Benzothiazole analogues: Synthesis, characterization, MO calculations with PM6 and DFT, in silico studies and in vitro antimalarial as DHFR inhibitors and antimicrobial activities

Article abstract of DOI:10.1016/j.bmc.2017.07.057

Benzothiazole analogues are of interest due to their potential activity against malarial and microbial infections. In search of suitable antimicrobial and antimalarial agents, we report here the synthesis, characterization and biological activities of benzothiazole analogues (J 1-J 10). The molecules were characterized by IR, Mass, ¹H NMR, ¹³C NMR and elemental analysis. The in vitro antimicrobial activity was investigated against pathogenic strains; the results were explained with the help of DFT and PM6 molecular orbital calculations. In vitro cytotoxicity and genotoxicity of the molecules were studied against S. pombe cells. In vitro antimalarial activity was studied. The active compounds J 1, J 2, J 3, J 5 and J 6 were further evaluated for enzyme inhibition efficacy against the receptor Pf-DHFR, computational and in vitro studies were carried out to examine their candidatures as lead dihydrofolate reductase inhibitors.

Full text of DOI:10.1016/j.bmc.2017.07.057

Accepted Manuscript
Benzothiazole analogues: synthesis, characterization, MO calculations with
PM6 and DFT, in silico studies and in vitro antimalarial as DHFR inhibitors and
antimicrobial activities
Sampark S. Thakkar, Parth Thakor, Arabinda Ray, Hiren Doshi, Vasudev R
Thakkar
PII:
S0968-0896(17)31475-X
http://dx.doi.org/10.1016/j.bmc.2017.07.057
BMC 13894
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
18 July 2017
26 July 2017
28 July 2017
Please cite this article as: Thakkar, S.S., Thakor, P., Ray, A., Doshi, H., Thakkar, V.R., Benzothiazole analogues:
synthesis, characterization, MO calculations with PM6 and DFT, in silico studies and in vitro antimalarial as DHFR
inhibitors and antimicrobial activities, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/
j.bmc.2017.07.057
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Benzothiazole analogues: synthesis, characterization, MO calculations with PM6 and
DFT, in silico studies and in vitro antimalarial as DHFR inhibitors and antimicrobial
activities
Sampark S. Thakkara, Parth Thakorb, Arabinda Raya*, Hiren Doshic, Vasudev R Thakkarb
a
Advanced Organic Chemistry Department, P. D. Patel Institute of Applied Sciences,
CHARUSAT, Changa-388421, Gujarat, India
b
P. G. Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar-388120,
Gujarat, India
c Madhav University, Abu Road, Sirohi- 307026, Rajasthan, India
* Corresponding author: Prof. Arabinda Ray, Advanced Organic Chemistry Department, P.
D. Patel Institute of Applied Sciences, CHARUSAT, Changa-388421, Gujarat, India. Tel.:
+91-9426397409, E-mail: arabinda24@yahoo.co.in
1

Highlights
•
•
•
Benzothiazole analogues were synthesized and characterized.
In vitro antimicrobial and antimalarial activities
Molecules were subjected to DFT and PM6 calculations to explain the antibacterial
activity.
•
In silico molecular docking and in vitro DHFR inhibiting assay
2

Abstract
Benzothiazole analogues are of interest due to their potential activity against malarial and
microbial infections. In search of suitable antimicrobial and antimalarial agents, we report
here the synthesis, characterization and biological activities of benzothiazole analogues (J 1-J
1
10). The molecules were characterized by IR, Mass, H NMR, 13C NMR and elemental
analysis. The in vitro antimicrobial activity was investigated against pathogenic strains; the
results were explained with the help of DFT and PM6 molecular orbital calculations. In vitro
cytotoxicity and genotoxicity of the molecules were studied against S. pombe cells. In vitro
antimalarial activity was studied. The active compounds J 1, J 2, J 3, J 5 and J 6 were
further evaluated for enzyme inhibition efficacy against the receptor Pf-DHFR,
computational and in vitro studies were carried out to examine their candidatures as lead
dihydrofolate reductase inhibitors.
Keywords benzothiazole; DFT; PM6; antimicrobial activity; molecular docking; antimalarial
activity; DHFR
1. Introduction
Malaria is one of the most disturbing infectious diseases in the world, due to their high
mortality and morbidity. The protozoan parasite Plasmodium falciparum (P. falciparum) is
responsible for their occurrence. It has been estimated that around 212 million people get
infected in subtropical and tropical countries and 429,000 deaths occur annually by malarial
infection [1]. To add to the problem, malaria parasites have acquired resistance against many
drugs causing serious problem. We do observe today, a dramatic increase in serious
infections due to microorganisms resistant to multiple antimicrobial agents and these have
become a problem of great concern [2, 3]. To overcome the threat of malarial infection, there
is an imperative need for development of new drugs with divergent and unique structures and
possibly with a different mechanism of action from that of the existing drugs. Also, recent
studies have demonstrated that amino acid changes within P. falciparum dihydrofolate
reductase (pf-DHFR) are associated with drug failures [4]. Hence DHFR enzyme has been
shown to be reliable and the best target to design new antimalarial drugs. Thus, the need for
the discovery of new lead structures and novel chemical entities which will act as anti-
malarial and anti-microbial agents becomes more relevant.
Heterocyclic compounds particularly those with oxygen, nitrogen and sulfur atoms have been
identified to have the most comprehensive spectrum of biological activities [5]. Further,
3

