive charge. Moreover, Mulliken net
charges showed that the H15 in BTM1 and H8A and H19A in BTD2 atom had higher
positive atomic charges [H15(0.276422), H8A (0.268028) and H19A (0.269725)] than the
other hydrogen atoms. This is due to the presence of an electronegative sulfur atom [S1 for
BTM1 and S2, S3 for BTD2] on the mercaptobenzothiazole moiety. Concerning Mulliken
charges of carbon in benzothiazole, except C-atoms [C8 for BTM1 and C2, C26 for BTD2]
near to N-atom, all the other carbons had negative charges. But in benzyl moiety, except
methyl-substituted carbon, all the other carbons had negative values. In BTD2, benzyl ring
carbons had positive values and benzothiazole ring carbons had a negative value. This
indicated the donor and acceptor character of carbon.
The presence of a high negative charge on carbon and the net positive charge on C/H-
atom could suggest the formation of intermolecular interactions in solid forms [36-38].
Mulliken charges of atoms on each benzothiazole ring (I and II) in BTD2 were not similar.
This confirmed its symmetrical structure with a slight distortion.
3.4. In Silicomolecular Docking Studies for Anticancer Activity

Ten different orientations of BTM1 and BTD2 to the receptor EGFR tyrosine kinase
protein were carried out. The docked compounds occupied the same binding site pocket as
occupied by Iressa in the crystal structure as shown in Fig.8. The binding affinities of both
the compounds were higher than that of the standard Iressa [19-22]. Weak intermolecular
interactions (hydrogen bonds and hydrophobic interactions) are crucial players in stabilizing
energetically-favoured ligands, in an open conformational surrounding of protein structures
[23]. The hydrophobic interactions of the best orientations are presented in Table 5. BTD2
complex showed thirteen hydrophobic interactions with a more binding affinity (-9.2
Kcal/mol) than BTM1complex (nine hydrophobic interactions with-8.0Kcal/mol) and Iressa
(eleven hydrophobic interactions with-7.2Kcal/mol). BTM1 and BTD2 formed several
interesting binding interactions with Lue718, Leu792, Val726, Lys745, and Leu844. Above
all, BTD2 was found to be a crucial molecule in its interaction with His805, Tyr801, and
Gly796. This revealed that the title molecules were capable enough to form some vital
interactions with the amino acid residues of EGFR and favored the potentiality of molecules
as anti-cancer agents.
This result is compared with the recent report in benzothiazole derivatives with
receptor EGFR tyrosine kinase protein [20]. Molecular docking clearly showed the final
proposed potential of benzothiazole derivatives in the formation of some binding interactions.
Intermolecular H-bonding was only observed in their phenoxy substituents containing amino-
and amide functional groups and hydrophobic interactions were observed in benzothiazole
moiety. This showed that intermolecular H-bonding was absent due to the absence of
hydrophilic groups (amide, amine, etc.) in our compounds. Instead of using hydrophobic
methyl groups, substituent with hydrophilic groups like acid, amide, amine, etc. in benzyl
moieties in BTM1 and BTD2 may improve the binding with the receptor EGFR tyrosine
kinase protein.