benzothiazole are highly important scaffolds in bioorganic and medicinal chemistry with
application in drug discovery. This core has shown numerous biological activities such as
anticancer [6-9], antiHIV [6], antioxidant [10], anticonvulsant [11], trypanocidal agent [12],
antitumor [13-16], antimicrobial [17], COX inhibitor [18], hypoglycemic [19], antidiabetic
[20], antituberculosis [6, 21], antiurease [22] and inhibitor of α-glucosidase [23]. There are
also some well-known drugs such as ethoxzolamide (glaucoma, ulcers and as a diuretic),
riluzole (anti-convulsant), pramipexole (Parkinson's disease), pharminox and phortress (anti-
tumor) having benzothiazole moiety. Because of their various biological activities such as
antimicrobial [24-26], antitubercular [27-29], antiHIV [29], anticancer [29], antitumor [30],
antiinflammatory [31], analgesic [31] and anticonvulsing [32, 33], compounds with Schiff
base moiety are of great interest. Consequently, Schiff bases with benzothiazole moiety are
expected to be good pharmaceutical agents.
The aim of the study is to develop new heterocyclic compounds as therapeutic agents;
accordingly we synthesized and characterized benzothiazole analogues having five member
and six member (benzene) rings. This was done to study the effect of the ring size and
substituent in the ring on the efficacy of the biological activity. The pharmacokinetic
parameters of compounds J 1-J 10 were evaluated. Also, in vitro antimicrobial activity on
different pathogenic bacterial and fungal strains, cytotoxicity and genotoxicity on
Schizosaccharomyces pombe (S. pombe) cells on cellular level were investigated. The fission
yeast S. pombe is an important model organism for the study of eukaryotic molecular and
cellular biology [34]. As eukaryotes, these yeasts can be used to study processes that are
conserved from yeast to humans but are absent from bacteria, such as organelle biogenesis
and cytoskeletal organization or to study mechanisms such as transcription, translation and
DNA replication, in which the eukaryotic components and processes are significantly
different from those of their bacterial counterparts [35]. The data of antimicrobial activity
were correlated with molecular orbital calculations using PM6 and density functional theory
(DFT) methods. The importance of DFT calculation to explain biological activity has been
demonstrated in a recent publication [36]. In addition, the antimalarial activity of compounds
J 1-J 10 were evaluated on P. falciparum strain. In silico molecular docking of active
compounds J 1, J 2, J 3, J 5 and J 6 was performed on wild-type Plasmodium falciparum
dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) complex (PDB ID: 4DPD) and their
in vitro DHFR enzyme inhibition activity was also studied.
4

2. Experimental
All starting materials and reagents were purchased from Sigma-Aldrich, Merck and Loba
Chemie. Thin layer chromatography (TLC) was performed on silica gel G60 F254 (Merck)
plates and eluted with the mobile phase n-hexane: ethyl acetate (70:30 %v/v). Melting points
were recorded on automated melting point system and are uncorrected. IR spectra were
obtained on a Thermo Scientific, Nicolet 6700 spectrophotometer using KBr pellet disk
method. NMR spectra were documented on Bruker 400 MHz using DMSO-d6 as solvent and
TMS as an internal standard. Chemical shifts are reported in parts per million (δ in ppm).
Mass spectra were recorded on Waters Micromass Q-Tof Micro. Elemental analysis was
performed on the Thermo Scientific Flash 2000.
2.1 Synthesis of benzo[d]thiazol-2-amine (2-ABT): Aniline (6.5 g, 69.8 mmol) and
NH4SCN (5.312 g, 69.8 mmol) were dissolved in glacial acetic acid (80 ml) and cooled to
0°C with stirring. Bromine (3.57 ml, 69.8 mmol dissolved in 20 ml of glacial acetic acid) was
added drop wise with stirring to the reaction mixture, while maintaining a temperature
between -3 °C to 0 °C (Scheme 1) [37]. The mixture was kept overnight to allow it to attain
room temperature. A cold solution of 10% NaOH was added with stirring until the pH of the
solution became 11 and the mixture was extracted with ethyl acetate. The organic layer was
washed with a saturated NaHCO3 solution and brine and finally washed with water. The
solvent was evaporated under vacuum. The residue was crystallized using methanol to give 8
g (75%) product. Light brown solid; M.P.: 124 °C; IR (KBr) (cm-1): 685, 741, 1446, 1528,
1
1589, 1644, 3056, 3271, 3396; EIMS (m/z): 151 (M+1); H NMR (DMSO-d6, 400 MHz) δ
(ppm): 6.990 (s, 1H, Ar-H), 7.190 (s, 1H, Ar-H), 7.398 (s, 3H, Ar-H and -NH2), 7.566 (s, 1H,
13
Ar-H); C NMR (DMSO-d6, 100 MHz) δ (ppm): 117.6, 120.4, 120.7, 125.1, 130.9, 152.5,
166.5; Anal Calcd. For C7H6N2S: C, 55.98; H, 4.04; N, 18.65; S, 21.35 %. Found: C, 55.92,
H, 4.02; N, 18.58, S, 21.40 %.
2.2 General Procedure for the synthesis of Schiff bases (J 1-J 10): Different aldehydes (5
mmol) were taken in a 50 ml flask and dissolved in 10 ml DMF, followed by addition of
catalytic amount of acetic acid. 2-aminobenzothiazole (2-ABT) (5 mmol) was added pinch
wise to the solution with stirring (Schemes 2 & 3). The contents of the flask were stirred at
room temperature for 6 h. After stirring, the reaction mixture was poured into cold water. The
precipitate was filtered, washed with cold water and purified by recrystallization from
methanol.
5

N-(benzo[d]thiazol-2-yl)-1-(3-nitrophenyl)methanimine (J 1): (0.900 g, 64%); Light yellow
solid; M.P.: 148 °C; IR (KBr) (cm-1): 696, 761, 780, 1349, 1434, 1532, 1573, 1601, 3085;
1
EIMS (m/z): 284 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.474 (t, 1H, Ar-H),
7.563 (t, 1H, Ar-H), 7.899 (t, 1H, Ar-H), 8.004 (d, 1H, Ar-H), 8.120 (d, 1H, Ar-H), 8.475 (d,
13
1H, Ar-H), 8.543 (d, 1H, Ar-H), 8.905 (s, 1H, Ar-H), 9.399 (s, 1H, Schiff Base); C NMR
(DMSO-d6, 100 MHz) δ (ppm): 122.5, 122.8, 124.1, 125.5, 126.8, 127.3, 130.8, 134.3,
135.6, 135.9, 148.2, 151.2, 165.7, 170.5; Anal Calcd. for C14H9N3O2S: C, 59.35; H, 3.20; N,
14.83; S, 11.32 %. Found: C, 59.42, H, 3.21; N, 14.77, S, 11.34 %.
N-(benzo[d]thiazol-2-yl)-1-(4-chlorophenyl)methanimine (J 2): (0.966 g, 71%); Light yellow
solid; M.P.: 145 °C; IR (KBr) (cm-1): 647, 690, 756, 821, 1434, 1536, 1591, 1616, 3052;
1
EIMS (m/z): 273 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.430 (t, 1H, Ar-H),
7.523 (t, 1H, Ar-H), 7.654 (d, 2H, Ar-H), 7.953 (d, 1H, Ar-H), 8.061 (d, 1H, Ar-H), 8.126 (d,
2H, Ar-H), 9.212 (s, 1H, Schiff Base); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 122.2,
122.6, 125.2, 126.5, 129.2, 131.5, 133.2, 134.2, 138.2, 151.2, 166.0, 170.9; Anal Calcd. for
C14H9ClN2S: C, 61.65; H, 3.33; N, 10.27; S, 11.75 %. Found: C, 61.70, H, 3.34; N, 10.29, S,
11.71 %.
N-(benzo[d]thiazol-2-yl)-1-(2-nitrophenyl)methanimine (J 3): (0.877 g, 62%); Light yellow
solid; M.P.: 150 °C; IR (KBr) (cm-1): 693, 748, 1347, 1442, 1530, 1597, 1628, 3051; EIMS
1
(m/z): 284 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.053 (t, 1H, Ar-H), 7.226 (t,
1H, Ar-H), 7.414 (t, 1H, Ar-H), 7.598 (t, 1H, Ar-H), 7.659 (d, 1H, Ar-H), 7.752 (d, 1H, Ar-
H), 7.859 (d, 1H, Ar-H), 7.988 (s, 1H, Ar-H), 9.080 (s, 1H, Schiff Base); 13C NMR (DMSO-
d6, 100 MHz) δ (ppm): 122.5, 122.9, 124.1, 125.4, 126.6, 128.2, 130.9, 133.9, 134.2, 134.4,
148.6, 151.2, 165.7, 170.9; Anal Calcd. for C14H9N3O2S: C, 59.35; H, 3.20; N, 14.83; S,
11.32 %. Found: C, 59.29, H, 3.19; N, 14.89, S, 11.30 %.
N-(benzo[d]thiazol-2-yl)-1-(3,4-dichlorophenyl)methanimine (J 4): (0.997 g, 65%); Light
yellow solid; M.P.: 154 °C; IR (KBr) (cm-1): 651, 693, 759, 875, 1435, 1549, 1581, 1611,
1
3044; EIMS (m/z): 307 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.410 (t, 1H, Ar-
H), 7.521 (t, 1H, Ar-H), 7.801 (d, 1H, Ar-H), 7.952 (d, 1H, Ar-H), 8.043 (m, 2H, Ar-H),
13
8.292 (d, 1H, Ar-H), 9.208 (s, 1H, Schiff Base); C NMR (DMSO-d6, 100 MHz) δ (ppm):
122.1, 122.7, 125.2, 126.5, 129.3, 131.23, 131.27, 132.2, 134.3 134.8, 136.0, 151.1, 164.6,
170.4; Anal Calcd. for C14H8Cl2N2S: C, 54.74; H, 2.63; N, 9.12; S, 10.44 %. Found: C, 54.75,
H, 2.62; N, 9.16, S, 10.47 %.
6

N-(benzo[d]thiazol-2-yl)-1-(4-bromophenyl)methanimine (J 5): (1.093 g, 69%); Light yellow
solid; M.P.: 143 °C; IR (KBr) (cm-1): 636, 687, 754, 822, 1435, 1561, 1583, 1617, 3059;
1
EIMS (m/z): 318 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.408 (t, 1H, Ar-H),
7.501 (t, 1H, Ar-H), 7.744 (d, 2H, Ar-H), 7.934 (q, 2H, Ar-H), 8.019 (d, 2H, Ar-H), 9.159 (s,
1H, Schiff Base); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 121.8, 122.5, 125.0, 126.3,
127.4, 131.4, 132.0, 133.4, 134.2, 151.2, 165.5, 170.8; Anal Calcd. for C14H9BrN2S: C,
53.01; H, 2.86; N, 8.83; S, 10.11 %. Found: C, 53.09, H, 2.87; N, 8.79, S, 10.07 %.
4-((benzo[d]thiazol-2-ylimino)methyl)phenol (J 6): (0.914 g, 72%); Light yellow solid; M.P.:
149 °C; IR (KBr) (cm-1): 690, 751, 837, 1310, 1438, 1520, 1575, 1604, 3028, 3427; EIMS
1
(m/z): 255 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 6.955 (d, 2H, Ar-H), 7.362 (t,
1H, Ar-H), 7.465 (t, 1H, Ar-H), 7.880 (d, 1H, Ar-H), 7.942 (q, 3H, Ar-H), 8.990 (s, 1H,
13
Schiff Base), 10.410 (b, 1H, -OH); C NMR (DMSO-d6, 100 MHz) δ (ppm): 116.0, 121.7,
122.1, 124.5, 125.6, 126.1, 132.4, 133.7, 151.3, 162.8, 165.9, 171.8; Anal Calcd. for
C14H10N2OS: C, 66.12; H, 3.96; N, 11.02; S, 12.61 %. Found: C, 66.10, H, 3.97; N, 10.99, S,
12.62 %.
N-(benzo[d]thiazol-2-yl)-1-(4-fluorophenyl)methanimine (J 7): (0.845 g, 66%); Light yellow
solid; M.P.: 142 °C; IR (KBr) (cm-1): 670, 757, 819, 1066, 1437, 1511, 1598, 1617, 3058;
1
EIMS (m/z): 257 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.391 (m, 4H, Ar-H),
7.943 (q, 2H, Ar-H), 8.172 (s, 2H, Ar-H), 9.194 (s, 1H, Schiff Base); 13C NMR (DMSO-d6,
100 MHz) δ (ppm): 122.1, 122.5, 125.0, 126.4, 128.7, 131.1, 132.6, 133.3, 134.1, 151.2,
165.7, 171.1; Anal Calcd. for C14H9FN2S: C, 65.61; H, 3.54; N, 10.93; S, 12.51 %. Found: C,
65.7, H, 3.53; N, 10.88, S, 12.49 %.
N-(benzo[d]thiazol-2-yl)-1-(furan-2-yl)methanimine (J 8): (0.718 g, 63%); Light yellow
solid; M.P.: 123 °C; IR (KBr) (cm-1): 678, 738, 1454, 1447, 1526, 1562, 1644, 2856, 2924,
1
3060; EIMS (m/z): 229 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 6.971 (t, 1H, Ar-
H), 7.009 (t, 1H, Ar-H), 7.226 (t, 1H, Ar-H), 7.349 (d, 1H, Ar-H), 7.439 (d, 1H, Ar-H), 7.555
13
(d, 1H, Ar-H), 7.609 (d, 1H, Ar-H), 9.346 (s, 1H, Schiff Base); C NMR (DMSO-d6, 100
MHz) δ (ppm): 120.3, 121.3, 122.2, 125.0, 126.3, 128.4, 134.0, 136.9, 140.7, 151.6, 166.3,
170.9; Anal Calcd. for C12H8N2OS: C, 63.14; H, 3.53; N, 12.27; S, 14.04 %. Found: C, 63.22,
H, 3.52; N, 12.22, S, 14.00 %.
7