3.5.DNA Binding Studies Using Spectrophotometric Titration Method
Electronic absorption spectroscopy is an effective method to determine the binding
mode and intensity of organic molecules with CT-DNA. The intrinsic binding constant Kb for
BTM1 was calculated as 11.18 x 104M−1 which is smaller than that of BTD2 (5.09x 104M−1),
indicating that both compounds were moderate binders to CT-DNA (vide supporting
information, Fig. 9s). There was another typical observation from the UV absorption
spectrum of each compound that there was an increase in absorption for the compounds while
increasing the DNA concentration, i.e., hyperchromic shifts have been observed for the
compounds (Figure 9). The absorption spectrum showed two significant peaks at 230 nm
(hyperchromism with2 nm red-shift) and 309 nm (hyperchromism) in BTM1 and258 nm
(hyperchromism with 2 nm red-shift) in BTD2.
This hyperchromic effect is usually observed when the interactions of the
chromophore (compound) with DNA are electrostatic or partially intercalative. This can
result in the decoiling of DNA helix thereby losing their H-bonds with the neighbour strand.
Therefore, this decoiled DNA single-strand might absorb the ultra-violet light due to the
presence of electrostatic atoms; and thus result in hyperchromocity [62].
3.6. In vitro Antimicrobial Activity
The two benzothiazole compounds, BTM1 and BTD2 were screened against some
microorganisms in order to evaluate their antimicrobial activities. From Table 6, it is clear
that with the minimum inhibitory concentration (500 µg/mL), the compounds could kill the
microorganisms. BTM1’s response against Bacillus subtilis, was vital, and the inhibition was
found as 78% and BTD2 showed 58% when compared with gentamicin (standard drug) and
reported benzothiazole derivatives (50%) [63, 64]. Similarly, in the inhibition study
against Staphylococcus aureus, BTM1 showed higher activity with 96% and BTD2 was

moderately active with 56% when compared to the inhibition with the standard drug and 2-
mercaptobenzothiazole derivatives (50%) [65].
Antifungal screening of BTM1 exhibited moderate activity against Candida
albicans with 45% inhibition when compared with the standard drug amphotericin
B. BTD2 showed no inhibition against Candida albicans. BTM1 showed higher activity
against Aspergillus niger (94%) whereas no activity was found in BTD2 when compared
with the antimicrobial activity of the standard drug and mercaptobenzothiazole derivatives
(46%) [66]. The antimicrobial activity depends upon the concentration of the title compounds
used. As evident from Table 6, inhibition against bacteria and fungi increased with a rise in
the concentration of the compounds. The higher activity of BTM1 could be because of the
lower stability and higher polarity than BTD2 which is confirmed by the DFT studies.
4.Conclusion
In this study, 2-(benzylthio)benzothiazoles (BTM1 and BTD2) were prepared and
characterised by FT-IR, NMR, and SCXRD. SCXRD. This indicated that the benzothiazole
ring was perpendicular to the mesitylene moiety in BTM1. In BTD2, the two benzothiazole
rings adopted a cis conformation with reference to the durene ring and had a symmetrical
arrangement with a slight distortion. The benzothiazole ring was connected in the clockwise
direction in BTM1 and benzothiazole rings were in the counter-clockwise directions in
BTD2. Hirshfeld surface and 2D fingerprint plot analysis displayed the percentage
contributions of weak intermolecular interactions and showed the extent of molecular
association. DFT calculations exposed that BTD2 had higher stability, electron affinity,
ionization potential, and nucleophilicity; and lower dipole moment than BTM1. Mulliken
charges on benzothiazole rings in BTD2 also confirmed the symmetrical arrangement with a
slight distortion.

Molecular docking studies has revealed that the compound BTD2 showed thirteen
hydrophobic interactions with the receptor EGFR tyrosine kinase protein and featured a
higher binding affinity than BTM1 and Iressa (standard drug). Antimicrobial studies revealed
that BTM1 showed good activity against the bacteria (Bacillus subtilis and Staphylococcus
aureus) and fungi (Candida albicans and Aspergillus niger). BTD2 showed moderate activity
against bacteria and no activity against fungi. The presence and importance of weak
interactions like H-bonds (CH…N, sp3-, and sp2- CH…π), π-π stackings, and Van der Waals
forces were confirmed by SCXRD, Hirshfeld surface analysis, DFT, molecular docking,
DNA protection, and anti-microbial studies. The extension of this work is ongoing.
Declaration of competing interests
The authors declare that they have no known competing for financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
Authors (CD, RNA, and RDN) thank Popes‟ College, TN, India for the support of this work.
We thank SAIF, Cochin and MK University, Madurai for spectral studies.
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