N-(benzo[d]thiazol-2-yl)-1-(thiophen-2-yl)methanimine (J 9): (0.732 g, 60%); Light yellow
solid; M.P.: 120 °C; IR (KBr) (cm-1): 686, 740, 1446, 1454, 1529, 1577, 1642, 2852, 2923,
1
3056; EIMS (m/z): 245 (M+1); H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.003 (t, 1H, Ar-
H), 7.198 (t, 1H, Ar-H), 7.250 (t, 1H, Ar-H), 7.492 (d, 1H, Ar-H), 7.527 (d, 1H, Ar-H), 7.838
13
(d, 1H, Ar-H), 7.894 (d, 1H, Ar-H), 9.254 (s, 1H, Schiff Base); C NMR (DMSO-d6, 100
MHz) δ (ppm): 120.8, 121.7, 122.5, 125.2, 126.3, 128.6, 134.3, 136.9, 140.7, 151.3, 166.6,
170.9; Anal Calcd. for C12H8N2S2: C, 58.99; H, 3.30; N, 11.47; S, 26.24 %. Found: C, 58.90,
H, 3.31; N, 11.42, S, 26.33 %.
N-(benzo[d]thiazol-2-yl)-1-(1H-pyrrol-2-yl)methanimine (J 10): (0.670 g, 59%); Light
yellow solid; M.P.: 121 °C; IR (KBr) (cm-1) νmax: 664, 746, 1439, 1453, 1534, 1564, 1650,
2852, 2922, 3064, 3246; EIMS (m/z): 228 (M+1); 1H NMR (DMSO-d6, 400 MHz) δ (ppm):
6.997 (t, 1H, Ar-H), 7.109 (t, 1H, Ar-H), 7.149 (t, 1H, Ar-H), 7.385 (d, 1H, Ar-H), 7.552 (d,
1H, Ar-H), 7.733 (d, 1H, Ar-H), 7.805 (d, 1H, Ar-H), 9.582 (s, 1H, Schiff Base), 10.337 (b,
1H, -NH pyrrole); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 120.5, 121.7, 121.9, 125.1,
125.5, 128.6, 134.2, 137.0, 140.5, 152.0, 166.3, 171.1;Anal Calcd. for C12H9N3S: C, 63.41;
H, 3.99; N, 18.49; S, 14.11 %. Found: C, 63.37, H, 3.98; N, 18.41, S, 14.16 %.
3. Results and discussion
3.1 Chemistry
The IR data of the molecules were compared with the standard IR frequencies [38]. The
molecule 2-ABT showed two peaks at 3396 and 3271 cm-1 due to asymmetric and symmetric
stretching of -NH2 group. The bending of -NH2 group was assigned at 1644 cm-1 in this
molecule. All the molecules synthesized showed stretching of -C-S, -C=N and -CH
(aromatic) at ~690, ~1580 and ~3050 cm-1 respectively for benzothiazole ring. The
compounds J 1 and J 3 showed two bands near ~1532 and ~1348 cm-1 due to asymmetric and
symmetric stretching of -NO2 group. The peak at ~650 cm-1 in J 2 and J 4 was attributed to
stretching of C-Cl group. The molecule J 6 showed a broad peak at 3427 cm-1 due to -OH
stretching.
The 1H NMR resonances due to one aromatic hydrogen and -NH2 moiety in 2-ABT appeared
at 7.398 ppm as singlet corresponding to 3H. For compounds (J 1-J 10), the aromatic protons
appeared between ~7.0-8.9 ppm. The new peak at ~9.0 (singlet) in the Schiff bases is due to -
CH in -N=CH-. The 13C NMR of compounds (2-ABT and J 1-J 10) showed peaks in the
range of 120-148 ppm due to the presence of aromatic C-atoms. The C-atom of -N=CH-
8

moiety in Schiff bases was seen at ~170 ppm for molecules J 1-J 10. The IR, MS, NMR and
elemental analysis data for all the compounds are reported in the experimental section.
Scheme 1. Method for the preparation of benzo[d]thiazol-2-amine
Scheme 2. Method for the preparation of Schiff Base
Scheme 3. Method for the preparation of Schiff Base
3.2 In silico pharmacokinetic evaluation
The pharmacokinetic parameters are important for an orally administered drug. This phase
includes absorption from G. I. track into the blood supply. There are many other factors such
as Caco2, % HIA etc. that affect drug’s survival as it travels to the target. The in silico study
to evaluate pharmacokinetic parameters was carried out by VlifeMDS 4.6 and ADMET
software and the data obtained are presented in Table 1 and Table 2 respectively.
9

Table 1. Evaluation of pharmacokinetic parameters of molecules J 1-J 10 using VlifeMDS
4.6
Compound Mol.
logSf
PSAg Volume
HBAb HBDc RotBd ClogPe
ID(s)
J 1
Wt.a
log(mol/l) (°A2) (°A3)
283.04
272.02
283.04
305.98
315.97
254.05
256.05
228.04
244.01
227.05
5
3
5
3
3
4
3
4
4
3
0
0
0
0
0
1
0
0
0
1
3
2
3
2
2
2
2
2
2
2
3.89
4.88
3.77
5.47
5.02
3.91
4.44
3.39
4.04
3.12
-5.05
-5.59
-4.74
-6.22
-5.78
-4.49
-5.15
-3.76
-4.29
-3.67
56.39 245.16
18.13 236.64
56.09 244.54
18.13 251.01
18.13 241.31
35.74 230.00
18.13 225.37
25.88 205.42
19.15 217.66
28.69 207.54
J 2
J 3
J 4
J 5
J 6
J 7
J 8
J 9
J 10
a = Molecular Weight ≤ 500 (g/mol) [39]; b = Hydrogen Bond Acceptor ≤ 10 [39]; c =
Hydrogen Bond Donor ≤ 5 [39]; d = Rotatable Bonds ≤ 10 [39]; e = ClogP ≤ 5 [40]; f; Water
Solubility range -0.5 to -6.5 (mol/l) [40]; g = Polar Surface Area ≤ 140°A2 [40].
The values of pharmacokinetic parameters (Table 1) like molecular weight, HBA and RotB
suggested that none of the molecules J 1-J 10 violate Lipinski's rule [39, 40], as all calculated
parameters were within the expected threshold values. The logP value for molecules J 1-J 3
and J 6-J 10 was within the range of 3.12-4.88 except for the molecules J 4 (5.47) and J 5
(5.02). Additionally, PSA and water solubility (logS) parameters are important in the
membrane penetration, absorption and distribution of drugs respectively; the value of logS
was in the acceptable range (logS = -6.22 to -3.67) and the PSA value for all the molecules J
1-J 10 synthesized was in the range of ≤ 140°A2.
10

Table 2. Evaluation of pharmacokinetic parameters of molecules J 1-J 10 using ADMET
Compound
ID(s)
J 1
Caco2
MDCK
(nm/sec)
1.5174
1.3537
2.1190
0.3998
0.0689
3.6104
2.2352
244.377
6.3615
257.966
hERG
BBB
% HIA
%PPB
(nm/sec)
inhibition
0.0310 16.182
0.5621 58.351
1.4523 8.202
0.3938 57.990
0.5788 56.909
0.9333 46.773
0.4458 55.262
2.7176 31.348
2.7339 26.827
2.1905 23.097
98.472
97.395
98.472
97.605
97.530
96.310
97.412
98.609
99.030
92.714
91.506
91.133
89.012
92.601
94.031
89.167
90.462
92.662
94.580
91.234
Low Risk
Medium Risk
Low Risk
J 2
J 3
Medium Risk
Medium Risk
Medium Risk
Medium Risk
Medium Risk
Medium Risk
Medium Risk
J 4
J 5
J 6
J 7
J 8
J 9
J 10
BBB (Blood Brain Barrier) [41]: High absorption CNS >2.0, Middle absorption CNS 2.0-0.1,
Low absorption to CNS <0.1; Caco2 [41]: High permeability >70, Middle permeability 4-70,
Low permeability <4; %HIA (Human Intestinal Absorbance) [41]: Well absorbed
compounds 70-100%, Moderately absorbed compounds 20-70%, Poorly absorbed compounds
0-20%; %PPB (Plasma Protein Binding) [42]: Strongly Bound >90%, Weakly Bound <90%,
MDCK [42]: Higher permeability >500, Medium Permeability 25-500, lower permeability
<25.
The parameters obtained from ADMET were compared with those reported for comparable
molecules [41, 42]. The low value for BBB in molecule J 1 suggested low permeability of
this molecule to CNS. The molecules J 3, J 6, J 9 and J 10 have a high value of BBB
indicating more ability of these molecules to cross blood brain barrier and consequently
greater transport of them to CNS.
The high %HIA values suggested very well absorption of the molecules J 1-J 10 in the
intestine. All the compounds had good Caco2 permeability, except J 1 and J 3. The higher
values of %PPB implied that the compounds J 1-J 10 can bind strongly to plasma protein.
Excretion of the drugs by kidney is represented by the values of MDCK. The molecules J 1-J
3, J 6, J 7 and J 9 had reasonably good MDCK permeability, while J 8 and J 10 had very
high permeability. hERG is best known for its contribution to the electrical activity of the
11

heart that coordinates the heart's beating. In the present investigation, the molecules J 1 and J
3 had low hERG inhibition risk, while J 2 and J 4-J 10 had moderate risk.
3.3 In vitro antimicrobial activity
The compounds synthesized appeared to be antibacterial agents and did not show any
significant activity against fungi. Here we attempted to explain the antibacterial activity of
these compounds with their ability to accept electrons. Since the energy of the LUMO can
guide about the relative ability of the molecules to be electron acceptors, we attempted to
correlate the antibacterial activity with the energy of the LUMOs.
Table 3. Energy of HOMO and LUMO from Molecular Orbital Calculations using PM6 and
DFT
Energy (PM6) eV Energy (DFT) eV
Compound
ID(s)
HOMO LUMO HOMO LUMO
J 1
J 2
J 3
J 4
J 5
J 6
J 7
J 8
J 9
J 10
-9.2275 -1.7895 -6.6447 -3.2363
-9.0649 -1.5070 -6.3717 -2.7532
-9,0930 -1.7842 -6.5501 -3.3173
-9.1400 -1.6448 -6.4842 -2.8875
-9.0771 -1.5716 -6.3700 -2.7670
-8.8940 -1.3205 -6.0874 -2.4455
-9.0493 -1.4431 -6.3480 -2.6660
-9.0094 -1.4734 -6.1493 -2.5709
-8.9607 -1.4954 -6.1743 -2.6625
-8.8457 -1.3149 -5.9297 -2.3660
The results of both PM6 and DFT calculations (Table 3) show that the molecules J 1 and J 3
have the most stable LUMOs, indicating that these are better electron acceptors. The
activities of J 1 and J 3 against certain bacterial strains were encouraging (Table 4).
Interestingly, as per DFT calculation J 3 is a slightly better electron acceptor than J 1.
12

Table 4. In vitro antimicrobial activity of compounds J 1-J 10 (MICs, mM)
Gram-positive bacteria Gram-negative bacteria Fungi
Compound
S.A.
S.P.
E.C.
P.A.
C.A.
MTCC
227
A.C.
MTCC
1323
0.883
1.838
NA
ID(s)
MTCC
96
MTCC
442
MTCC
443
MTCC
1688
0.221
0.460
0.707
0.409
0.396
0.394
0.976
0.877
0.410
1.101
0.286
0.155
0.075
0.031
NT
0.707
0.460
0.353
0.409
0.791
0.984
0.244
0.877
0.410
0.551
0.716
0.155
0.151
0.031
NT
0.883
0.368
0.221
0.327
0.633
1.968
0.391
0.439
0.410
0.440
0.286
0.155
0.151
0.031
NT
0.883
0.735
0.442
0.409
0.198
0.394
0.781
0.548
0.256
0.881
0.286
0.155
0.075
0.031
NT
1.767
3.676
3.533
NA
J 1
J 2
J 3
J 4
J 5
J 6
J 7
J 8
J 9
J 10
A
1.634
NA
NA
NA
NA
1.953
4.385
4.098
2.202
NT
1.953
NA
NA
4.404
NT
NT
NT
B
NT
NT
C
NT
NT
D
0.108
1.420
0.108
0.284
E
NT
NT
NT
NT
F
S.A.: Staphylococcus aureus; S.P.: Streptococcus pyogenes; E.C.: Escherichia coli; P.A.:
Pseudomonas aeruginosa; C.A.: Candida albicans; A.C.: Aspergillus clavatus; A:
Ampicillin; B: Chloramphenicol; C: Ciprofloxacin; D: Norfloxacin; E: Nystatin; F:
Greseofulvin; NA: Not active; NT: Not tested
The overall antibacterial activity of J 5 is poor in comparison to J 3, although against
bacterial strain E.C., J 5 is more active than J 3. In comparison to J 3, J 5 is a poor electron
acceptor (Table 3). It is interesting to note here that the highest LUMO electron density in J
5 (0.4499 by PM6 and 0.4665 by DFT) is more than the same in J 3 (0.3010 by PM6 and
0.2078 by DFT). The molecules J 6 and J 10 have less stable LUMO and hence are poor
acceptor of electrons. Of these two molecules J 6 is a better electron acceptor and
consequently is a slightly better antibacterial agent than J 10 (Table 3).
13

It may be instructive to compare activities of molecules J 6 and J 7. The latter is a better
acceptor than J 6 and consequently the overall antibacterial propensity of J 7 is better than
that in J 6. The molecule J 10 has the lowest antibacterial activity in this series. The least
activity of J 10 is traced to its highest LUMO energy that makes it a poor acceptor of
electrons.
3.4 Cytotoxicity study
The cytotoxicity of compounds (J 1-J 10) was tested using bioassay of S. pombe cells at the
cellular level. The results are shown in Table 5. It can be inferred from the results that the
toxic nature of the compounds (J 1-J 10) caused cell death. The toxicity was found to vary
with the type of substituent present and concentrations of compounds (J 1-J 10). It has been
observed that cytotoxicity increases with increasing the concentration of compounds (J 1-J
10).
Table 5. Effect of compounds on percentage cell viability of S. pombe cells at different
concentrations with standard deviation for three independent experiments
Percentage cell viability at different concentrations
Compound
ID (s)
2 µg/ml
88.67
0.58
4 µg/ml
84.00
1.00
6 µg/ml
79.33
0.58
8 µg/ml
74.67
1.53
10 µg/ml
70.33
0.58
-
-
-
-
-
-
-
-
J 1
J 2
J 3
J 4
J 5
J 6
J 7
J 8
89.33
0.58
87.00
1.00
82.67
0.58
78.00
1.00
75.33
0.58
84.33
0.58
82.00
1.53
79.67
0.58
77.00
1.00
72.33
0.58
89.00
1.00
84.33
0.58
81.00
1.00
77.00
1.00
73.33
0.58
77.33
0.58
73.33
0.58
72.33
0.58
68.33
0.58
66.33
0.58
87.00
1.00
75.00
1.00
69.33
1.15
68.33
0.58
65.33
0.58
86.33
1.53
81.67
0.58
73.00
1.00
72.33
1.53
71.67
0.58
78.67
1.53
73.00
1.00
64.00
1.00
62.33
0.58
61.67
0.58
14

86.00
1.00
81.67
0.58
80.67
0.58
73.00
1.00
67.00
1.00
-
-
J 9
87.00
1.00
82.33
0.58
74.33
0.58
71.67
0.58
69.67
0.58
J 10
94.67
1.53
-
-
-
-
-
-
-
-
-
-
Untreated
DMSO
93.67
1.53
The compound J 8 (R1 = -O) showed maximum percentage cell viability (61.67) at 10 µg/ml
concentration, while the compounds J 5 (R = 4-Br), J 6 (R = 4-OH), J 9 (R1 = -S) and J 10
(R1 = -NH) have almost similar percentage cell viability (~66) at 10 µg/ml concentration
(Table 5). The molecules J 1 (R = 3-NO2), J 3 (R = 2-NO2), J 4 (R = 3, 4-di Cl) and J 7 (R =
4-F) exhibited similar cell viability (~72) at 10 µg/ml concentration. The cell viability results
of compound J 2 (R = 4-Cl) suggested that it was least effective amongst all (Table 5).
Herewith, it may be concluded from the results that in general compounds with five member
ring attached to benzothiazole moiety were more effective than those of six member ring
(benzene). However it is to be noted that J 5 and J 6 having respectively 4-Br and 4-OH
substituted benzene ring were also effective.
3.5 Genotoxicity study
Results of cytotoxicity prompted us to find out whether compounds (J 1-J 10) have any
effect on the integrity of DNA. The effect of compounds (J 1-J 10) on the DNA integrity of
S. pombe cells was carried out by the isolation of treated and untreated S. pombe cells and
subjected them to electrophoresis on 1% agarose gel followed by visualization. DNA damage
was found as smear in benzothiazole analogues (J 1-J 10) treated S. pombe cells while, in
untreated S. pombe cells it was observed as an intact band without smearing (Fig. 1). The
results showed that compounds (J 1-J 10) affected the integrity of DNA of S. pombe cells.
Smearing of DNA suggested that the damage has occurred due to the toxic nature of the
compounds. This result is in agreement with the results of cytotoxicity that benzothiazole
derivatives (J 1-J 10) have entered into the cell and affected the integrity of DNA.
15

Fig. 1 Effect of the compounds (J 1-J 10) on the integrity of DNA isolated from S. Pombe
cells
3.6 In vitro antimalarial activity
The compounds J 1-J 10, Chloroquine and Pyrimethamine were evaluated for their
antimalarial screening against P. falciparum strain. All the experiments were performed in
duplicate and a mean value of IC50 is reported in Table 6.
Table 6. In vitro antimalarial activity of benzothiazole analogues (J 1-J 10) (IC50, µM)
Compound ID(s)
IC50 (µM)
J 1
0.201 0.032
0.353 0.065
0.396 0.051
0.980 0.076
0.288 0.021
0.386 0.097
1.484 0.111
1.864 0.199
1.016 0.099
1.907 0.165
0.063 0.009
1.005 0.053
J 2
J 3
J 4
J 5
J 6
J 7
J 8
J 9
J 10
Chloroquine
Pyrimethamine
16

The compound J 1 having -NO2 at m-position in benzene ring was found to be the most
effective against P. falciparum strain (Table 6). Also, the compounds J 2 (R = 4-Cl), J 3 (R
= 2-Cl), J 5 (R = 4-Br) and J 6 (R = 4-OH) required quite less concentration for treating P.
falciparum strain. On the other hand, compounds J 4 (R = 3, 4- diCl), J 7 (R = 4-F), J 8 (R1 =
-O) and J 9 (R1 = -S) and J 10 (R1 = -NH) had IC 50 values higher than that of standard drugs
(Table 6).
3.7 In silico docking study
To rationalize the potency of the active compounds (J 1, J 2, J 3, J 5, J 6) as antimalarial
agents, these compounds were docked against P. falciparum dihydrofolate reductase (PDB
ID: 4DPD) obtained from protein data bank (RCSB). The binding energies obtained for
ligands (J 1, J 2, J 3, J 5, J 6) and standard drugs Chloroquine & Pyrimethamine are
presented in Table 7.
Table 7. Docking results of benzothiazole analogues and standard drugs against P.
falciparum (PDB ID: 4DPD)
Binding
Energy
Intermol
Energy
Internal
Energy
Torsional Unbound
Energy Energy
Compound
ID(s)
(kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)
-7.79
-7.36
-7.23
-7.36
-7.16
-4.24
-6.10
-8.69
-7.96
-8.12
-7.96
-8.05
-6.63
-7.00
-0.21
-0.17
-0.32
-0.18
-0.14
-1.06
-0.26
0.89
0.60
0.89
0.60
0.89
2.39
0.89
-0.21
-0.17
-0.32
-0.18
-0.14
-1.06
-0.26
J 1
J 2
J 3
J 5
J 6
Chloroquine
Pyrimethamine
A potential interaction was observed between the active molecules and the Pf-DHFR enzyme.
The 3D diagrams (Fig. 2a-8a) showed the binding sites of all the ligands within the receptor
4DPD. The molecules interacted with the active sites (amino acids) of receptor 4DPD
through H-bond, van der Waals interaction, π-anion, π-donor H-bond, π-sulphur, C-H bond,
π-sigma, amide-π-stacked, π- lone pair and many more (Fig. 2b-8b). As shown in Table 7,
binding energies of the molecules against the active sites indicated higher affinity of the
molecules to bind with the protein leading to its inhibition. The binding energies of the
17

molecules (J 1, J 2, J 3, J 5, J 6) were found to be ranging from -7.16 to -7.79 kcal/mol and
were better compared to standard drugs Chloroquine and Pyrimethamine (Table 7). The O-
atom of -NO2 group in J 1 interacted with active sites of enzyme by attractive charges with
LYS A:601 and LYS A:373 at distances of 3.43 °A and 2.76 °A respectively (Fig. 2b). The
N-atom of -NO2 showed unfavourable type of bonding with these amino acids. The S-atom of
benzothiazole ring formed π-sulphur bond with PHE A:32 (5.83°A). Additionally, this ring
formed π-sigma bond with VAL A:31 at distance of 3.93 °A (Fig. 2b). As shown in Fig. 3b,
the benzothiazole ring, aromatic ring and Cl-atom in J 2 interacted with enzyme by alkyl and
π-alkyl type of bonding with VAL B:195, PHE B:58 and ILE B:14 at different distances.
Moreover these rings formed π-sigma bond with LEU B:40 (3.96 °A & 3.89 °A) and ALA
B:16 (3.90 °A). Amide-π-stacked type of bonding observed between benzothiazole ring of J
3 and HIS B:308 (3.85 °A & 3.55 °A) (Fig. 4b). Conventional H-bond was formed between
O-atom of -NO2 group in J 3 and ASN B:303 at 3.07 °A. In ligand J 5, aromatic ring
interacted with receptor by forming π-alkyl, alkyl and π-π T shaped type of bonds with ALA
B:16 (4.10 °A), ILE B:14 (5.00 °A) and PHE B:18 (4.41 °A) respectively, while the
benzothiazole ring formed π-donor H-bond with SER B:108 (3.59 °A) and π-alkyl bond with
LEU B:40 (5.02 °A) (Fig. 5b). The O-atom of -OH group formed two conventional hydrogen
bonds with GLY B:44 and ASP B:194 at distances of 2.85 °A and 1.88 °A respectively. Also,
van der Waals, π-sigma & π-alkyl type of interactions were indicated with different amino
acids of chain B of receptor with J 6 (Fig. 6b). The Cl-atom in Chloroquine (Fig. 7b)
interacted through conventional H-bond with THR A:391 at distance 2.90 °A. Furthermore
this Cl-atom formed π-alkyl bond with PHE A:42 (4.95 °A), TYR A:441 (5.49 °A) & TYR
A:448 (4.59 °A). In the case of Pyrimethamine, van der Waals, carbon-hydrogen bond, π-
alkyl, alkyl etc. type of interactions were observed between different amino acids present in
chain A of 4DPD receptor and ligand as shown in Fig. 8b. The results of docking study
indicated that the ligand molecules are selective DHFR inhibitors.
18

(b)
(a)
Fig 2. (a) 3D model of ligand J 1 bonded with active site of 4DPD receptor. (b) 2D model of
interaction between amino acid residues and J 1 with types of bond and bond lengths.
(a) (b)
Fig 3. (a) 3D model of ligand J 2 bonded with active site of 4DPD receptor. (b) 2D model of
interaction between amino acid residues and J 2 with types of bond and bond lengths.
(b)
(a)
Fig 4. (a) 3D model of ligand J 3 bonded with active site of 4DPD receptor. (b) 2D model of
interaction between amino acid residues and J 3 with types of bond and bond lengths.
19

(b)
(a)
Fig 5. (a) 3D model of ligand J 5 bonded with active site of 4DPD receptor. (b) 2D model of
interaction between amino acid residues and J 5 with types of bond and bond lengths.
(a) (b)
Fig 6. (a) 3D model of ligand J 6 bonded with active site of 4DPD receptor. (b) 2D model of
interaction between amino acid residues and J 6 with types of bond and bond lengths.
(b)
(a)
Fig 7. (a) 3D model of ligand Chloroquine bonded with active site of 4DPD receptor. (b) 2D
model of interaction between amino acid residues and Chloroquine with types of bond and
bond lengths.
20

(b)
(a)
Fig 8. (a) 3D model of ligand Pyrimethamine bonded with active site of 4DPD receptor. (b)
2D model of interaction between amino acid residues and Pyrimethamine with types of
bond and bond lengths.
3.8 DHFR Inhibition assay:
The potent compounds (J 1, J 2, J 3, J 5 & J 6) and standard drugs (Chloroquine &
Pyrimethamine) were found to be active against P. falciparum strain and were further
evaluated for inhibitory efficacy against bovine liver DHFR enzyme. The average IC50 (µM)
values are presented with standard deviation for three independent experiments in Table 8.
Table 8. DHFR enzyme inhibition assay
Compound ID(s)
IC50 (µM)
0.0312 0.008
0.0415 0.004
0.0531 0.012
0.0455 0.009
0.0512 0.003
0.0301 0.005
J 1
J 2
J 3
J 5
J 6
Chloroquine
Pyrimethamine 0.1007 0.012
The IC50 values of J 1, J 2, J 3, J 5 & J 6 indicated that all compounds were potent DHFR
inhibitors when compared to Pyrimethamine (Table 8). The compound containing m-
nitrobenzene (J 1; IC50 = 0.0312 0.008 µM) was found to be most potent inhibitor, while
the compounds J 2 (R = 4-Cl) and J 5 (R = 4-Br) showed moderate potency. The compounds
J 3 (R = 2-NO2) and J 6 (R = 4-OH) had poor inhibitory property compared to other
compounds (J 1, J 2 & J 5) and Chloroquine, but were still better inhibitor of DHFR
enzyme than Pyrimethamine (Table 8). The results suggested that the analogues were active
antimalarial compounds inhibiting dihydrofolate reductase enzyme.
21

4. Conclusion
Analogues of benzothiazole have been synthesized and characterized. To avoid late stage
failure, it is important to study the preliminary pharmacokinetic parameters. The results of in
silico pharmacokinetic data suggested that all the molecules might be considered as drug. So
in vitro antimicrobial, cytotoxicity and genotoxicity on S. pombe cells and antimalarial on P.
falciparum strain of these analogues were investigated. It was found that the antibacterial
activity could be correlated to energies of the LUMO and maximum LUMO electron density
derived from DFT and PM6 calculations. All the compounds possessed moderate to good
inhibitory potency against bacterial strains and did not show any antifungal activities. The
compounds J 5, J 6 and J 8-J 10 were found to possess maximum toxicity against S. pombe
cells at cellular level. All the compounds J 1-J 10 showed smearing type pattern on agarose
gel due to toxic nature of these compounds. The compounds J 1, J 2, J 3, J 5 and J 6 where
substituent is attached to phenyl ring were found to be most potent against P. falciparum
strain. The in silico molecular docking study of these active compounds were performed and
all the compounds showed good interaction with the binding sites of Pf-DHFR with great
ease. The antimalarial efficacy was further proved by in vitro DHFR enzyme inhibition study.
All the compounds showed potent DHFR inhibitory activity with IC50 in the range of 0.0312
to 0.0531 µM. This study could identify new antibacterial and antimalarial agents which
could be used as lead molecules for further research and development of antibacterial and
antimalarial agents.
Acknowledgement The authors thank the authorities of Charotar University of Science &
Technology (CHARUSAT), Changa, Gujarat, India for providing the facilities to carry out
this investigation. They also thank Dr. Devendra Tiwari, School of Chemistry, University of
Bristol, U.K. for his help in molecular orbital study. Mr. Parth Thakor is thankful to DST
INSPIRE program for the fellowship.
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